WO2004098515A2 - Nucleic acids and corresponding proteins entitled 109p1d4 useful in treatment and detection of cancer - Google Patents

Nucleic acids and corresponding proteins entitled 109p1d4 useful in treatment and detection of cancer Download PDF

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WO2004098515A2
WO2004098515A2 PCT/US2004/013568 US2004013568W WO2004098515A2 WO 2004098515 A2 WO2004098515 A2 WO 2004098515A2 US 2004013568 W US2004013568 W US 2004013568W WO 2004098515 A2 WO2004098515 A2 WO 2004098515A2
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protein
cancer
amino acid
cell
peptide
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PCT/US2004/013568
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French (fr)
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WO2004098515A3 (en
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Arthur B. Raitano
Pia M. Challita-Eid
Wangmao Ge
Juan J. Perez-Villar
Steven B. Kanner
Aya Jakobovits
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Agensys, Inc.
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Priority to AU2004235755A priority Critical patent/AU2004235755A1/en
Priority to JP2006514206A priority patent/JP2007525183A/en
Priority to EP04760669A priority patent/EP1622571A4/en
Priority to CA2522994A priority patent/CA2522994C/en
Publication of WO2004098515A2 publication Critical patent/WO2004098515A2/en
Priority to AU2008212020A priority patent/AU2008212020B2/en
Publication of WO2004098515A3 publication Critical patent/WO2004098515A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants

Definitions

  • the invention described herein relates to genes and their encoded proteins, termed 109P1D4 and variants thereof, expressed in certain cancers, and to diagnostic and therapeutic methods and compositions useful in the management of cancers that express 109P1D4.
  • Cancer is the second leading cause of human death next to coronary disease. Worldwide, millions of people die from cancer every year. In the United States alone, as reported by the American Cancer Society, cancer causes the death of well over a half-million people annually, with over 1.2 million new cases diagnosed per year. While deaths from heart disease have been declining significantly, those resulting from cancer generally are on the rise. In the early part of the next century, cancer is predicted to become the leading cause of death.
  • carcinomas of the lung, prostate, breast, colon, pancreas, and ovary represent the primary causes of cancer death. These and virtually all other carcinomas share a common lethal feature. With very few exceptions, metastatic disease from a carcinoma is fatal. Moreover, even for those cancer patients who initially survive their primary cancers, common experience has shown that their lives are dramatically altered. Many cancer patients experience strong anxieties driven by the awareness of the potential for recurrence or treatment failure. Many cancer patients experience physical debilitations following treatment. Furthermore, many cancer patients experience a recurrence.
  • prostate cancer is the fourth most prevalent cancer in men. In North America and Northern Europe, it is by far the most common cancer in males and is the second leading cause of cancer death in men. In the United States alone, well over 30,000 men die annually of this disease - second only to lung cancer. Despite the magnitude of these figures, there is still no effective treatment for metastatic prostate cancer. Surgical prostatectomy, radiation therapy, hormone ablation therapy, surgical castration and chemotherapy continue to be the main treatment modalities. Unfortunately, these treatments are ineffective for many and are often associated with undesirable consequences.
  • prostate tumor marker that can accurately detect early-stage, localized tumors remains a significant limitation in the diagnosis and management of this disease.
  • serum prostate specific antigen (PSA) assay has been a very useful tool, however its specificity and general utility is widely regarded as lacking in several important respects.
  • Progress in identifying additional specific markers for prostate cancer has been improved by the generation of prostate cancer xenografts that can recapitulate different stages of the disease in mice.
  • the LAPC (Los Angeles Prostate Cancer) xenografts are prostate cancer xenografts that have survived passage in severe combined immune deficient (SCID) mice and have exhibited the capacity to mimic the transition from androgen dependence to androgen independence (Klein ef al., 1997, Nat. Med. 3:402). More recently identified prostate cancer markers include PCTA-1 (Su ef al., 1996, Proc. Natl. Acad. Sci. USA 93: 7252), prostate-specific membrane (PSM) antigen (Pinto et al., Clin Cancer Res 1996 Sep 2 (9): 1445- 51), STEAP (Hubert, ef al., Proc Natl Acad Sci U S A. 1999 Dec 7; 96(25): 14523-8) and prostate stem cell antigen (PSCA) (Reiter ef al., 1998, Proc. Natl. Acad. Sci. USA 95: 1735).
  • PCTA-1 Pur ef al., 1996, Proc
  • Renal cell carcinoma accounts for approximately 3 percent of adult malignancies. Once adenomas reach a diameter of 2 to 3 cm, malignant potential exists. In the adult, the two principal malignant renal tumors are renal cell adenocarcinoma and transitional cell carcinoma of the renal pelvis or ureter. The incidence of renal cell adenocarcinoma is estimated at more than 29,000 cases in the United States, and more than 11,600 patients died of this disease in 1998. Transitional cell carcinoma is less frequent, with an incidence of approximately 500 cases per year in the United States.
  • bladder cancer represents approximately 5 percent in men (fifth most common neoplasm) and 3 percent in women (eighth most common neoplasm). The incidence is increasing slowly, concurrent with an increasing older population. In 1998, there was an estimated 54,500 cases, including 39,500 in men and 15,000 in women. The age-adjusted incidence in the United States is 32 per 100,000 for men and eight per 100,000 in women. The historic male/female ratio of 3:1 may be decreasing related to smoking patterns in women. There were an estimated 11 ,000 deaths from bladder cancer in 1998 (7,800 in men and 3,900 in women). Bladder cancer incidence and mortality strongly increase with age and will be an increasing problem as the population becomes more elderly.
  • bladder cancers recur in the bladder.
  • Bladder cancer is managed with a combination of transurethral resection of the bladder (TUR) and intravesical chemotherapy or immunotherapy.
  • TUR transurethral resection of the bladder
  • the multifocal and recurrent nature of bladder cancer points out the limitations of TUR.
  • Most muscle-invasive cancers are not cured by TUR alone. Radical cystectomy and urinary diversion is the most effective means to eliminate the cancer but carry an undeniable impact on urinary and sexual function. There continues to be a significant need for treatment modalities that are beneficial for bladder cancer patients.
  • Treatment options for lung and bronchial cancer are determined by the type and stage of the cancer and include surgery, radiation therapy, and chemotherapy. For many localized cancers, surgery is usually the treatment of choice. Because the disease has usually spread by the time it is discovered, radiation therapy and chemotherapy are often needed in combination with surgery. Chemotherapy alone or combined with radiation is the treatment of choice for small cell lung cancer; on this regimen, a large percentage of patients experience remission, which in some cases is long lasting. There is however, an ongoing need for effective treatment and diagnostic approaches for lung and bronchial cancers.
  • treatment of breast cancer may involve lumpectomy (local removal of the tumor) and removal of the lymph nodes under the arm; mastectomy (surgical removal of the breast) and removal of the lymph nodes under the arm; radiation therapy; chemotherapy; or hormone therapy.
  • lumpectomy local removal of the tumor
  • mastectomy surgical removal of the breast
  • radiation therapy chemotherapy
  • hormone therapy chemotherapy
  • two or more methods are used in combination.
  • Numerous studies have shown that, for early stage disease, long-term survival rates after lumpectomy plus radiotherapy are similar to survival rates after modified radical mastectomy.
  • Significant advances in reconstruction techniques provide several options for breast reconstruction after mastectomy. Recently, such reconstruction has been done at the same time as the mastectomy.
  • DCIS ductal carcinoma in situ
  • Surgery, radiation therapy, and chemotherapy are treatment options for ovarian cancer.
  • Surgery usually includes the removal of one or both ovaries, the fallopian tubes (salpingo-oophorectomy), and the uterus (hysterectomy).
  • the fallopian tubes salivary-oophorectomy
  • the uterus hematoma-oophorectomy
  • pancreatic cancer There were an estimated 28,300 new cases of pancreatic cancer in the United States in 2000. Over the past 20 years, rates of pancreatic cancer have declined in men. Rates among women have remained approximately constant but may be beginning to decline. Pancreatic cancer caused an estimated 28,200 deaths in 2000 in the United States. Over the past 20 years, there has been a slight but significant decrease in mortality rates among men (about -0.9% per year) while rates have increased slightly among women.
  • pancreatic cancer Surgery, radiation therapy, and chemotherapy are treatment options for pancreatic cancer. These treatment options can extend survival and/or relieve symptoms in many patients but are not likely to produce a cure for most. There is a significant need for additional therapeutic and diagnostic options for pancreatic cancer.
  • the present invention relates to a gene, designated 109P1D4, that has now been found to be over-expressed in the cancer(s) listed in Table I.
  • Northern blot expression analysis of 109P1D4 gene expression in normal tissues shows a restricted expression pattern in adult tissues.
  • the nucleotide ( Figure 2) and amino acid ( Figure 2, and Figure 3) sequences of 109P1D4 are provided.
  • tissue-related profile of 109P1D4 in normal adult tissues combined with the over-expression observed in the tissues listed in Table I, shows that 109P1 D4 is aberrantly over-expressed in at least some cancers, and thus serves as a useful diagnostic, prophylactic, prognostic, and/or therapeutic target for cancers of the tissue(s) such as those listed in Table I.
  • the invention provides polynucleotides corresponding or complementary to all or part of the 109P1D4 genes, mRNAs, and/or coding sequences, preferably in isolated form, including polynucleotides encoding 109P1D4-related proteins and fragments of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25 contiguous amino acids; at least 30, 35, 40, 5, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100 or more than 100 contiguous amino acids of a 109P1D4-related protein, as well as the peptides/proteins themselves; DNA, RNA, DNA/RNA hybrids, and related molecules, polynucleotides or oligonucleotides complementary or having at least a 90% homology to the 109P1 D4 genes or mRNA sequences or parts thereof, and polynucleotides or oligonucleotides that hybridize to the 109P1D4 genes
  • 109P1 D4 Recombinant DNA molecules containing 109P1 D4 polynucleotides, cells transformed or transduced with such molecules, and host-vector systems for the expression of 109P1 D4 gene products are also provided.
  • the invention further provides antibodies that bind to 109P1D4 proteins and polypeptide fragments thereof, including polyclonal and monoclonal antibodies, murine and other mammalian antibodies, chimeric antibodies, humanized and fully human antibodies, and antibodies labeled with a detectable marker or therapeutic agent.
  • the entire nucleic acid sequence of Figure 2 is not encoded and/or the entire amino acid sequence of Figure 2 is not prepared.
  • the entire nucleic acid sequence of Figure 2 is encoded and/or the entire amino acid sequence of Figure 2 is prepared, either of which are in respective human unit dose forms.
  • the invention further provides methods for detecting the presence and status of 109P1 D4 polynucleotides and proteins in various biological samples, as well as methods for identifying cells that express 109P1 D4.
  • a typical embodiment of this invention provides methods for monitoring 109P1 D4 gene products in a tissue or hematology sample having or suspected of having some form of growth dysregulation such as cancer.
  • the invention further provides various immunogenic or therapeutic compositions and strategies for treating cancers that express 109P1D4 such as cancers of tissues listed in Table I, including therapies aimed at inhibiting the transcription, translation, processing or function of 109P1 D4 as well as cancer vaccines.
  • the invention provides compositions, and methods comprising them, for treating a cancer that expresses 109P1 D4 in a human subject wherein the composition comprises a carrier suitable for human use and a human unit dose of one or more than one agent that inhibits the production or function of 109P1 D4.
  • the carrier is a uniquely human carrier.
  • the agent is a moiety that is immunoreactive with 109P1 D4 protein.
  • Non-limiting examples of such moieties include, but are not limited to, antibodies (such as single chain, monoclonal, polyclonal, humanized, chimeric, or human antibodies), functional equivalents thereof (whether naturally occurring or synthetic), and combinations thereof.
  • the antibodies can be conjugated to a diagnostic or therapeutic moiety.
  • the agent is a small molecule as defined herein.
  • the agent comprises one or more than one peptide which comprises a cytotoxic T lymphocyte (CTL) epitope that binds an HLA class I molecule in a human to elicit a CTL response to 109P1 D4 and/or one or more than one peptide which comprises a helper T lymphocyte (HTL) epitope which binds an HLA class II molecule in a human to elicit an HTL response.
  • CTL cytotoxic T lymphocyte
  • HTL helper T lymphocyte
  • the peptides of the invention may be on the same or on one or more separate polypeptide molecules.
  • the agent comprises one or more than one nucleic acid molecule that expresses one or more than one of the CTL or HTL response stimulating peptides as described above.
  • the one or more than one nucleic acid molecule may express a moiety that is immunologically reactive with 109P1D4 as described above.
  • the one or more than one nucleic acid molecule may also be, or encodes, a molecule that inhibits production of 109P1D4.
  • Non-limiting examples of such molecules include, but are not limited to, those complementary to a nucleotide sequence essential for production of 109P1 D4 (e.g. antisense sequences or molecules that form a triple helix with a nucleotide double helix essential for 109P1D4 production) or a ribozyme effective to lyse 109P1D4 mRNA.
  • HLA Peptide Tables respective to its parental protein, e.g., variant 1, variant 2, etc.
  • HLA Peptide Tables respective to its parental protein, e.g., variant 1, variant 2, etc.
  • search Peptides in Table VII Generally, a unique Search Peptide is used to obtain HLA peptides of a particular for a particular variant. The position of each Search Peptide relative to its respective parent molecule is listed in Table VII.
  • a Search Peptide begins at position "X"
  • a particular Search Peptide begins at position 150 of its parental molecule, one must add 150 - 1, i.e., 149 to each HLA peptide amino acid position to calculate the position of that amino acid in the parent molecule.
  • One embodiment of the invention comprises an HLA peptide, that occurs at least twice in Tables VIII-XXI and XXII to XLIX collectively, or an oligonucleotide that encodes the HLA peptide.
  • Another embodiment of the invention comprises an HLA peptide that occurs at least once in Tables VIII-XXI and at least once in tables XXII to XLIX, or an oligonucleotide that encodes the HLA peptide.
  • antibody epitopes which comprise a peptide regions, or an oligonucleotide encoding the peptide region, that has one two, three, four, or five of the following characteristics: i) a peptide region of at least 5 amino acids of a particular peptide of Figure 3, in any whole number increment up to the full length of that protein in Figure 3, that includes an amino acid position having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Hydrophilicity profile of Figure 5; ii) a peptide region of at least 5 amino acids of a particular peptide of Figure 3, in any whole number increment up to the full length of that protein in Figure 3, that includes an amino acid position having a value equal to or less than 0.5, 0.4, 0.3, 0.2, 0.1, or having a value equal to 0.0, in the Hydropathicity profile of Figure 6; iii) a peptide region of at least 5
  • Figure 1 The 109P1D4 SSH sequence of 192 nucleotides.
  • FIG. 2A The cDNA and amino acid sequence of 109P1 D4 variant 1 (also called “109P1D4 v.1" or “109P1D4 variant 1”) is shown in Figure 2A, The start methionine is underlined. The open reading frame extends from nucleic acid
  • 109P1D4 variant 2 also called “109P1D4 v.2"
  • Figure 2B The cDNA and amino acid sequence of 109P1D4 variant 2 (also called “109P1D4 v.2”) is shown in Figure 2B.
  • the codon for the start methionine is underlined.
  • the open reading frame extends from nucleic acid 503-3667 including the stop codon.
  • 109P1 D4 variant 3 (also called “109P1 D4 v.3") is shown in Figure 2C.
  • the codon for the start methionine is underlined.
  • the open reading frame extends from nucleic acid 846-4889 including the stop codon.
  • 109P1D4 variant 4 also called “109P1D4 v.4"
  • Figure 2D The cDNA and amino acid sequence of 109P1D4 variant 4 (also called "109P1D4 v.4") is shown in Figure 2D.
  • the codon for the start methionine is underlined.
  • the open reading frame extends from nucleic acid 846-4859 including the stop codon.
  • 109P1D4 variant 5 also called “109P1D4 v.5"
  • Figure 2E The cDNA and amino acid sequence of 109P1D4 variant 5 (also called “109P1D4 v.5") is shown in Figure 2E.
  • the codon for the start methionine is underlined.
  • the open reading frame extends from nucleic acid 846-4778 including the stop codon.
  • 109P1D4 variant 6 also called “109P1D4 v.6"
  • Figure 2F The cDNA and amino acid sequence of 109P1D4 variant 6 (also called “109P1D4 v.6”) is shown in Figure 2F.
  • the codon for the start methionine is underlined.
  • the open reading frame extends from nucleic acid 614-3727 including the stop codon.
  • 109P1D4 variant 7 also called “109P1D4 v.7"
  • Figure 2G The cDNA and amino acid sequence of 109P1D4 variant 7 (also called “109P1D4 v.7”) is shown in Figure 2G.
  • the codon for the start methionine is underlined.
  • the open reading frame extends from nucleic acid 735-3881 including the stop codon.
  • 109P1D4 variant 8 (also called “109P1D4 v.8") is shown in Figure 2H.
  • the codon for the start methionine is underlined.
  • the open reading frame extends from nucleic acid 735-4757 including the stop codon.
  • the cDNA and amino acid sequence of 109P1D4 variant 9 (also called “109P1D4 v.9") is shown in Figure 21.
  • the codon for the start methionine is underlined.
  • the open reading frame extends from nucleic acid 514-3627 including the stop codon.
  • the amino acid sequence of 109P1D4 v.7 is shown in Figure 3G; it has 1048 amino acids.
  • H The amino acid sequence of 109P1D4 v.8 is shown in Figure 3H; it has 1340 amino acids.
  • I The amino acid sequence of 109P1D4 v.9 is shown in Figure 31; it has 1037 amino acids.
  • 109P1D4 includes all variants thereof, including those shown in Figures 2, 3, 10, 11, and 12 unless the context clearly indicates otherwise.
  • Figure 6 Hydropathicity amino acid profile of 109P1D4 v.l-v.9 determined by computer algorithm sequence analysis using the method of Kyte and Doolittle (Kyte J, Doolittle R.F., 1982. J. Mol. Biol. 157:105-132) accessed on the ProtScale website located on the World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.
  • Figure 7 Percent accessible residues amino acid profile of 109P1 D4 v.l-v.9 determined by computer algorithm sequence analysis using the method of Janin (Janin J., 1979 Nature 277:491-492) accessed on the ProtScale website located on the World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.
  • Figure 8 Average flexibility amino acid profile of 109P1D4 v.l-v.9 determined by computer algorithm sequence analysis using the method of Bhaskaran and Ponnuswamy (Bhaskaran R., and Ponnuswamy P.K., 1988. Int. J. Pept. Protein Res. 32:242-255) accessed on the ProtScale website located on the World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.
  • Beta-turn amino acid profile of 109P1 D4 v.1 -v.9 determined by computer algorithm sequence analysis using the method of Deleage and Roux (Deleage, G., Roux B. 1987 Protein Engineering 1 :289-294) accessed on the ProtScale website located on the World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.
  • FIG. 10 Structure of transcript variants of 109P1D4.
  • Variants 109P1D4 v.2 through v.9 were transcript variants of v.1.
  • Variant v.2 shared middle portion of v.1 sequence (the 3' portion of exon 1 and 5' portion of exon 2).
  • Variant v.6 was similar to v.2 but added an extra exon between exons 1 and 2 of v.2.
  • V.3 shared exon 1 and 5' portion of exon 2 with v.1 with five additional exons downstream.
  • v.3 deleted exon 4 of v.3 while v.5 deleted exons 3 and 4 of v.3.
  • Variant v.5 lacked exons 3 and 4.
  • PCD11 This gene (called PCD11) is located in sex chromosomes X and Y. Ends of exons in the transcripts are marked above the boxes. Potential exons of this gene are shown in order as on the human genome. Poly A tails and single nucleotide differences are not shown in the figure. Lengths of introns and exons are not proportional.
  • FIG. 11 Schematic alignment of protein variants of 109P1D4.
  • Variants 109P1D4 v.2 through v.9 were proteins translated from the corresponding transcript variants. All these protein variants shared a common portion of the sequence, i.e., 3-1011 of v.1, except for a few amino acids different in this segment resulted from SNP in the transcripts.
  • Variant v.6 and v.9 were the same except for two amino acids at 906 and 1001.
  • Variant v.8 was almost the same as v.5, except for the N-terminal end, and a 2-aa deletion at 1117-8. Single amino acid difference was not shown. Numbers in parentheses corresponded to positions in variant v.3.
  • Figures 13(J)-(R) top panels Schematic representation of the probability of existence of transmembrane regions of 109P1D4 variants based on the TMpred algorithm of Hofmann and Stoffel which utilizes TMBASE (K. Hofmann, W. Stoffel. TMBASE - A database of membrane spanning protein segments Biol. Chem. Hoppe-Seyler 374:166, 1993).
  • Figures 13(J)-(R) bottom panels Schematic representation of the probability of the existence of transmembrane regions of 109P1D4 variants based on the TMHMM algorithm of Sonnhammer, von Heijne, and Krogh (Erik L.L Sonnhammer, Gunnar von Heijne, and Anders Krogh: A hidden Markov model for predicting transmembrane helices in protein sequences.
  • the TMpred and TMHMM algorithms are accessed from the ExPasy molecular biology server located on the World Wide Web at (.expasy.ch/tools/).
  • RNA was extracted from peripheral blood lymphocytes, cord blood isolated from normal individuals, and from lymphoma patient cancer specimens. Northern blots with 10 ⁇ g of total RNA were probed with the 109P1D4 sequence. Size standards in kilobases are on the side. Results show expression of 109P1D4 in lymphoma patient specimens but not in the normal blood cells tested.
  • Figure 15 Expression of 109P1D4 by RT-PCR.
  • First strand cDNA was prepared from vital pool 1 (liver, lung and kidney), vital pool 2 (pancreas, colon and stomach), prostate cancer pool, bladder cancer pool, kidney cancer pool, colon cancer pool, lung cancer pool, ovary cancer pool, breast cancer pool, cancer metastasis pool, and pancreas cancer pool. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 109P1 D4, was performed at 30 cycles of amplification. Results show strong expression of 109P1 D4 in all cancer pools tested. Very low expression was detected in the vital pools.
  • FIG. 16 Expression of 109P1D4 in normal tissues. Two multiple tissue northern blots (Clontech), both with 2 ⁇ g of mRNA/lane, were probed with the 109P1D4 SSH fragment. Size standards in kilobases (kb) are indicated on the side. Results show expression of approximately 10 kb 109P1D4 transcript in ovary. Weak expression was also detected in placenta and brain, but not in the other normal tissues tested.
  • FIG. 1 Expression of 109P1 D4 in human cancer cell lines.
  • RNA was extracted from a number of human prostate and bone cancer cell lines. Northern blots with 10 ⁇ g of total RNA/lane were probed with the 109P1D4 SSH fragment. Size standards in kilobases (kb) are indicated on (he side. Results show expression of 109P1D4 in LAPC-9AD, LAPC-9AI, LNCaP prostate cancer cell lines, and in the bone cancer cell lines, SK-ES-1 and RD-ES.
  • Figure 18A 109P1D4 Expression in Human Normal Tissues. An cDNA dot blot containing 76 different samples from human tissues was analyzed using a 109P1D4 SSH probe. Expression was only detected in multiple areas of the brain, placenta, ovary, and fetal brain, amongst all tissues tested.
  • Figure 18B Expression of 109P1D4 in patient cancer specimens, Expression of 109P1 D4 was assayed in a panel of human cancers (T) and their respective matched normal tissues (N) on RNA dot blots. Upregulated expression of 109P1D4 in tumors compared to normal tissues was observed in uterus, lung and stomach. The expression detected in normal adjacent tissues (isolated from diseased tissues) but not in normal tissues (isolated from healthy donors) may indicate that these tissues are not fully normal and that 109P1D4 may be expressed in early stage tumors.
  • RNA was extracted from normal lung, prostate cancer xenograft LAPC-9AD, bone cancer cell line RD-ES, and lung cancer patient tumors. Northern blots with 10 ⁇ g of total RNA were probed with 109P1D4. Size standards in kilobases are on the side. Results show strong expression of 109P1D4 in lung tumor tissues as well as the RD-ES cell line, but not in normal lung.
  • FIG. 20 Expression of soluble secreted Tag5 109P1 D4 in 293T cells.
  • 293T cells were transfected with either an empty vector or with the Tag ⁇ secretion vector encoding the extracellular domain (ECD; amino acids 24-812) of 109P1 D4 variant 1 fused to a Myc/His epitope Tag. 2 days later, cells and media harvested and analyzed for expression of the recombinant Tag ⁇ 109P1D4 protein by SDS-PAGE followed by anti-His epitope tag Western blotting.
  • An arrow indicates the immunoreactive band corresponding to the 109P1D4 ECD present in the media and the lysate from Tag ⁇ 109P1D4 transfected cells.
  • FIG. 21 Expression of 109P1 D4 protein in 293T cells.
  • 293T cells were transfected with either an empty vector or with pCDNA3.1 vector encoding the full length cDNA of 109P1 D4 variant 1 fused to a Myc/His epitope Tag. 2 days later, cells were harvested and analyzed for expression of 109P1D4 variant 1 protein by SDS-PAGE followed by anti-His epitope tag Western blotting.
  • An arrow indicates the immunoreactive band corresponding to the full length 109P1D4 variant 1 protein expressed in cells transfected with the 109P1D4 vector but not in control cells.
  • FIG 22 Tyrosine phosphorylation of 109P1D4 after pervanadate treatment
  • 293T cells were transfected with the neomycin resistance gene alone or with 109P1 D4 in pSR ⁇ vector. Twenty four hours after transfection, the cells were either left in 10% serum or grown in 0.1 % serum overnight. The cells were then left untreated or were treated with 200 ⁇ M pervanadate (1:1 mixture of Na3V04 and H2O2) for 30 minutes. The cells were lysed in Triton X-100, and the 109P1D4 protein was immunoprecipitated with anti-His monoclonal antibody.
  • the immunoprecipitates were run on SDS-PAGE and then Western blotted with either anti-phosphotyrosine (upper panel) or anti-His (lower panel).
  • the 109P1D4 protein is phosphorylated on tyrosine in response to pervanadate treatment, and a large amount of the protein moves to the insoluble fraction following pervanadate-induced activation.
  • FIG. 23 Effect of 109P1D4 RNAi on cell proliferation.
  • LNCaP cells were transfected with Lipofectamine 2000 alone or with siRNA oligonucleotides.
  • the siRNA oligonucleotides included a negative control, Luc4, specific for Luciferase, a positive control, Eg5, specific for the mitotic spindle protein Eg5, or three siRNAs specific for the 109P1D4 protein, 109P1 D4.a, 109P1 D4.c and 109P1 D4.d at 20 nM concentration. Twenty four hours after transfection, the cells were pulsed with 3 H-thymidine and incorporation was measured after 72 hours. All three siRNAs to 109P1D4 inhibited the proliferation of LNCaP cells, indicating that 109P1D4 expression is important for the cell growth pathway of these cancer cells.
  • Vaccine Compositions Comprising DC Pulsed with CTL and/or HTL Peptides
  • prostate cancer and “locally advanced disease” mean prostate cancers that have extended through the prostate capsule, and are meant to include stage C disease under the American Urological Association (AUA) system, stage C1 - C2 disease under the Whitmore-Jewett system, and stage T3 - T4 and N+ disease under the TNM (tumor, node, metastasis) system.
  • AUA American Urological Association
  • stage C1 - C2 disease under the Whitmore-Jewett system
  • TNM tumor, node, metastasis
  • surgery is not recommended for patients with locally advanced disease, and these patients have substantially less favorable outcomes compared to patients having clinically localized (organ-confined) prostate cancer.
  • Locally advanced disease is clinically identified by palpable evidence of induration beyond the lateral border of the prostate, or asymmetry or induration above the prostate base.
  • Locally advanced prostate cancer is presently diagnosed pathologically following radical prostatectomy if the tumor invades or penetrates the prostatic capsule, extends into the surgical margin, or invades the seminal
  • “Altering the native glycosylation pattern” is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence 109P1D4 (either by removing the underlying glycosylation site or by deleting the glycosylation by chemical and/or enzymatic means), and/or adding one or more glycosylation sites that are not present in the native sequence 109P1D4.
  • the phrase includes qualitative changes in the glycosylation of the native proteins, involving a change in the nature and proportions of the various carbohydrate moieties present.
  • an analog refers to a molecule which is structurally similar or shares similar or corresponding attributes with another molecule (e.g. a 109P1D4-related protein).
  • a 109P1D4-related protein e.g. an analog of a 109P1D4 protein can be specifically bound by an antibody or T cell that specifically binds to 109P1 D4.
  • Antibody is used in the broadest sense. Therefore, an “antibody” can be naturally occurring or man-made such as monoclonal antibodies produced by conventional hybridoma technology.
  • Anti-109P1D4 antibodies comprise monoclonal and polyclonal antibodies as well as fragments containing the antigen-binding domain and/or one or more complementarity determining regions of these antibodies.
  • an “antibody fragment” is defined as at least a portion of the variable region of the immunoglobulin molecule that binds to its target, i.e., the antigen-binding region. In one embodiment it specifically covers single anti-109P1D4 antibodies and clones thereof (including agonist, antagonist and neutralizing antibodies) and anti-109P1D4 antibody compositions with polyepitopic specificity.
  • codon optimized sequences refers to nucleotide sequences that have been optimized for a particular host species by replacing any codons having a usage frequency of less than about 20%, Nucleotide sequences that have been optimized for expression in a given host species by elimination of spurious polyadenylation sequences, elimination of exon/intron splicing signals, elimination of transposon-like repeats and/or optimization of GC content in addition to codon optimization are referred to herein as an "expression enhanced sequences.”
  • a “combinatorial library” is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis by combining a number of chemical "building blocks” such as reagents.
  • a linear combinatorial chemical library such as a polypeptide (e.g., mutein) library, is formed by combining a set of chemical building blocks called amino acids in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound).
  • Numerous chemical compounds are synthesized through such combinatorial mixing of chemical building blocks (Gallop et al., J. Med. Chem. 37(9): 1233-1251 (1994)).
  • combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Patent No. 5,010,175, Furka, Pept. Prot. Res. 37:487-493 (1991), Houghton et al., Nature, 354:84-88 (1991)), peptoids (PCT Publication No WO 91/19735), encoded peptides (PCT Publication WO 93/20242), random bio- oligomers (PCT Publication WO 92/00091), benzodiazepines (U.S. Pat. No.
  • oligocarbarnates Cho, et al., Science 261:1303 (1993)
  • peptidyl phosphonates Campbell et al., J. Org. Chem. 59:658 (1994)
  • nucleic acid libraries see, e.g., Stratagene, Corp.
  • peptide nucleic acid libraries see, e.g., U.S.
  • Patent 5,539,083) antibody libraries (see, e.g., Vaughn et al., Nature Biotechnology 14(3): 309-314 (1996), and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al., Science 274:1520-1522 (1996), and U.S. Patent No. 5,593,853), and small organic molecule libraries (see, e.g., benzodiazepines, Baum, C&EN, Jan 18, page 33 (1993); isoprenoids, U.S. Patent No. 5,569,588; thiazolidinones and metathiazanones, U.S. Patent No. 5,549,974; pyrrolidines, U.S. Patent Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. Patent No. 5,506, 337; benzodiazepines, U.S. Patent No. 5,288,514; and the like).
  • antibody libraries see
  • cytotoxic agent refers to a substance that inhibits or prevents the expression activity of cells, function of cells and/or causes destruction of cells.
  • the term is intended to include radioactive isotopes chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof.
  • cytotoxic agents include, but are not limited to auristatins, auromycins, maytansinoids, yttrium, bismuth, ricin, ricin A-chain, combrestatin, duocarmycins, dolostatins, doxorubicin, daunorubicin, taxol, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, aipha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, Sapaonaria offio
  • the “gene product” is sometimes referred to herein as a protein or mRNA.
  • a “gene product of the invention” is sometimes referred to herein as a "cancer amino acid sequence", “cancer protein”, “protein of a cancer listed in Table I", a “cancer mRNA”, “mRNA of a cancer listed in Table I”, etc.
  • the cancer protein is encoded by a nucleic acid of Figure 2.
  • the cancer protein can be a fragment, or alternatively, be the full-length protein to the fragment encoded by the nucleic acids of Figure 2.
  • a cancer amino acid sequence is used to determine sequence identity or similarity.
  • the sequences are naturally occurring allelic variants of a protein encoded by a nucleic acid of Figure 2.
  • the sequences are sequence variants as further described herein.
  • high throughput screening systems are commercially available (see, e.g., Amersham Biosciences, Piscataway, NJ; Zymark Corp., Hopkinton, MA; Air Technical Industries, Mentor, OH; Beckman Instruments, Inc. Fullerton, CA; Precision Systems, Inc., Natick, MA; etc.). These systems typically automate entire procedures, including all sample and reagent pipetting, liquid dispensing, timed incubations, and final readings of the microplate in detector(s) appropriate for the assay. These configurable systems provide high throughput and rapid start up as well as a high degree of flexibility and customization. The manufacturers of such systems provide detailed protocols for various high throughput systems. Thus, e.g., Zymark Corp. provides technical bulletins describing screening systems for detecting the modulation of gene transcription, ligand binding, and the like.
  • homolog refers to a molecule which exhibits homology to another molecule, by for example, having sequences of chemical residues that are the same or similar at corresponding positions.
  • HLA Human Leukocyte Antigen
  • HLA Human Leukocyte Antigen
  • MHC Major Histocompatibility Complex
  • hybridize used in the context of polynucleotides, are meant to refer to conventional hybridization conditions, preferably such as hybridization in 50% formamide/6XSSC/0.1 % SDS/100 ⁇ g/ml ssDNA, in which temperatures for hybridization are above 37 degrees C and temperatures for washing in 0.1XSSC/0.1 % SDS are above 55 degrees C.
  • isolated or “biologically pure” refer to material which is substantially or essentially free from components which normally accompany the material as it is found in its native state.
  • isolated peptides in accordance with the invention preferably do not contain materials normally associated with the peptides in their in situ environment.
  • a polynucleotide is said to be “isolated” when it is substantially separated from contaminant polynucleotides that correspond or are complementary to genes other than the 109P1 D4 genes or that encode polypeptides other than 109P1 D4 gene product or fragments thereof.
  • a skilled artisan can readily employ nucleic acid isolation procedures to obtain an isolated 109P1 D4 polynucleotide.
  • a protein is said to be "isolated,” for example, when physical, mechanical or chemical methods are employed to remove the 109P1D4 proteins from cellular constituents that are normally associated with the protein.
  • a skilled artisan can readily employ standard purification methods to obtain an isolated 109P1D4 protein.
  • an isolated protein can be prepared by chemical means.
  • mammal refers to any organism classified as a mammal, including mice, rats, rabbits, dogs, cats, cows, horses and humans. In one embodiment of the invention, the mammal is a mouse. In another embodiment of the invention, the mammal is a human.
  • metastatic prostate cancer and “metastatic disease” mean prostate cancers that have spread to regional lymph nodes or to distant sites, and are meant to include stage D disease under the AUA system and stage TxNxM ⁇ under the TNM system.
  • surgery is generally not indicated for patients with metastatic disease, and hormonal (androgen ablation) therapy is a preferred treatment modality.
  • Patients with metastatic prostate cancer eventually develop an androgen-refractory state within 12 to 18 months of treatment initiation. Approximately half of these androgen-refractory patients die within 6 months after developing that status.
  • the most common site for prostate cancer metastasis is bone. Prostate cancer bone metastases are often osteoblastic rather than osteolytic (i.e., resulting in net bone formation).
  • Bone metastases are found most frequently in the spine, followed by the femur, pelvis, rib cage, skull and humerus. Other common sites for metastasis include lymph nodes, lung, liver and brain. Metastatic prostate cancer is typically diagnosed by open or laparoscopic pelvic lymphadenectomy, whole body radionuclide scans, skeletal radiography, and/or bone lesion biopsy.
  • modulator or “test compound” or “drug candidate” or grammatical equivalents as used herein describe any molecule, e.g., protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide, etc., to be tested for the capacity to directly or indirectly alter the cancer phenotype or the expression of a cancer sequence, e.g., a nucleic acid or protein sequences, or effects of cancer sequences (e.g., signaling, gene expression, protein interaction, etc.)
  • a modulator will neutralize the effect of a cancer protein of the invention.
  • neutralize is meant that an activity of a protein is inhibited or blocked, along with the consequent effect on the cell.
  • a modulator will neutralize the effect of a gene, and its corresponding protein, of the invention by normalizing levels of said protein.
  • modulators alter expression profiles, or expression profile nucleic acids or proteins provided herein, or downstream effector pathways.
  • the modulator suppresses a cancer phenotype, e.g. to a normal tissue fingerprint.
  • a modulator induced a cancer phenotype.
  • a plurality of assay mixtures is run in parallel with different agent concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration or below the level of detection.
  • Modulators, drug candidates or test compounds encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 100 and less than about 2,500 Daltons. Preferred small molecules are less than 2000, or less than 1500 or less than 1000 or less than 500 D.
  • Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups.
  • the candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • Modulators also comprise biomolecules such as peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Particularly preferred are peptides.
  • One class of modulators are peptides, for example of from about five to about 35 amino acids, with from about five to about 20 amino acids being preferred, and from about 7 to about 15 being particularly preferred.
  • the cancer modulalory protein is soluble, includes a non-transmembrane region, and/or, has an N- terminal Cys to aid in solubility.
  • the C-terminus of the fragment is kept as a free acid and the N-terminus is a free amine to aid in coupling, i.e., to cysteine.
  • a cancer protein of the invention is conjugated to an immunogenic agent as discussed herein.
  • the cancer protein is conjugated to BSA.
  • the peptides of the invention e.g., of preferred lengths, can be linked to each other or to other amino acids to create a longer peptide/protein.
  • modulatory peptides can be digests of naturally occurring proteins as is outlined above, random peptides, or "biased" random peptides.
  • peptide/protein-based modulators are antibodies, and fragments thereof, as defined herein.
  • Modulators of cancer can also be nucleic acids.
  • Nucleic acid modulating agents can be naturally occurring nucleic acids, random nucleic acids, or "biased" random nucleic acids. For example, digests of prokaryotic or eukaryotic genomes can be used in an approach analogous to that outlined above for proteins.
  • the term "monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the antibodies comprising the population are identical except for possible naturally occurring mutations that are present in minor amounts.
  • a "motif, as in biological motif of a 109P1 D4-related protein, refers to any pattern of amino acids forming part of the primary sequence of a protein, that is associated with a particular function (e.g. protein-protein interaction, protein-DNA interaction, etc) or modification (e.g. that is phosphorylated, glycosylated or amidated), or localization (e.g. secretory sequence, nuclear localization sequence, etc.) or a sequence that is correlated with being immunogenic, either humorally or cellularly.
  • a motif can be either contiguous or capable of being aligned to certain positions that are generally correlated with a certain function or property.
  • motif refers to the pattern of residues in a peptide of defined length, usually a peptide of from about 8 to about 13 amino acids for a class I HLA motif and from about 6 to about 25 amino acids for a class II HLA motif, which is recognized by a particular HLA molecule.
  • Peptide motifs for HLA binding are typically different for each protein encoded by each human HLA allele and differ in the pattern of the primary and secondary anchor residues.
  • a “pharmaceutical excipient” comprises a material such as an adjuvant, a carrier, pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservative, and the like.
  • “Pharmaceutically acceptable” refers to a non-toxic, inert, and/or composition that is physiologically compatible with humans or other mammals.
  • polynucleotide means a polymeric form of nucleotides of at least 10 bases or base pairs in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide, and is meant to include single and double stranded forms of DNA and/or RNA. In the art, this term if often used interchangeably with “oligonucleotide”.
  • a polynucleotide can comprise a nucleotide sequence disclosed herein wherein thymidine (T), as shown for example in Figure 2, can also be uracil (U); this definition pertains to the differences between the chemical structures of DNA and RNA, in particular the observation that one of the four major bases in RNA is uracil (U) instead of thymidine (T).
  • T thymidine
  • U uracil
  • polypeptide means a polymer of at least about 4, 5, 6, 7, or 8 amino acids. Throughout the specification, standard three letter or single letter designations for amino acids are used. In the art, this term is often used interchangeably with “peptide” or "protein”.
  • HLA "primary anchor residue” is an amino acid at a specific position along a peptide sequence which is understood to provide a contact point between the immunogenic peptide and the HLA molecule.
  • One to three, usually two, primary anchor residues within a peptide of defined length generally defines a "motif for an immunogenic peptide. These residues are understood to fit in close contact with peptide binding groove of an HLA molecule, with their side chains buried in specific pockets of the binding groove.
  • the primary anchor residues for an HLA class I molecule are located at position 2 (from the amino terminal position) and at the carboxyl terminal position of a 8, 9, 10, 11, or 12 residue peptide epitope in accordance with the invention.
  • the primary anchor residues of a peptide binds an HLA class II molecule are spaced relative to each other, rather than to the termini of a peptide, where the peptide is generally of at least 9 amino acids in length.
  • the primary anchor positions for each motif and supermotif are set forth in Table IV.
  • analog peptides can be created by altering the presence or absence of particular residues in the primary and/or secondary anchor positions shown in Table IV. Such analogs are used to modulate the binding affinity and/or population coverage of a peptide comprising a particular HLA motif or supermotif.
  • Radioisotopes include, but are not limited to the following (non-limiting exemplary uses are also set forth):
  • Actinium-227 Parent of Radium-223 (Ra-223) which is an alpha emitter used to treat metastases in the skeleton (AC-227) resulting from cancer (i.e., breast and prostate cancers), and cancer radioimmunotherapy
  • Cobalt-60 Radiation source for radiotherapy of cancer, for food irradiators, and for sterilization of medical
  • Copper-67' Beta/gamma emitter used in cancer radioimmunotherapy and diagnostic studies (i.e., breast and
  • Osteoporosis detection diagnostic imaging, tracer drugs, brain cancer treatment, radiolabeling, lodine-125 tumor imaging, mapping of receptors in the brain, interstitial radiation therapy, brachytherapy for (1-125) treatment of prostate cancer, determination of glomerular filtration rate (GFR), determination of plasma volume, detection of deep vein thrombosis of the legs
  • thyroid function evaluation thyroid disease detection, treatment of thyroid cancer as well as other lodine-131 non-malignant thyroid diseases (i.e., Graves disease, goiters, and hyperthyroidism), treatment of (1-131) leukemia, lymphoma, and other forms of cancer (e.g., breast cancer) using radioimmunotherapy lridium-192 Brachytherapy, brain and spinal cord tumor treatment, treatment of blocked arteries (i.e., (lr-192) arteriosclerosis and restenosis), and implants for breast and prostate tumors
  • Tc-99m Parent of Technetium-99m which is used for imaging the brain, liver, lungs, heart, and
  • Tc-99m is the most widely used radioisotope used for diagnostic imaging (Mo-99) of various cancers and diseases involving the brain, heart, liver, lungs; also used in detection of deep vein thrombosis of the legs
  • Polycythemia rubra vera blood cell disease
  • leukemia treatment bone cancer
  • Phosphorus-32 diagnosis/treatment colon, pancreatic, and liver cancer treatment; radiolabeling nucleic acids for (P-32) in vitro research, diagnosis of superficial tumors, treatment of blocked arteries (i.e., arteriosclerosis and restenosis), and intracavity therapy
  • Phosphorus-33 Leukemia treatment bone disease diagnosis/treatment, radiolabeling, and treatment of blocked
  • Rhenium-186 Bone cancer pain relief, rheumatoid arthritis treatment, and diagnosis and treatment of lymphoma
  • Radiotracer used in brain studies, imaging of adrenal cortex by gamma-scintigraphy, lateral
  • Bone cancer pain relief multiple myeloma treatment, and osteoblastic therapy
  • Rhenium-188 (Re-188) which is used for cancer diagnostics/treatment, bone cancer
  • a gamma-emitting label for Yttrium-90 which is used for cancer radioimmunotherapy (i.e.,
  • randomized or grammatical equivalents as herein applied to nucleic acids and proteins is meant that each nucleic acid and peptide consists of essentially random nucleotides and amino acids, respectively. These random peptides (or nucleic acids, discussed herein) can incorporate any nucleotide or amino acid al any position.
  • the synthetic process can be designed to generate randomized proteins or nucleic acids, to allow the formation of all or most of the possible combinations over the length of the sequence, thus forming a library of randomized candidate bioactive proteinaceous agents.
  • a library is "fully randomized,” with no sequence preferences or constants at any position.
  • the library is a "biased random” library. That is, some positions within the sequence either are held constant, or are selected from a limited number of possibilities.
  • the nucleotides or amino acid residues are randomized within a defined class, e.g., of hydrophobic amino acids, hydrophilic residues, sterically biased (either small or large) residues, towards the creation of nucleic acid binding domains, the creation of cysteines, for cross-linking, prolines for SH-3 domains, serines, threonines, tyrosines or histidines for phosphorylation sites, etc., or to purines, etc.
  • a "recombinant" DNA or RNA molecule is a DNA or RNA molecule that has been subjected to molecular manipulation in vitro.
  • Non-limiting examples of small molecules include compounds that bind or interact with 109P1 D4, ligands including hormones, neuropeptides, chemokines, odorants, phospholipids, and functional equivalents thereof that bind and preferably inhibit 109P1 D4 protein function.
  • Such non-limiting small molecules preferably have a molecular weight of less than about 10 kDa, more preferably below about 9, about 8, about 7, about 6, about 5 or about 4 kDa.
  • small molecules physically associate with, or bind, 109P1 D4 protein; are not found in naturally occurring metabolic pathways; and/or are more soluble in aqueous than non-aqueous solutions
  • “Stringency” of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured nucleic acid sequences to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature that can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel ef al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).
  • “Stringent conditions” or “high stringency conditions”, as defined herein, are identified by, but not limited to, those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50°C; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1 % Ficoll/0.1 % polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42 °C; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCI, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 ⁇ g/ml), 0.1 % S
  • Modely stringent conditions are described by, but not limited to, those in Sambrook ef al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and %SDS) less stringent than those described above.
  • washing solution and hybridization conditions e.g., temperature, ionic strength and %SDS
  • moderately stringent conditions is overnight incubation at 37°C in a solution comprising: 20% formamide, 5 x SSC (150 mM NaCI, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 37-50°C.
  • 5 x SSC 150 mM NaCI, 15 mM trisodium citrate
  • 50 mM sodium phosphate pH 7.6
  • 5 x Denhardt's solution 10% dextran sulfate
  • 20 mg/mL denatured sheared salmon sperm DNA followed by washing the filters in 1 x SSC at about 37-50°C.
  • the skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.
  • HLA-supermotif is a peptide binding specificity shared by HLA molecules encoded by two or more HLA alleles. Overall phenotypic frequencies of HLA-supertypes in different ethnic populations are set forth in Table IV (F). The non- limiting constituents of various supetypes are as follows:
  • A2 AO201 A*0202, A*0203, A*0204, A* 0205, A*0206, A*6802, A*6901, A*0207
  • A3 A3, A11 , A31 , A*3301 , A*6801 , A*0301 , A*1101 , A*3101
  • B7 B7, B*3501-03, B*51, B*5301, B*5401, B*5501, B*5502, B*5601, 13*6701, B7801, B*0702, B*5101, B*5602
  • B44 B*3701, B*4402, B*4403, B*60 (B*4001), B61 (B*4006)
  • A24 A*24, A*30, A*2403, A*2404, A*3002, A*3003
  • B27 B*1401-02, B*1503, B*1509, B*1510, B*1518, B*3801-02, B*3901, B*3902, B*3903-04, B 801-02, B*7301, B*2701-08
  • B58 B*1516, B*1517, B*5701, B*5702, B58
  • B62 B*4601. B52, B*1501 (B62), B*1502 (B7 ⁇ ), B*1513 (B77) Calculated population coverage afforded by different HLA-supertype combinations are set forth in Table IV (G).
  • to treat or "therapeutic” and grammatically related terms, refer to any improvement of any consequence of disease, such as prolonged survival, less morbidity, and/or a lessening of side effects which are the byproducts of an alternative therapeutic modality; full eradication of disease is not required.
  • transgenic animal e.g., a mouse or rat
  • transgene is an animal having cells that contain a transgene, which transgene was introduced into the animal or an ancestor of the animal at a prenatal, e.g., an embryonic stage.
  • transgene is a DNA that is integrated into the genome of a cell from which a transgenic animal develops.
  • an HLA or cellular immune response "vaccine” is a composition that contains or encodes one or more peptides of the invention.
  • vaccines such as a cocktail of one or more individual peptides; one or more peptides of the invention comprised by a polyepitopic peptide; or nucleic acids that encode such individual peptides or polypeptides, e.g., a minigene that encodes a polyepitopic peptide.
  • the "one or more peptides” can include any whole unit integer from 1-150 or more, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 or more peptides of the invention.
  • the peptides or polypeptides can optionally be modified, such as by lipidation, addition of targeting or other sequences.
  • HLA class I peptides of the invention can be admixed with, or linked to, HLA class II peptides, to facilitate activation of both cytotoxic T lymphocytes and helper T lymphocytes.
  • HLA vaccines can also comprise peptide-pulsed antigen presenting cells, e.g., dendritic cells.
  • variant refers to a molecule that exhibits a variation from a described type or norm, such as a protein that has one or more different amino acid residues in the corresponding position(s) of a specifically described protein (e.g. the 109P1D4 protein shown in Figure 2 or Figure 3.
  • An analog is an example of a variant protein.
  • Splice isoforms and single nucleotides polymorphisms (SNPs) are further examples of variants.
  • the "109P1D4-related proteins” of the invention include those specifically identified herein, as well as allelic variants, conservative substitution variants, analogs and homologs that can be isolated/generated and characterized without undue experimentation following the methods outlined herein or readily available in the art.
  • Fusion proteins that combine parts of different 109P1 D4 proteins or fragments thereof, as well as fusion proteins of a 109P1 D4 protein and a heterologous polypeptide are also included.
  • Such 109P1D4 proteins are collectively referred to as the 109P1D4-related proteins, the proteins of the invention, or 109P1D4.
  • 109P1D4-related protein refers to a polypeptide fragment or a 109P1D4 protein sequence of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25 amino acids; or, at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, or 576 or more amino acids.
  • One aspect of the invention provides polynucleotides corresponding or complementary to all or part of a 109P1 D4 gene, mRNA, and/or coding sequence, preferably in isolated form, including polynucleotides encoding a 109P1 D4-related protein and fragments thereof, DNA, RNA, DNA/RNA hybrid, and related molecules, polynucleotides or oligonucleotides complementary to a 109P1D4 gene or mRNA sequence or a part thereof, and polynucleotides or oligonucleotides that hybridize to a 109P1D4 gene, mRNA, or to a 109P1D4 encoding polynucleotide (collectively, "109P1D4 polynucleotides").
  • T can also be U in Figure 2.
  • Embodiments of a 109P1D4 polynucleotide include: a 109P1D4 polynucleotide having the sequence shown in Figure 2, the nucleotide sequence of 109P1D4 as shown in Figure 2 wherein T is U; at least 10 contiguous nucleotides of a polynucleotide having the sequence as shown in Figure 2; or, at least 10 contiguous nucleotides of a polynucleotide having the sequence as shown in Figure 2 where T is U.
  • embodiments of 109P1D4 nucleotides comprise, without limitation:
  • V a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 2D, from nucleotide residue number 846 through nucleotide residue number 4859, including the stop codon, wherein T can also be U;
  • VI a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 2E, from nucleotide residue number 846 through nucleotide residue number 4778, including the stop codon, wherein T can also be U;
  • VII a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 2F, from nucleotide residue number 614 through nucleotide residue number 3727, including the stop codon, wherein T can also be U;
  • VIII a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 2G, from nucleotide residue number 735 through nucleotide residue number 3881, including the stop codon, wherein T can also be U;
  • (IX) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 2H, from nucleotide residue number 735 through nucleotide residue number 4757, including the stop codon, wherein T can also be U;
  • (X) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 21, from nucleotide residue number 514 through nucleotide residue number 3627, including the stop codon, wherein T can also be U;
  • XI a polynucleotide that encodes a 109P1 D4-related protein that is at least 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100% homologous to an entire amino acid sequence shown in Figure 2A-I;
  • XII a polynucleotide that encodes a 109P1 D4-related protein that is at least 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to an entire amino acid sequence shown in Figure 2A-I;
  • (XIV) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35 amino acids of a peptide of Figures 3A in any whole number increment up to 1021 that includes at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of Figure 5;
  • (XV) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35 amino acids of a peptide of Figure 3A in any whole number increment up to 1021 that includes 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of Figure 6;
  • (XVI) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of Figure 3A in any whole number increment up to 1021 that includes 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of Figure 7;
  • (XVII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35 amino acids of a peptide of Figure 3A in any whole number increment up to 1021 that includes 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of Figure 8;
  • (XVI II) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of Figure 3A in any whole number increment up to 1021 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of Figure 9;
  • (XIX) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35 amino acids of a peptide of Figure 3B, 3C, and/or 3D in any whole number increment up to 1054, 1347, and/or 1337 respectively that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of Figure 5;
  • (XX) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35 amino acids of a peptide of Figure 3B, 3C, and/or 3D in any whole number increment up to 1054, 1347, and/or 1337 respectively that includes 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of Figure 6;
  • (XXI) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35 amino acids of a peptide of Figure 3B, 3C, and or 3D in any whole number increment up to 1054, 1347, and/or 1337 respectively that includes 1, 2, 3, , 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of Figure 7;
  • (XXII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of Figure 3B, 3C, and/or 3D in any whole number increment up to 1054, 1347, and/or 1337 respectively that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of Figure 8;
  • (XXIII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11 , 12, 13, 1 , 15, 1 ⁇ , 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35 amino acids of a peptide of Figure 3B, 3C, and/or 3D in any whole number increment up to 1054, 1347, and/or 1337 respectively that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of Figure 9;
  • (XXIV) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of Figure 3E, 3F, 3G, 3H and/or 31 in any whole number increment up to 1310, 1037, 1048, 1340, and/or 1037 respectively that includes 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of Figure 5;
  • (XXV) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of Figure 3E, 3F, 3G, 3H and/or 31 in any whole number increment up to 1310, 1037, 1048, 1340, and/or 1037 respectively that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of Figure 6; (XXVI) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of Figure 3E, 3F, 3G
  • (XXVI I) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of Figure 3E, 3F, 3G, 3H and/or 31 in any whole number increment up to 1310, 1037, 1048, 1340, and/or 1037 respectively that includes 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of Figure 8;
  • (XXVIII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35 amino acids of a peptide of Figure 3E, 3F, 3G, 3H, and/or 31 in any whole number increment up to 1310, 1037, 1048, 1340, and/or 1037 respectively that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of Figure 9;
  • (XXIX) a polynucleotide that is fully complementary to a polynucleotide of any one of (l)-(XXVIII);
  • (XXX) a polynucleotide that is fully complementary to a polynucleotide of any one of (l)-(XXIX);
  • (XXXII) a composition comprising a polynucleotide of any of (l)-(XXX) or peptide of (XXXI) together with a pharmaceutical excipient and/or in a human unit dose form;
  • (XXXIII) a method of using a polynucleotide of any (l)-(XXX) or peptide of (XXXI) or a composition of (XXXII) in a method to modulate a cell expressing 109P1D4;
  • (XXXIV) a method of using a polynucleotide of any (l)-(XXX) or peptide of (XXXI) or a composition of (XXXII) in a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressing 109P1D4;
  • (XXXV) a method of using a polynucleotide of any (l)-(XXX) or peptide of (XXXI) or a composition of (XXXII) in a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressing 109P1D4, said cell from a cancer of a tissue listed in Table I;
  • (XXXVI) a method of using a polynucleotide of any (l)-(XXX) or peptide of (XXXI) or a composition of (XXXII) in a method to diagnose, prophylax, prognose, or treat a a cancer;
  • (XXXVII) a method of using a polynucleotide of any (l)-(XXX) or peptide of (XXXI) or a composition of (XXXII) in a method to diagnose, prophylax, prognose, or treat a a cancer of a tissue listed in Table I; and;
  • (XXXVIII) a method of using a polynucleotide of any (l)-(XXX) or peptide of (XXXI) or a composition of (XXXII) in a method to identify or characterize a modulator of a cell expressing 109P1 D4.
  • Typical embodiments of the invention disclosed herein include 109P1D4 polynucleotides that encode specific portions of 109P1D4 mRNA sequences (and those which are complementary to such sequences) such as those that encode the proteins and/or fragments thereof, for example: .
  • representative embodiments of the invention disclosed herein include: polynucleotides and their encoded peptides themselves encoding about amino acid 1 to about amino acid 10 of the 109P1D4 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 10 to about amino acid 20 of the 109P1D4 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 20 to about amino acid 30 of the 109P1D4 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 30 to about amino acid 40 of the 109P1D4 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 40 to about amino acid 50 of the 109P1D4 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 50 to about amino acid 60 of the 109P1 D4 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 60 to about amino acid 70 of the
  • polynucleotides encoding portions of the amino acid sequence (of about 10 amino acids), of amino acids, 100 through the carboxyl terminal amino acid of the 109P1 D4 protein are embodiments of the invention. Wherein it is understood that each particular amino acid position discloses that position plus or minus five amino acid residues.
  • Polynucleotides encoding relatively long portions of a 109P1 D4 protein are also within the scope of the invention.
  • polynucleotides encoding from about amino acid 1 (or 20 or 30 or 40 etc.) to about amino acid 20, (or 30, or 40 or 50 etc.) of the 109P1 D4 protein "or variant" shown in Figure 2 or Figure 3 can be generated by a variety of techniques well known in the art.
  • These polynucleotide fragments can include any portion of the 109P1D4 sequence as shown in Figure 2.
  • Additional illustrative embodiments of the invention disclosed herein include 109P1D4 polynucleotide fragments encoding one or more of the biological motifs contained within a 109P1D4 protein "or variant" sequence, including one or more of the motif-bearing subsequences of a 109P1 D4 protein "or variant” set forth in Tables VIII-XXI and XXII-XLIX.
  • typical polynucleotide fragments of the invention encode one or more of the regions of 109P1D4 protein or variant that exhibit homology to a known molecule.
  • typical polynucleotide fragments can encode one or more of the 109P1D4 protein or variant N-glycosylation sites, cAMP and cGMP-dependent protein kinase phosphorylation sites, casein kinase II phosphorylation sites or N-myristoylation site and amidation sites.
  • HLA Peptide Tables a unique Search Peptide is used to obtain HLA peptides for a particular variant.
  • the position of each Search Peptide relative to its respective parent molecule is listed in Table VII.
  • a Search Peptide begins at position "X"
  • a particular Search Peptide begins at position 150 of its parental molecule, one must add 150 - 1, i.e., 149 to each HLA peptide amino acid position to calculate the position of that amino acid in the parent molecule.
  • the polynucleotides of the preceding paragraphs have a number of different specific uses.
  • the human 109P1 D4 gene maps to the chromosomal location set forth in the Example entitled "Chromosomal Mapping of 109P1 D4.”
  • polynucleotides that encode different regions of the 109P1D4 proteins are used to characterize cytogenetic abnormalities of this chromosomal locale, such as abnormalities that are identified as being associated with various cancers.
  • cytogenetic abnormalities of this chromosomal locale such as abnormalities that are identified as being associated with various cancers.
  • a variety of chromosomal abnormalities including rearrangements have been identified as frequent cytogenetic abnormalities in a number of different cancers (see e.g.
  • polynucleotides encoding specific regions of the 109P1D4 proteins provide new tools that can be used to delineate, with greater precision than previously possible, cytogenetic abnormalities in the chromosomal region that encodes 109P1 D4 that may contribute to the malignant phenotype.
  • these polynucleotides satisfy a need in the art for expanding the sensitivity of chromosomal screening in order to identify more subtle and less common chromosomal abnormalities (see e.g. Evans ef al., Am. J. Obstet. Gynecol 171(4): 1055-1057 (1994)).
  • 109P1D4 was shown to be highly expressed in prostate and other cancers
  • 109P1D4 polynucleotides are used in methods assessing the status of 109P1D4 gene products in normal versus cancerous tissues.
  • polynucleotides that encode specific regions of the 109P1D4 proteins are used to assess the presence of perturbations (such as deletions, insertions, point mutations, or alterations resulting in a loss of an antigen etc.) in specific regions of the 109P1D4 gene, such as regions containing one or more motifs.
  • Exemplary assays include both RT-PCR assays as well as single-strand conformation polymorphism (SSCP) analysis (see, e.g., Marrogi et al, J. Cutan. Pathol. 26(8): 369-378 (1999), both of which utilize polynucleotides encoding specific regions of a protein to examine these regions within the protein.
  • SSCP single-strand conformation polymorphism
  • nucleic acid related embodiments of the invention disclosed herein are genomic DNA, cDNAs, ribozymes, and antisense molecules, as well as nucleic acid molecules based on an alternative backbone, or including alternative bases, whether derived from natural sources or synthesized, and include molecules capable of inhibiting the RNA or protein expression of 109P1 D4.
  • antisense molecules can be RNAs or other molecules, including peptide nucleic acids (PNAs) or non-nucleic acid molecules such as phosphorothioate derivatives that specifically bind DNA or RNA in a base pair-dependent manner.
  • PNAs peptide nucleic acids
  • non-nucleic acid molecules such as phosphorothioate derivatives that specifically bind DNA or RNA in a base pair-dependent manner.
  • a skilled artisan can readily obtain these classes of nucleic acid molecules using the 109P1D4 polynucleotides and polynucleotide sequences disclosed herein.
  • Antisense technology entails the administration of exogenous oligonucleotides that bind to a target polynucleotide located within the cells.
  • the term "antisense” refers to the fact that such oligonucleotides are complementary to their intracellular targets, e.g., 109P1 D4. See for example, Jack Cohen, Oligodeoxynucleotides, Antisense Inhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1:1-5 (1988).
  • the 109P1D4 antisense oligonucleotides of the present invention include derivatives such as S-oligonucleotides (phosphorothioate derivatives or S-oligos, see, Jack Cohen, supra), which exhibit enhanced cancer cell growth inhibitory action.
  • S-oligos are isoelectronic analogs of an oligonucleotide (O-oligo) in which a nonbridging oxygen atom of the phosphate group is replaced by a sulfur atom.
  • the S-oligos of the present invention can be prepared by treatment of the corresponding O-oligos with 3H-1.2- benzodithiol-3-one-1,1 -dioxide, which is a sulfur transfer reagent. See, e.g., Iyer, R. P. ef al., J. Org. Chem. 55:4693-4698 (1990); and Iyer, R. P. ef al., J. Am. Chem. Soc. 112:1253-1254 (1990).
  • Additional 109P1D4 antisense oligonucleotides of the present invention include morpholino antisense oligonucleotides known in the art (see, e.g., Partridge ef al., 1996, Antisense & Nucleic Acid Drug Development 6: 169-175).
  • the 109P1 D4 antisense oligonucleotides of the present invention typically can be RNA or DNA that is complementary to and stably hybridizes with the first 100 5' codons or last 100 3' codons of a 109P1D4 genomic sequence or the corresponding mRNA. Absolute complementarity is not required, although high degrees of complementarity are preferred. Use of an oligonucleotide complementary to this region allows for the selective hybridization to 109P1 D4 mRNA and not to mRNA specifying other regulatory subunits of protein kinase.
  • 109P1 D4 antisense oligonucleotides of the present invention are 15 to 30-mer fragments of the antisense DNA molecule that have a sequence that hybridizes to 109P1 D4 mRNA.
  • 109P1 D4 antisense oligonucleotide is a 30-mer oligonucleotide that is complementary to a region in the first 10 5' codons or last 10 3' codons of 109P1D4.
  • the antisense molecules are modified to employ ribozymes in the inhibition of 109P1 D4 expression, see, e.g., L. A. Couture & D. T. Stinchcomb; Trends Genet 12: 510-515 (1996).
  • nucleotides of the invention include primers and primer pairs, which allow the specific amplification of polynucleotides of the invention or of any specific parts thereof, and probes that selectively or specifically hybridize to nucleic acid molecules of the invention or to any part thereof.
  • Probes can be labeled with a detectable marker, such as, for example, a radioisotope, fluorescent compound, bioluminescent compound, a chemiluminescent compound, metal chelator or enzyme.
  • a detectable marker such as, for example, a radioisotope, fluorescent compound, bioluminescent compound, a chemiluminescent compound, metal chelator or enzyme.
  • Such probes and primers are used to detect the presence of a 109P1D4 polynucleotide in a sample and as a means for detecting a cell expressing a 109P1D4 protein.
  • probes include polypeptides comprising all or part of the human 109P1 D4 cDNA sequence shown in Figure 2.
  • primer pairs capable of specifically amplifying 109P1D4 mRNAs are also described in the Examples.
  • primers and probes can be prepared based on the sequences provided herein and used effectively to amplify and/or detect a 109P1 D4 mRNA.
  • the 109P1 D4 polynucleotides of the invention are useful for a variety of purposes, including but not limited to their use as probes and primers for the amplification and/or detection of the 109P1D4 gene(s), mRNA(s), or fragments thereof; as reagents for the diagnosis and/or prognosis of prostate cancer and other cancers; as coding sequences capable of directing the expression of 109P1D4 polypeptides; as tools for modulating or inhibiting the expression of the 109P1D4 gene(s) and/or translation of the 109P1 D4 transcript(s); and as therapeutic agents.
  • the present invention includes the use of any probe as described herein to identify and isolate a 109P1 D4 or 109P1 D4 related nucleic acid sequence from a naturally occurring source, such as humans or other mammals, as well as the isolated nucleic acid sequence perse, which would comprise all or most of the sequences found in the probe used. II A4.) Isolation of 109P1D4-Encoding Nucleic Acid Molecules
  • the 109P1 D4 cDNA sequences described herein enable the isolation of other polynucleotides encoding 109P1 D4 gene product(s), as well as the isolation of polynucleotides encoding 109P1D4 gene product homologs, alternatively spliced isoforms, allelic variants, and mutant forms of a 109P1 D4 gene product as well as polynucleotides that encode analogs of 109P1 D4-related proteins.
  • Various molecular cloning methods that can be employed to isolate full length cDNAs encoding a 109P1 D4 gene are well known (see, for example, Sambrook, J.
  • a 109P1 D4 cDNA (e.g., Figure 2) or a portion thereof can be synthesized and used as a probe to retrieve overlapping and full-length cDNAs corresponding to a 109P1D4 gene.
  • a 109P1D4 gene itself can be isolated by screening genomic DNA libraries, bacterial artificial chromosome libraries (BACs), yeast artificial chromosome libraries (YACs), and the like, with 109P1D4 DNA probes or primers.
  • the invention also provides recombinant DNA or RNA molecules containing a 109P1D4 polynucleotide, a fragment, analog or homologue thereof, including but not limited to phages, plasmids, phagemids, cosmids, YACs, BACs, as well as various viral and non-viral vectors well known in the art, and cells transformed or transfected with such recombinant DNA or RNA molecules. Methods for generating such molecules are well known (see, for example, Sambrook ef al., 1989, supra).
  • the invention further provides a host-vector system comprising a recombinant DNA molecule containing a 109P1 D4 polynucleotide, fragment, analog or homologue thereof within a suitable prokaryotic or eukaryotic host cell.
  • suitable eukaryotic host cells include a yeast cell, a plant cell, or an animal cell, such as a mammalian cell or an insect cell (e.g., a baculovirus-infectible cell such as an Sf9 or HighFive cell).
  • suitable mammalian cells include various prostate cancer cell lines such as DU145 and TsuPrl , other transfectable or transducible prostate cancer cell lines, primary cells (PrEC), as well as a number of mammalian cells routinely used for the expression of recombinant proteins (e.g., COS, CHO, 293, 293T cells). More particularly, a polynucleotide comprising the coding sequence of 109P1D4 or a fragment, analog or homolog thereof can be used to generate 109P1 D4 proteins or fragments thereof using any number of host-vector systems routinely used and widely known in the art.
  • 109P1 D4 A wide range of host-vector systems suitable for the expression of 109P1 D4 proteins or fragments thereof are available, see for example, Sambrook etal., 1989, supra; Current Protocols in Molecular Biology, 1995, supra).
  • Preferred vectors for mammalian expression include but are not limited to pcDNA 3.1 myc-His-tag (Invitrogen) and the retroviral vector pSR ⁇ tkneo (Muller ef al. , 1991 , MCB 11 : 1785).
  • 109P1 D4 can be expressed in several prostate cancer and non-prostate cell lines, including for example 293, 293T, rat-1, NIH 3T3 and TsuPrl.
  • the host-vector systems of the invention are useful for the production of a 109P1 D4 protein or fragment thereof. Such host-vector systems can be employed to study the functional properties of 109P1D4 and 109P1D4 mutations or analogs.
  • Recombinant human 109P1 D4 protein or an analog or homolog or fragment thereof can be produced by mammalian cells transfected with a construct encoding a 109P1 D4-related nucleotide.
  • 293T cells can be transfected with an expression plasmid encoding 109P1 D4 or fragment, analog or homolog thereof, a 109P1 D4-related protein is expressed in the 293T cells, and the recombinant 109P1 D4 protein is isolated using standard purification methods (e.g., affinity purification using anti-109P1D4 antibodies).
  • a 109P1D4 coding sequence is subcloned into the retroviral vector pSR ⁇ MSVtkneo and used to infect various mammalian cell lines, such as NIH 3T3, TsuPrl, 293 and rat-1 in order to establish 109P1 D4 expressing cell lines.
  • mammalian cell lines such as NIH 3T3, TsuPrl, 293 and rat-1
  • Various other expression systems well known in the art can also be employed.
  • Expression constructs encoding a leader peptide joined in frame to a 109P1D4 coding sequence can be used for the generation of a secreted form of recombinant 109P1 D4 protein.
  • codon preferences for a specific species are calculated, for example, by utilizing codon usage tables available on the INTERNET such as at URL dna.affrc.go.jp/ ⁇ nakamura/codon.html.
  • Additional sequence modifications are known to enhance protein expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon/intron splice site signals, transposon-like repeats, and/or other such well-characterized sequences that are deleterious to gene expression.
  • the GC content of the sequence is adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. Where possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures.
  • Other useful modifications include the addition of a translational initiation consensus sequence at the start of the open reading frame, as described in Kozak, Mol. Cell Biol., 9:5073-5080 (1989).
  • 109P1 D4-related proteins Another aspect of the present invention provides 109P1 D4-related proteins.
  • Specific embodiments of 109P1 D4 proteins comprise a polypeptide having all or part of the amino acid sequence of human 109P1 D4 as shown in Figure 2 or Figure 3.
  • embodiments of 109P1D4 proteins comprise variant, homolog or analog polypeptides that have alterations in the amino acid sequence of 109P1D4 shown in Figure 2 or Figure 3.
  • Embodiments of a 109P1 D4 polypeptide include: a 109P1 D4 polypeptide having a sequence shown in Figure 2, a peptide sequence of a 109P1 D4 as shown in Figure 2 wherein T is U; at least 10 contiguous nucleotides of a polypeptide having the sequence as shown in Figure 2; or, at least 10 contiguous peptides of a polypeptide having the sequence as shown in Figure 2 where T is U.
  • embodiments of 109P1D4 peptides comprise, without limitation:
  • (V) a protein that comprises at least one peptide set forth in Tables VIII-XXI, collectively, which peptide is also set forth in Tables XXII to XLIX, collectively, optionally with a proviso that it is not an entire protein of Figure 2;
  • VII a protein that comprises at least two peptides selected from the peptides set forth in Tables VIII to XLIX collectively, with a proviso that the protein is not a contiguous sequence from an amino acid sequence of Figure 2;
  • (VIII) a protein that comprises at least one peptide selected from the peptides set forth in Tables VIII-XXI; and at least one peptide selected from the peptides set forth in Tables XXII to XLIX, with a proviso that the protein is not a contiguous sequence from an amino acid sequence of Figure 2;
  • (IX) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35 amino acids of a protein of Figure 3A, 3B, 3C, 3D and/or 3E in any whole number increment up to 1021 , 1054, 1347, 1337, and/or 1310 respectively that includes at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of Figure 5;
  • (X) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35 amino acids of a protein of Figure 3A, 3B, 3C, 3D, and/or 3E, in any whole number increment up to 1021, 1054, 1347, 1337, and/or 1310 respectively respectively that includes at least al least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
  • (XI) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35 amino acids of a protein of Figure 3A, 3B, 3C, 3D, and/or 3E, in any whole number increment up to 1021 , 1054, 1347, 1337, and/or 1310 respectively respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
  • (XI I) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of Figure 3A, 3B, 3C, 3D, and/or 3E, in any whole number increment up to 1021, 1054, 1347, 1337, and/or 1310 respectively respectively that includes at least at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
  • (XIV) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35 amino acids of a protein of Figure 3F, 3G, 3H, and/or 31, in any whole number increment up to 1037, 1048, 1340, and/or 1037 respectively that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of Figure 5;
  • (XV) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of Figure 3F, 3G, 3H, and/or 31 in any whole number increment up to 1037, 1048, 1340, and/or 1037 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of Figure 6;
  • (XVI) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35 amino acids of a protein of Figure 3F, 3G, 3H, and/or 31 in any whole number increment up to 1037, 1048, 1340, and/or 1037 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of Figure 7;
  • (XVII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of Figure 3F, 3G, 3H, and/or 31 in any whole number increment up to 1037, 1048, 1340, and/or 1037 respectively that includes at least at least 1, 2, 3, 4, 5, 6, , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of Figure 8;
  • (XVIII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, amino acids of a protein of Figure 3F, 3G, 3H, and/or 31 in any whole number increment up to 1037, 1048, 1340, and/or 1037 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of Figure 9;
  • XXVII a peptide which comprises one two, three, four, or five of the following characteristics, or an oligonucleotide encoding such peptide: i) a region of at least 5 amino acids of a particular peptide of Figure 3, in any whole number increment up to the full length of that protein in Figure 3, that includes an amino acid position having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Hydrophilicity profile of Figure 5; ii) a region of at least 5 amino acids of a particular peptide of Figure 3, in any whole number increment up to the full length of that protein in Figure 3, that includes an amino acid position having a value equal to or less than 0.5, 0.4, 0.3, 0.2, 0.1 , or having a value equal to 0.0, in the Hydropathicity profile of Figure 6; iii) a region of at least 5 amino acids of a particular peptide of Figure 3, in any whole number increment up to
  • (XXVIII) a composition comprising a peptide of (l)-(XXVII) or an antibody or binding region thereof together with a pharmaceutical excipient and/or in a human unit dose form.
  • (XXIX) a method of using a peptide of (l)-(XXVII), or an antibody or binding region thereof or a composition of (XXVIII) in a method to modulate a cell expressing 109P1D4,;
  • (XXX) a method of using a peptide of (l)-(XXVII) or an antibody or binding region thereof or a composition of (XXVIII) in a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressing 109P1D4;
  • (XXXI) a method of using a peptide of (l)-(XXVII) or an antibody or binding region thereof or a composition (XXVIII) in a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressing 109P1D4, said cell from a cancer of a tissue listed in Table I;
  • (XXXII) a method of using a peptide of (l)-(XXVII) or an antibody or binding region thereof or a composition of (XXVIII) in a method to diagnose, prophylax, prognose, or treat a a cancer;
  • (XXXIII) a method of using a peptide of (l)-(XXVII) or an antibody or binding region thereof or a composition of (XXVIII) in a method to diagnose, prophylax, prognose, or treat a a cancer of a tissue listed in Table I; and;
  • (XXXIV) a method of using a a peptide of (l)-(XXVII) or an antibody or binding region thereof or a composition (XXVIII) in a method to identify or characterize a modulator of a cell expressing 109P1 D4
  • Typical embodiments of the invention disclosed herein include 109P1D4 polynucleotides that encode specific portions of 109P1D4 mRNA sequences (and those which are complementary to such sequences) such as those that encode the proteins and/or fragments thereof, for example:
  • allelic variants of human 109P1 D4 share a high degree of structural identity and homology (e.g., 90% or more homology).
  • allelic variants of a 109P1 D4 protein contain conservative amino acid substitutions within the 109P1 D4 sequences described herein or contain a substitution of an amino acid from a corresponding position in a homologue of 109P1 D4.
  • One class of 109P1 D4 allelic variants are proteins that share a high degree of homology with at least a small region of a particular 109P1 D4 amino acid sequence, but further contain a radical departure from the sequence, such as a non-conservative substitution, truncation, insertion or frame shift.
  • Proteins of the invention can comprise 1 , 2, 3, , 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 conservative substitutions. Such changes include substituting any of isoleucine (I), valine (V), and leucine (L) for any other of these hydrophobic amino acids; aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q) for asparagine (N) and vice versa; and serine (S) for threonine (T) and vice versa.
  • isoleucine I
  • V valine
  • L leucine
  • Such changes include substituting any of isoleucine (I), valine (V), and leucine (L) for any other of these hydrophobic amino acids; aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q) for asparagine (N) and vice versa; and serine (S) for threonine (T) and vice versa.
  • substitutions can also be considered conservative, depending on the environment of the particular amino acid and its role in the three- dimensional structure of the protein.
  • G glycine
  • A alanine
  • V valine
  • M Methionine
  • L Lysine
  • K arginine
  • R arginine
  • Embodiments of the invention disclosed herein include a wide variety of art-accepted variants or analogs of 109P1D4 proteins such as polypeptides having amino acid insertions, deletions and substitutions.
  • 109P1D4 variants can be made using methods known in the art such as site-directed mutagenesis, alanine scanning, and PCR mutagenesis.
  • Site- directed mutagenesis (Carter et al., Nucl. Acids Res., 73:4331 (1986); Zoller ef al., Nucl.
  • Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence that is involved in a specific biological activity such as a protein-protein interaction.
  • preferred scanning amino acids are relatively small, neutral amino acids.
  • amino acids include alanine, glycine, serine, and cysteine.
  • Alanine is typically a preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta- carbon and is less likely to alter the main-chain conformation of the variant. Alanine is also typically preferred because it is the most common amino acid. Further, it is frequently found in both buried and exposed positions (Creighton, 77?e Proteins, (W.H. Freeman & Co., N.Y); Chothia, J. Mol. Biol., 150:1 (1976)). If alanine substitution does not yield adequate amounts of variant, an isosteric amino acid can be used.
  • 109P1D4 variants, analogs or homologs have the distinguishing attribute of having at least one epitope that is "cross reactive" with a 109P1D4 protein having an amino acid sequence of Figure 3.
  • cross reactive means that an antibody or T cell that specifically binds to a 109P1D4 variant also specifically binds to a 109P1 D4 protein having an amino acid sequence set forth in Figure 3.
  • a polypeptide ceases to be a variant of a protein shown in Figure 3, when it no longer contains any epitope capable of being recognized by an antibody or T cell that specifically binds to the starting 109P1D4 protein.
  • 109P1D4-related protein variants share 70%, 75%, 80%, 85% or 90% or more similarity with an amino acid sequence of Figure 3, or a fragment thereof.
  • Another specific class of 109P1 D4 protein variants or analogs comprises one or more of the 109P1D4 biological motifs described herein or presently known in the art.
  • analogs of 109P1D4 fragments that have altered functional (e.g. immunogenic) properties relative to the starting fragment. It is to be appreciated that motifs now or which become part of the art are to be applied to the nucleic or amino acid sequences of Figure 2 or Figure 3.
  • embodiments of the claimed invention include polypeptides containing less than the full amino acid sequence of a 109P1 D4 protein shown in Figure 2 or Figure 3.
  • representative embodiments of the invention comprise peptides/proteins having any 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15 or more contiguous amino acids of a 109P1D4 protein shown in Figure 2 or Figure 3.
  • representative embodiments of the invention disclosed herein include polypeptides consisting of about amino acid 1 to about amino acid 10 of a 109P1 D4 protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 10 to about amino acid 20 of a 109P1D4 prote siirn shown in Figure 2 or Figur • e 3, polypeptides consi ;ing of about amino acid 20 to about amino acid 30 of a 109P1D4 prote siiin shown in Figure 2 or Figun ⁇ e 3 .
  • polypeptides consi ing of about amino acid 30 to about amino acid 40 of a 109P1D4 prote siiin shown in Figure 2 or Figur polypeptides consi :ing of about amino acid 40 to about amino acid 50 of a 109P1D4 prote siiin shown in Figure 2 or Figur e 3 ; polypeptides consi :ing of about amino acid 50 to about amino acid 60 of a 109P1D4 prote eiirn shown in Figure 2 or Figur ⁇ e 3, polypeptides consi :ing of about amino acid 60 to about amino acid 70 of a 109P1D4 prote siiin shown in Figure 2 or Figur • e 3.
  • polypeptides consi ing of about amino acid 70 to about amino acid 80 of a 109P1 D4 prote siiin shown in Figure 2 or Figur polypeptides consi :ing of about amino acid 80 to about amino acid 90 of a 109P1 D4 prote siiin shown in Figure 2 or Figur e 3 polypeptides consi ing of about amino acid 90 to about amino acid 100 of a 109P1D4 protein shown in Figure 2 or Figure 3, etc. throughout the entirety of a 109P1 D4 amino acid sequence.
  • polypeptides consisting of about amino acid 1 (or 20 or 30 or 40 etc.) to about amino acid 20, (or 130, or 140 or 150 etc.) of a 109P1D4 protein shown in Figure 2 or Figure 3 are embodiments of the invention. It is to be appreciated that the starting and stopping positions in this paragraph refer to the specified position as well as that position plus or minus 5 residues.
  • 109P1D4-related proteins are generated using standard peptide synthesis technology or using chemical cleavage methods well known in the art. Alternatively, recombinant methods can be used to generate nucleic acid molecules that encode a 109P1D4-related protein. In one embodiment, nucleic acid molecules provide a means to generate defined fragments of a 109P1 D4 protein (or variants, homologs or analogs thereof). HLA.) Motif-bearing Protein Embodiments
  • Additional illustrative embodiments of the invention disclosed herein include 109P1D4 polypeptides comprising the amino acid residues of one or more of the biological motifs contained within a 109P1 D4 polypeptide sequence set forth in Figure 2 or Figure 3.
  • Various motifs are known in the art, and a protein can be evaluated for the presence of such motifs by a number of publicly available Internet sites (see, e.g., URL addresses: pfam.wustl.edu/; searchlauncher.bcm.tmc.edu/seq- search/struc-predict.html; psort.ims.u-tokyo.ac.jp/; cbs.dtu.dk/; ebi.ac.uk/interpro/scan.html; expasy.cn/tools/scnpsit1.html; EpimatrixTM and EpimerTM, Brown University, brown.edu/Research/TB-HIV_Lab/ep
  • Motif bearing subsequences of all 109P1 D4 variant proteins are set forth and identified in Tables VIII-XXI and XXII- XLIX.
  • Table V sets forth several frequently occurring motifs based on pfam searches (see URL address pfam.wustl.edu/). The columns of Table V list (1) motif name abbreviation, (2) percent identity found amongst the different member of the motif family, (3) motif name or description and (4) most common function; location information is included if the motif is relevant for location.
  • Polypeptides comprising one or more of the 109P1D4 motifs discussed above are useful in elucidating the specific characteristics of a malignant phenotype in view of the observation that the 109P1 D4 motifs discussed above are associated with growth dysregulation and because 109P1D4 is overexpressed in certain cancers (See, e.g., Table I).
  • Casein kinase II, cAMP and camp-dependent protein kinase, and Protein Kinase C are enzymes known to be associated with the development of the malignant phenotype (see e.g.
  • Amidation is another protein modification also associated with cancer and cancer progression (see e.g. Treston ef al., J. Natl. Cancer Inst. Monogr. (13): 169-175 (1992)).
  • proteins of the invention comprise one or more of the immunoreactive epitopes identified in accordance with art-accepted methods, such as the peptides set forth in Tables VIII-XXI and XXII-XLIX.
  • CTL epitopes can be determined using specific algorithms to identify peptides within a 109P1D4 protein that are capable of optimally binding to specified HLA alleles (e.g., Table IV; EpimatrixTM and EpimerTM, Brown University, URL brown.edu/Research/TB- HIV_Lab/epimatrix/epimatrix.html; and BIMAS, URL bimas.dcrt.nih.gov/.)
  • processes for identifying peptides that have sufficient binding affinity for HLA molecules and which are correlated with being immunogenic epitopes are well known in the art, and are carried out without undue experimentation.
  • processes for identifying peptides that are immunogenic epitopes are well known in the
  • epitopes in order to modulate immunogenicity. For example, one begins with an epitope that bears a CTL or HTL motif (see, e.g., the HLA Class I and HLA Class II motifs/supermotifs of Table IV).
  • the epitope is analoged by substituting out an amino acid at one of the specified positions, and replacing it with another amino acid specified for that position.
  • residues defined in Table IV one can substitute out a deleterious residue in favor of any other residue, such as a preferred residue; substitute a less- preferred residue with a preferred residue; or substitute an originally-occurring preferred residue with another preferred residue. Substitutions can occur at primary anchor positions or at other positions in a peptide; see,, e.g., Table IV.
  • polypeptides comprising combinations of the different motifs set forth in Table VI, and/or, one or more of the predicted CTL epitopes of Tables VIII-XXI and XXII-XLIX, and/or, one or more of the predicted HTL epitopes of Tables XL I-XLIX, and/or, one or more of the T cell binding motifs known in the art.
  • Preferred embodiments contain no insertions, deletions or substitutions either within the motifs or within the intervening sequences of the polypeptides.
  • embodiments which include a number of either N-terminal and/or C-terminal amino acid residues on either side of these motifs may be desirable (to, for example, include a greater portion of the polypeptide architecture in which the motif is located).
  • the number of N-terminal and/or C-terminal amino acid residues on either side of a motif is between about 1 to about 100 amino acid residues, preferably 5 to about 50 amino acid residues.
  • 109P1 D4-related proteins are embodied in many forms, preferably in isolated form.
  • a purified 109P1 D4 protein molecule will be substantially free of other proteins or molecules that impair the binding of 109P1 D4 to antibody, T cell or other ligand.
  • the nature and degree of isolation and purification will depend on the intended use.
  • Embodiments of a 109P1 D4- related proteins include purified 109P1D4-related proteins and functional, soluble 109P1D4-related proteins.
  • a functional, soluble 109P1D4 protein or fragment thereof retains the ability to be bound by antibody, T cell or other ligand.
  • the invention also provides 109P1D4 proteins comprising biologically active fragments of a 109P1D4 amino acid sequence shown in Figure 2 or Figure 3.
  • Such proteins exhibit properties of the starting 109P1D4 protein, such as the ability to elicit the generation of antibodies that specifically bind an epitope associated with the starting 109P1 D4 protein; to be bound by such antibodies; to elicit the activation of HTL or CTL; and/or, to be recognized by HTL or CTL that also specifically bind to the starting protein.
  • 109P1 D4-related polypeptides that contain particularly interesting structures can be predicted and/or identified using various analytical techniques well known in the art, including, for example, the methods of Chou-Fasman, Garnier-Robson, Kyte- Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis, or based on i munogenicity. Fragments that contain such structures are particularly useful in generating subunit-specific anti-109P1 D4 antibodies or T cells or in identifying cellular factors that bind to 109P1D4. For example, hydrophilicity profiles can be generated, and immunogenic peptide fragments identified, using the method of Hopp, T.P. and Woods, K.R., 1981, Proc. Natl.
  • Hydropathicity profiles can be generated, and immunogenic peptide fragments identified, using the method of Kyte, J. and Doolittle, R.F., 1982, J. Mol. Biol. 157:105-132. Percent (%) Accessible Residues profiles can be generated, and immunogenic peptide fragments identified, using the method of Janin J., 1979, Nature 277:491-492. Average Flexibility profiles can be generated, and immunogenic peptide fragments identified, using the method of Bhaskaran R., Ponnuswamy P.K., 1988, int. J. Pept. Protein Res. 32:242-255. Beta-turn profiles can be generated, and immunogenic peptide fragments identified, using the method of Deleage, G contend Roux B., 1987, Protein Engineering 1:289-294.
  • CTL epitopes can be determined using specific algorithms to identify peptides within a 109P1D4 protein that are capable of optimally binding to specified HLA alleles (e.g., by using the SYFPEITHI site at World Wide Web URL syfpeithi.bmi- heidelberg.com/; the listings in Table IV(A)-(E); EpimatrixTM and EpimerTM, Brown University, URL (brown.edu/Research/TB- HIV_Lab/epimatrix/epimatrix.html); and BIMAS, URL bimas.dcrt.nih.gov ).
  • peptide epitopes from 109P1D4 that are presented in the context of human MHC Class I molecules, e.g., HLA-A1, A2, A3, A11, A24, B7 and B35 were predicted (see, e.g., Tables VIII-XXI, XXII-XLIX).
  • the complete amino acid sequence of the 109P1D4 protein and relevant portions of other variants i.e., for HLA Class I predictions 9 flanking residues on either side of a point mutation or exon juction, and for HLA Class II predictions 14 flanking residues on either side of a point mutation or exon junction corresponding to that variant, were entered into the HLA Peptide Motif Search algorithm found in the Bioinformatics and Molecular Analysis Section (BIMAS) web site listed above; in addition to the site SYFPEITHI, at URL syfpeithi.bmi- heidelberg.com/.
  • BIMAS Bioinformatics and Molecular Analysis Section
  • HLA peptide motif search algorithm was developed by Dr. Ken Parker based on binding of specific peptide sequences in the groove of HLA Class I molecules, in particular HLA-A2 (see, e.g., Falk etal., Nature 351: 290-6 (1991); Hunt ef a/., Science 255:1261-3 (1992); Parker ef al., J. Immunol. 149:3580-7 (1992); Parker ef a/., J. Immunol. 152:163-75 (1994)).
  • This algorithm allows location and ranking of 8-mer, 9-mer, and 10-mer peptides from a complete protein sequence for predicted binding to HLA-A2 as well as numerous other HLA Class I molecules.
  • HLA class I binding peptides are 8-, 9-, 10 or 11-mers.
  • the epitopes preferably contain a leucine (L) or methionine (M) at position 2 and a valine (V) or leucine (L) at the C-terminus (see, e.g., Parker ef at, J. Immunol. 149:3580-7 (1992)).
  • Selected results of 109P1D4 predicted binding peptides are shown in Tables VIII-XXI and XXII-XLIX herein.
  • Tables VIII- XXI and XXII-XLVII selected candidates, 9-mers and 10-mers, for each family member are shown along with their location, the amino acid sequence of each specific peptide, and an estimated binding score.
  • Tables XLVI-XLIX selected candidates, 15-mers, for each family member are shown along with their location, the amino acid sequence of each specific peptide, and an estimated binding score.
  • the binding score corresponds to the estimated half time of dissociation of complexes containing the peptide at 37°C at pH 6.5. Peptides with the highest binding score are predicted to be the most tightly bound to HLA Class I on the cell surface for the greatest period of time and thus represent the best immunogenic targets for T-cell recognition.
  • every epitope predicted by the BIMAS site, EpimerTM and EpimatrixTM sites, or specified by the HLA class I or class II motifs available in the art or which become part of the art such as set forth in Table IV (or determined using World Wide Web site URL syfpeithi.bmi-heidelberg.com/, or BIMAS, bimas.dcrt.nih.gov/) are to be "applied” to a 109P1D4 protein in accordance with the invention.
  • “applied” means that a 109P1D4 protein is evaluated, e.g., visually or by computer-based patterns finding methods, as appreciated by those of skill in the relevant art.
  • Every subsequence of a 109P1D4 protein of 8, 9, 10, or 11 amino acid residues that bears an HLA Class I motif, or a subsequence of 9 or more amino acid residues that bear an HLA Class II motif are within the scope of the invention.
  • 109P1D4 can be conveniently expressed in cells (such as 293T cells) transfected with a commercially available expression vector such as a CMV-driven expression vector encoding 109P1D4 with a C-terminal 6XHis and MYC tag (pcDNA3.1/mycHIS, Invitrogen or Tag ⁇ , GenHunter Corporation, Nashville TN).
  • the Tag5 vector provides an IgGK secretion signal that can be used to facilitate the production of a secreted 109P1 D4 protein in transfected cells.
  • the secreted HIS-tagged 109P1D4 in the culture media can be purified, e.g., using a nickel column using standard techniques.
  • 109P1 D4-related proteins such as covalent modifications are included within the scope of this invention.
  • One type of covalent modification includes reacting targeted amino acid residues of a 109P1D4 polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues of a 109P1 D4 protein.
  • Another type of covalent modification of a 109P1 D4 polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of a protein of the invention.
  • Another type of covalent modification of 109P1D4 comprises linking a 109P1D4 polypeptide to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.
  • PEG polyethylene glycol
  • polypropylene glycol polypropylene glycol
  • polyoxyalkylenes polyoxyalkylenes
  • the 109P1D4-related proteins of the present invention can also be modified to form a chimeric molecule comprising 109P1D4 fused to another, heterologous polypeptide or amino acid sequence.
  • a chimeric molecule can be synthesized chemically or recombinantly.
  • a chimeric molecule can have a protein of the invention fused to another tumor- associated antigen or fragment thereof.
  • a protein in accordance with the invention can comprise a fusion of fragments of a 109P1D4 sequence (amino or nucleic acid) such that a molecule is created that is not, through its length, directly homologous to the amino or nucleic acid sequences shown in Figure 2 or Figure 3.
  • Such a chimeric molecule can comprise multiples of the same subsequence of 109P1D4.
  • a chimeric molecule can comprise a fusion of a 109P1D4-related protein with a polyhistidine epitope tag, which provides an epitope to which immobilized nickel can selectively bind, with cytokines or with growth factors.
  • the epitope tag is generally placed at the amino- or carboxyl- terminus of a 109P1 D4 protein.
  • the chimeric molecule can comprise a fusion of a 109P1 D4-related protein with an immunoglobulin or a particular region of an immunoglobulin.
  • the chimeric molecule also referred to as an "immunoadhesin"
  • a fusion could be to the Fc region of an IgG molecule.
  • the Ig fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of a 109P1 D4 polypeptide in place of at least one variable region within an Ig molecule.
  • the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge, CHI, CH2 and CH3 regions of an IgGI molecule.
  • the proteins of the invention have a number of different specific uses. As 109P1D4 is highly expressed in prostate and other cancers, 109P1D4-related proteins are used in methods that assess the status of 109P1D4 gene products in normal versus cancerous tissues, thereby elucidating the malignant phenotype. Typically, polypeptides from specific regions of a 109P1D4 protein are used to assess the presence of perturbations (such as deletions, insertions, point mutations etc.) in those regions (such as regions containing one or more motifs).
  • perturbations such as deletions, insertions, point mutations etc.
  • Exemplary assays utilize antibodies or T cells targeting 109P1 D4-related proteins comprising the amino acid residues of one or more of the biological motifs contained within a 109P1 D4 polypeptide sequence in order to evaluate the characteristics of this region in normal versus cancerous tissues or to elicit an immune response to the epitope.
  • 109P1D4-related proteins that contain the amino acid residues of one or more of the biological motifs in a 109P1 D4 protein are used to screen for factors that interact with that region of 109P1D4.
  • 109P1D4 protein fragments/subsequences are particularly useful in generating and characterizing domain-specific antibodies (e.g., antibodies recognizing an extracellular or intracellular epitope of a 109P1 D4 protein), for identifying agents or cellular factors that bind to 109P1 D4 or a particular structural domain thereof, and in various therapeutic and diagnostic contexts, including but not limited to diagnostic assays, cancer vaccines and methods of preparing such vaccines.
  • domain-specific antibodies e.g., antibodies recognizing an extracellular or intracellular epitope of a 109P1 D4 protein
  • Proteins encoded by the 109P1 D4 genes, or by analogs, homologs or fragments thereof, have a variety of uses, including but not limited to generating antibodies and in methods for identifying ligands and other agents and cellular constituents that bind to a 109P1D4 gene product.
  • Antibodies raised against a 109P1D4 protein or fragment thereof are useful in diagnostic and prognostic assays, and imaging methodologies in the management of human cancers characterized by expression of 109P1D4 protein, such as those listed in Table I. Such antibodies can be expressed intracellularly and used in methods of treating patients with such cancers.
  • 109P1D4-related nucleic acids or proteins are also used in generating HTL or CTL responses.
  • 109P1 D4 proteins are used, including but not limited to various types of radioimmunoassays, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunofluorescent assays (ELIFA), immunocytochemical methods, and the like.
  • Antibodies can be labeled and used as immunological imaging reagents capable of detecting 109P1D4-expressing cells (e.g., in radioscintigraphic imaging methods).
  • 109P1D4 proteins are also particularly useful in generating cancer vaccines, as further described herein.
  • Another aspect of the invention provides antibodies that bind to 109P1 D4-related proteins.
  • Preferred antibodies specifically bind to a 109P1 D4-related protein and do not bind (or bind weakly) to peptides or proteins that are not 109P1 D4- related proteins under physiological conditions.
  • physiological conditions include: 1) phosphate buffered saline; 2) Tris-buffered saline containing 25mM Tris and 150 mM NaCI; or normal saline (0.9% NaCI); 4) animal serum such as human serum; or, 5) a combination of any of 1 ) through 4); these reactions preferably taking place at pH 7.5, alternatively in a range of pH 7.0 to 8.0, or alternatively in a range of pH 6.5 to 8.5; also, these reactions taking place at a temperature between 4°C to 37°C.
  • antibodies that bind 109P1D4 can bind 109P1D4-related proteins such as the homologs or analogs thereof.
  • 109P1 D4 antibodies of the invention are particularly useful in cancer (see, e.g., Table I) diagnostic and prognostic assays, and imaging methodologies. Similarly, such antibodies are useful in the treatment, diagnosis, and/or prognosis of other cancers, to the extent 109P1 D4 is also expressed or overexpressed in these other cancers. Moreover, intracellularly expressed antibodies (e.g., single chain antibodies) are therapeutically useful in treating cancers in which the expression of 109P1D4 is involved, such as advanced or metastatic prostate cancers.
  • the invention also provides various immunological assays useful for the detection and quantification of 109P1 D4 and mutant 109P1 D4-related proteins.
  • Such assays can comprise one or more 109P1 D4 antibodies capable of recognizing and binding a 109P1D4-related protein, as appropriate.
  • These assays are performed within various immunological assay formats well known in the art, including but not limited to various types of radioimmunoassays, enzyme-linked im unosorbent assays (ELISA), enzyme-linked immunofluorescent assays (ELIFA), and the like.
  • Immunological non-antibody assays of the invention also comprise T cell immunogenicity assays (inhibitory or stimulatory) as well as major histocompatibility complex (MHC) binding assays.
  • T cell immunogenicity assays inhibitory or stimulatory
  • MHC major histocompatibility complex
  • immunological imaging methods capable of detecting prostate cancer and other cancers expressing 109P1D4 are also provided by the invention, including but not limited to radioscintigraphic imaging methods using labeled 109P1D4 antibodies.
  • assays are clinically useful in the detection, monitoring, and prognosis of 109P1D4 expressing cancers such as prostate cancer.
  • 109P1 D4 antibodies are also used in methods for purifying a 109P1 D4-re!ated protein and for isolating 109P1 D4 homologues and related molecules.
  • a method of purifying a 109P1D4-related protein comprises incubating a 109P1 D4 antibody, which has been coupled to a solid matrix, with a lysate or other solution containing a 109P1 D4-related protein under conditions that permit the 109P1 D4 antibody to bind to the 109P1 D4-related protein; washing the solid matrix to eliminate impurities; and eluting the 109P1D4-related protein from the coupled antibody.
  • Other uses of 109P1D4 antibodies in accordance with the invention include generating anti-idiotypic antibodies that mimic a 109P1D4 protein.
  • antibodies can be prepared by immunizing a suitable mammalian host using a 109P1 D4-related protein, peptide, or fragment, in isolated or immunoconjugated form (Antibodies: A Laboratory Manual, CSH Press, Eds., Harlow, and Lane (1988); Harlow, Antibodies, Cold Spring Harbor Press, NY (1989)).
  • fusion proteins of 109P1D4 can also be used, such as a 109P1D4 GST-fusion protein.
  • a GST fusion protein comprising all or most of the amino acid sequence of Figure 2 or Figure 3 is produced, then used as an immunogen to generate appropriate antibodies.
  • a 109P1D4-related protein is synthesized and used as an immunogen.
  • naked DNA immunization techniques known in the art are used (with or without purified 109P1 D4-related protein or 109P1D4 expressing cells) to generate an immune response to the encoded immunogen (for review, see Donnelly ef a/., 1997, Ann. Rev. Immunol. 15: 617-648).
  • the amino acid sequence of a 109P1D4 protein as shown in Figure 2 or Figure 3 can be analyzed to select specific regions of the 109P1 D4 protein for generating antibodies.
  • hydrophobicity and hydrophilicity analyses of a 109P1 D4 amino acid sequence are used to identify hydrophilic regions in the 109P1D4 structure. Regions of a 109P1D4 protein that show immunogenic structure, as well as other regions and domains, can readily be identified using various other methods known in the art, such as Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis. Hydrophilicity profiles can be generated using the method of Hopp, T.P. and Woods, K.R., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824-
  • Hydropathicity profiles can be generated using the method of Kyte, J. and Doolittle, R.F., 1982, J. Mol. Biol. 157:105-
  • Percent (%) Accessible Residues profiles can be generated using the method of Janin J., 1979, Nature 277:491-492.
  • Beta-turn profiles can be generated using the method of Deleage, G., Roux B., 1987, Protein
  • Methods for preparing a protein or polypeptide for use as an immunogen are well known in the art. Also well known in the art are methods for preparing immunogenic conjugates of a protein with a carrier, such as BSA, KLH or other carrier protein. In some circumstances, direct conjugation using, for example, carbodumide reagents are used; in other instances linking reagents such as those supplied by Pierce Chemical Co., Rockford, IL, are effective. Administration of a 109P1D4 immunogen is often conducted by injection over a suitable time period and with use of a suitable adjuvant, as is understood in the art. During the immunization schedule, titers of antibodies can be taken to determine adequacy of antibody formation.
  • 109P1D4 monoclonal antibodies can be produced by various means well known in the art.
  • immortalized cell lines that secrete a desired monoclonal antibody are prepared using the standard hybridoma technology of Kohler and Milstein or modifications that immortalize antibody-producing B cells, as is generally known.
  • Immortalized cell lines that secrete the desired antibodies are screened by immunoassay in which the antigen is a 109P1D4-related protein.
  • the appropriate immortalized cell culture is identified, the cells can be expanded and antibodies produced either from in vitro cultures or from ascites fluid.
  • the antibodies or fragments of the invention can also be produced, by recombinant means. Regions that bind specifically to the desired regions of a 109P1 D4 protein can also be produced in the context of chimeric or complementarity- determining region (CDR) grafted antibodies of multiple species origin. Humanized or human 109P1 D4 antibodies can also be produced, and are preferred for use in therapeutic contexts.
  • CDR complementarity- determining region
  • Fully human 109P1D4 monoclonal antibodies can be generated using cloning technologies employing large human Ig gene combinatorial libraries (i.e., phage display) (Griffiths and Hoogenboom, Building an in vitro immune system: human antibodies from phage display libraries. In: Protein Engineering of Antibody Molecules for Prophylactic and Therapeutic Applications in Man, Clark, M. (Ed.), Nottingham Academic, pp 45-64 (1993); Burton and Barbas, Human Antibodies from combinatorial libraries. Id-, PP 65-82).
  • Fully human 109P1D4 monoclonal antibodies can also be produced using transgenic mice engineered to contain human immunoglobulin gene loci as described in PCT Patent Application W098/24893, Kucherlapati and Jakobovits etal., published December 3, 1997 (see also, Jakobovits, 1998, Exp. Opin. Invest. Drugs 7(4): 607-614; U.S. patents 6,162,963 issued 19 December 2000; 6,150,584 issued 12 November 2000; and, 6,114698 issued ⁇ September 2000). This method avoids the in vitro manipulation required with phage display technology and efficiently produces high affinity authentic human antibodies.
  • Reactivity of 109P1 D4 antibodies with a 109P1 D4-related protein can be established by a number of well known means, inciuding Western blot, i munoprecipitation, ELISA, and FACS analyses using, as appropriate, 109P1D4-related proteins, 109P1 D4-expressing cells or extracts thereof.
  • a 109P1 D4 antibody or fragment thereof can be labeled with a detectable marker or conjugated to a second molecule. Suitable detectable markers include, but are not limited to, a radioisotope, a fluorescent compound, a bioluminescent compound, chemiluminescent compound, a metal chelator or an enzyme.
  • bi-specific antibodies specific for two or more 109P1 D4 epitopes are generated using methods generally known in the art.
  • Homodimeric antibodies can also be generated by cross-linking techniques known in the art (e.g., Wolff ef al., Cancer Res. ⁇ 3: 2560-2566).
  • compositions of the invention induce a therapeutic or prophylactic immune responses in very broad segments of the worldwide population.
  • immunology-related technology For an understanding of the value and efficacy of compositions of the invention that induce cellular immune responses, a brief review of immunology-related technology is provided.
  • a complex of an HLA molecule and a peptidic antigen acts as the ligand recognized by HLA-restricted T cells (Buus, S. et al., Cell 47:1071, 1986; Babbitt, B. P. ef al., Nature 317:359, 1985; Townsend, A. and Bodmer, H., Annu. Rev. Immunol. 7:601, 1989; Germain, R. N., Annu. Rev. Immunol. 11:403, 1993).
  • class I and class II allele-specific HLA binding motifs allows identification of regions within a protein that are correlated with binding to particular HLA antigen(s).
  • candidates for epitope-based vaccines have been identified; such candidates can be further evaluated by HLA-peptide binding assays to determine binding affinity and/or the time period of association of the epitope and its corresponding HLA molecule. Additional confirmatory work can be performed to select, amongst these vaccine candidates, epitopes with preferred characteristics in terms of population coverage, and/or immunogenicity.
  • HLA transgenic mice see, e.g., Wentworth, P. A. et al., J. Immunol. 26;97, 1996; Wentworth, P. A. etal., Int. Immunol. 8:651, 1996; Alexander, J. etal., J. Immunol. 159:4753, 1997).
  • peptides in incomplete Freund's adjuvant are administered subcutaneously to HLA transgenic mice.
  • splenocytes are removed and cultured in vitro in the presence of test peptide for approximately one week.
  • Peptide-specific T cells are detected using, e.g., a 51 Cr-release assay involving peptide sensitized target cells and target cells expressing endogenously generated antigen.
  • recall responses are detected by culturing PBL from subjects that have been exposed to the antigen due to disease and thus have generated an immune response "naturally", or from patients who were vaccinated against the antigen.
  • PBL from subjects are cultured in vitro for 1-2 weeks in the presence of test peptide plus antigen presenting cells (APC) to allow activation of "memory" T cells, as compared to "naive” T cells.
  • APC antigen presenting cells
  • T cell activity is detected using assays including 51 Cr release in olving peptide-sensitized targets, T cell proliferation, or lymphokine release.
  • Nucleic acids that encode a 109P1D4-related protein can also be used to generate either transgenic animals or "knock out" animals that, in turn, are useful in the development and screening of therapeutically useful reagents.
  • cDNA encoding 109P1 D4 can be used to clone genomic DNA that encodes 109P1D4.
  • the cloned genomic sequences can then be used to generate transgenic animals containing cells that express DNA that encode 109P1 D4.
  • Methods for generating transgenic animals, particularly animals such as mice or rats have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866 issued 12 April 1988, and 4,870,009 issued 26 September 1989.
  • particular cells would be targeted for 109P1D4 transgene incorporation with tissue-specific enhancers.
  • Transgenic animals that include a copy of a transgene encoding 109P1 D4 can be used to examine the effect of increased expression of DNA that encodes 109P1D4. Such animals can be used as tester animals for reagents thought to confer protection from, for example, pathological conditions associated with its overexpression.
  • an animal is treated with a reagent and a reduced incidence of a pathological condition, compared to untreated animals that bear the transgene, would indicate a potential therapeutic intervention for the pathological condition.
  • non-human homologues of 109P1 D4 can be used to construct a 109P1 D4 "knock out" animal that has a defective or altered gene encoding 109P1 D4 as a result of homologous recombination between the endogenous gene encoding 109P1D4 and altered genomic DNA encoding 109P1D4 introduced into an embryonic cell of the animal.
  • cDNA that encodes 109P1 D4 can be used to clone genomic DNA encoding 109P1 D4 in accordance with established techniques.
  • a portion of the genomic DNA encoding 109P1D4 can be deleted or replaced with another gene, such as a gene encoding a selectable marker that can be used to monitor integration.
  • flanking DNA typically, several kilobases of unaltered flanking DNA (both at the 5' and 3' ends) are included in the vector (see, e.g., Thomas and Capecchi, Cejl, ⁇ .:503 (1987) for a description of homologous recombination vectors).
  • the vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced DNA has homologously reco bined with the endogenous DNA are selected (see, e.g., Li ef al, Cell, 69:915 (1992)). The selected cells are then injected into a blastocyst of an animal
  • aggregation chimeras see, e.g., Bradley, in Teratocarcinomas and Embryonic Stem Cells: A
  • a chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal, and the embryo brought to term to create a "knock out" animal.
  • Progeny harboring the homologously recombined DNA in their germ cells can be identified by standard techniques and used to breed animals in which all cells of the animal contain the homologously recombined DNA.
  • Knock out animals can be characterized, for example, for their ability to defend against certain pathological conditions or for their development of pathological conditions due to absence of a 109P1D4 polypeptide.
  • Another aspect of the present invention relates to methods for detecting 109P1D4 polynucleotides and 109P1D4- related proteins, as well as methods for identifying a cell that expresses 109P1 D4.
  • the expression profile of 109P1 D4 makes it a diagnostic marker for metastasized disease. Accordingly, the status of 109P1 D4 gene products provides information useful for predicting a variety of factors including susceptibility to ad anced stage disease, rate of progression, and/or tumor aggressiveness.
  • the status of 109P1D4 gene products in patient samples can be analyzed by a variety protocols that are well known in the art including immunohistochemical analysis, the variety of Northern blotting techniques including in situ hybridization, RT-PCR analysis (for example on laser capture micro-dissected samples), Western blot analysis and tissue array analysis.
  • the invention provides assays for the detection of 109P1D4 polynucleotides in a biological sample, such as serum, bone, prostate, and other tissues, urine, semen, cell preparations, and the like.
  • Detectable 109P1D4 polynucleotides include, for example, a 109P1 D4 gene or fragment thereof, 109P1 D4 mRNA, alternative splice variant 109P1 D4 mRNAs, and recombinant DNA or RNA molecules that contain a 109P1 D4 polynucleotide.
  • a number of methods for amplifying and/or detecting the presence of 109P1 D4 polynucleotides are well known in the art and can be employed in the practice of this aspect of the invention.
  • a method for detecting a 109P1 D4 mRNA in a biological sample comprises producing cDNA from the sample by reverse transcription using at least one primer; amplifying the cDNA so produced using a 109P1 D4 polynucleotides as sense and antisense primers to amplify 109P1D4 cDNAs therein; and detecting the presence of the amplified 109P1D4 cDNA.
  • the sequence of the amplified 109P1D4 cDNA can be determined.
  • a method of detecting a 109P1 D4 gene in a biological sample comprises first isolating genomic DNA from the sample; amplifying the isolated genomic DNA using 109P1 D4 polynucleotides as sense and antisense primers; and detecting the presence of the amplified 109P1 D4 gene.
  • Any number of appropriate sense and antisense probe combinations can be designed from a 109P1D4 nucleotide sequence (see, e.g., Figure 2) and used for this purpose.
  • the invention also provides assays for detecting the presence of a 109P1 D4 protein in a tissue or other biological sample such as serum, semen, bone, prostate, urine, cell preparations, and the like.
  • Methods for detecting a 109P1D4-related protein are also well known and include, for example, immunoprecipitation, immunohistochemical analysis, Western blot analysis, molecular binding assays, ELISA, ELIFA and the like.
  • a method of detecting the presence of a 109P1 D4-related protein in a biological sample comprises first contacting the sample with a 109P1 D4 antibody, a 109P1 D4-reactive fragment thereof, or a recombinant protein containing an antigen-binding region of a 109P1 D4 antibody; and then detecting the binding of 109P1D4-related protein in the sample.
  • an assay for identifying a cell that expresses a 109P1 D4 gene comprises detecting the presence of 109P1 D4 mRNA in the cell.
  • Methods for the detection of particular mRNAs in cells include, for example, hybridization assays using complementary DNA probes (such as in situ hybridization using labeled 109P1D4 riboprobes, Northern blot and related techniques) and various nucleic acid amplification assays (such as RT-PCR using complementary primers specific for 109P1 D4, and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like).
  • an assay for identifying a cell that expresses a 109P1 D4 gene comprises delecting the presence of 109P1 D4-related protein in the cell or secreted by the cell.
  • Various methods for the detection of proteins are well known in the art and are employed for the detection of 109P1 D4-related proteins and cells that express 109P1D4-related proteins.
  • 109P1 D4 expression analysis is also useful as a tool for identifying and evaluating agents that modulate 109P1 D4 gene expression.
  • 109P1 D4 expression is significantly upregulated in prostate cancer, and is expressed in cancers of the tissues listed in Table I. Identification of a molecule or biological agent that inhibits 109P1 D4 expression or over- expression in cancer cells is of therapeutic value.
  • such an agent can be identified by using a screen that quantifies 109P1D4 expression by RT-PCR, nucleic acid hybridization or antibody binding.
  • Oncogenesis is known to be a multistep process where cellular growth becomes progressively dysregulated and cells progress from a normal physiological state to precancerous and then cancerous states (see, e.g., Alers ef al, Lab Invest. 77(5): 437-438 (1997) and Isaacs ef al, Cancer Surv. 23: 19-32 (1995)).
  • examining a biological sample for evidence of dysregulated cell growth allows for early detection of such aberrant physiology, before a pathologic state such as cancer has progressed to a stage that therapeutic options are more limited and or the prognosis is worse.
  • the status of 109P1 D4 in a biological sample of interest can be compared, for example, to the status of 109P1D4 in a corresponding normal sample (e.g. a sample from that individual or alternatively another individual that is not affected by a pathology).
  • a corresponding normal sample e.g. a sample from that individual or alternatively another individual that is not affected by a pathology.
  • An alteration in the status of 109P1D4 in the biological sample provides evidence of dysregulated cellular growth.
  • a predetermined normative value such as a predetermined normal level of mRNA expression (see, e.g., Grever ef al., J. Comp. Neurol. 1996 Dec 9; 376(2): 306-14 and U.S. Patent No. 5,837,601) to compare 109P1D4 status in a sample.
  • status in this context is used according to its art accepted meaning and refers to the condition or state of a gene and its products.
  • skilled artisans use a number of parameters to evaluate the condition or state of a gene and its products. These include, but are not limited to the location of expressed gene products (including the location of 109P1 D4 expressing cells) as well as the level, and biological activity of expressed gene products (such as 109P1D4 mRNA, polynucleotides and polypeptides).
  • an alteration in the status of 109P1D4 comprises a change in the location of 109P1D4 and/or 109P1D4 expressing cells and/or an increase in 109P1D4 mRNA and/or protein expression.
  • 109P1 D4 status in a sample can be analyzed by a number of means well known in the art, including without limitation, immunohistochemical analysis, in situ hybridization, RT-PCR analysis on laser capture micro-dissected samples, Western blot analysis, and tissue array analysis.
  • Typical protocols for evaluating the status of a 109P1 D4 gene and gene products are found, for example in Ausubel et al. eds., 1996, Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4 (Southern Blotting), 1 ⁇ (Immunoblotting) and 18 (PCR Analysis).
  • 109P1D4 in a biological sample is evaluated by various methods utilized by skilled artisans including, but not limited to genomic Southern analysis (to examine, for example perturbations in a 109P1D4 gene), Northern analysis and/or PCR analysis of 109P1D4 mRNA (to examine, for example alterations in the polynucleotide sequences or expression levels of 109P1D4 mRNAs), and, Western and/or immunohistochemical analysis (to examine, for example alterations in polypeptide sequences, alterations in polypeptide localization within a sample, alterations in expression levels of 109P1D4 proteins and/or associations of 109P1D4 proteins with polypeptide binding partners).
  • genomic Southern analysis to examine, for example perturbations in a 109P1D4 gene
  • Northern analysis and/or PCR analysis of 109P1D4 mRNA to examine, for example alterations in the polynucleotide sequences or expression levels of 109P1D4 mRNAs
  • Detectable 109P1D4 polynucleotides include, for example, a 109P1D4 gene or fragment thereof, 109P1 D4 mRNA, alternative splice variants, 109P1 D4 mRNAs, and recombinant DNA or RNA molecules containing a 109P1D4 polynucleotide.
  • the expression profile of 109P1 D4 makes it a diagnostic marker for local and/or melastasized disease, and provides information on the growth or oncogenic potential of a biological sample.
  • the status of 109P1 D4 provides information useful for predicting susceptibility to particular disease stages, progression, and/or tumor aggressiveness.
  • the invention provides methods and assays for determining 109P1 D4 status and diagnosing cancers that express 109P1 D4, such as cancers of the tissues listed in Table I.
  • 109P1 D4 mRNA is so highly expressed in prostate and other cancers relative to normal prostate tissue
  • assays that evaluate the levels of 109P1D4 mRNA transcripts or proteins in a biological sample can be used to diagnose a disease associated with 109P1D4 dysregulation, and can provide prognostic information useful in defining appropriate therapeutic options.
  • the expression status of 109P1D4 provides information including the presence, stage and location of dysplastic, precancerous and cancerous cells, predicting susceptibility to various stages of disease, and/or for gauging tumor aggressiveness. Moreover, the expression profile makes it useful as an imaging reagent for metastasized disease. Consequently, an aspect of the invention is directed to the various molecular prognostic and diagnostic methods for examining the status of 109P1 D4 in biological samples such as those from individuals suffering from, or suspected of suffering from a pathology characterized by dysregulated cellular growth, such as cancer.
  • the status of 109P1D4 in a biological sample can be examined by a number of well-known procedures in the art.
  • the status of 109P1D4 in a biological sample taken from a specific location in the body can be examined by evaluating the sample for the presence or absence of 109P1 D4 expressing cells (e.g. those that express 109P1 D4 mRNAs or proteins).
  • This examination can provide evidence of dysregulated cellular growth, for example, when 109P1D4-expressing cells are found in a biological sample that does not normally contain such cells (such as a lymph node), because such alterations in the status of 109P1D4 in a biological sample are often associated with dysregulated cellular growth.
  • one indicator of dysregulated cellular growth is the metastases of cancer cells from an organ of origin (such as the prostate) to a different area of the body (such as a lymph node).
  • evidence of dysregulated cellular growth is important for example because occult lymph node metastases can be detected in a substantial proportion of patients with prostate cancer, and such metastases are associated with known predictors of disease progression (see, e.g., Murphy ef a/., Prostate 42(4): 316-317 (2000);Su ef al, Semin. Surg. Oncol. 18(1): 17-28 (2000) and Freeman et al, J Urol 1995 Aug 154(2 Pt 1):474-8).
  • the invention provides methods for monitoring 109P1D4 gene products by determining the status of 109P1D4 gene products expressed by cells from an individual suspected of having a disease associated with dysregulated cell growth (such as hyperplasia or cancer) and then comparing the status so determined to the status of 109P1D4 gene products in a corresponding normal sample.
  • the presence of aberrant 109P1 D4 gene products in the test sample relative to the normal sample provides an indication of the presence of dysregulated cell growth within the cells of the individual.
  • the invention provides assays useful in determining the presence of cancer in an individual, comprising detecting a significant increase in 109P1D4 mRNA or protein expression in a test cell or tissue sample relative to expression levels in the corresponding normal cell or tissue.
  • the presence of 109P1 D4 mRNA can, for example, be evaluated in tissues including but not limited to those listed in Table I.
  • the presence of significant 109P1D4 expression in any of these tissues is useful to indicate the emergence, presence and/or severity of a cancer, since the corresponding normal tissues do not express 109P1 D4 mRNA or express it at lower levels.
  • 109P1 D4 status is determined at the protein level rather than at the nucleic acid level.
  • a method comprises determining the level of 109P1 D4 protein expressed by cells in a test tissue sample and comparing the level so determined to the level of 109P1D4 expressed in a corresponding normal sample.
  • the presence of 109P1D4 protein is evaluated, for example, using immunohistochemical methods.
  • 109P1D4 antibodies or binding partners capable of detecting 109P1 D4 protein expression are used in a variety of assay formats well known in the art for this purpose.
  • These perturbations can include insertions, deletions, substitutions and the like.
  • Such evaluations are useful because perturbations in the nucleotide and amino acid sequences are observed in a large number of proteins associated with a growth dysregulated phenotype (see, e.g., Marrogi ef al, 1999, J. Cutan. Pathol. 26(8):369-378).
  • a mutation in the sequence of 109P1D4 may be indicative of the presence or promotion of a tumor.
  • Such assays therefore have diagnostic and predictive value where a mutation in 109P1 D4 indicates a potential loss of function or increase in tumor growth.
  • a 109P1D4 gene Aberrant demethylation and/or hypermethylation of CpG islands in gene 5' regulatory regions frequently occurs in immortalized and transformed cells, and can result in altered expression of various genes.
  • promoter hypermethylation of the pi-class glutathione S-transferase (a protein expressed in normal prostate but not expressed in >90% of prostate carcinomas) appears to permanently silence transcription of this gene and is the most frequently detected genomic alteration in prostate carcinomas (De Marzo et al, Am. J. Pathol. 155(6): 1985-1992 (1999)).
  • methylation-sensitive restriction enzymes that cannot cleave sequences that contain methylated CpG sites to assess the methylation status of CpG islands.
  • MSP methylation specific PCR
  • MSP methylation specific PCR
  • This procedure involves initial modification of DNA by sodium bisulfite (which will convert all unmethylated cytosines to uracil) followed by amplification using primers specific for methylated versus unmethylated DNA, Protocols involving methylation interference can also be found for example in Current Protocols In Molecular Biology, Unit 12, Frederick M. Ausubel ef al. eds., 1996.
  • Gene amplification is an additional method for assessing the status of 109P1D4.
  • Gene amplification is measured in a sample directly, for example, by conventional Southern blotting or Northern blotting to quantitate the transcription of mRNA (Thomas, 1980, Proc. Natl. Acad. Sci. USA, 77:5201-5205), dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein.
  • antibodies are employed that recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn are labeled and the assay carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.
  • Biopsied tissue or peripheral blood can be conveniently assayed for the presence of cancer cells using for example, Northern, dot blot or RT-PCR analysis to detect 109P1 D4 expression.
  • the presence of RT-PCR amplifiable 109P1 D4 mRNA provides an indication of the presence of cancer.
  • RT-PCR assays are well known in the art. RT-PCR detection assays for tumor cells in peripheral blood are currently being evaluated for use in the diagnosis and management of a number of human solid tumors, in the prostate cancer field, these include RT-PCR assays for the detection of cells expressing PSA and PSM (Verkaik ef al, 1997, Urol. Res.25:373-384; Ghossein ef al, 1995, J. Clin. Oncol. 13:1195-2000; Heston ef al, 1995, Clin. Chem.41:1687- 1688).
  • a further aspect of the invention is an assessment of the susceptibility that an individual has for developing cancer.
  • a method for predicting susceptibility to cancer comprises detecting 109P1 D4 mRNA or 109P1 D4 protein in a tissue sample, its presence indicating susceptibility to cancer, wherein the degree of 109P1 D4 mRNA expression correlates to the degree of susceptibility, in a specific embodiment, the presence of 109P1 D4 in prostate or other tissue is examined, with the presence of 109P1 D4 in the sample providing an indication of prostate cancer susceptibility (or the emergence or existence of a prostate tumor).
  • 109P1 D4 nucleotide and amino acid sequences in a biological sample, in order to identify perturbations in the structure of these molecules such as insertions, deletions, substitutions and the like.
  • the presence of one or more perturbations in 109P1 D4 gene products in the sample is an indication of cancer susceptibility (or the emergence or existence of a tumor).
  • the invention also comprises methods for gauging tumor aggressiveness.
  • a method for gauging aggressiveness of a tumor comprises determining the level of 109P1 D4 mRNA or 109P1 D4 protein expressed by tumor cells, comparing the level so determined to the level of 109P1 D4 mRNA or 109P1 D4 protein expressed in a corresponding normal tissue taken from the same individual or a normal tissue reference sample, wherein the degree of 109P1D4 mRNA or 109P1D4 protein expression in the tumor sample relative to the normal sample indicates the degree of aggressiveness.
  • aggressiveness of a tumor is evaluated by determining the extent to which 109P1 D4 is expressed in the tumor cells, with higher expression levels indicating more aggressive tumors.
  • Another embodiment is the evaluation of the integrity of 109P1 D4 nucleotide and amino acid sequences in a biological sample, in order to identify perturbations in the structure of these molecules such as insertions, deletions, substitutions and the like. The presence of one or more perturbations indicates more aggressive tumors.
  • methods for observing the progression of a malignancy in an individual over time comprise determining the level of 109P1D4 mRNA or 109P1D4 protein expressed by cells in a sample of the tumor, comparing the level so determined to the level of 109P1 D4 mRNA or 109P1 D4 protein expressed in an equivalent tissue sample taken from the same individual at a different time, wherein the degree of 109P1 D4 mRNA or 109P1 D4 protein expression in the tumor sample over time provides information on the progression of the cancer.
  • the progression of a cancer is evaluated by determining 109P1 D4 expression in the tumor cells over time, where increased expression over time indicates a progression of the cancer. Also, one can evaluate the integrity 109P1D4 nucleotide and amino acid sequences in a biological sample in order to identify perturbations in the structure of these molecules such as insertions, deletions, substitutions and the like, where the presence of one or more perturbations indicates a progression of the cancer.
  • Another embodiment of the invention is directed to methods for observing a coincidence between the expression of 109P1 D4 gene and 109P1 D4 gene products (or perturbations in 109P1 D4 gene and 109P1 D4 gene products) and a factor that is associated with malignancy, as a means for diagnosing and prognosticating the status of a tissue sample.
  • factors associated with malignancy can be utilized, such as the expression of genes associated with malignancy (e.g.
  • Methods for observing a coincidence between the expression of 109P1D4 gene and 109P1D4 gene products (or perturbations in 109P1D4 gene and 109P1D4 gene products) and another factor that is associated with malignancy are useful, for example, because the presence of a set of specific factors that coincide with disease provides information crucial for diagnosing and prognosticating the status of a tissue sample.
  • methods for observing a coincidence between the expression of 109P1 D4 gene and 109P1 D4 gene products (or perturbations in 109P1D4 gene and 109P1D4 gene products) and another factor associated with malignancy entails detecting the overexpression of 109P1D4 mRNA or protein in a tissue sample, detecting the overexpression of PSA mRNA or protein in a tissue sample (or PSCA or PSM expression), and observing a coincidence of 109P1 D4 mRNA or protein and PSA mRNA or protein overexpression (or PSCA or PSM expression).
  • the expression of 109P1 D4 and PSA mRNA in prostate tissue is examined, where the coincidence of 109P1D4 and PSA mRNA overexpression in the sample indicates the existence of prostate cancer, prostate cancer susceptibility or the emergence or status of a prostate tumor.
  • Standard methods for the detection and quantification of 109P1 D4 mRNA include in situ hybridization using labeled 109P1 D4 riboprobes, Northern blot and related techniques using 109P1D4 polynucleotide probes, RT-PCR analysis using primers specific for 109P1D4, and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like.
  • semi- quantitative RT-PCR is used to detect and quantify 109P1 D4 mRNA expression.
  • primers capable of amplifying 109P1 D4 can be used for this purpose, including but not limited to the various primer sets specifically described herein.
  • polyclonal or monoclonal antibodies specifically reactive with the wild-type 109P1 D4 protein can be used in an immunohistochemical assay of biopsied tissue.
  • the 109P1D4 protein and nucleic acid sequences disclosed herein allow a skilled artisan to identify proteins, small molecules and other agents that interact with 109P1 D4, as well as pathways activated by 109P1 D4 via any one of a variety of art accepted protocols.
  • one can utilize one of the so-called interaction trap systems also referred to as the "two-hybrid assay".
  • molecules interact and reconstitute a transcription factor which directs expression of a reporter gene, whereupon the expression of the reporter gene is assayed.
  • Other systems identify protein-protein interactions in vivo through reconstitution of a eukaryotic transcriptional activator, see, e.g., U.S. Patent Nos.
  • peptide libraries can be screen peptide libraries to identify molecules that interact with 109P1 D4 protein sequences.
  • peptides that bind to 109P1D4 are identified by screening libraries that encode a random or controlled collection of amino acids.
  • Peptides encoded by the libraries are expressed as fusion proteins of bacteriophage coat proteins, the bacteriophage particles are then screened against the 109P1D4 protein(s).
  • peptides having a wide variety of uses are thus identified without any prior information on the structure of the expected ligand or receptor molecule.
  • Typical peptide libraries and screening methods that can be used to identify molecules that interact with 109P1 D4 protein sequences are disclosed for example in U.S. Patent Nos. 5,723,286 issued 3 March 1998 and 5,733,731 issued 31 March 1998.
  • cell lines that express 109P1D4 are used to identify protein-protein interactions mediated by 109P1D4. Such interactions can be examined using immunoprecipitation techniques (see, e.g., Hamilton B.J., et al. Biochem. Biophys. Res. Commun. 1999, 261 :646-51).
  • 109P1D4 protein can be immunoprecipitated from 109P1D4- expressing cell lines using anti-109P1D4 antibodies.
  • antibodies against His-tag can be used in a cell line engineered to express fusions of 109P1D4 and a His-tag (vectors mentioned above).
  • the immunoprecipitated complex can be examined for protein association by procedures such as Western blotting, 35 S-methionine labeling of proteins, protein microsequencing, silver staining and two-dimensional gel electrophoresis.
  • Small molecules and ligands that interact with 109P1D4 can be identified through related embodiments of such screening assays. For example, small molecules can be identified that interfere with protein function, including molecules that interfere with 109P1D4's ability to mediate phosphorylation and de-phosphorylation, interaction with DNA or RNA molecules as an indication of regulation of cell cycles, second messenger signaling or tumorigenesis.
  • small molecules that modulate 109P1D4-related ion channel, protein pump, or cell communication functions are identified and used to treat patients that have a cancer that expresses 109P1 D4 (see, e.g., Hille, B., Ionic Channels of Excitable Membranes 2 nd Ed., Sinauer Assoc, Sunderland, MA, 1992).
  • ligands that regulate 109P1D4 function can be identified based on their ability to bind 109P1D4 and activate a reporter construct. Typical methods are discussed for example in U.S. Patent No. 5,928,868 issued 27 July 1999, and include methods for forming hybrid ligands in which at least one ligand is a small molecule.
  • cells engineered to express a fusion protein of 109P1D4 and a DNA-binding protein are used to co-express a fusion protein of a hybrid ligand/small molecule and a cDNA library transcriptional activator protein.
  • the cells further contain a reporter gene, the expression of which is conditioned on the proximity of the first and second fusion proteins to each other, an event that occurs only if the hybrid ligand binds to target sites on both hybrid proteins.
  • Those cells that express the reporter gene are selected and the unknown small molecule or the unknown ligand is identified. This method provides a means of identifying modulators, which activate or inhibit 109P1D4.
  • An embodiment of this invention comprises a method of screening for a molecule that interacts with a 109P1 D4 amino acid sequence shown in Figure 2 or Figure 3, comprising the steps of contacting a population of molecules with a 109P1D4 amino acid sequence, allowing the population of molecules and the 109P1D4 amino acid sequence to interact under conditions that facilitate an interaction, determining the presence of a molecule that interacts with the 109P1 D4 amino acid sequence, and then separating molecules that do not interact with the 109P1 D4 amino acid sequence from molecules that do.
  • the method further comprises purifying, characterizing and identifying a molecule that interacts with the 109P1D4 amino acid sequence. The identified molecule can be used to modulate a function performed by 109P1 D4.
  • the 109P1 D4 amino acid sequence is contacted with a library of peptides.
  • 109P1 D4 as a protein that is normally expressed in a restricted set of tissues, but which is also expressed in cancers such as those listed in Table I, opens a number of therapeutic approaches to the treatment of such cancers.
  • targeted antitumor therapies have been useful even when the targeted protein is expressed on normal tissues, even vital normal organ tissues.
  • a vital organ is one that is necessary to sustain life, such as the heart or colon.
  • a non-vital organ is one that can be removed whereupon the individual is still able to survive. Examples of non-vital organs are ovary, breast, and prostate.
  • Herceptin® is an FDA approved pharmaceutical that has as its active ingredient an antibody which is immunoreactive with the protein variously known as HER2, HER2/neu, and erb-b-2. It is marketed by Genentech and has been a commercially successful antitumor agent. Herceptin sales reached almost $400 million in 2002. Herceptin is a treatment for HER2 positive metastatic breast cancer.
  • HER2 is not limited to such tumors.
  • the same protein is expressed in a number of normal tissues.
  • HER2/neu is present in normal kidney and heart, thus these tissues are present in all human recipients of Herceptin.
  • the presence of HER2/neu in normal kidney is also confirmed by Latif, Z., et al., B.J.U. International (2002) 89:5-9.
  • both protein and mRNA are produced in benign renal tissues.
  • HER2/neu protein was strongly overexpressed in benign renal tissue.
  • Herceptin is a very useful, FDA approved, and commercially successful drug.
  • the effect of Herceptin on cardiac tissue, i.e., "cardiotoxicity,” has merely been a side effect to treatment. When patients were treated with Herceptin alone, significant cardiotoxicity occurred in a very low percentage of patients.
  • kidney tissue is indicated to exhibit normal expression, possibly even higher expression than cardiac tissue, kidney has no appreciable Herceptin side effect whatsoever.
  • kidney tissue is indicated to exhibit normal expression, possibly even higher expression than cardiac tissue, kidney has no appreciable Herceptin side effect whatsoever.
  • cardiac tissue has manifested any appreciable side effect at all.
  • EGFR epidermal growth factor receptor
  • therapeutic approaches that inhibit the activity of a 109P1 D4 protein are useful for patients suffering from a cancer that expresses 109P1D4.
  • These therapeutic approaches generally fall into two classes.
  • One class comprises various methods for inhibiting the binding or association of a 109P1 D4 protein with its binding partner or with other proteins.
  • Another class comprises a variety of methods for inhibiting the transcription of a 109P1D4 gene or translation of 109P1D4 mRNA.
  • the invention provides cancer vaccines comprising a 109P1D4-related protein or 109P1D4-related nucleic acid.
  • cancer vaccines prevent and/or treat 109P1 D4-expressing cancers with minimal or no effects on non-target tissues.
  • the use of a tumor antigen in a vaccine that generates humoral and/or cell-mediated immune responses as anti-cancer therapy is well known in the art and has been employed in prostate cancer using human PSMA and rodent PAP immunogens (Hodge etal, 1995, Int. J. Cancer 63:231-237; Fong etal, 1997, J. Immunol. 159:3113-3117).
  • Such methods can be readily practiced by employing a 109P1D4-related protein, or a 109P1D4-encoding nucleic acid molecule and recombinant vectors capable of expressing and presenting the 109P1 D4 immunogen (which typically comprises a number of antibody or T cell epitopes).
  • Skilled artisans understand that a wide variety of vaccine systems for delivery of immunoreactive epitopes are known in the art (see, e.g., Heryln ef a/., Ann Med 1999 Feb 31(1):66-78; Maruyama ef al, Cancer Immunol Immunother 2000 Jun 49(3):123-32) Briefly, such methods of generating an immune response (e.g.
  • a 109P1D4 immunogen contains a biological motif, see e.g., Tables VIII-XXI and XXII-XLIX, or a peptide of a size range from 109P1 D4 indicated in Figure 5, Figure 6, Figure 7, Figure 8, and Figure 9.
  • Such vaccine compositions can include, for example, lipopeptides (e.g.,Viliello, A. ef al, J. Clin. Invest. 95:341 , 1996), peptide compositions encapsulated in poly(DL-lactide-co-glycolide) ("PLG”) microspheres (see, e.g., Eldridge, ef a/., Molec. Immunol.
  • lipopeptides e.g.,Viliello, A. ef al, J. Clin. Invest. 95:341 , 1996)
  • PLG poly(DL-lactide-co-glycolide)
  • Toxin-targeted delivery technologies also known as receptor mediated targeting, such as those of Avant Immunotherapeutics, Inc. (Needham, Massachusetts) may also be used.
  • the vaccine compositions of the invention can also be used in conjunction with other treatments used for cancer, e.g., surgery, chemotherapy, drug therapies, radiation therapies, efc. including use in combination with immune adjuvants such as IL-2, IL-12, GM-CSF, and the like.
  • CTL epitopes can be determined using specific algorithms to identify peptides within 109P1 D4 protein that bind corresponding HLA alleles (see e.g., Table IV; EpimerTM and EpimatrixTM, Brown University (URL brown.edu/Research/TB- HIV_Lab/epimatrix/epimatrix.html); and, BIMAS, (URL bimas.dcrt.nih.gov/; SYFPEITHI at URL syfpeithi.bmi-heidelberg.com/).
  • a 109P1D4 immunogen contains one or more amino acid sequences identified using techniques well known in the art, such as the sequences shown in Tables VIII-XXI and XXII-XLIX or a peptide of 8, 9, 10 or 11 amino acids specified by an HLA Class I motif/supermotif (e.g., Table IV (A), Table IV (D), or Table IV (E)) and/or a peptide of at least 9 amino acids that comprises an HLA Class II motif/supermotif (e.g., Table IV (B) or Table IV (C)).
  • HLA Class I motif/supermotif e.g., Table IV (A), Table IV (D), or Table IV (E)
  • HLA Class II motif/supermotif e.g., Table IV (B) or Table IV (C)
  • the HLA Class I binding groove is essentially closed ended so that peptides of only a particular size range can fit into the groove and be bound, generally HLA Class I epitopes are 8, 9, 10, or 11 amino acids long.
  • the HLA Class II binding groove is essentially open ended; therefore a peptide of about 9 or more amino acids can be bound by an HLA Class II molecule. Due to the binding groove differences between HLA Class I and II, HLA Class I motifs are length specific, i.e., position two of a Class I motif is the second amino acid in an amino to carboxyl direction of the peptide.
  • HLA Class II epitopes are often 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25 amino acids long, or longer than 25 amino acids.
  • Methods of generating an immune response in a mammal comprise exposing the mammal's immune system to an immunogenic epitope on a protein (e.g. a 109P1D4 protein) so that an immune response is generated.
  • a protein e.g. a 109P1D4 protein
  • a typical embodiment consists of a method for generating an immune response to 109P1D4 in a host, by contacting the host with a sufficient amount of al least one 109P1 D4 B cell or cytotoxic T-cell epitope or analog thereof; and at least one periodic interval thereafter re-contacting the host with the 109P1 D4 B cell or cytotoxic T-cell epitope or analog thereof.
  • a specific embodiment consists of a method of generating an immune response against a 109P1 D4- related protein or a man-made multiepitopic peptide comprising: administering 109P1D4 immunogen (e.g.
  • a 109P1D4 protein or a peptide fragment thereof, a 109P1D4 fusion protein or analog etc. in a vaccine preparation to a human or another mammal.
  • vaccine preparations further contain a suitable adjuvant (see, e.g., U.S. Patent No. 6,146,635) or a universal helper epitope such as a PADRETM peptide (Epimmune Inc., San Diego, CA; see, e.g., Alexander ef al, J. Immunol. 2000 164(3); 164(3): 1625-1633; Alexander ef al, Immunity 1994 1(9): 751-761 and Alexander ef al, Immunol. Res. 1998 18(2): 79-92).
  • a suitable adjuvant see, e.g., U.S. Patent No. 6,146,635
  • a universal helper epitope such as a PADRETM peptide (Epimmune Inc., San Diego, CA; see,
  • An alternative method comprises generating an immune response in an individual against a 109P1D4 immunogen by: administering in vivo to muscle or skin of the individual's body a DNA molecule that comprises a DNA sequence that encodes a 109P1D4 immunogen, the DNA sequence operatively linked to regulatory sequences which control the expression of the DNA sequence; wherein the DNA molecule is taken up by cells, the DNA sequence is expressed in the cells and an immune response is generated against the immunogen (see, e.g., U.S. Patent No. 5,962,428).
  • a genetic vaccine facilitator such as anionic lipids; saponins; lectins; estrogenic compounds; hydroxylated lower alkyls; dimethyl sulfoxide; and urea is also administered.
  • an antiidiotypic antibody can be administered that mimics 109P1D4, in order to generate a response to the target antigen.
  • Vaccine compositions of the invention include nucleic acid-mediated modalities.
  • DNA or RNA that encode protein(s) of the invention can be administered to a patient.
  • Genetic immunization methods can be employed to generate prophylactic or therapeutic humoral and cellular immune responses directed against cancer cells expressing 109P1D4.
  • Constructs comprising DNA encoding a 109P1D4-related protein/immunogen and appropriate regulatory sequences can be injected directly into muscle or skin of an individual, such that the cells of the muscle or skin take-up the construct and express the encoded 109P1 D4 protein/immunogen.
  • a vaccine comprises a 109P1D4-related protein.
  • 109P1 D4-related protein immunogen results in the generation of prophylactic or therapeutic humoral and cellular immunity against cells that bear a 109P1D4 protein.
  • Various prophylactic and therapeutic genetic immunization techniques known in the art can be used (for review, see information and references published at Internet address genweb.com). Nucleic acid-based delivery is described, for instance, in Wolff ef. a/., Science 247:1466 (1990) as well as U.S. Patent Nos. 6,680,859; 5,589,466; 5,804,666; 5,739,118; 5,736,524; 5,679,647; WO 98/04720.
  • DNA-based delivery technologies include "naked DNA”, facilitated (bupivicaine, polymers, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated (“gene gun”) or pressure-mediated delivery (see, e.g., U.S. Patent No. 5,922,687).
  • proteins of the invention can be expressed via viral or bacterial vectors.
  • viral gene delivery systems that can be used in the practice of the invention include, but are not limited to, vaccinia, fowlpox, canarypox, adenovirus, influenza, poliovirus, adeno-associated virus, lentivirus, and Sindbis virus (see, e.g., Restifo, 1996, Curr. Opin. Immunol. 8:658-663; Tsang ef al. J. Natl. Cancer Inst. 87:982-990 (1995)).
  • Non-viral delivery systems can also be employed by introducing naked DNA encoding a 109P1D4-related protein into the patient (e.g., intramuscularly or intradermally) to induce an anti-tumor response.
  • Vaccinia virus is used, for example, as a vector to express nucleotide sequences that encode the peptides of the invention. Upon introduction into a host, the recombinant vaccinia virus expresses the protein immunogenic peptide, and thereby elicits a host immune response.
  • Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Patent No. 4,722,848.
  • Another vector is BCG (Bacille Cal ette Guerin). BCG vectors are described in Stover ef al, Nature 351:456-460 (1991).
  • BCG vectors are described in Stover ef al, Nature 351:456-460 (1991).
  • a wide variety of other vectors useful for therapeutic administration or immunization of the peptides of the invention e.g. adeno and adeno-associated virus vectors, retroviral vectors, Salmonella typhi vectors, detoxified anthrax to
  • gene delivery systems are used to deliver a 109P1D4-related nucleic acid molecule.
  • the full- length human 109P1 D4 cDNA is employed.
  • 109P1 D4 nucleic acid molecules encoding specific cytotoxic T lymphocyte (CTL) and/or antibody epitopes are employed.
  • APCs antigen presenting cells
  • DC dendritic cells
  • D4 antigen 109P1 D4 antigen to a patient's immune system.
  • Dendritic cells express MHC class I and II molecules, B7 co-stimulator, and IL-12, and are thus highly specialized antigen presenting cells.
  • PSMA prostate-specific membrane antigen
  • dendritic cells can be used to present 109P1D4 peptides to T cells in the context of MHC class I or II molecules.
  • autologous dendritic cells are pulsed with 109P1 D4 peptides capable of binding to MHC class I and/or class II molecules.
  • dendritic cells are pulsed with the complete 109P1D4 protein.
  • Yet another embodiment involves engineering the overexpression of a 109P1D4 gene in dendritic cells using various implementing vectors known in the art, such as adenovirus (Arthur ef a/., 1997, Cancer Gene Ther. 4:17-25), retrovirus (Henderson et al, 1996, Cancer Res.
  • Cells that express 109P1D4 can also be engineered to express immune modulators, such as GM- CSF, and used as immunizing agents.
  • 109P1 D4 is an attractive target for antibody-based therapeutic strategies.
  • a number of antibody strategies are known in the art for targeting both extracellular and intracellular molecules (see, e.g., complement and ADCC mediated killing as well as the use of intrabodies).
  • 109P1D4 is expressed by cancer cells of various lineages relative to corresponding normal cells, systemic administration of 109P1 D4-immunoreactive compositions are prepared that exhibit excellent sensitivity without toxic, non-specific and/or non-target effects caused by binding of the immunoreactive composition to non-target organs and tissues.
  • Antibodies specifically reactive with domains of 109P1D4 are useful to treat 109P1D4-expressing cancers systemically, either as conjugates with a toxin or therapeutic agent, or as naked antibodies capable of inhibiting cell proliferation or function.
  • 109P1D4 antibodies can be introduced into a patient such "that the antibody binds to 109P1D4 and modulates a function, such as an interaction with a binding partner, and consequently mediates destruction of the tumor cells and/or inhibits the growth of the tumor cells.
  • Mechanisms by which such antibodies exert a therapeutic effect can include complement-mediated cytolysis, antibody-dependent cellular cytotoxicity, modulation of the physiological function of 109P1 D4, inhibition of ligand binding or signal transduction pathways, modulation of tumor cell differentiation, alteration of tumor angiogenesis factor profiles, and/or apoptosis.
  • antibodies can be used to specifically target and bind immunogenic molecules such as an immunogenic region of a 109P1 D4 sequence shown in Figure 2 or Figure 3.
  • cytotoxic agents see, e.g., Slevers ef al. Blood 93:11 3678- 3684 (June 1, 1999)
  • cytotoxic and/or therapeutic agents When cytotoxic and/or therapeutic agents are delivered directly to cells, such as by conjugating them to antibodies specific for a molecule expressed by that cell (e.g. 109P1 D4), the cytotoxic agent will exert its known biological effect (i.e. cytotoxicity) on those cells.
  • compositions and methods for using antibody-cytotoxic agent conjugates to kill cells are known in the art.
  • typical methods entail administering to an animal having a tumor a biologically effective amount of a conjugate comprising a selected cytotoxic and/or therapeutic agent linked to a targeting agent (e.g. an anti- 109P1D4 antibody) that binds to a marker (e.g. 109P1D4) expressed, accessible to binding or localized on the cell surfaces.
  • a targeting agent e.g. an anti- 109P1D4 antibody
  • a marker e.g. 109P1D4 expressed, accessible to binding or localized on the cell surfaces.
  • a typical embodiment is a method of delivering a cytotoxic and/or therapeutic agent to a cell expressing 109P1D4, comprising conjugating the cytotoxic agent to an antibody that immunospecifically binds to a 109P1 D4 epitope, and, exposing the cell to the antibody-agent conjugate.
  • Another illustrative embodiment is a method of treating an individual suspected of suffering from metastasized cancer, comprising a step of administering parenterally to said individual a pharmaceutical composition comprising a therapeutically effective amount of an antibody conjugated to a cytotoxic and/or therapeutic agent.
  • Cancer immunotherapy using anti-109P1D4 antibodies can be done in accordance with various approaches that have been successfully employed in the treatment of other types of cancer, including but not limited to colon cancer (Arlen ef a/., 1998, Crit. Rev. Immunol. 18:133-138), multiple myeloma (Ozaki ef al, 1997, Blood 90:3179-3186, Tsunenari et al, 1997, Blood 90:2437-2444), gastric cancer (Kasprzyk ef a/., 1992, Cancer Res. 52:2771-2776), B-cell lymphoma (Funakoshi et al, 1996, J. Immunother. Emphasis Tumor Immunol.
  • leukemia Zhong et al., 1996, Leuk. Res. 20:681-689
  • colorectal cancer Moun ef al, 1994, Cancer Res. 64:6160-6166; Velders ef al, 1995, Cancer Res. 56:4398-4403
  • breast cancer Shepard ef a/., 1991, J. Clin. Immunol. 11:117-127.
  • Some therapeutic approaches involve conjugation of naked antibody to a toxin or radioisotope, such as the conjugation of Y 91 or I 131 to anti-CD20 antibodies (e.g., ZevalinTM, IDEC Pharmaceuticals Corp.
  • 109P1D4 antibodies can be administered in conjunction with radiation, chemotherapy or hormone ablation.
  • antibodies can be conjugated to a toxin such as calicheamicin (e.g., MylotargTM, Wyeth-Ayerst, Madison, NJ, a recombinant humanized lgG kappa antibody conjugated to antitumor antibiotic calicheamicin) or a maytansinoid (e.g., taxane-based Tumor-Activated Prodrug, TAP, platform, ImmunoGen, Cambridge, MA, also see e.g., US Patent 5,416,064).
  • a toxin such as calicheamicin (e.g., MylotargTM, Wyeth-Ayerst, Madison, NJ, a recombinant humanized lgG kappa antibody conjugated to antitumor antibiotic calicheamicin) or a maytansinoid (e.g., taxane-based Tumor-Activated Prodrug, TAP, platform, ImmunoGen, Cambridge, MA, also see e.g., US Patent
  • antibody therapy can be particularly appropriate in advanced or metastatic cancers.
  • Treatment with the antibody therapy of the invention is indicated for patients who have received one or more rounds of chemotherapy.
  • antibody therapy of the invention is combined with a chemotherapeutic or radiation regimen for patients who have not received chemotherapeutic treatment.
  • antibody therapy can enable the use of reduced dosages of concomitant chemotherapy, particularly for patients who do not tolerate the toxicity of the chemotherapeutic agent very well.
  • Fan et al. (Cancer Res. 53:4637-4642, 1993), Prewett et al. (International J. of Onco. 9:217-224, 1996), and Hancock et al. (Cancer Res. 51 :4576-4680, 1991) describe the use of various antibodies together with chemotherapeutic agents.
  • antibody therapy can be particularly appropriate in advanced or metastatic cancers. Treatment with the antibody therapy of the invention is indicated for patients who have received one or more rounds of chemotherapy. Alternatively, antibody therapy of the invention is combined with a chemotherapeutic or radiation regimen for patients who have not received chemotherapeutic treatment. Additionally, antibody therapy can enable the use of reduced dosages of concomitant chemotherapy, particularly for patients who do not tolerate the toxicity of the chemotherapeutic agent very well.
  • Cancer patients can be evaluated for the presence and level of 109P1D4 expression, preferably using immunohistochemical assessments of tumor tissue, quantitative 109P1 D4 imaging, or other techniques that reliably indicate the presence and degree of 109P1 D4 expression. Immunohistochemical analysis of tumor biopsies or surgical specimens is preferred for this purpose. Methods for immunohistochemical analysis of tumor tissues are well known in the art.
  • Anti-109P1D4 monoclonal antibodies that treat prostate and other cancers include those that initiate a potent immune response against the tumor or those that are directly cytotoxic.
  • anti-109P1D4 monoclonal antibodies can elicit tumor cell lysis by either complement-mediated or antibody-dependent cell cytotoxicity (ADCC) mechanisms, both of which require an intact Fc portion of the immunoglobulin molecule for interaction with effector cell Fc receptor sites on complement proteins.
  • ADCC antibody-dependent cell cytotoxicity
  • anti-109P1D4 mAbs that exert a direct biological effect on tumor growth are useful to treat cancers that express 109P1 D4.
  • Mechanisms by which directly cytotoxic mAbs act include: inhibition of cell growth, modulation of cellular differentiation, modulation of tumor angiogenesis factor profiles, and the induction of apoptosis.
  • the mechanism(s) by which a particular anti-109P1 D4 mAb exerts an anti-tumor effect is evaluated using any number of in vitro assays that evaluate cell death such as ADCC, ADMMC, complement-mediated cell lysis, and so forth, as is generally known in the art.
  • preferred monoclonal antibodies used in the therapeutic methods of the invention are those that are either fully human or humanized and that bind specifically to the target 109P1D4 antigen with high affinity but exhibit low or no antigenicity in the patient.
  • Therapeutic methods of the invention contemplate the administration of single anti-109P1 D4 mAbs as well as combinations, or cocktails, of different mAbs.
  • Such mAb cocktails can have certain advantages inasmuch as they contain mAbs that target different epitopes, exploit different effector mechanisms or combine directly cytotoxic mAbs with mAbs that rely on immune effector functionality. Such mAbs in combination can exhibit synergistic therapeutic effects.
  • anti- 109P1 D4 mAbs can be administered concomitantly with other therapeutic modalities, including but not limited to various chemotherapeutic agents, androgen-blockers, immune modulators (e.g., lL-2, GM-CSF), surgery or radiation.
  • the anti- 109P1 D4 mAbs are administered in their "naked" or unconjugated form, or can have a therapeutic agent(s) conjugated to them.
  • Anti-109P1 D4 antibody formulations are administered via any route capable of delivering the antibodies to a tumor cell.
  • Routes of administration include, but are not limited to, intravenous, intraperitoneal, intramuscular, intratumor, intradermal, and the like.
  • Treatment generally involves repeated administration of the anti-109P1D4 antibody preparation, via an acceptable route of administration such as intravenous injection (IV), typically at a dose in (he range of about 0.1, .2, .3, .4, .5, .6, .7, .8, .9., 1, 2, 3, 4, ⁇ , 6, 7, 8, 9, 10, 15, 20, or 25 mg/kg body weight.
  • IV intravenous injection
  • doses in the range of 10-1000 mg mAb per week are effective and well tolerated.
  • an initial loading dose of approximately 4 mg/kg patient body weight IV, followed by weekly doses of about 2 mg/kg IV of the anti- 109P1D4 mAb preparation represents an acceptable dosing regimen.
  • the initial loading dose is administered as a 90-minute or longer infusion.
  • the periodic maintenance dose is administered as a 30 minute or longer infusion, provided the initial dose was well tolerated.
  • various factors can influence the ideal dose regimen in a particular case.
  • Such factors include, for example, the binding affinity and half life of the Ab or mAbs used, the degree of 109P1 D4 expression in the patient, the extent of circulating shed 109P1 D4 antigen, the desired steady-state antibody concentration level, frequency of treatment, and the influence of chemotherapeutic or other agents used in combination with the treatment method of the invention, as well as the health status of a particular patient.
  • patients should be evaluated for the levels of 109P1D4 in a given sample (e.g. the levels of circulating 109P1D4 antigen and/or 109P1D4 expressing cells) in order to assist in the determination of the most effective dosing regimen, etc.
  • a given sample e.g. the levels of circulating 109P1D4 antigen and/or 109P1D4 expressing cells
  • Such evaluations are also used for monitoring purposes throughout therapy, and are useful to gauge therapeutic success in combination with the evaluation of other parameters (for example, urine cytology and/or ImmunoCyt levels in bladder cancer therapy, or by analogy, serum PSA levels in prostate cancer therapy).
  • Anti-idiotypic anti-109P1D4 antibodies can also be used in anti-cancer therapy as a vaccine for inducing an immune response to cells expressing a 109P1D4-related protein.
  • the generation of anti-idiotypic antibodies is well known in the art; this methodology can readily be adapted to generate anti-idiotypic anti-109P1D4 antibodies that mimic an epitope on a 109P1D4-related protein (see, for example, Wagner ef al, 1997, Hybridoma 16: 33-40; Foon ef al, 1995, J. Clin. Invest. 96:334-342; Herlyn etal, 1996, Cancer Immunol. Immunother. 43:65-76).
  • Such an anti-idiotypic antibody can be used in cancer vaccine strategies.
  • Vaccines and methods of preparing vaccines that contain an immunogenically effective amount of one or more HLA-binding peptides as described herein are further embodiments of the invention.
  • vaccines in accordance with the invention encompass compositions of one or more of the claimed peptides.
  • a peptide can be present in a vaccine individually.
  • the peptide can exist as a homopolymer comprising multiple copies of the same peptide, or as a heteropolymer of various peptides.
  • Polymers have the advantage of increased immunological reaction and, where different peptide epitopes are used to make up the polymer, the additional ability to induce antibodies and/or CTLs that react with different antigenic determinants of the pathogenic organism or tumor-related peptide targeted for an immune response.
  • the composition can be a naturally occurring region of an antigen or can be prepared, e.g., recombinantly or by chemical synthesis.
  • Carriers that can be used with vaccines of the invention are well known in the art, and include, e.g., thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly L-lysine, poly L-glutamic acid, influenza, hepatitis B virus core protein, and the like.
  • the vaccines can contain a physiologically tolerable (i.e., acceptable) diluent such as water, or saline, preferably phosphate buffered saline.
  • the vaccines also typically include an adjuvant.
  • Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum are examples of materials well known in the art. Additionally, as disclosed herein, CTL responses can be primed by conjugating peptides of the invention to lipids, such as tripalmitoyl-S-glycerylcysteinlyseryl- serine (P3CSS). Moreover, an adjuvant such as a synthetic cytosine-phosphorothiolated-guanine-containing (CpG) oligonucleotides has been found to increase CTL responses 10- to 100-fold. (see, e.g. Davila and Celis, J. Immunol. 165:539-547 (2000))
  • the immune system of the host Upon immunization with a peptide composition in accordance with the invention, via injection, aerosol, oral, transdermal, transmucosal, infrapleural, intrathecal, or other suitable routes, the immune system of the host responds to the vaccine by producing large amounts of CTLs and/or HTLs specific for the desired antigen. Consequently, the host becomes at least partially immune to later development of cells that express or overexpress 109P1D4 antigen, or derives at least some therapeutic benefit when the antigen was tumor-associated.
  • class I peptide components may be desirable to combine with components that induce or facilitate neutralizing antibody and or helper T cell responses directed to the target antigen.
  • a preferred embodiment of such a composition comprises class I and class II epitopes in accordance with the invention.
  • An alternative embodiment of such a composition comprises a class I and/or class II epitope in accordance with the invention, along with a cross reactive HTL epitope such as PADRETM (Epimmune, San Diego, CA) molecule (described e.g., in U.S. Patent Number 5,736,142).
  • a vaccine of the invention can also include antigen-presenting cells (APC), such as dendritic cells (DC), as a vehicle to present peptides of the invention.
  • APC antigen-presenting cells
  • DC dendritic cells
  • Vaccine compositions can be created in vitro, following dendritic cell mobilization and harvesting, whereby loading of dendritic cells occurs in vitro.
  • dendritic cells are transfected, e.g., with a mi ⁇ igene in accordance with the invention, or are pulsed with peptides.
  • the dendritic cell can then be administered to a patient to elicit immune responses in vivo.
  • Vaccine compositions either DNA- or peptide-based, can also be administered in vivo in combination with dendritic cell mobilization whereby loading of dendritic cells occurs in vivo.
  • the following principles are utilized when selecting an array of epitopes for inclusion in a polyepitopic composition for use in a vaccine, or for selecting discrete epitopes to be included in a vaccine and/or to be encoded by nucleic acids such as a minigene. It is preferred that each of the following principles be balanced in order to make the selection.
  • the multiple epitopes to be incorporated in a given vaccine composition may be, but need not be, contiguous in sequence in the native antigen from which the epitopes are derived.
  • Epitopes are selected which, upon administration, mimic immune responses that have been observed to be correlated with tumor clearance.
  • this includes 3-4 epitopes that come from at least one tumor associated antigen (TAA).
  • TAA tumor associated antigen
  • HLA Class II a similar rationale is employed; again 3-4 epitopes are selected from at least one TAA (see, e.g., Rosenberg ef a/., Science 278:1447-1450).
  • Epitopes from one TAA may be used in combination with epitopes from one or more additional TAAs to produce a vaccine that targets tumors with varying expression patterns of frequently-expressed TAAs.
  • Epitopes are selected that have the requisite binding affinity established to be correlated with immunogenicity: for HLA Class I an ICso of 500 nM or less, often 200 nM or less; and for Class II an ICso of 1000 nM or less.
  • Sufficient supermotif bearing-peptides, or a sufficient array of allele-specific motif-bearing peptides, are selected to give broad population coverage. For example, it is preferable to have at least 80% population coverage.
  • a Monte Carlo analysis a statistical evaluation known in the art, can be employed to assess the breadth, or redundancy of, population coverage.
  • nested epitopes are epitopes referred to as "nested epitopes.” Nested epitopes occur where at least two epitopes overlap in a given peptide sequence.
  • a nested peptide sequence can comprise B cell, HLA class I and/or HLA class II epitopes.
  • a general objective is to provide the greatest number of epitopes per sequence.
  • an aspect is to avoid providing a peptide that is any longer than the amino terminus of the amino terminal epitope and the carboxyl terminus of the carboxyl terminal epitope in the peptide.
  • a multi-epitopic sequence such as a sequence comprising nested epitopes, it is generally important to screen the sequence in order to insure that it does not have pathological or other deleterious biological properties.
  • a polyepitopic protein is created, or when creating a minigene, an objective is to generate the smallest peptide that encompasses the epitopes of interest. This principle is similar, if not the same as that employed when selecting a peptide comprising nested epitopes. However, with an artificial polyepitopic peptide, the size minimization objective is balanced against the need to integrate any spacer sequences between epitopes in the polyepitopic protein.
  • Spacer amino acid residues can, for example, be introduced to avoid junctional epitopes (an epitope recognized by the immune system, not present in the target antigen, and only created by the man-made juxtaposition of epitopes), or to facilitate cleavage between epitopes and thereby enhance epitope presentation.
  • Junctional epitopes are generally to be avoided because the recipient may generate an immune response to that non-native epitope. Of particular concern is a junctional epitope that is a "dominant epitope.” A dominant epitope may lead to such a zealous response that immune responses to other epitopes are diminished or suppressed.
  • potential peptide epitopes can also be selected on the basis of their conservancy.
  • a criterion for conservancy may define that the entire sequence of an HLA class I binding peptide or the entire 9-mer core of a class II binding peptide be conserved in a designated percentage of the sequences evaluated for a specific protein antigen.
  • Nucleic acids encoding the peptides of the invention are a particularly useful embodiment of the invention. Epitopes for inclusion in a minigene are preferably selected according to the guidelines set forth in the previous section.
  • a preferred means of administering nucleic acids encoding the peptides of the invention uses minigene constructs encoding a peptide comprising one or multiple epitopes of the invention.
  • muiti-epitope inigenes The use of muiti-epitope inigenes is described below and in, Ishioka ef al, J. Immunol 162:3915-3925, 1999; An, L. and Whitton, J. L, J. Virol. 71:2292, 1997; Thomson, S. A. et al, J. Immunol. 157:822, 1996; Whitton, J. L. et al, J. Virol. 67:348, 1993; Hanke, R. ef al, Vaccine 16:426, 1998.
  • a multi-epitope DNA plasmid encoding supermotif- and/or motif-bearing epitopes derived 109P1D4, the PADRE® universal helper T cell epitope or multiple HTL epitopes from 109P1D4 (see e.g., Tables VIII-XXI and XXII to XLIX), and an endoplasmic reticulum-translocating signal sequence can be engineered.
  • a vaccine may also comprise epitopes that are derived from other TAAs.
  • the immunogenicity of a multi-epitopic minigene can be confirmed in transgenic mice to evaluate the magnitude of CTL induction responses against the epitopes tested. Further, the immunogenicity of DNA-encoded epitopes in vivo can be correlated with the in vitro responses of specific CTL lines against target cells transfected with the DNA plasmid. Thus, these experiments can show that the minigene serves to both: 1.) generate a CTL response and 2.) that the induced CTLs recognized cells expressing the encoded epitopes.
  • the amino acid sequences of the epitopes may be reverse translated.
  • a human codon usage table can be used to guide the codon choice for each amino acid.
  • These epitope-encoding DNA sequences may be directly adjoined, so that when translated, a continuous polypeptide sequence is created.
  • additional elements can be incorporated into the minigene design.
  • amino acid sequences that can be reverse translated and included in the minigene sequence include: HLA class I epitopes, HLA class II epitopes, antibody epitopes, a ubiquitination signal sequence, and/or an endoplasmic reticulum targeting signal.
  • HLA presentation of CTL and HTL epitopes may be improved by including synthetic (e.g. poly-alanine) or naturally-occurring flanking sequences adjacent to the CTL or HTL epitopes; these larger peptides comprising the epitope(s) are within the scope of the invention.
  • the minigene sequence may be converted to DNA by assembling oligonucleotides that encode the plus and minus strands of the minigene. Overlapping oligonucleotides (30-100 bases long) may be synthesized, phosphorylated, purified and annealed under appropriate conditions using well known techniques. The ends of the oligonucleotides can be joined, for example, using T4 DNA ligase. This synthetic minigene, encoding the epitope polypeptide, can then be cloned into a desired expression vector.
  • Standard regulatory sequences well known to those of skill in the art are preferably included in the vector to ensure expression in the target cells.
  • a promoter with a down-stream cloning site for minigene insertion a polyadenylation signal for efficient transcription termination; an E. coli origin of replication; and an £ coli selectable marker (e.g. ampicillin or kanamycin resistance).
  • Numerous promoters can be used for this purpose, e.g., the human cytomegalovirus (hCMV) promoter. See, e.g., U.S. Patent Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.
  • introns are required for efficient gene expression, and one or more synthetic or naturally-occurring introns could be incorporated into the transcribed region of the minigene.
  • mRNA stabilization sequences and sequences for replication in mammalian cells may also be considered for increasing minigene expression.
  • the minigene is cloned into the polylinker region downstream of the promoter.
  • This plasmid is transformed into an appropriate £ coli strain, and DNA is prepared using standard techniques. The orientation and DNA sequence of the minigene, as well as all other elements included in the vector, are confirmed using restriction mapping and DNA sequence analysis.
  • Bacterial cells harboring the correct plasmid can be stored as a master cell bank and a working cell bank.
  • immunostimulatory sequences appear to play a role in the immunogenicity of DNA vaccines. These sequences may be included in the vector, outside the minigene coding sequence, if desired to enhance immunogenicity.
  • a bi-cistronic expression vector which allows production of both the minigene-encoded epitopes and a second protein (included to enhance or decrease immunogenicity) can be used.
  • proteins or polypeptides that could beneficially enhance the immune response if co-expressed include cytokines (e.g., IL-2, IL-12, GM- CSF), cytokine-inducing molecules (e.g., LelF), costimulatory molecules, or for HTL responses, pan-DR binding proteins (PADRETM, Epimmune, San Diego, CA).
  • Helper (HTL) epitopes can be joined to intracellular targeting signals and expressed separately from expressed CTL epitopes; this allows direction of the HTL epitopes to a cell compartment different than that of the CTL epitopes. If required, this could facilitate more efficient entry of HTL epitopes into the HLA class II pathway, thereby improving HTL induction.
  • immunosuppressive molecules e.g. TGF- ⁇
  • TGF- ⁇ immunosuppressive molecules
  • Therapeutic quantities of plasmid DNA can be produced for example, by fermentation in £ coli, followed by purification. Aliquots from the working cell bank are used to inoculate growth medium, and grown to saturation in shaker flasks or a bioreactor according to well-known techniques. Plasmid DNA can be purified using standard bioseparation technologies such as solid phase anion-exchange resins supplied by QIAGEN, Inc. (Valencia, California). If required, supercoiled DNA can be isolated from the open circular and linear forms using gel electrophoresis or other methods.
  • Purified plasmid DNA can be prepared for injection using a variety of formulations. The simplest of these is reconstitution of lyophilized DNA in sterile phosphate-buffer saline (PBS). This approach, known as "naked DNA,” is currently being used for intramuscular (IM) administration in clinical trials. To maximize the immunotherapeutic effects of minigene DNA vaccines, an alternative method for formulating purified plasmid DNA may be desirable. A variety of methods have been described, and new techniques may become available.
  • Cationic lipids, glycolipids, and fusogenic liposomes can also be used in the formulation (see, e.g., as described by WO 93/24640; Mannino & Gould-Fogerite, BioTechniques 6(7): 682 (1988); U.S. Pat No. 5,279,833; WO 91/06309; and Feigner, ef al, Proc. Nat'IAcad. Sci. USA 84:7413 (1987).
  • peptides and compounds referred to collectively as protective, interactive, non-condensing compounds could also be complexed to purified plasmid DNA to influence variables such as stability, intramuscular dispersion, or trafficking to specific organs or cell types.
  • Target cell sensitization can be used as a functional assay for expression and HLA class I presentation of minigene-encoded CTL epitopes.
  • the plasmid DNA is introduced into a mammalian cell line that is suitable as a target for standard CTL chromium release assays.
  • the transfection method used will be dependent on the final formulation. Electroporation can be used for "naked" DNA, whereas cationic lipids allow direct in vitro transfection.
  • a plasmid expressing green fluorescent protein (GFP) can be co-transfected to allow enrichment of transfected cells using fluorescence activated cell sorting (FACS).
  • FACS fluorescence activated cell sorting
  • HTL epitopes are then chromium-51 ( 51 Cr) labeled and used as target cells for epitope-specific CTL lines; cytolysis, detected by 51 Cr release, indicates both production of, and HLA presentation of, minigene-encoded CTL epitopes. Expression of HTL epitopes may be evaluated in an analogous manner using assays to assess HTL activity.
  • In vivo immunogenicity is a second approach for functional testing of minigene DNA formulations.
  • Transgenic mice expressing appropriate human HLA proteins are immunized with the DNA product.
  • the dose and route of administration are formulation dependent (e.g., IM for DNA in PBS, intraperitoneal (i.p.) for lipid-complexed DNA).
  • Twenty-one days after immunization splenocytes are harvested and restimulated for one week in the presence of peptides encoding each epitope being tested. Thereafter, for CTL effector cells, assays are conducted for cytolysis of peptide-loaded, 51 Cr-labeled target cells using standard techniques.
  • Lysis of target cells that were sensitized by HLA loaded with peptide epitopes, corresponding to minigene-encoded epitopes, demonstrates DNA vaccine function for in vivo induction of CTLs. Immunogenicity of HTL epitopes is confirmed in transgenic mice in an analogous manner.
  • nucleic acids can be administered using ballistic delivery as described, for instance, in U.S. Patent No. 5,204,253.
  • particles comprised solely of DNA are administered.
  • DNA can be adhered to particles, such as gold particles.
  • Minigenes can also be delivered using other bacterial or viral delivery systems well known in the art, e.g., an expression construct encoding epitopes of the invention can be incorporated into a viral vector such as vaccinia.
  • Vaccine compositions comprising CTL peptides of the invention can be modified, e.g., analoged, to provide desired attributes, such as improved serum half life, broadened population coverage or enhanced immunogenicity.
  • the ability of a peptide to induce CTL activity can be enhanced by linking the peptide to a sequence which contains at least one epitope that is capable of inducing a T helper cell response.
  • a CTL peptide can be directly linked to a T helper peptide, often CTL epitope/HTL epitope conjugates are linked by a spacer molecule.
  • the spacer is typically comprised of relatively small, neutral molecules, such as amino acids or amino acid mimetics, which are substantially uncharged under physiological conditions.
  • the spacers are typically selected from, e.g., Ala, Gly, or other neutral spacers of nonpolar amino acids or neutral polar amino acids, it will be understood that the optionally present spacer need not be comprised of the same residues and thus may be a hetero- or homo-oligomer. When present, the spacer will usually be at least one or two residues, more usually three to six residues and sometimes 10 or more residues.
  • the CTL peptide epitope can be linked to the T helper peptide epitope either directly or via a spacer either at the amino or carboxy terminus of the CTL peptide. The amino terminus of either the immunogenic peptide or the T helper peptide may be acylated.
  • the T helper peptide is one that is recognized by T helper cells present in a majority of a genetically diverse population. This can be accomplished by selecting peptides that bind to many, most, or all of the HLA class II molecules.
  • Examples of such amino acid bind many HLA Class II molecules include sequences from antigens such as fefamvs toxoid at positions 830-843 QYIKANSKFIGITE; (SEQ ID NO: 40), Plasmodium falciparum circumsporozoite (CS) protein at positions 378-398 DIEKKIAKMEKASSVFNWNS; (SEQ ID NO: 41), and Streptococcus 18kD protein at positions 116-131 GAVDSILGGVATYGAA; (SEQ ID NO: 42).
  • Other examples include peptides bearing a DR 1-4-7 supermotif, or either of the DR3 motifs.
  • An alternative of a pan-DR binding epitope comprises all "L” natural amino acids and can be provided in the form of nucleic acids that encode the epitope.
  • HTL peptide epitopes can also be modified to alter their biological properties. For example, they can be modified to include D-amino acids to increase their resistance to proteases and thus extend their serum half life, or they can be conjugated to other molecules such as lipids, proteins, carbohydrates, and the like to increase their biological activity.
  • a T helper peptide can be conjugated to one or more palmitic acid chains at either the amino or carboxyl termini.
  • compositions of the invention at least one component which primes B lymphocytes or T lymphocytes.
  • Lipids have been identified as agents capable of priming CTL in vivo.
  • palmitic acid residues can be attached to the ⁇ -and ⁇ - amino groups of a lysine residue and then linked, e.g., via one or more linking residues such as Gly, Gly-Gly-, Ser, Ser-Ser, or the like, to an immunogenic peptide.
  • the lipidated peptide can then be administered either directly in a micelle or particle, incorporated into a liposome, or emulsified in an adjuvant, e.g., incomplete Freund's adjuvant.
  • a particularly effective immunogenic composition comprises palmitic acid attached to ⁇ - and ⁇ - amino groups of Lys, which is attached via linkage, e.g., Ser-Ser, to the amino terminus of the immunogenic peptide.
  • £ coli lipoproteins such as tripalmitoyl-S- glycerylcysteinlyseryl- serine (P3CSS) can be used to prime virus specific CTL when covalently attached to an appropriate peptide (see, e.g., Deres, etal, Nature 342:561, 1989).
  • Peptides of the invention can be coupled to P3CSS, for example, and the lipopeptide administered to an individual to prime specifically an immune response to the target antigen.
  • two such compositions can be combined to more effectively elicit both humoral and cell-mediated responses.
  • Vaccine Compositions Comprising DC Pulsed with CTL and/or HTL Peptides
  • An embodiment of a vaccine composition in accordance with the invention comprises ex vivo administration of a cocktail of epitope-bearing peptides to PBMC, or isolated DC therefrom, from the patient's blood.
  • a pharmaceutical to facilitate harvesting of DC can be used, such as ProgenipoielinTM (Pharmacia-Monsanto, St. Louis, MO) or GM-CSF/IL-4. After pulsing the DC with peptides and prior to reinfusion into patients, the DC are washed to remove unbound peptides.
  • a vaccine comprises peptide-pulsed DCs which present thenoied peptide epitopes complexed with HLA molecules on their surfaces.
  • the DC can be pulsed ex vivo with a cocktail of peptides, some of which stimulate CTL responses to 109P1D4.
  • a helper T cell (HTL) peptide such as a natural or artificial loosely restricted HLA Class II peptide, can be included to facilitate the CTL response.
  • HTL helper T cell
  • a vaccine in accordance with the invention is used to treat a cancer which expresses or overexpresses 109P1D4.
  • Antigenic 109P1 D4-related peptides are used to elicit a CTL and/or HTL response ex vivo, as well.
  • the resulting CTL or HTL cells can be used to treat tumors in patients that do not respond to other conventional forms of therapy, or will not respond to a therapeutic vaccine peptide or nucleic acid in accordance with the invention.
  • Ex vivo CTL or HTL responses to a particular antigen are induced by incubating in tissue culture the patient's, or genetically compatible, CTL or HTL precursor cells together with a source of antigen-presenting cells (APC), such as dendritic cells, and the appropriate immunogenic peptide.
  • APC antigen-presenting cells
  • the cells After an appropriate incubation time (typically about 7-28 days), in which the precursor cells are activated and expanded into effector cells, the cells are infused back into the patient, where they will destroy (CTL) or facilitate destruction (HTL) of their specific target cell (e.g., a tumor cell).
  • CTL destroy
  • HTL facilitate destruction
  • Transfected dendritic cells may also be used as antigen presenting cells.
  • compositions of the invention are typically used to treat and/or prevent a cancer that expresses or overexpresses 109P1D4.
  • peptide and/or nucleic acid compositions are administered to a patient in an amount sufficient to elicit an effective B cell, CTL and/or HTL response to the antigen and to cure or at least partially arrest or slow symptoms and/or complications.
  • An amount adequate to accomplish this is defined as "therapeutically effective dose.” Amounts effective for this use will depend on, e.g., the particular composition administered, the manner of administration, the stage and severity of the disease being treated, the weight and general state of health of the patient, and the judgment of the prescribing physician.
  • the immunogenic peptides of the invention are generally administered to an individual already bearing a tumor that expresses 109P1D4.
  • the peptides or DNA encoding them can be administered individually or as fusions of one or more peptide sequences.
  • Patients can be treated with the immunogenic peptides separately or in conjunction with other treatments, such as surgery, as appropriate.
  • administration should generally begin at the first diagnosis of 109P1 D4-associated cancer. This is followed by boosting doses until at least symptoms are substantially abated and for a period thereafter.
  • the embodiment of the vaccine composition i.e., including, but not limited to embodiments such as peptide cocktails, polyepitopic polypeptides, minigenes, or TAA-specific CTLs or pulsed dendritic cells
  • delivered to the patient may vary according to the stage of the disease or the patient's health status. For example, in a patient with a tumor that expresses 109P1 D4, a vaccine comprising 109P1 D4-specific CTL may be more efficacious in killing tumor cells in patient with advanced disease than alternative embodiments.
  • compositions which stimulate helper T cell responses can also be given in accordance with this embodiment of the invention.
  • the dosage for an initial therapeutic immunization generally occurs in a unit dosage range where the lower value is about 1, 5, 50, 500, or 1,000 ⁇ g and the higher value is about 10,000; 20,000; 30,000; or 50,000 ⁇ g.
  • Dosage values for a human typically range from about 500 ⁇ g to about 50,000 ⁇ g per 70 kilogram patient.
  • Boosting dosages of between about 1.0 ⁇ g to about 60,000 ⁇ g of peptide pursuant to a boosting regimen over weeks to months may be administered depending upon the patient's response and condition as determined by measuring the specific activity of CTL and HTL obtained from the patient's blood. Administration should continue until at least clinical symptoms or laboratory tests indicate that the neoplasia, has been eliminated or reduced and for a period thereafter.
  • the dosages, routes of administration, and dose schedules are adjusted in accordance with methodologies known in the art.
  • the peptides and compositions of the present invention are employed in serious disease states, that is, life-threatening or potentially life threatening situations.
  • life-threatening or potentially life threatening situations in certain embodiments, it is possible and may be felt desirable by the treating physician to administer substantial excesses of these peptide compositions relative to these stated dosage amounts.
  • the vaccine compositions of the invention can also be used purely as prophylactic agents.
  • the dosage for an initial prophylactic immunization generally occurs in a unit dosage range where the lower value is about 1, 5, 60, 600, or 1000 ⁇ g and the higher value is about 10,000; 20,000; 30,000; or 60,000 ⁇ g.
  • Dosage values for a human typically range from about 500 ⁇ g to about 50,000 ⁇ g per 70 kilogram patient. This is followed by boosting dosages of between about 1.0 ⁇ g to about 50,000 ⁇ g of peptide administered at defined intervals from about four weeks to six months after the initial administration of vaccine.
  • the immunogenicity of the vaccine can be assessed by measuring the specific activity of CTL and HTL obtained from a sample of the patient's blood.
  • compositions for therapeutic treatment are intended for parenteral, topical, oral, nasal, intrathecal, or local (e.g. as a cream or topical ointment) administration.
  • the pharmaceutical compositions are administered parentally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly.
  • compositions for parenteral administration which comprise a solution of the immunogenic peptides dissolved or suspended in an acceptable carrier, preferably an aqueous carrier.
  • aqueous carriers may be used, e.g., water, buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid and the like. These compositions may be sterilized by conventional, well-known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration.
  • compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservatives, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanola ine oleate, etc.
  • auxiliary substances such as pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservatives, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanola ine oleate, etc.
  • concentration of peptides of the invention in the pharmaceutical formulations can vary widely, i.e., from less than about 0.1 %, usually at or at least about 2% to as much as 20% to 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, efc, in accordance with the particular mode of administration selected.
  • a human unit dose form of a composition is typically included in a pharmaceutical composition that comprises a human unit dose of an acceptable carrier, in one embodiment an aqueous carrier, and is administered in a volume/quantity that is known by those of skill in the art to be used for administration of such compositions to humans (see, e.g., Remington's Pharmaceutical Sciences, 17 th Edition, A. Gennaro, Editor, Mack Publishing Co., Easton, Pennsylvania, 1985).
  • a peptide dose for initial immunization can be from about 1 to about 50,000 ⁇ g, generally 100-5,000 ⁇ g, for a 70 kg patient.
  • an initial immunization may be performed using an expression vector in the form of naked nucleic acid administered IM (or SC or ID) in the amounts of 0.5-5 mg at multiple sites.
  • the nucleic acid (0.1 to 1000 ⁇ g) can also be administered using a gene gun.
  • a booster dose is then administered.
  • the booster can be recombinant fowlpox virus administered at a dose of 5-10 7 to 5x10 9 pfu.
  • a treatment generally involves repeated administration of the anti-109P1D4 antibody preparation, via an acceptable route of administration such as intravenous injection (IV), typically at a dose in the range of about 0.1 to about 10 mg/kg body weight.
  • IV intravenous injection
  • doses in the range of 10-500 mg mAb per week are effective and well tolerated.
  • an initial loading dose of approximately 4 mg/kg patient body weight IV, followed by weekly doses of about 2 mg/kg IV of the anti- 109P1 D4 mAb preparation represents an acceptable dosing regimen.
  • various factors can influence the ideal dose in a particular case.
  • Such factors include, for example, half life of a composition, the binding affinity of an Ab, the immunogenicity of a substance, the degree of 109P1 D4 expression in the patient, the extent of circulating shed 109P1D4 antigen, the desired steady-state concentration level, frequency of treatment, and the influence of chemotherapeutic or other agents used in combination with the treatment method of the invention, as well as the health status of a particular patient.
  • Non-limiting preferred human unit doses are, for example, ⁇ OO ⁇ g - 1 mg, 1 mg - 50mg, 50mg - 100mg, 100mg - 200mg, 200mg - 300mg, 400mg - ⁇ OOmg, 500mg - 600mg, 600mg - 700mg, 700mg - 800mg, 800mg - 900mg, 900mg - 1g, or 1mg - 700mg.
  • the dose is in a range of 2-5 mg/kg body weight, e.g., with follow on weekly doses of 1-3 mg/kg; 0.5mg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10mg/kg body weight followed, e.g., in two, three or four weeks by weekly doses; 0.5 - 10mg/kg body weight, e.g., followed in two, three or four weeks by weekly doses; 225, 250, 275, 300, 325, 350, 375, 400mg m 2 of body area weekly; 1-600mg m z of body area weekly; 225-400mg m 2 of body area weekly; these does can be followed by weekly doses for 2, 3, 4, ⁇ , 6, 7, 8, 9, 19, 11 , 12 or more weeks.
  • human unit dose forms of polynucleotides comprise a suitable dosage range or effective amount that provides any therapeutic effect.
  • a therapeutic effect depends on a number of factors, including the sequence of the polynucleotide, molecular weight of the polynucleotide and route of administration. Dosages are generally selected by the physician or other health care professional in accordance with a variety of parameters known in the art, such as severity of symptoms, history of the patient and the like.
  • a dosage range may be selected from, for example, an independently selected lower limit such as about 0.1, 0.25, 0.5, 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 mg/kg up to an independently selected upper limit, greater than the lower limit, of about 60, 80, 100, 200, 300, 400, 500, 750, 1000, 1600, 2000, 3000, 4000, 6000, 6000, 7000, 8000, 9000 or 10,000 mg/kg.
  • an independently selected lower limit such as about 0.1, 0.25, 0.5, 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 mg/kg up to an independently selected upper limit, greater than the lower limit, of about 60, 80, 100, 200, 300, 400, 500, 750, 1000, 1600, 2000, 3000, 4000, 6000, 6000, 7000, 8000, 9000 or 10,000 mg/kg.
  • a dose may be about any of the following: 0.1 to 100 mg/kg, 0.1 to 60 mg/kg, 0.1 to 25 mg/kg, 0.1 to 10 mg/kg, 1 to 500 mg/kg, 100 to 400 mg/kg, 200 to 300 mg/kg, 1 to 100 mg/kg, 100 to 200 mg/kg, 300 to 400 mg/kg, 400 to 500 mg/kg, 500 to 1000 mg/kg, 500 to 5000 mg/kg, or 500 to 10,000 mg/kg.
  • parenteral routes of administration may require higher doses of polynucleotide compared to more direct application to the nucleotide to diseased tissue, as do polynucleotides of increasing length.
  • human unit dose forms of T-cells comprise a suitable dosage range or effective amount that provides any therapeutic effect.
  • a therapeutic effect depends on a number of factors. Dosages are generally selected by the physician or other health care professional in accordance with a variety of parameters known in the art, such as severity of symptoms, history of the patient and the like.
  • a dose may be about 10 4 cells to about 10 6 cells, about 10 6 cells to about 10 8 cells, about 10 3 to about 10 11 cells, or about 10 B to about 5 x 10TM cells.
  • a dose may also about 10 6 cells/m 2 to about 10 10 cells/m 2 , or about 10 6 cells/m 2 to about 10 8 cells/m 2 .
  • Proteins(s) of the invention, and/or nucleic acids encoding the protein(s), can also be administered via liposomes, which may also serve to: 1) target the proteins(s) to a particular tissue, such as lymphoid tissue; 2) to target selectively to diseases cells; or, 3) to increase the half-life of the peptide composition.
  • liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like.
  • the peptide to be delivered is incorporated as part of a liposome, alone or in conjunction with a molecule which binds to a receptor prevalent among lymphoid cells, such as monoclonal antibodies which bind to the CD45 antigen, or with other therapeutic or immunogenic compositions.
  • liposomes either filled or decorated with a desired peptide of the invention can be directed to the site of lymphoid cells, where the liposomes then deliver the peptide compositions.
  • Liposomes for use in accordance with the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol.
  • lipids are generally guided by consideration of, e.g., liposome size, acid lability and stability of the liposomes in the blood stream.
  • a variety of methods are available for preparing liposomes, as described in, e.g., Szoka, ef a/., Ann. Rev. Biophys. Bioeng. 9:467 (1980), and U.S. Patent Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.
  • a ligand to be incorporated into the liposome can include, e.g., antibodies or fragments thereof specific for cell surface determinants of the desired immune system cells.
  • a liposome suspension containing a peptide may be administered intravenously, locally, topically, efc. in a dose which varies according to, infer a//a, the manner of administration, the peptide being delivered, and the stage of the disease being treated.
  • nontoxic solid carriers may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10- 95% of active ingredient, that is, one or more peptides of the invention, and more preferably at a concentration of 25%-7 ⁇ %.
  • immunogenic peptides are preferably supplied in finely divided form along with a surfactant and propellant. Typical percentages of peptides are about 0.01 %-20% by weight, preferably about 1%-10%.
  • the surfactant must, of course, be nontoxic, and preferably soluble in the propellant.
  • Representative of such agents are the esters or partial esters of fatty acids containing from about 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride.
  • Mixed esters such as mixed or natural glycerides may be employed.
  • the surfactant may constitute about 0.1 %-20% by weight of the composition, preferably about 0.25-6%.
  • the balance of the composition is ordinarily propellant.
  • a carrier can also be included, as desired, as with, e.g., lecithin for intranasal delivery.
  • 109P1 D4 polynucleotides, polypeptides, reactive cytotoxic T cells (CTL), reactive helper T cells (HTL) and anti-polypeptide antibodies are used in well known diagnostic, prognostic and therapeutic assays that examine conditions associated with dysregulated cell growth such as cancer, in particular the cancers listed in Table I (see, e.g., both its specific pattern of tissue expression as well as its overexpression in certain cancers as described for example in the Example entitled "Expression analysis of 109P1 D4 in normal tissues, and patient specimens").
  • 109P1D4 can be analogized to a prostate associated antigen PSA, the archetypal marker that has been used by medical practitioners for years to identify and monitor the presence of prostate cancer (see, e.g., Merrill ef al, J. Urol. 163(2): 503-5120 (2000); Polascik ef al, J. Urol. Aug; 162(2):293-306 (1999) and Fortier ef al., J. Nat. Cancer Inst. 91(19): 1635- 1640(1999)).
  • PSA prostate associated antigen PSA
  • 109P1D4 polynucleotides and polypeptides as well as 109P1D4 polynucleotide probes and anti-109P1D4 antibodies used to identify the presence of these molecules
  • 109P1D4 polynucleotide probes and anti-109P1D4 antibodies used to identify the presence of these molecules allows skilled artisans to utilize these molecules in methods that are analogous to those used, for example, in a variety of diagnostic assays directed to examining conditions associated with cancer.
  • Typical embodiments of diagnostic methods which utilize the 109P1D4 polynucleotides, polypeptides, reactive T cells and antibodies are analogous to those methods from well-established diagnostic assays, which employ, e.g., PSA polynucleotides, polypeptides, reactive T cells and antibodies.
  • PSA polynucleotides are used as probes (for example in Northern analysis, see, e.g., Sharief ef al, Biochem. Mol. Biol. Int. 33(3) :567-74(1994)) and primers (for example in PCR analysis, see, e.g., Okegawa etal, J. Urol.
  • the 109P1 D4 polynucleotides described herein can be utilized in the same way to detect 109P1D4 overexpression or the metastasis of prostate and other cancers expressing this gene.
  • PSA polypeptides are used to generate antibodies specific for PSA which can then be used to observe the presence and/or the level of PSA proteins in methods to monitor PSA protein overexpression (see, e.g., Stephan et al, Urology 65(4):560-3 (2000)) or the metastasis of prostate cells (see, e.g., Alanen et al, Pathol. Res. Pract. 192(3):233-7 (1996)), the 109P1D4 polypeptides described herein can be utilized to generate antibodies for use in detecting 109P1 D4 overexpression or the metastasis of prostate cells and cells of other cancers expressing this gene.
  • metastases involves the movement of cancer cells from an organ of origin (such as the lung or prostate gland etc.) to a different area of the body (such as a lymph node)
  • assays which examine a biological sample for the presence of cells expressing 109P1D4 polynucleotides and/or polypeptides can be used to provide evidence of metastasis.
  • tissue that does not normally contain 109P1D4-expressing cells lymph node
  • 109P1D4-expressing cells such as the 109P1D4 expression seen in LAPC4 and LAPC9
  • 109P1D4 polynucleotides and/or polypeptides can be used to provide evidence of cancer, for example, when cells in a biological sample that do not normally express 109P1D4 or express 109P1D4 at a different level are found to express 109P1D4 or have an increased expression of 109P1D4 (see, e.g., the 109P1D4 expression in the cancers listed in Table I and in patient samples etc. shown in the accompanying Figures).
  • artisans may further wish to generate supplementary evidence of metastasis by testing the biological sample for the presence of a second tissue restricted marker (in addition to 109P1D4) such as PSA, PSCA etc. (see, e.g., Alanen ef al, Pathol. Res. Pract. 192(3): 233- 237 (1996)).
  • immunohistochemistry to identify the presence of a 109P1 D4 polypeptide within a tissue section can indicate an altered state of certain cells within that tissue. It is well understood in the art that the ability of an antibody to localize to a polypeptide that is expressed in cancer cells is a way of diagnosing presence of disease, disease stage, progression and/or tumor aggressiveness. Such an antibody can also detect an altered distribution of the polypeptide within the cancer cells, as compared to corresponding non-malignant tissue.
  • the 109P1 D4 polypeptide and immunogenic compositions are also useful in view of the phenomena of altered subcellular protein localization in disease states. Alteration of cells from normal to diseased state causes changes in cellular morphology and is often associated with changes in subcellular protein localization/distribution. For example, cell membrane proteins that are expressed in a polarized manner in normal cells can be altered in disease, resulting in distribution of the protein in a non-polar manner over the whole cell surface.
  • normal breast epithelium is either negative for Her2 protein or exhibits only a basolateral distribution whereas malignant cells can express the protein over the whole cell surface (De Potter, et al, International Journal of Cancer, 44; 969-974 (1989): McCormick, et al, 117; 935-943 (2002)).
  • distribution of the protein may be altered from a surface only localization to include diffuse cytoplasmic expression in the diseased state. Such an example can be seen with MUC1 (Diaz, etal, The Breast Journal, 7: 40-45 (2001)).
  • the 109P1D4 protein and immune responses related thereto are very useful. Accordingly, the ability to determine whether alteration of subcellular protein localization occurred for 24P4C12 make the 109P1D4 protein and immune responses related thereto very useful.
  • Use of the 109P1D4 compositions allows those skilled in the art to make important diagnostic and therapeutic decisions.
  • Immunohistochemical reagents specific to 109P1D4 are also useful to detect metastases of tumors expressing 109P1D4 when the polypeptide appears in tissues where 109P1D4 is not normally produced.
  • 109P1D4 polypeptides and antibodies resulting from immune responses thereto are useful in a variety of important contexts such as diagnostic, prognostic, preventative and/or therapeutic purposes known to those skilled in the art.
  • PSA polynucleotide fragments and polynucleotide variants are employed by skilled artisans for use in methods of monitoring PSA
  • 109P1D4 polynucleotide fragments and polynucleotide variants are used in an analogous manner.
  • typical PSA polynucleotides used in methods of monitoring PSA are probes or primers which consist of fragments of the PSA cDNA sequence.
  • primers used to PCR amplify a PSA polynucleotide must include less than the whole PSA sequence to function in the polymerase chain reaction.
  • PCR reactions In the context of such PCR reactions, skilled artisans generally create a variety of different polynucleotide fragments that can be used as primers in order to amplify different portions of a polynucleotide of interest or to optimize amplification reactions (see, e.g., Caetano-Anolles, G. Biotechniques 25(3): 472-476, 478-480 (1998); Robertson etal, Methods Mol. Biol. 98:121-154 (1998)).
  • variant polynucleotide sequences are typically used as primers and probes for the corresponding mRNAs in PCR and Northern analyses (see, e.g., Sawai ef a/., Fetal Diagn. Ther. 1996 Nov-Dec 11(6):407-13 and Current Protocols In Molecular Biology, Volume 2, Unit 2, Frederick M. Ausubel ef a/, eds., 1995)).
  • Polynucleotide fragments and variants are useful in this context where they are capable of binding to a target polynucleotide sequence (e.g., a 109P1 D4 polynucleotide shown in Figure 2 or variant thereof) under conditions of high stringency.
  • a target polynucleotide sequence e.g., a 109P1 D4 polynucleotide shown in Figure 2 or variant thereof
  • PSA polypeptides which contain an epitope that can be recognized by an antibody or T cell that specifically binds to that epitope are used in methods of monitoring PSA.
  • 109P1 D4 polypeptide fragments and polypeptide analogs or variants can also be used in an analogous manner.
  • This practice of using polypeptide fragments or polypeptide variants to generate antibodies is typical in the art with a wide variety of systems such as fusion proteins being used by practitioners (see, e.g., Current Protocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubel etal. eds., 1995).
  • each epitope(s) functions to provide the architecture with which an antibody or T cell is reactive.
  • polypeptide fragments that can be used in order to generate immune responses specific for different portions of a polypeptide of interest (see, e.g., U.S. Patent No. 5,840,501 and U.S. Patent No. 5,939,533).
  • a polypeptide comprising one of the 109P1D4 biological motifs discussed herein or a motif-bearing subsequence which is readily identified by one of skill in the art based on motifs available in the art.
  • Polypeptide fragments, variants or analogs are typically useful in this context as long as they comprise an epitope capable of generating an antibody or T cell specific for a target polypeptide sequence (e.g. a 109P1D4 polypeptide shown in Figure 3).
  • the 109P1D4 polynucleotides and polypeptides exhibit specific properties that make them useful in diagnosing cancers such as those listed in Table I.
  • Diagnostic assays that measure the presence of 109P1 D4 gene products in order to evaluate the presence or onset of a disease condition described herein, such as prostate cancer, are used to identify patients for preventive measures or further monitoring, as has been done so successfully with PSA.
  • these materials satisfy a need in the art for molecules having similar or complementary characteristics to PSA in situations where, for example, a definite diagnosis of metastasis of prostatic origin cannot be made on the basis of a test for PSA alone (see, e.g., Alanen ef al, Pathol. Res. Pract. 192(3): 233-237 (1996)), and consequently, materials such as 109P1D4 polynucleotides and polypeptides (as well as the 109P1D4 polynucleotide probes and anti- 109P1D4 antibodies used to identify the presence of these molecules) need to be employed to confirm a metastases of prostatic origin.
  • the 109P1D4 polynucleotides disclosed herein have a number of other utilities such as their use in the identification of oncogenetic associated chromosomal abnormalities in the chromosomal region to which the 109P1D4 gene maps (see the Example entitled "Chromosomal Mapping of 109P1D4" below).
  • the 109P1D4-related proteins and polynucleotides disclosed herein have other utilities such as their use in the forensic analysis of tissues of unknown origin (see, e.g., Takahama K Forensic Sci Int 1996 Jun 28;80(1-2): 63-9).
  • 109P1D4-related proteins or polynucleotides of the invention can be used to treat a pathologic condition characterized by the over-expression of 109P1 D4.
  • the amino acid or nucleic acid sequence of Figure 2 or Figure 3, or fragments of either can be used to generate an immune response to a 109P1D4 antigen.
  • Antibodies or other molecules that react with 109P1 D4 can be used to modulate the function of this molecule, and thereby provide a therapeutic benefit.
  • the invention includes various methods and compositions for inhibiting the binding of 109P1 D4 to its binding partner or its association with other protein(s) as well as methods for inhibiting 109P1D4 function.
  • a recombinant vector that encodes single chain antibodies that specifically bind to 109P1 D4 are introduced into 109P1D4 expressing cells via gene transfer technologies. Accordingly, the encoded single chain anti- 109P1D4 antibody is expressed intracellularly, binds to 109P1D4 protein, and thereby inhibits its function.
  • Methods for engineering such intracellular single chain antibodies are well known.
  • intracellular antibodies also known as "intrabodies” are specifically targeted to a particular compartment within the cell, providing control over where the inhibitory activity of the treatment is focused. This technology has been successfully applied in the art (for review, see Richardson and Marasco, 1995, TIBTECH vol. 13).
  • Intrabodies have been shown to virtually eliminate the expression of otherwise abundant cell surface receptors (see, e.g., Richardson ef al, 1995, Proc. Natl. Acad. Sci. USA 92: 3137-3141; Beerli ef al, 1994, J. Biol. Chem. 289: 23931-23936; Deshane ef al, 1994, Gene Ther. 1: 332-337).
  • Single chain antibodies comprise the variable domains of the heavy and light chain joined by a flexible linker polypeptide, and are expressed as a single polypeptide.
  • single chain antibodies are expressed as a single chain variable region fragment joined to the light chain constant region.
  • Well-known intracellular trafficking signals are engineered into recombinant polynucleotide vectors encoding such single chain antibodies in order to target precisely the intrabody to the desired intracellular compartment.
  • intrabodies targeted to the endoplasmic reticulum (ER) are engineered to incorporate a leader peptide and, optionally, a C-terminal ER retention signal, such as the KDEL amino acid motif.
  • Intrabodies intended to exert activity in the nucleus are engineered to include a nuclear localization signal.
  • Lipid moieties are joined to intrabodies in order to tether the intrabody to the cytosolic side of the plasma membrane.
  • Intrabodies can also be targeted to exert function in the cytosol.
  • cytosolic intrabodies are used to sequester factors within the cytosol, thereby preventing them from being transported to their natural cellular destination.
  • intrabodies are used to capture 109P1D4 in the nucleus, thereby preventing its activity within the nucleus.
  • Nuclear targeting signals are engineered into such 109P1 D4 intrabodies in order to achieve the desired targeting.
  • Such 109P1D4 intrabodies are designed to bind specifically to a particular 109P1D4 domain.
  • cytosolic intrabodies that specifically bind to a 109P1D4 protein are used to prevent 109P1D4 from gaining access to the nucleus, thereby preventing it from exerting any biological activity within the nucleus (e.g., preventing 109P1 D4 from forming transcription complexes with other factors).
  • the transcription of the intrabody is placed under the regulatory control of an appropriate tumor-specific promoter and/or enhancer.
  • an appropriate tumor-specific promoter and/or enhancer In order to target intrabody expression specifically to prostate, for example, the PSA promoter and/or promoter/enhancer can be utilized (See, for example, U.S. Patent No. 5,919,652 issued 6 July 1999).
  • recombinant molecules bind to 109P1D4 and thereby inhibit 109P1D4 function.
  • these recombinant molecules prevent or inhibit 109P1D4 from accessing/binding to its binding partner(s) or associating with other protein(s).
  • Such recombinant molecules can, for example, contain the reactive partis) of a 109P1 D4 specific antibody molecule.
  • the 109P1 D4 binding domain of a 109P1 D4 binding partner is engineered into a dimeric fusion protein, whereby the fusion protein comprises two 109P1D4 ligand binding domains linked to the Fc portion of a human IgG, such as human lgG1.
  • Such IgG portion can contain, for example, the CH2 and CH3 domains and the hinge region, but not the CH1 domain.
  • Such dimeric fusion proteins are administered in soluble form to patients suffering from a cancer associated with the expression of 109P1 D4, whereby the dimeric fusion protein specifically binds to 109P1 D4 and blocks 109P1 D4 interaction with a binding partner.
  • Such dimeric fusion proteins are further combined into multimeric proteins using known antibody linking technologies.
  • the present invention also comprises various methods and compositions for inhibiting the transcription of the 109P1 D4 gene. Similarly, the invention also provides methods and compositions for inhibiting the translation of 109P1 D4 mRNA into protein.
  • a method of inhibiting the transcription of the 109P1D4 gene comprises contacting the 109P1D4 gene with a 109P1 D4 antisense polynucleotide.
  • a method of inhibiting 109P1 D4 mRNA translation comprises contacting a 109P1D4 mRNA with an antisense polynucleotide.
  • a 109P1 D4 specific ribozyme is used to cleave a 109P1D4 message, thereby inhibiting translation.
  • Such antisense and ribozyme based methods can also be directed to the regulatory regions of the 109P1D4 gene, such as 109P1D4 promoter and/or enhancer elements.
  • proteins capable of inhibiting a 109P1 D4 gene transcription factor are used to inhibit 109P1 D4 mRNA transcription.
  • the various polynucleotides and compositions useful in the aforementioned methods have been described above.
  • the use of antisense and ribozyme molecules to inhibit transcription and translation is well known in the art.
  • Gene transfer and gene therapy technologies can be used to deliver therapeutic polynucleotide molecules to tumor cells synthesizing 109P1D4 (i.e., antisense, ribozyme, polynucleotides encoding intrabodies and other 109P1D4 inhibitory molecules).
  • 109P1D4 i.e., antisense, ribozyme, polynucleotides encoding intrabodies and other 109P1D4 inhibitory molecules.
  • a number of gene therapy approaches are known in the art.
  • Recombinant vectors encoding 109P1 D4 antisense polynucleotides, ribozymes, factors capable of interfering with 109P1 D4 transcription, and so forth, can be delivered to target tumor cells using such gene therapy approaches.
  • the above therapeutic approaches can be combined with any one of a wide variety of surgical, chemotherapy or radiation therapy regimens.
  • the therapeutic approaches of the invention can enable the use of reduced dosages of chemotherapy (or other therapies) and/or less frequent administration, an advantage for all patients and particularly for those that do not tolerate the toxicity of the chemotherapeutic agent well.
  • the anti-tumor activity of a particular composition can be evaluated using various in vitro and in vivo assay systems.
  • In vitro assays that evaluate therapeutic activity include cell growth assays, soft agar assays and other assays indicative of tumor promoting activity, binding assays capable of determining the extent to which a therapeutic composition will inhibit the binding of 109P1 D4 to a binding partner, etc.
  • a 109P1 D4 therapeutic composition can be evaluated in a suitable animal model.
  • xenogenic prostate cancer models can be used, wherein human prostate cancer explants or passaged xenograft tissues are introduced into immune compromised animals, such as nude or SCID mice (Klein et al, 1997, Nature Medicine 3: 402-408).
  • PCT Patent Application W098/16628 and U.S. Patent 6,107,540 describe various xenograft models of human prostate cancer capable of recapitulating the development of primary tumors, micrometastasis, and the formation of osteoblastic metastases characteristic of late stage disease. Efficacy can be predicted using assays that measure inhibition of tumor formation, tumor regression or metastasis, and the like.
  • xenografts from tumor bearing mice treated with the therapeutic composition can be examined for the presence of apoptotic foci and compared to untreated control xenograft-bearing mice. The extent to which apoptotic foci are found in the tumors of the treated mice provides an indication of the therapeutic efficacy of the composition.
  • Suitable carriers include any material that when combined with the therapeutic composition retains the anti-tumor function of the therapeutic composition and is generally non-reactive with the patient's immune system. Examples include, but are not limited to, any of a number of standard pharmaceutical carriers such as sterile phosphate buffered saline solutions, bacteriostatic water, and the like (see, generally, Remington's Pharmaceutical Sciences 16 th Edition, A. Osal., Ed., 1980).
  • Therapeutic formulations can be solubilized and administered via any route capable of delivering the therapeutic composition to the tumor site.
  • Potentially effective routes of administration include, but are not limited to, intravenous, parenteral, intraperitoneal, intramuscular, intratumor, intradermal, intraorgan, orthotopic, and the like.
  • a preferred formulation for intravenous injection comprises the therapeutic composition in a solution of preserved bacteriostatic water, sterile unpreserved water, and/or diluted in polyvinylchloride or polyethylene bags containing 0.9% sterile Sodium Chloride for Injection, USP.
  • Therapeutic protein preparations can be lyophilized and stored as sterile powders, preferably under vacuum, and then reconstituted in bacteriostatic water (containing for example, benzyl alcohol preservative) or in sterile water prior to injection.
  • Dosages and administration protocols for the treatment of cancers using the foregoing methods will vary with the method and the target cancer, and will generally depend on a number of other factors appreciated in the art.
  • screening is performed to identify modulators that induce or suppress a particular expression profile, suppress or induce specific pathways, preferably generating the associated phenotype thereby.
  • having identified differentially expressed genes important in a particular state screens are performed to identify modulators that alter expression of individual genes, either increase or decrease.
  • screening is performed to identify modulators that alter a biological function of the expression product of a differentially expressed gene. Again, having identified the importance of a gene in a particular state, screens are performed to identify agents that bind and/or modulate the biological activity of the gene product.
  • screens are done for genes that are induced in response to a candidate agent.
  • identifying a modulator one that suppresses a cancer expression pattern leading to a normal expression pattern, or a modulator of a cancer gene that leads to expression of the gene as in normal tissue
  • a screen is performed to identify genes that are specifically modulated in response to the agent. Comparing expression profiles between normal tissue and agent-treated cancer tissue reveals genes that are not expressed in normal tissue or cancer tissue, but are expressed in agent treated tissue, and vice versa.
  • agent-specific sequences are identified and used by methods described herein for cancer genes or proteins. In particular these sequences and the proteins they encode are used in marking or identifying agent- treated cells.
  • antibodies are raised against the agent-induced proteins and used to target novel therapeutics to the treated cancer tissue sample.
  • Proteins, nucleic acids, and antibodies of the invention are used in screening assays.
  • the cancer-associated proteins, antibodies, nucleic acids, modified proteins and cells containing these sequences are used in screening assays, such as evaluating the effect of drug candidates on a "gene expression profile," expression profile of polypeptides or alteration of biological function.
  • the expression profiles are used, preferably in conjunction with high throughput screening techniques to allow monitoring for expression profile genes after treatment with a candidate agent (e.g., Davis, GF, et al, J Biol Screen 7:69 (2002); Zlokamik, et al., Science 279:84-8 (1998); Heid, Genome Res 6:986- 94,1996).
  • a candidate agent e.g., Davis, GF, et al, J Biol Screen 7:69 (2002); Zlokamik, et al., Science 279:84-8 (1998); Heid, Genome Res 6:986- 94,1996).
  • the cancer proteins, antibodies, nucleic acids, modified proteins and cells containing the native or modified cancer proteins or genes are used in screening assays. That is, the present invention comprises methods for screening for compositions which modulate the cancer phenotype or a physiological function of a cancer protein of the invention. This is done on a gene itself or by evaluating the effect of drug candidates on a "gene expression profile" or biological function. In one embodiment, expression profiles are used, preferably in conjunction with high throughput screening techniques to allow monitoring after treatment with a candidate agent, see Zlokamik, supra.
  • a variety of assays are executed directed to the genes and proteins of the invention. Assays are run on an individual nucleic acid or protein level. That is, having identified a particular gene as up regulated in cancer, test compounds are screened for the ability to modulate gene expression or for binding to the cancer protein of the invention. "Modulation" in this context includes an increase or a decrease in gene expression. The preferred amount of modulation will depend on the original change of the gene expression in normal versus tissue undergoing cancer, with changes of at least 10%, preferably 50%, more preferably 100-300%, and in some embodiments 300-1000% or greater.
  • a gene exhibits a 4-fold increase in cancer tissue compared to normal tissue, a decrease of about four-fold is often desired; similarly, a 10-fold decrease in cancer tissue compared to normal tissue a target value of a 10-fold increase in expression by the test compound is often desired.
  • Modulators that exacerbate the type of gene expression seen in cancer are also useful, e.g., as an upregulated target in further analyses.
  • the amount of gene expression is monitored using nucleic acid probes and the quantification of gene expression levels, or, alternatively, a gene product itself is monitored, e.g., through the use of antibodies to the cancer protein and standard immunoassays. Proteomics and separation techniques also allow for quantification of expression.
  • gene expression monitoring i.e., an expression profile
  • Such profiles will typically involve one or more of the genes of Figure 2.
  • cancer nucleic acid probes are attached to biochips to detect and quantify cancer sequences in a particular cell.
  • PCR can be used.
  • a series e.g., wells of a microtiter plate, can be used with dispensed primers in desired wells. A PCR reaction can then be performed and analyzed for each well.
  • Expression monitoring is performed to identify compounds that modify the expression of one or more cancer- associated sequences, e.g., a polynucleotide sequence set out in Figure 2.
  • a test modulator is added to the cells prior to analysis.
  • screens are also provided to identify agents that modulate cancer, modulate cancer proteins of the invention, bind to a cancer protein of the invention, or interfere with the binding of a cancer protein of the invention and an antibody or other binding partner.
  • high throughput screening methods involve providing a library containing a large number of potential therapeutic compounds (candidate compounds). Such "combinatorial chemical libraries" are then screened in one or more assays to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional "lead compounds,” as compounds for screening, or as therapeutics.
  • combinatorial libraries of potential modulators are screened for an ability to bind to a cancer polypeptide or to modulate activity.
  • new chemical entities with useful properties are generated by identifying a chemical compound (called a "lead compound") with some desirable property or activity, e.g., inhibiting activity, creating variants of the lead compound, and evaluating the property and activity of those variant compounds.
  • HTS high throughput screening
  • gene expression monitoring is conveniently used to test candidate modulators (e.g., protein, nucleic acid or small molecule).
  • candidate modulators e.g., protein, nucleic acid or small molecule.
  • the target sequence is prepared using known techniques. For example, a sample is treated to lyse the cells, using known lysis buffers, electroporation, etc., with purification and/or amplification such as PCR performed as appropriate. For example, an in vitro transcription with labels covalently attached to the nucleotides is performed. Generally, the nucleic acids are labeled with biotin-FITC or PE, or with cy3 or cy5.
  • the target sequence can be labeled with, e.g., a fluorescent, a chemiluminescent, a chemical, or a radioactive signal, to provide a means of detecting the target sequence's specific binding to a probe.
  • the label also can be an enzyme, such as alkaline phosphatase or horseradish peroxidase, which when provided with an appropriate substrate produces a product that is detected.
  • the label is a labeled compound or small molecule, such as an enzyme inhibitor, that binds but is not catalyzed or altered by the enzyme.
  • the label also can be a moiety or compound, such as, an epitope tag or biotin which specifically binds to streptavidin.
  • the streptavidin is labeled as described above, thereby, providing a detectable signal for the bound target sequence. Unbound labeled streptavidin is typically removed prior to analysis.
  • these assays can be direct hybridization assays or can comprise "sandwich assays", which include the use of multiple probes, as is generally outlined in U.S. Patent Nos. 5, 681,702; 5,597,909; 5,545,730; 5,594,117; 6,691,584; 5,571,670; 5,580,731; 5,571,670; 5,691,584; 5,624,802; 5,635,352; 5,594,118; 5,359,100; 5,124, 246; and 5,681,697.
  • the target nucleic acid is prepared as outlined above, and then added to the biochip comprising a plurality of nucleic acid probes, under conditions that allow the formation of a hybridization complex.
  • hybridization conditions are used in the present invention, including high, moderate and low stringency conditions as outlined above.
  • the assays are generally run under stringency conditions which allow formation of the label probe hybridization complex only in the presence of target.
  • Stringency can be controlled by altering a step parameter that is a thermodynamic variable, including, but not limited to, temperature, formamide concentration, salt concentration, chaotropic salt concentration pH, organic solvent concentration, etc. These parameters may also be used to control non-specific binding, as is generally outlined in U.S. Patent No. 5,681,697. Thus, it can be desirable to perform certain steps at higher stringency conditions to reduce non-specific binding.
  • the reactions outlined herein can be accomplished in a variety of ways. Components of the reaction can be added simultaneously, or sequentially, in different orders, with preferred embodiments outlined below.
  • the reaction may include a variety of other reagents. These include salts, buffers, neutral proteins, e.g. albumin, detergents, etc. which can be used to facilitate optimal hybridization and detection, and/or reduce nonspecific or background interactions. Reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti- icrobial agents, etc., may also be used as appropriate, depending on the sample preparation methods and purity of the target.
  • the assay data are analyzed to determine the expression levels of individual genes, and changes in expression levels as between states, forming a gene expression profile.
  • the invention provides methods identify or screen for a compound that modulates the activity of a cancer-related gene or protein of the invention.
  • the methods comprise adding a test compound, as defined above, to a cell comprising a cancer protein of the invention.
  • the cells contain a recombinant nucleic acid that encodes a cancer protein of the invention.
  • a library of candidate agents is tested on a plurality of cells.
  • the assays are evaluated in the presence or absence or previous or subsequent exposure of physiological signals, e.g. hormones, antibodies, peptides, antigens, cytokines, growth factors, action potentials, pharmacological agents including chemotherapeutics, radiation, carcinogenics, or other cells (i.e., cell-cell contacts).
  • physiological signals e.g. hormones, antibodies, peptides, antigens, cytokines, growth factors, action potentials, pharmacological agents including chemotherapeutics, radiation, carcinogenics, or other cells (i.e., cell-cell contacts).
  • the determinations are made at different stages of the cell cycle process. In this way, compounds that modulate genes or proteins of the invention are identified. Compounds with pharmacological activity are able to enhance or interfere with the activity of the cancer protein of the invention. Once identified, similar structures are evaluated to identify critical structural features of the compound.
  • a method of modulating (e.g., inhibiting) cancer cell division comprises administration of a cancer modulator.
  • a method of modulating (e.g., inhibiting) cancer is provided; the method comprises administration of a cancer modulator.
  • methods of treating cells or individuals with cancer are provided; the method comprises administration of a cancer modulator.
  • a method for modulating the status of a cell that expresses a gene of the invention comprises such art-accepted parameters such as growth, proliferation, survival, function, apoptosis, senescence, location, enzymatic activity, signal transduction, etc. of a cell.
  • a cancer inhibitor is an antibody as discussed above.
  • the cancer inhibitor is an antisense molecule.
  • a variety of cell growth, proliferation, and metastasis assays are known to those of skill in the art, as described herein.
  • the assays to identify suitable modulators are amenable to high throughput screening. Preferred assays thus detect enhancement or inhibition of cancer gene transcription, inhibition or enhancement of polypeptide expression, and inhibition or enhancement of polypeptide activity.
  • modulators evaluated in high throughput screening methods are proteins, often naturally occurring proteins or fragments of naturally occurring proteins.
  • proteins e.g., cellular extracts containing proteins, or random or directed digests of proteinaceous cellular extracts, are used.
  • libraries of proteins are made for screening in the methods of the invention.
  • Particularly preferred in this embodiment are libraries of bacterial, fungal, viral, and mammalian proteins, with the latter being preferred, and human proteins being especially preferred.
  • Particularly useful test compound will be directed to the class of proteins to which the target belongs, e.g., substrates for enzymes, or ligands and receptors.
  • Normal cells require a solid substrate to attach and grow. When cells are transformed, they lose this phenotype and grow detached from the substrate. For example, transformed cells can grow in stirred suspension culture or suspended in semi-solid media, such as semi-solid or soft agar. The transformed cells, when transfected with tumor suppressor genes, can regenerate normal phenotype and once again require a solid substrate to attach to and grow. Soft agar growth or colony formation in assays are used to identify modulators of cancer sequences, which when expressed in host cells, inhibit abnormal cellular proliferation and transformation. A modulator reduces or eliminates the host cells' ability to grow suspended in solid or semisolid media, such as agar.
  • Normal cells typically grow in a flat and organized pattern in cell culture until they touch other cells. When the cells touch one another, they are contact inhibited and stop growing. Transformed cells, however, are not contact inhibited and continue to grow to high densities in disorganized foci. Thus, transformed cells grow to a higher saturation density than corresponding normal cells. This is detected morphologically by the formation of a disoriented monolayer of cells or cells in foci.
  • labeling index with ( 3 H)-thymidine at saturation density is used to measure density limitation of growth, similarly an MTT or Alamar blue assay will reveal proliferation capacity of cells and the the ability of modulators to affect same. See Freshney (1994), supra.
  • Transformed cells when transfected with tumor suppressor genes, can regenerate a normal phenotype and become contact inhibited and would grow to a lower density.
  • labeling index with 3 H)-thymidine at saturation density is a preferred method of measuring density limitation of growth.
  • Transformed host cells are transfected with a cancer-associated sequence and are grown for 24 hours at saturation density in non-limiting medium conditions. The percentage of cells labeling with ( 3 H)-thymidine is determined by incorporated cpm.
  • a modulator reduces or eliminates contact independent growth, and returns the cells to a normal phenotype.
  • Transformed cells have lower serum dependence than their normal counterparts (see, e.g., Temin, J. Natl. Cancer Inst. 37:167-175 (1966); Eagle et al., J. Exp. Med 131:836-879 (1970)); Freshney, supra. This is in part due to release of various growth factors by the transformed cells.
  • the degree of growth factor or serum dependence of transformed host cells can be compared with that of control. For example, growth factor or serum dependence of a cell is monitored in methods to identify and characterize compounds that modulate cancer-associated sequences of the invention.
  • Tumor cells release an increased amount of certain factors (hereinafter "tumor specific markers") than their normal counterparts.
  • tumor specific markers plasminogen activator (PA) is released from human glioma at a higher level than from normal brain cells (see, e.g., Gullino, Angiogenesis, Tumor Vascularization, and Potential Interference with Tumor Growth, in Biological Responses in Cancer, pp. 178-184 (Mihich (ed.) 1985)).
  • Tumor Angiogenesis Factor TAF
  • TAF Tumor Angiogenesis Factor
  • the degree of invasiveness into Matrigel or an extracellular matrix constituent can be used as an assay to identify and characterize compounds that modulate cancer associated sequences.
  • Tumor cells exhibit a positive correlation between malignancy and invasiveness of cells into Matrigel or some other extracellular matrix constituent.
  • tumorigenic cells are typically used as host cells. Expression of a tumor suppressor gene in these host cells would decrease invasiveness of the host cells. Techniques described in Cancer Res. 1999; 59:6010; Freshney (1994), supra, can be used. Briefly, the level of invasion of host cells is measured by using filters coated with Matrigel or some other extracellular matrix constituent.
  • Penetration into the gel, or through to the distal side of the filter, is rated as invasiveness, and rated histologically by number of cells and distance moved, or by prelabeling the cells with 125 1 and counting the radioactivity on the distal side of the filter or bottom of the dish. See, e.g., Freshney (1984), supra.
  • Transgenic organisms are prepared in a variety of art-accepted ways. For example, knock-out transgenic organisms, e.g., mammals such as mice, are made, in which a cancer gene is disrupted or in which a cancer gene is inserted. Knock-out transgenic mice are made by insertion of a marker gene or other heterologous gene into the endogenous cancer gene site in the mouse genome via homologous recombination. Such mice can also be made by substituting the endogenous cancer gene with a mutated version of the cancer gene, or by mutating the endogenous cancer gene, e.g., by exposure to carcinogens.
  • transgenic chimeric animals e.g., mice
  • a DNA construct is introduced into the nuclei of embryonic stem cells.
  • Cells containing the newly engineered genetic lesion are injected into a host mouse embryo, which is re- implanted into a recipient female. Some of these embryos develop into chimeric mice that possess germ cells some of which are derived from the mutant cell line. Therefore, by breeding the chimeric mice it is possible to obtain a new line of mice containing the introduced genetic lesion (see, e.g., Capecchi et al., Science 244:1288 (1989)).
  • Chimeric mice can be derived according to US Patent 6,365,797, issued 2 April 2002; US Patent 6,107,540 issued 22 August 2000; Hogan et al., Manipulating the Mouse Embryo: A laboratory Manual, Cold Spring Harbor Laboratory (1988) and Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, Robertson, ed., IRL Press, Washington, D.C., (1987).
  • various immune-suppressed or immune-deficient host animals can be used.
  • a genetically athymic "nude” mouse see, e.g., Giovanella et al., J. Natl. Cancer Inst. 52:921 (1974)
  • SCID mouse see, e.g., Giovanella et al., J. Natl. Cancer Inst. 52:921 (1974)
  • SCID mouse e.g., a thymectornized mouse
  • an irradiated mouse see, e.g., Bradley et al., Br. J. Cancer 38:263 (1978); Selby et al., Br. J. Cancer 41:52 (1980)
  • Transplantable tumor cells typically about 10 6 cells injected into isogenic hosts produce invasive tumors in a high proportion of cases, while normal cells of similar origin will not.
  • cells expressing cancer-associated sequences are injected subcutaneously or orthotopically. Mice are then separated into groups, including control groups and treated experimental groups) e.g. treated with a modulator). After a suitable length of time, preferably 4-8 weeks, tumor growth is measured (e.g., by volume or by its two largest dimensions, or weight) and compared to the control. Tumors that have statistically significant reduction (using, e.g., Student's T test) are said to have inhibited growth.
  • Assays to identify compounds with modulating activity can be performed in vitro.
  • a cancer polypeptide is first contacted with a potential modulator and incubated for a suitable amount of time, e.g., from 0.5 to 48 hours.
  • the cancer polypeptide levels are determined in vitro by measuring the level of protein or mRNA.
  • the level of protein is measured using immunoassays such as Western blotting, ELISA and the like with an antibody that selectively binds to the cancer polypeptide or a fragment thereof.
  • amplification e.g., using PCR, LCR, or hybridization assays, e. g., Northern hybridization, RNAse protection, dot blotting, are preferred.
  • the level of protein or mRNA is detected using directly or indirectly labeled detection agents, e.g., fluorescently or radioactively labeled nucleic acids, radioactively or enzymatically labeled antibodies, and the like, as described herein.
  • a reporter gene system can be devised using a cancer protein promoter operably linked to a reporter gene such as luciferase, green fluorescent protein, CAT, or P-gal.
  • the reporter construct is typically transfected into a cell. After treatment with a potential modulator, the amount of reporter gene transcription, translation, or activity is measured according to standard techniques known to those of skill in the art (Davis GF, supra; Gonzalez, J. & Negulescu, P. Curr. Opin. Biotechnol. 1998: 9:624).
  • in vitro screens are done on individual genes and gene products. That is, having identified a particular differentially expressed gene as important in a particular state, screening of modulators of the expression of the gene or the gene product itself is performed.
  • screening for modulators of expression of specific gene(s) is performed. Typically, the expression of only one or a few genes is evaluated. In another embodiment, screens are designed to first find compounds that bind to differentially expressed proteins. These compounds are then evaluated for the ability to modulate differentially expressed activity. Moreover, once initial candidate compounds are identified, variants can be further screened to better evaluate structure activity relationships.
  • a purified or isolated gene product of the invention is generally used.
  • antibodies are generated to a protein of the invention, and immunoassays are run to determine the amount and/or location of protein.
  • cells comprising the cancer proteins are used in the assays.
  • the methods comprise combining a cancer protein of the invention and a candidate compound such as a ligand, and determining the binding of the compound to the cancer protein of the invention.
  • a cancer protein of the invention utilizes the human cancer protein; animal models of human disease of can also be developed and used.
  • other analogous mammalian proteins also can be used as appreciated by those of skill in the art.
  • variant or derivative cancer proteins are used.
  • the cancer protein of the invention, or the ligand is non-diffusibly bound to an insoluble support.
  • the support can, e.g., be one having isolated sample receiving areas (a microtiter plate, an array, etc.).
  • the insoluble supports can be made of any composition to which the compositions can be bound, is readily separated from soluble material, and is otherwise compatible with the overall method of screening.
  • the surface of such supports can be solid or porous and of any convenient shape.
  • suitable insoluble supports include microtiter plates, arrays, membranes and beads. These are typically made of glass, plastic (e.g., polystyrene), polysaccharide, nylon, nitrocellulose, or TeflonTM, etc. Microtiter plates and arrays are especially convenient because a large number of assays can be carried out simultaneously, using small amounts of reagents and samples. The particular manner of binding of the composition to the support is not crucial so long as it is compatible with the reagents and overall methods of the invention, maintains the activity of the composition and is nondiffusable.
  • Preferred methods of binding include the use of antibodies which do not sterically block either the ligand binding site or activation sequence when attaching the protein to the support, direct binding to "sticky" or ionic supports, chemical crosslinking, the synthesis of the protein or agent on the surface, etc. Following binding of the protein or ligand/binding agent to the support, excess unbound material is removed by washing. The sample receiving areas may then be blocked through incubation with bovine serum albumin (BSA), casein or other innocuous protein or other moiety.
  • BSA bovine serum albumin
  • Binding agents include specific antibodies, non-natural binding agents identified in screens of chemical libraries, peptide analogs, etc.
  • assays to identify agents that have a low toxicity for human cells.
  • a wide variety of assays can be used for this purpose, including proliferation assays, cAMP assays, labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, functional assays (phosphorylation assays, etc.) and the like.
  • a determination of binding of the test compound (ligand, binding agent, modulator, etc.) to a cancer protein of the invention can be done in a number of ways.
  • the test compound can be labeled, and binding determined directly, e.g., by attaching all or a portion of the cancer protein of the invention to a solid support, adding a labeled candidate compound (e.g., a fluorescent label), washing off excess reagent, and determining whether the label is present on the solid support.
  • a labeled candidate compound e.g., a fluorescent label
  • washing off excess reagent e.g., a fluorescent label
  • Various blocking and washing steps can be utilized as appropriate.
  • only one of the components is labeled, e.g., a protein of the invention or ligands labeled.
  • more than one component is labeled with different labels, e.g., I 125 , for the proteins and a fluorophor for the compound.
  • Proximity reagents e.g., quenching or energy transfer reagents are also useful.
  • the binding of the "test compound” is determined by competitive binding assay with a "competitor.”
  • the competitor is a binding moiety that binds to the target molecule (e.g., a cancer protein of the invention). Competitors include compounds such as antibodies, peptides, binding partners, ligands, etc. Under certain circumstances, the competitive binding between the test compound and the competitor displaces the test compound.
  • the test compound is labeled. Either the test compound, the competitor, or both, is added to the protein for a time sufficient to allow binding. Incubations are performed at a temperature that facilitates optimal activity, typically between four and 40°C.
  • Incubation periods are typically optimized, e.g., to facilitate rapid high throughput screening; typically between zero and one hour will be sufficient. Excess reagent is generally removed or washed away. The second component is then added, and the presence or absence of the labeled component is followed, to indicate binding.
  • the competitor is added first, followed by the test compound.
  • Displacement of the competitor is an indication that the test compound is binding to the cancer protein and thus is capable of binding to, and potentially modulating, the activity of the cancer protein, in this embodiment, either component can be labeled.
  • the presence of label in the post-test compound wash solution indicates displacement by the test compound.
  • the presence of the label on the support indicates displacement.
  • the test compound is added first, with incubation and washing, followed by the competitor.
  • the absence of binding by the competitor indicates that the test compound binds to the cancer protein with higher affinity than the competitor.
  • the presence of the label on the support, coupled with a lack of competitor binding indicates that the test compound binds to and thus potentially modulates the cancer protein of the invention.
  • the competitive binding methods comprise differential screening to identity agents that are capable of modulating the activity of the cancer proteins of the invention.
  • the methods comprise combining a cancer protein and a competitor in a first sample.
  • a second sample comprises a test compound, the cancer protein, and a competitor.
  • the binding of the competitor is determined for both samples, and a change, or difference in binding between the two samples indicates the presence of an agent capable of binding to the cancer protein and potentially modulating its activity. That is, if the binding of the competitor is different in the second sample relative to the first sample, the agent is capable of binding to the cancer protein.
  • differential screening is used to identify drug candidates that bind to the native cancer protein, but cannot bind to modified cancer proteins.
  • the structure of the cancer protein is modeled and used in rational drug design to synthesize agents that interact with that site, agents which generally do not bind to site-modified proteins.
  • drug candidates that affect the activity of a native cancer protein are also identified by screening drugs for the ability to either enhance or reduce the activity of such proteins.
  • Positive controls and negative controls can be used in the assays.
  • control and test samples are performed in at least triplicate to obtain statistically significant results. Incubation of all samples occurs for a time sufficient to allow for the binding of the agent to the protein. Following incubation, samples are washed free of non-specifically bound material and the amount of bound, generally labeled agent determined. For example, where a radiolabel is employed, the samples can be counted in a scintillation counter to determine the amount of bound compound.
  • a variety of other reagents can be included in the screening assays. These include reagents like salts, neutral proteins, e.g. albumin, detergents, etc. which are used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., can be used. The mixture of components is added in an order that provides for the requisite binding.
  • Polynucleotide modulators of cancer can be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand-binding molecule, as described in WO 91/04753.
  • Suitable ligand-binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors.
  • conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or block entry of the sense or antisense oligonucleotide or its conjugated version into the cell.
  • a polynucleotide modulator of cancer can be introduced into a cell containing the target nucleic acid sequence, e.g., by formation of a polynucleotide-lipid complex, as described in WO 90/10448. It is understood that the use of antisense molecules or knock out and knock in models may also be used in screening assays as discussed above, in addition to methods of treatment.
  • the activity of a cancer-associated protein is down-regulated, or entirely inhibited, by the use of antisense polynucleotide or inhibitory small nuclear RNA (snRNA), i.e., a nucleic acid complementary to, and which can preferably hybridize specifically to, a coding mRNA nucleic acid sequence, e.g., a cancer protein of the invention, mRNA, or a subsequence thereof. Binding of the antisense polynucleotide to the mRNA reduces the translation and/or stability of the mRNA.
  • snRNA inhibitory small nuclear RNA
  • antisense polynucleotides can comprise naturally occurring nucleotides, or synthetic species formed from naturally occurring subunits or their close homologs. Antisense polynucleotides may also have altered sugar moieties or inter-sugar linkages. Exemplary among these are the phosphorothioate and other sulfur containing species which are known for use in the art. Analogs are comprised by this invention so long as they function effectively to hybridize with nucleotides of the invention. See, e.g., Isis Pharmaceuticals, Carlsbad, CA; Sequitor, Inc., Natick, MA.
  • antisense polynucleotides can readily be synthesized using recombinant means, or can be synthesized in vitro. Equipment for such synthesis is sold by several vendors, including Applied Biosystems. The preparation of other oligonucleotides such as phosphorothioates and alkylated derivatives is also well known to those of skill in the art.
  • Antisense molecules as used herein include antisense or sense oligonucleotides.
  • Sense oligonucleotides can, e.g., be employed to block transcription by binding to the anti-sense strand.
  • the antisense and sense oligonucleotide comprise a single stranded nucleic acid sequence (either RNA or DNA) capable of binding to target mRNA (sense) or DNA (antisense) sequences for cancer molecules.
  • Antisense or sense oligonucleotides, according to the present invention comprise a fragment generally at least about 12 nucleotides, preferably from about 12 to 30 nucleotides.
  • ribozymes can be used to target and inhibit transcription of cancer- associated nucleotide sequences.
  • a ribozyme is an RNA molecule that catalytically cleaves other RNA molecules.
  • Different kinds of ribozymes have been described, including group I ribozymes, hammerhead ribozymes, hairpin ribozymes, RNase P, and axhead ribozymes (see, e.g., Castanotto et al., Adv. in Pharmacology 25: 289-317 (1994) for a general review of the properties of different ribozymes).
  • hairpin ribozymes are described, e.g., in Hampel et al., Nucl. Acids Res. 18:299-304 (1990); European Patent Publication No. 0360257; U.S. Patent No. 5,254,678.
  • Methods of preparing are well known to those of skill in the art (see, e.g., WO 94/26877; Ojwang et al., Proc. Natl. Acad. Sci. USA 90:6340-6344 (1993); Yamada et al., Human Gene Therapy 1:39-45 (1994); Leavitt et al., Proc. Natl. Acad Sci. USA 92:699- 703 (1995); Leavitt et al., Human Gene Therapy 5: 1151-120 (1994); and Yamada et al., Virology 205: 121-126 (1994)).
  • a test compound is administered to a population of cancer cells, which have an associated cancer expression profile.
  • administration or “contacting” herein is meant that the modulator is added to the cells in such a manner as to allow the modulator to act upon the cell, whether by uptake and intracellular action, or by action at the cell surface.
  • a nucleic acid encoding a proteinaceous agent i.e., a peptide
  • a viral construct such as an adenoviral or retroviral construct
  • expression of the peptide agent is accomplished, e.g., PCT US97/01019.
  • Regulatable gene therapy systems can also be used.
  • the cells are washed if desired and are allowed to incubate under preferably physiological conditions for some period.
  • the cells are then harvested and a new gene expression profile is generated.
  • cancer tissue is screened for agents that modulate, e.g., induce or suppress, the cancer phenotype.
  • a change in at least one gene, preferably many, of the expression profile indicates that the agent has an effect on cancer activity.
  • altering a biological function or a signaling pathway is indicative of modulator activity.
  • screens are done to assess genes or gene products. That is, having identified a particular differentially expressed gene as important in a particular state, screening of modulators of either the expression of the gene or the gene product itself is performed.
  • Measurements of cancer polypeptide activity, or of the cancer phenotype are performed using a variety of assays. For example, the effects of modulators upon the function of a cancer polypeptide(s) are measured by examining parameters described above. A physiological change that affects activity is used to assess the influence of a test compound on the polypeptides of this invention.
  • a variety of effects can be assesses such as, in the case of a cancer associated with solid tumors, tumor growth, tumor metastasis, neovascularization, hormone release, transcriptional changes to both known and uncharacterized genetic markers (e.g., by Northern blots), changes in cell metabolism such as cell growth or pH changes, and changes in intracellular second messengers such as cGNIP.
  • the invention provides methods for identifying cells containing variant cancer genes, e.g., determining the presence of, all or part, the sequence of at least one endogenous cancer gene in a cell. This is accomplished using any number of sequencing techniques.
  • the invention comprises methods of identifying the cancer genotype of an individual, e.g., determining all or part of the sequence of at least one gene of the invention in the individual. This is generally done in at least one tissue of the individual, e.g., a tissue set forth in Table I, and may include the evaluation of a number of tissues or different samples of the same tissue.
  • the method may include comparing the sequence of the sequenced gene to a known cancer gene, i.e., a wild-type gene to determine the presence of family members, homologies, mutations or variants.
  • the sequence of all or part of the gene can then be compared to the sequence of a known cancer gene to determine if any differences exist. This is done using any number of known homology programs, such as BLAST, Bestftt, etc.
  • the presence of a difference in the sequence between the cancer gene of the patient and the known cancer gene correlates with a disease state or a propensity for a disease state, as outlined herein.
  • the cancer genes are used as probes to determine the number of copies of the cancer gene in the genome.
  • the cancer genes are used as probes to determine the chromosomal localization of the cancer genes.
  • Information such as chromosomal localization finds use in providing a diagnosis or prognosis in particular when chromosomal abnormalities such as translocations, and the like are identified in the cancer gene locus.
  • kits are within the scope of the invention.
  • kits can comprise a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in the method, along with a label or insert comprising instructions for use, such as a use described herein.
  • the container(s) can comprise a probe that is or can be detectably labeled.
  • probe can be an antibody or polynucleotide specific for a protein or a gene or message of the invention, respectively.
  • kits can also have containers containing nucleotide(s) for amplification of the target nucleic acid sequence.
  • Kits can comprise a container comprising a reporter, such as a biotin- binding protein, such as avidin or streptavidin, bound to a reporter molecule, such as an enzymatic, fluorescent, or radioisotope label; such a reporter can be used with, e.g., a nucleic acid or antibody.
  • the kit can include all or part of the amino acid sequences in Figure 2 or Figure 3 or analogs thereof, or a nucleic acid molecule that encodes such amino acid sequences.
  • the kit of the invention will typically comprise the container described above and one or more other containers associated therewith that comprise materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes; carrier, package, container, vial and/or tube labels listing contents and/or instructions for use, and package inserts with instructions for use.
  • a label can be present on or with the container to indicate that the composition is used for a specific therapy or non- therapeutic application, such as a prognostic, prophylactic, diagnostic or laboratory application, and can also indicate directions for either in vivo or in vitro use, such as those described herein. Directions and or other information can also be included on an insert(s) or label(s) which is included with or on the kit.
  • the label can be on or associated with the container.
  • a label a can be on a container when letters, numbers or other characters forming the label are molded or etched into the container itself; a label can be associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert.
  • the label can indicate that the composition is used for diagnosing, treating, prophylaxing or prognosing a condition, such as a neoplasia of a tissue set forth in Table I.
  • kits and “article of manufacture” can be used as synonyms.
  • an article(s) of manufacture containing compositions such as amino acid sequence(s), small molecule(s), nucleic acid sequence(s), and/or antibody(s), e.g., materials useful for the diagnosis, prognosis, prophylaxis and/or treatment of neoplasias of tissues such as those set forth in Table I is provided.
  • the article of manufacture typically comprises at least one container and at least one label. Suitable containers include, for example, bottles, vials, syringes, and test tubes.
  • the containers can be formed from a variety of materials such as glass, metal or plastic.
  • the container can hold amino acid sequence(s), small mol ⁇ cule(s), nucleic acid sequence(s), cell population(s) and/or antibody(s).
  • the container holds a polynucleotide for use in examining the mRNA expression profile of a cell, together with reagents used for this purpose.
  • a container comprises an antibody, binding fragment thereof or specific binding protein for use in evaluating protein expression of) 09P1 D4 in cells and tissues, or for relevant laboratory, prognostic, diagnostic, prophylactic and therapeutic purposes; indications and/or directions for such uses can be included on or with such container, as can reagents and other compositions or tools used for these purposes.
  • a container comprises materials for eliciting a cellular or humoral immune response, together with associated indications and/or directions.
  • a container comprises materials for adoptive immunotherapy, such as cytotoxic T cells (CTL) or helper T cells (HTL), together with associated indications and/or directions; reagents and other compositions or tools used for such purpose can also be included.
  • CTL cytotoxic T cells
  • HTL helper T cells
  • the container can alternatively hold a composition that is effective for treating, diagnosis, prognosing or prophylaxing a condition and can have a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • the active agents in the composition can be an antibody capable of specifically binding 109P1D4 and modulating the function of 109P1D4.
  • the article of manufacture can further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution and/or dextrose solution. It can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, stirrers, needles, syringes, and/or package inserts with indications and/or instructions for use.
  • a pharmaceutically-acceptable buffer such as phosphate-buffered saline, Ringer's solution and/or dextrose solution.
  • It can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, stirrers, needles, syringes, and/or package inserts with indications and/or instructions for use.
  • Example 1 SSH-Generated Isolation of cDNA Fragment of the 109P1 D4 Gene
  • SSH Suppression Subtractive Hybridization
  • the patient cancer and normal tissues were purchased from different sources such as the NDRI (Philadelphia, PA).
  • mRNA for some normal tissues were purchased from different companies such as Clontech, Palo Alto, CA.
  • RNA samples were homogenized in Trizol reagent (Life Technologies, Gibco BRL) using 10 ml/ g tissue to isolate total RNA.
  • Poly A RNA was purified from total RNA using Qiagen's Oiigotex mRNA Mini and Midi kits. Total and mRNA were quantified by spectrophotometric analysis (O.D. 260/280 nm) and analyzed by gel electrophoresis.
  • SSH Suppression Subtractive Hybridization
  • the 109P1D4 SSH sequence was derived from cDNA subtraction of LNCaP stimulated with mibolerone minus LNCaP in the absence of androgen.
  • the SSH DNA sequence ( Figure 1) was identified.
  • the cDNA derived from androgen-deprived LNCaP cells was used as the source of the "driver” cDNA, while the cDNA from androgen-stimulated LNCaP cells was used as the source of the "tester” cDNA.
  • Double stranded cDNAs corresponding to tester and driver cDNAs were synthesized from 2 ⁇ g of poly(A) + RNA isolated from the relevant xenograft tissue, as described above, using CLONTECH's PCR-Select cDNA Subtraction Kit and 1 ⁇ g of oligonucleotide DPNCDN as primer. First- and second- strand synthesis were carried out as described in the Kit's user manual protocol (CLONTECH Protocol No. PT1117-1, Catalog No. K1804-1). The resulting cDNA was digested with Dpn II for 3 hrs at 37°C. Digested cDNA was extracted with phenol/chloroform (1:1) and ethanol precipitated.
  • Tester cDNA was generated by diluting 1 ⁇ l of Dpn II digested cDNA from the relevant tissue source (see above) (400 ng) in 5 ⁇ l of water. The diluted cDNA (2 ⁇ l, 160 ng) was then ligated to 2 ⁇ l of Adaptor 1 and Adaptor 2 (10 ⁇ M), in separate ligation reactions, in a total volume of 10 ⁇ l at 16°C overnight, using 400 ⁇ l of T4 DNA ligase (CLONTECH). Ligation was terminated with 1 ⁇ l of 0.2 M EDTA and heating at 72°C for 5 min.
  • the first hybridization was performed by adding 1.5 ⁇ l (600 ng) of driver cDNA to each of two tubes containing 1.5 ⁇ l (20 ng) Adaptor 1- and Adaptor 2- ligated tester cDNA. In a final volume of 4 ⁇ l, the samples were overlaid with mineral oil, denatured in an MJ Research thermal cycler at 98°C for 1.5 minutes, and then were allowed to hybridize for 8 hrs at 68°C. The two hybridizations were then mixed together with an additional 1 ⁇ l of fresh denatured driver cDNA and were allowed to hybridize overnight at 68°C. The second hybridization was then diluted in 200 ⁇ l of 20 mM Hepes, pH 8.3, 50 mM NaCI, 0.2 mM EDTA, heated at 70°C for 7 min. and stored at -20°C.
  • PCR Amplification, Cloning and Sequencing of Gene Fragments Generated from SSH To amplify gene fragments resulting from SSH reactions, two PCR amplifications were performed. In the primary PCR reaction 1 ⁇ l of the diluted final hybridization mix was added to 1 ⁇ l of PCR primer 1 (10 ⁇ M), 0.5 ⁇ l dNTP mix (10 ⁇ M), 2.5 ⁇ l 10 x reaction buffer (CLONTECH) and 0.5 ⁇ l 50 x Advantage cDNA polymerase Mix (CLONTECH) in a final volume of 25 ⁇ l.
  • PCR 1 was conducted using the following conditions: 75°C for 5 min., 94°C for 25 sec, then 27 cycles of 94°C for 10 sec, 66°C for 30 sec, 72°C for 1.5 min. Five separate primary PCR reactions were performed for each experiment. The products were pooled and diluted 1:10 with water. For the secondary PCR reaction, 1 ⁇ l from the pooled and diluted primary PCR reaction was added to the same reaction mix as used for PCR 1, except that primers NP1 and NP2 (10 ⁇ M) were used instead of PCR primer 1.
  • PCR 2 was performed using 10-12 cycles of 94°C for 10 sec, 68°C for 30 sec, and 72°C for 1.5 minutes. The PCR products were analyzed using 2% agarose gel electrophoresis.
  • PCR products were inserted into pCR2.1 using the T/A vector cloning kit (Invitrogen). Transformed £ coli were subjected to blue/white and ampicillin selection. White colonies were picked and arrayed into 96 well plates and were grown in liquid culture overnight. To identify inserts, PCR amplification was performed on 1 ⁇ l of bacterial culture using the conditions of PCR1 and NP1 and NP2 as primers. PCR products were analyzed using 2% agarose gel electrophoresis.
  • Bacterial clones were stored in 20% glycerol in a 96 well format. Plasmid DNA was prepared, sequenced, and subjected to nucleic acid homology searches of the GenBank, dBest, and NCI-CGAP databases.
  • First strand cDNAs can be generated from 1 ⁇ g of mRNA with oligo (dT)12-18 priming using the Gibco-BRL Superscript Preamplification system. The manufacturer's protocol was used which included an incubation for 50 min at 42°C with reverse transcriptase followed by RNAse H treatment at 37°C for 20 min. After completing the reaction, the volume can be increased to 200 ⁇ l with water prior to normalization. First strand cDNAs from 16 different normal human tissues can be obtained from Clontech.
  • First strand cDNAs (5 ⁇ l) were amplified in a total volume of 50 ⁇ l containing 0.4 ⁇ M primers, 0.2 ⁇ M each dNTPs, 1X PCR buffer (Clontech, 10 mM Tris-HCL, 1.5 mM MgC , 50 mM KCl, pH8.3) and 1X Klentaq DNA polymerase (Clontech).
  • PCR Five ⁇ l of the PCR reaction can be removed at 18, 20, and 22 cycles and used for agarose gel electrophoresis.
  • PCR was performed using an MJ Research thermal cycler under the following conditions: Initial denaturation can be at 94°C for 15 sec, followed by a 18, 20, and 22 cycles of 94°C for 15, 65°C for 2 min, 72°C for 5 sec. A final extension at 72°C was carried out for 2 min.
  • the band intensities of the 283 base pair ⁇ -actin bands from multiple tissues were compared by visual inspection. Dilution factors for the first strand cDNAs were calculated to result in equal ⁇ -actin band intensities in all tissues after 22 cycles of PCR. Three rounds of normalization can be required to achieve equal band intensities in all tissues after 22 cycles of PCR.
  • the 109P1D4 SSH sequence of 192 bp ( Figure 1) exhibited homology to protocadherin 11 (PCDH11), a cell adhesion molecule related to the calcium dependent cadherins.
  • the human cDNA sequence encodes a 1021 amino acid protein with an N- terminal leader sequence and a transmembrane domain.
  • 109P1D4 v.1 of 4603bp was cloned from human prostate cancer xenograft LAPC-9AD cDNA library, revealing an ORF of 1021 amino acids ( Figure 2 and Figure 3).
  • Other variants (Transcript and SNP) of 109P1D4 were also identified and these are listed sequentially in Figure 2 and Figure 3.
  • Chromosomal localization can implicate genes in disease pathogenesis.
  • chromosome mapping approaches include fluorescent in situ hybridization (FISH), human/hamster radiation hybrid (RH) panels (Walter et al., 1994; Nature Genetics 7:22; Research Genetics, Huntsville Al), human-rodent somatic cell hybrid panels such as is available from the Coriell Institute (Camden, New Jersey), and genomic viewers utilizing BLAST homologies to sequenced and mapped genomic clones (NCBI, Bethesda, Maryland).
  • Therapeutic applications for a gene of Figure 2 include use as a small molecule therapy and/or a vaccine (T cell or antibody) target. Diagnostic applications for a gene of Figure 2 include use as a diagnostic marker for local and/or metastasized disease. The restricted expression of a gene of Figure 2 in normal tissues makes it useful as a tumor target for diagnosis and therapy. Expression analysis of a gene of Figure 2 provides information useful for predicting susceptibility to advanced stage disease, rate of progression, and/or tumor aggressiveness.
  • Expression status of a gene of Figure 2 in patient samples, tissue arrays and/or cell lines may be analyzed by: (i) immunohistochemical analysis; (ii) in situ hybridization; (iii) RT-PCR analysis on laser capture micro-dissected samples; (iv) Western blot analysis; and (v) Northern analysis.
  • Figure 14 shows expression of 109P1 D4 in lymphoma cancer patient specimens.
  • RNA was extracted from peripheral blood lymphocytes, cord blood isolated from normal individuals, and from lymphoma patient cancer specimens. Northern blots with 10 ⁇ g of total RNA were probed with the 109P1 D4 sequence. Size standards in kilobases are on the side. Results show expression of 109P1D4 in lymphoma patient specimens but not in the normal blood cells tested.
  • Figure 15 shows expression of 109P1D4 by RT-PCR.
  • First strand cDNA was prepared from vital pool 1 (liver, lung and kidney), vital pool 2 (pancreas, colon and stomach), prostate cancer pool, bladder cancer pool, kidney cancer pool, colon cancer pool, lung cancer pool, ovary cancer pool, breast cancer pool, cancer metastasis pool, and pancreas cancer pool. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 109P1 D4, was performed at 30 cycles of amplification. Results show strong expression of 109P1 D4 in all cancer pools tested. Very low expression was detected in the vital pools.
  • Figure 16 shows expression of 109P1D4 in normal tissues.
  • Two multiple tissue northern blots (Clontech), both with 2 ⁇ g of mRNA/lane, were probed with the 109P1D4 SSH fragment. Size standards in kilobases (kb) are indicated on the side. Results show expression of approximately 10 kb 109P1D4 transcript in ovary. Weak expression was also detected in placenta and brain, but not in the other normal tissues tested.
  • Figure 17 shows expression of 109P1D4 in human cancer cell lines.
  • RNA was extracted from a number of human prostate and bone cancer cell lines. Northern blots with 10 ⁇ g of total RNA/lane were probed with the 109P1 D4 SSH fragment. Size standards in kilobases (kb) are indicated on the side. Results show expression of 109P1D4 in LAPC-9AD, LAPC-9AI, LNCaP prostate cancer cell lines, and in the bone cancer cell lines, SK-ES-1 and RD-ES.
  • FIG. 18A Extensive expression of 109P1 D4 in normal tissues is shown in Figure 18A.
  • Figure 18B shows expression of 109P1D4 in patient cancer specimens.
  • Expression of 109P1D4 was assayed in a panel of human cancers (T) and their respective matched normal tissues (N) on RNA dot blots. Upregulated expression of 109P1D4 in tumors compared to normal tissues was observed in uterus, lung and stomach. The expression detected in normal adjacent tissues (isolated from diseased tissues) but not in normal tissues (isolated from healthy donors) may indicate that these tissues are not fully normal and that 109P1D4 may be expressed in early stage tumors.
  • Figure 19 shows 109P1D4 expression in lung cancer patient specimens.
  • RNA was extracted from normal fung, prostate cancer xenograft LAPC-9AD, bone cancer cell line RD-ES, and lung cancer patient tumors.
  • Northern blots with 10 ⁇ g of total RNA were probed with 109P1D4. Size standards in kilobases are on the side. Results show strong expression of 109P1D4 in lung tumor tissues as well as the RD-ES cell line, but not in normal lung.
  • 109P1D4 is a potential therapeutic target and a diagnostic marker for human cancers.
  • Transcript variants are variants of mature mRNA from the same gene which arise by alternative transcription or alternative splicing.
  • Alternative transcripts are transcripts from the same gene but start transcription at different points.
  • Splice variants are mRNA variants spliced differently from the same transcript, in eukaryotes, when a multi-exon gene is transcribed from genomic DNA, the initial RNA is spliced to produce functional mRNA, which has only exons and is used for translation into an amino acid sequence. Accordingly, a given gene can have zero to many alternative transcripts and each transcript can have zero to many splice variants.
  • Each transcript variant has a unique exon makeup, and can have different coding and/or non-coding (5' or 3' end) portions, from the original transcript.
  • Transcript variants can code for similar or different proteins with the same or a similar function or can encode proteins with different functions, and can be expressed in the same tissue at the same time, or in different tissues at the same time, or in the same tissue at different times, or in different tissues at different times. Proteins encoded by transcript variants can have similar or different cellular or extracellular localizations, e.g., secreted versus intracellular.
  • Transcript variants are identified by a variety of art-accepted methods. For example, alternative transcripts and splice variants are identified by full-length cloning experiment, or by use of full-length transcript and EST sequences. First, all human ESTs were grouped into clusters which show direct or indirect identity with each other. Second, ESTs in the same cluster were further grouped into sub-clusters and assembled into a consensus sequence. The original gene sequence is compared to the consensus sequence(s) or other full-length sequences. Each consensus sequence is a potential splice variant for that gene. Even when a variant is identified that is not a full-length clone, that portion of the variant is very useful for antigen generation and for further cloning of the full-length splice variant, using techniques known in the art.
  • Genomic-based transcript variant identification programs include FgenesH (A. Salamov and V. Solovyev, "Ab initio gene finding in Drosophila genomic DNA,” Genome Research. 2000 April; 10(4):516-22); Grail (URL compbio.ornl.gov/Grail-bin/EmptyGrailForm) and GenScan (URL genes.mit.edu/GENSCAN.html).
  • FgenesH A. Salamov and V. Solovyev, "Ab initio gene finding in Drosophila genomic DNA," Genome Research. 2000 April; 10(4):516-22
  • Grail URL compbio.ornl.gov/Grail-bin/EmptyGrailForm
  • GenScan URL genes.mit.edu/GENSCAN.html
  • PCR-based Validation Wellmann S, ef al, Specific reverse transcription-PCR quantification of vascular endothelial growth factor (VEGF) splice variants by LightCycler technology, Clin Chem. 2001 Apr;47(4):654-60; Jia, H.P., ef al, Discovery of new human beta- defensins using a geno ics-based approach, Gene. 2001 Jan 24; 263(1-2):211-8.
  • VEGF vascular endothelial growth factor
  • genomic regions are modulated in cancers.
  • the alternative transcripts or splice variants of the gene are modulated as well.
  • 109P1D4 has a particular expression profile related to cancer.
  • Alternative transcripts and splice variants of 109P1D4 may also be involved in cancers in the same or different tissues, thus serving as tumor-associated markers/antigens.
  • Table LI The boundaries of the exon in the original transcript, 109P1D4 v.1, were shown in Table LI.
  • transcript variant 109P1D4 v.3 has spliced out 2069-2395 from variant 109P1D4 v.1, as shown in Figure 12.
  • Variant 109P1D4 v.5 added one exon to the 5' and extended 2 bp to the 5' end and 288 bp to the 3' end of variant 109P1D4 v.1.
  • each different combination of exons in spatial order e.g. exon 1 of v.5 and exons 1 and 2 of v.3 or v.4, is a potential splice variant.
  • Tables Lll through LV are set forth on a variant-by-variant basis.
  • Tables Lll(a)-(h) show nucleotide sequence of the transcript variants.
  • Tables Llll(a)-(h) show the alignment of the transcript variants with nucleic acid sequence of 109P1 D4 v.1.
  • Tables LIV(a)-(h) lay out amino acid translation of the transcript variants for the identified reading frame orientation.
  • Tables LV(a)-(h) displays alignments of the amino acid sequence encoded by the splice variants with that of 109P1D4 V.1.
  • a Single Nucleotide Polymorphism is a single base pair variation in a nucleotide sequence at a specific location.
  • SNP Single Nucleotide Polymorphism
  • A/T refers to the specific base pair sequence of one or more locations in the genome of an individual.
  • Haplotype refers to the base pair sequence of more than one location on the same DNA molecule (or the same chromosome in higher organisms), often in the context of one gene or in the context of several tightly linked genes.
  • SNP that occurs on a cDNA is called cSNP. This cSNP may change amino acids of the protein encoded by the gene and thus change the functions of the protein.
  • SNP and/or combinations of alleles have many applications, including diagnosis of inherited diseases, determination of drug reactions and dosage, identification of genes responsible for diseases, and analysis of the genetic relationship between individuals (P. Nowotny, J. M. Kwon and A. M. Goate, " SNP analysis to dissect human traits," Curr. Opin. Neurobiol. 2001 Oct; 11(5):637-641; M. Pirmohamed and B. K. Park, "Genetic susceptibility to adverse drug reactions," Trends Pharmacol. Sci. 2001 Jun; 22(6):298-305; J. H. Riley, C. J.
  • SNP are identified by a variety of art-accepted methods (P. Bean, "The promising voyage of SNP target discovery,” Am. Clin. Lab. 2001 Oct-Nov; 20(9):18-20; K. M. Weiss, "In search of human variation,” Genome Res. 1998 Jul; 8(7):691- 697; M. M. She, “Enabling large-scale pharmacogenetic studies by high-throughput mutation detection and genotyping technologies," Clin. Chem. 2001 Feb; 47(2):164-172).
  • SNP can be identified by sequencing DNA fragments that show polymorphism by gel-based methods such as restriction fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE).
  • RFLP restriction fragment length polymorphism
  • DGGE denaturing gradient gel electrophoresis
  • SNP were identified in the original transcript, 109P4D4 v.1, and its variants (see Figure 2J and Figure 2K). These alleles of the SNP, though shown separately here, can occur in different combinations (haplotypes) and in any one of the transcript variants (such as 109P4D4 v.4 or v.5) that contains the site of the SNP.
  • Transcript variants v.4 and v.5 contained those SNP in the exons shared with variant v.3, and transcript variant v.9 contained all the SNP occurred in variant v.6 (see Figure 10).
  • 109P1D4 and 109P1D4 variants are cloned into any one of a variety of expression vectors known in the art.
  • One or more of the following regions of 109P1D4 variants are expressed: the full length sequence presented in Figures 2 and 3, or any 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from 109P1D4, variants, or analogs thereof.
  • pCRIl To generate 109P1D4 sense and anti-sense RNA probes for RNA in situ investigations, pCRII constructs (Invitrogen, Carlsbad CA) are generated encoding either all or fragments of the 109P1 D4 cDNA.
  • the pCRIl vector has Sp6 and T7 promoters flanking the insert to drive the transcription of 109P1 D4 RNA for use as probes in RNA in situ hybridization experiments. These probes are used to analyze the cell and tissue expression of 109P1 D4 at the RNA level.
  • Transcribed 109P1D4 RNA representing the cDNA amino acid coding region of the 109P1D4 gene is used in in vitro translation systems such as the TnTTM Coupled Reticulolysate System (Promega, Corp., Madison, Wl) to synthesize 109P1D4 protein.
  • TnTTM Coupled Reticulolysate System Promega, Corp., Madison, Wl
  • pGEX Constructs To generate recombinant 109P1D4 proteins in bacteria that are fused to the Glutathione S- transferase (GST) protein, all or parts of the 109P1D4 cDNA protein coding sequence are cloned into the pGEX family of GST-fusion vectors (Amersham Pharmacia Biotech, Piscataway, NJ). These constructs allow controlled expression of recombinant 109P1 D4 protein sequences with GST fused at the amino-terminus and a six histidine epitope (6X His) at the carboxyl-terminus.
  • GST Glutathione S- transferase
  • the GST and 6X His tags permit purification of the recombinant fusion protein from induced bacteria with the appropriate affinity matrix and allow recognition of the fusion protein with anti-GST and anti-His antibodies.
  • the 6X His tag is generated by adding 6 histidine codons to the cloning primer at the 3' end, e.g., of the open reading frame (ORF).
  • a proteolytic cleavage site such as the PreScissionTM recognition site in pGEX-6P-1, may be employed such that it permits cleavage of the GST tag from 109P1D4-related protein.
  • the ampicillin resistance gene and pBR322 origin permits selection and maintenance of the pGEX plasmids in £ coli.
  • pMAL Constructs To generate, in bacteria, recombinant 109P1D4 proteins that are fused to maltose-binding protein (MBP), all or parts of the 109P1D4 cDNA protein coding sequence are fused to the MBP gene by cloning into the pMAL-c2X and pMAL-p2X vectors (New England Biolabs, Beverly, MA). These constructs allow controlled expression of recombinant 109P1D4 protein sequences with MBP fused at the amino-terminus and a 6X His epitope tag at the carboxyl- terminus.
  • MBP maltose-binding protein
  • the MBP and 6X His tags permit purification of the recombinant protein from induced bacteria with the appropriate affinity matrix and allow recognition of the fusion protein with anti-MBP and anti-His antibodies.
  • the 6X His epitope tag is generated by adding 6 histidine codons to the 3' cloning primer.
  • a Factor Xa recognition site permits cleavage of the pMAL tag from 109P1D4.
  • the pMAL-c2X and pMAL-p2X vectors are optimized to express the recombinant protein in the cytoplasm or periplasm respectively. Periplasm expression enhances folding of proteins with disulfide bonds.
  • amino acids 24-419 of 109P1D4 variant 1 was cloned into the pMAL-c2X vector and was used to express the fusion protein.
  • pET Constructs To express 109P1D4 in bacterial cells, all or parts of the 109P1D4 cDNA protein coding sequence are cloned into the pET family of vectors (Novagen, Madison, Wl). These vectors allow tightly controlled expression of recombinant 109P1D4 protein in bacteria with and without fusion to proteins that enhance solubility, such as NusA and thioredoxin (Trx), and epitope tags, such as 6X His and S-Tag TM that aid purification and detection of the recombinant protein.
  • Nrx NusA and thioredoxin
  • epitope tags such as 6X His and S-Tag TM that aid purification and detection of the recombinant protein.
  • constructs are made utilizing pET NusA fusion system 43.1 such that regions of the 109P1D4 protein are expressed as amino-terminal fusions to NusA.
  • amino acids 24-419 and 24-815 were cloned into pET43.1 vector and used to express the fusion protein.
  • Yeast Constructs To express 109P1 D4 in the yeast species Saccharomyces cerevisiae for generation of recombinant protein and functional studies, all or parts of the 109P1 D4 cDNA protein coding sequence are cloned into the pESC family of vectors each of which contain 1 of 4 selectable markers, HIS3, TRP1, LEU2, and URA3 (Stratagene, La Jolla, CA). These vectors allow controlled expression from the same plasmid of up to 2 different genes or cloned sequences containing either FlagTM or Myc epitope tags in the same yeast cell. This system is useful to confirm protein-protein interactions of 109P1 D4.
  • telomere sequence is cloned into the pESP family of vectors. These vectors allow controlled high level of expression of a 109P1D4 protein sequence that is fused at either the amino terminus or at the carboxyl terminus to GST which aids purification of the recombinant protein.
  • a FlagTM epitope tag allows detection of the recombinant protein with anti- FlagTM antibody.
  • Example 8 Production of Recombinant 109P1D4 in Higher Eukaryotic Systems
  • 109P1D4 To express recombinant 109P1D4 in eukaryotic cells, the full or partial length 109P1D4 cDNA sequences were cloned into any one of a variety of expression vectors known in the art. One or more of the following regions of 109P1 D4 were expressed in these constructs, amino acids 1 to 1021 or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from 109P1D4 v.1; amino acids 1 to 1054, 1 to 1347, 1 to 1337, 1 to 1310, 1 to 1037, 1 to 1048, 1 to 1340 of v.2, v.3, v.4, v.5, v.6, v.7, and v.8 respectively; or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from 109P1D4 variants, or analogs thereof.
  • constructs can be transfected into any one of a wide variety of mammalian cells such as 293T cells.
  • Transfected 293T cell lysates can be probed with the anti-109P1D4 polyclonal serum, described herein.
  • pcDNA4/HisMax Constructs To express 109P1D4 in mammalian cells, a 109P1D4 ORF, or portions thereof, of 109P1D4 are cloned into pcDNA4/HisMax Version A (Invitrogen, Carlsbad, CA). Protein expression is driven from the cytomegalovirus (CMV) promoter and the SP16 translational enhancer. The recombinant protein has XpressTM and six histidine (6X His) epitopes fused to the amino-terminus.
  • CMV cytomegalovirus
  • 6X His six histidine
  • the pcDNA4/HisMax vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen.
  • BGH bovine growth hormone
  • the Zeocin resistance gene allows for selection of mammalian cells expressing the protein and the ampicillin resistance gene and ColE1 origin permits selection and maintenance of the plasmid in £ coli.
  • telomere sequence is cloned into pcDNA3.1/MycHis Version A (Invitrogen, Carlsbad, CA). Protein expression is driven from the cytomegalovirus (CMV) promoter. The recombinant proteins have the myc epitope and 6X His epitope fused to the carboxyl-terminus.
  • CMV cytomegalovirus
  • the pcDNA3.1/MycHis vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability, along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen.
  • BGH bovine growth hormone
  • the Neomycin resistance gene can be used, as it allows for selection of mammalian cells expressing the protein and the ampicillin resistance gene and ColE1 origin permits selection and maintenance of the plasmid in £ coli.
  • 109P1D4 v.1 The complete ORF of 109P1D4 v.1 was cloned into the pcDNA3.1/MycHis construct to generate 109P1D4.pcDNA3.1/MycHis.
  • pcDNA3.1/CT-GFP-TOPO Construct To express 109P1D4 in mammalian cells and to allow detection of the recombinant proteins using fluorescence, a 109P1D4 ORF, or portions thereof, with a consensus Kozak translation initiation site are cloned into pcDNA3.1/CT-GFP-TOPO (Invitrogen, CA). Protein expression is driven from the cytomegalovirus (CMV) promoter.
  • CMV cytomegalovirus
  • the recombinant proteins have the Green Fluorescent Protein (GFP) fused to the carboxyl-terminus facilitating non-invasive, in vivo detection and cell biology studies.
  • the pcDNA3.1CT-GFP-TOPO vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen.
  • BGH bovine growth hormone
  • the Neomycin resistance gene allows for selection of mammalian cells that express the protein, and the ampicillin resistance gene and ColE1 origin permits selection and maintenance of the plasmid in £ coli. Additional constructs with an amino- terminal GFP fusion are made in pcDNA3.1/NT-GFP-TOPO spanning the entire length of a 109P1D4 protein.
  • PAPtag A 109P1D4 ORF, or portions thereof, is cloned into pAPtag-5 (GenHunter Corp. Nashville, TN). This construct generates an alkaline phosphatase fusion at the carboxyl-terminus of a 109P1 D4 protein while fusing the lgG ⁇ signal sequence to the amino-terminus. Constructs are also generated in which alkaline phosphatase with an amino- terminal lgG signal sequence is fused to the amino-terminus of a 109P1D4 protein.
  • the resulting recombinant 109P1D4 proteins are optimized for secretion into the media of transfected mammalian cells and can be used to identify proteins such as ligands or receptors that interact with 109P1 D4 proteins.
  • Protein expression is driven from the CMV promoter and the recombinant proteins also contain myc and 6X His epitopes fused at the carboxyl-terminus that facilitates detection and purification.
  • the Zeocin resistance gene present in the vector allows for selection of mammalian cells expressing the recombinant protein and the ampicillin resistance gene permits selection of the plasmid in £ coli.
  • pTag ⁇ A 109P1D4 ORF, or portions thereof, were cloned into pTag-5.
  • This vector is similar to pAPtag but without the alkaline phosphatase fusion.
  • This construct generated 109P1D4 protein with an amino-terminal lgG ⁇ signal sequence and myc and 6X His epitope tags at the carboxyl-terminus that facilitate detection and affinity purification.
  • the resulting recombinant 109P1D4 protein was optimized for secretion into the media of transfected mammalian cells, and was used as immunogen or ligand to identify proteins such as ligands or receptors that interact with the 109P1D4 proteins. Protein expression is driven from the CMV promoter.
  • the Zeocin resistance gene present in the vector allows for selection of mammalian cells expressing the protein, and the ampicillin resistance gene permits selection of the plasmid in £ coli.
  • PsecFc A 109P1D4 ORF, or portions thereof, is also cloned into psecFc.
  • the psecFc vector was assembled by cloning the human immunoglobulin G1 (IgG) Fc (hinge, CH2, CH3 regions) into pSecTag2 (Invitrogen, California). This construct generates an lgG1 Fc fusion at the carboxyl-terminus of the 109P1 D4 proteins, while fusing the IgGK signal sequence to N-terminus.
  • 109P1D4 fusions utilizing the murine lgG1 Fc region are also used.
  • 109P1D4 proteins are optimized for secretion into the media of transfected mammalian cells, and can be used as immunogens or to identify proteins such as ligands or receptors that interact with 109P1 D4 protein. Protein expression is driven from the CMV promoter.
  • the hygromycin resistance gene present in the vector allows for selection of mammalian cells that express the recombinant protein, and the ampicillin resistance gene permits selection of the plasmid in £ coli.
  • pSR ⁇ Constructs To generate mammalian cell lines that express 109P1D4 constitutively, 109P1D4 ORF, or portions thereof, were cloned into pSR ⁇ constructs.
  • Amphotropic and ecotropic retroviruses were generated by transfection of pSR ⁇ constructs into the 293T-10A1 packaging line or co-transfection of pSR ⁇ and a helper plasmid (containing deleted packaging sequences) into the 293 cells, respectively.
  • the retrovirus is used to infect a variety of mammalian cell lines, resulting in the integration of the cloned gene, 109P1D4, into the host cell-lines. Protein expression is driven from a long terminal repeat (LTR).
  • LTR long terminal repeat
  • the Neomycin resistance gene present in the vector allows for selection of mammalian cells that express the protein, and the ampicillin resistance gene and ColE1 origin permit selection and maintenance of the plasmid in £ coli.
  • the retroviral vectors can thereafter be used for infection and generation of various ceil lines using, for example, PC3, NIH 3T3, TsuPrl, 293 or rat-1 cells.
  • Additional pSR ⁇ constructs are made that fuse an epitope tag such as the FLAGTM tag to the carboxyl-terminus of 109P1D4 sequences to allow detection using anti-Flag antibodies.
  • the FLAGTM sequence 5' GAT TAG AAG GAT GAC GAC GAT AAG 3' (SEQ ID NO: 56) is added to cloning primer at the 3' end of the ORF.
  • Additional pSR ⁇ constructs are made to produce both amino-terminal and carboxyl-terminal GFP and myc/6X His fusion proteins of the full- length 109P1D4 proteins.
  • Additional Viral Vectors Additional constructs are made for viral-mediated delivery and expression of 109P1D4.
  • High virus titer leading to high level expression of 109P1D4 is achieved in viral delivery systems such as adenoviral vectors and herpes amplicon vectors.
  • a 109P1D4 coding sequence or fragments thereof are amplified by PCR and subcloned into the AdEasy shuttle vector (Stratagene). Recombination and virus packaging are performed according to the manufacturer's instructions to generate adenoviral vectors.
  • 109P1 D4 coding sequences or fragments thereof are cloned into the HSV-1 vector (Imgenex) to generate herpes viral vectors.
  • the viral vectors are thereafter used for infection of various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.
  • telomeres To control expression of 109P1D4 in mammalian cells, coding sequences of 109P1D4, or portions thereof, are cloned into regulated mammalian expression systems such as the T-Rex System (Invitrogen), the GeneSwitch System (Invitrogen) and the tightly-regulated Ecdysone System (Stratagene). These systems allow the study of the temporal and concentration dependent effects of recombinant 109P1 D4. These vectors are thereafter used to control expression of 109P1D4 in various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.
  • 109P1D4 ORF To generate recombinant 109P1D4 proteins in a baculovirus expression system, 109P1D4 ORF, or portions thereof, are cloned into the baculovirus transfer vector pBlueBac 4.5 (Invitrogen), which provides a His-tag at the N-terminus. Specifically, pBlueBac-109P1D4 is co-transfected with helper plasmid pBac-N-Blue (Invitrogen) into SF9 (Spodoptera frugiperda) insect cells to generate recombinant baculovirus (see Invitrogen instruction manual for details). Baculovirus is then collected from cell supernatant and purified by plaque assay.
  • pBlueBac 4.5 Invitrogen
  • helper plasmid pBac-N-Blue Invitrogen
  • SF9 Spodoptera frugiperda
  • Recombinant 109P1 D4 protein is then generated by infection of HighFive insect cells (Invitrogen) with purified baculovirus.
  • Recombinant 109P1D4 protein can be detected using anti-109P1D4 or anti-His-tag antibody.
  • 109P1D4 protein can be purified and used in various cell-based assays or as immunogen to generate polyclonal and monoclonal antibodies specific for 109P1D4.
  • Figure(s) 5A-I, Figure 6A-I, Figure 7A-1, Figure 8A-I, and Figure 9A-I depict graphically five amino acid profiles of 109P1D4 variants 1 through 9, each assessment available by accessing the ProtScale website located on the World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) on the ExPasy molecular biology server.
  • Hydrophilicity (Figure 5), Hydropathicity ( Figure 6) and Percentage Accessible Residues ( Figure 7) profiles were used to determine stretches of hydrophilic amino acids (i.e., values greater than 0.5 on the Hydrophilicity and Percentage Accessible Residues profile, and values less than 0.5 on the Hydropathicity profile). Such regions are likely to be exposed to the aqueous environment, be present on the surface of the protein, and thus available for immune recognition, such as by antibodies.
  • Average Flexibility ( Figure 8) and Beta-turn ( Figure 9) profiles determine stretches of amino acids (i.e., values greater than 0.5 on the Beta-turn profile and the Average Flexibility profile) that are not constrained in secondary structures such as beta sheets and alpha helices. Such regions are also more likely to be exposed on the protein and thus accessible to immune recognition, such as by antibodies.
  • Antigenic sequences of the 109P1 D4 variant proteins indicated, e.g., by the profiles set forth in Figure 5, Figure 6, Figure 7, Figure 8, and/or Figure 9 are used to prepare immunogens, either peptides or nucleic acids that encode them, to generate therapeutic and diagnostic anti-109P1D4 antibodies.
  • the immunogen can be any 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more than 50 contiguous amino acids, or the corresponding nucleic acids that encode them, from the 109P1D4 protein variants listed in Figures 2 and 3.
  • peptide immunogens of the invention can comprise, a peptide region of at least 5 amino acids of Figures 2 and 3 in any whole number increment that includes an amino acid position having a value greater than 0.5 in the Hydrophilicity profiles of Figure 5; a peptide region of at least 5 amino acids of Figures 2 and 3 in any whole number increment that includes an amino acid position having a value less than 0.5 in the Hydropathicity profile of Figure 6 ; a peptide region of at least 5 amino acids of Figures 2 and 3 in any whole number increment that includes an amino acid position having a value greater than 0.5 in the Percent Accessible Residues profiles of Figure 7; a peptide region of at least 5 amino acids of Figures 2 and 3 in any whole number increment that includes an amino acid position having a value greater than 0.5 in the Average Flexibility profiles on Figure 8 ; and, a peptide region of at least 5 amino acids of Figures 2 and 3 in any whole number increment that includes an amino acid position having a value greater than 0.5 in the Beta-
  • All immunogens of the invention, peptide or nucleic acid can be embodied in human unit dose form, or comprised by a composition that includes a pharmaceutical excipient compatible with human physiology.
  • This analysis for protein variants 1 through 9 are shown in Figure 13A through 131 respectively.
  • the percent of structure for each variant comprised of alpha helix, extended strand, and random coil is also indicated.
  • Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant.
  • the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections.
  • computer algorithms are employed in design of immunogens that, based on amino acid sequence analysis contain characteristics of being antigenic and available for recognition by the immune system of the immunized host (see the Example entitled "Antigenicity Profiles and Secondary Structure").
  • Such regions would be predicted to be hydrophilic, flexible, in beta-turn conformations, and be exposed on the surface of the protein (see, e.g., Figure 5, Figure 6, Figure 7, Figure 8, or Figure 9 for amino acid profiles that indicate such regions of 109P1D4 protein variant 1).
  • recombinant bacterial fusion proteins or peptides containing hydrophilic, flexible, beta-turn regions of 109P1D4 protein variants are used as antigens to generate polyclonal antibodies in New Zealand White rabbits or monoclonal antibodies as described in the example entitled "Generation of 109P1D4 Monoclonal Antibodies (mAbs)".
  • mAbs Monoclonal Antibodies
  • such regions include, but are not limited to, amino acids 22-39, amino acids 67-108, amino acids 200-232, amino acids 454-499, amino acids 525-537, amino acids 640-660, amino acids 834-880, and amino acids 929-942.
  • the immunizing agent it is useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized.
  • immunogenic proteins include, but are not limited to, keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor.
  • KLH keyhole limpet hemocyanin
  • serum albumin serum albumin
  • bovine thyroglobulin bovine thyroglobulin
  • soybean trypsin inhibitor soybean trypsin inhibitor.
  • peptides encoding amino acids 77-90 and amino acids 929-942 of 109P1D4 variant 1 were synthesized, conjugated to KLH, and used to immunize separate rabbits.
  • the immunizing agent may include all or portions of the 109P1 D4 variant proteins, analogs or fusion proteins thereof.
  • the 109P1D4 variant 1 amino acid sequence can be fused using recombinant DNA techniques to any one of a variety of fusion protein partners that are well known in the art, such as glutathione-S-transferase (GST) and HIS tagged fusion proteins.
  • GST glutathione-S-transferase
  • amino acids 24-419 of 109P1D4 variant 1 was fused to NUSa using recombinant techniques and the pET43.1 expression vector, expressed, purified and used to immunize a rabbit.
  • Such fusion proteins are purified from induced bacteria using the appropriate affinity matrix.
  • recombinant bacterial fusion proteins that may be employed include maltose binding protein, LacZ, thioredoxin, NusA, or an immunoglobulin constant region (see the section entitled “Production of 109P1D4 in Prokaryotic Systems” and Current Protocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubul et al. eds., 1995; Linsley, P.S., Brady, W., Urnes, M., Grosmaire, L, Damle, N strictly and Ledbetter, J.(1991) J.Exp. Med. 174, 561-566).
  • mammalian expressed protein antigens are also used. These antigens are expressed from mammalian expression vectors such as the Tag5 and Fc-fusion vectors (see the section entitled "Production of Recombinant 109P1D4 in Eukaryotic Systems"), and retain post-translational modifications such as glycosylations found in native protein.
  • amino acids 24-812 of 109P1 D4 variant 1 was cloned into the Tag5 mammalian secretion vector, and expressed in 293T cells (See Figure 20).
  • the recombinant protein is purified by metal chelate chromatography from tissue culture supernatants of 293T cells stably expressing the recombinant vector.
  • the purified Tag ⁇ 109P1D4 protein is then used as immunogen.
  • adjuvants include, but are not limited to, complete Freund's adjuvant (CFA) and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
  • CFA complete Freund's adjuvant
  • MPL-TDM adjuvant monophosphoryl Lipid A, synthetic trehalose dicorynomycolate
  • rabbits are initially immunized subcutaneously with up to 200 ⁇ g, typically 100-200 ⁇ g, of fusion protein or peptide conjugated to KLH mixed in complete Freund's adjuvant (CFA). Rabbits are then injected subcutaneously every two weeks with up to 200 ⁇ g, typically 100-200 ⁇ g, of the immunogen in incomplete Freund's adjuvant (IFA). Test bleeds are taken approximately 7-10 days following each immunization and used to monitor the titer of the antiserum by ELISA.
  • CFA complete Freund's adjuvant
  • 109P1D4 variant 1 cDNA is cloned into pCDNA 3.1 myc-his expression vector (Invitrogen, see the Example entitied "Production of Recombinant 109P1D4 in Eukaryotic Systems").
  • cell lysates are probed with the anti-109P1D4 serum to determine specific reactivity to denatured 109P1D4 protein using the Western blot technique.
  • Probing with anti-His antibody serves as a positive control for expression of 109P1D4 in the transfected cells (See Figure 21).
  • the immune serum is tested by fluorescence microscopy, flow cytometry and immunoprecipitatio ⁇ against 293T and other recombinant 109P1 D4- expressing cells to determine specific recognition of native protein.
  • Western blot, immunoprecipitation, fluorescent microscopy, and flow cytometric techniques using cells that endogenously express 109P1 D4 are also carried out to test reactivity and specificity.
  • Anti-serum from rabbits immunized with 109P1D4 variant fusion proteins are purified by depletion of antibodies reactive to the fusion partner sequence by passage over an affinity column containing the fusion partner either alone or in the context of an irrelevant fusion protein.
  • antiserum derived from a NUSa- 109P1D4 variant 1 fusion protein is first purified by passage over a column of MBP protein covalently coupled to AffiGel matrix (BioRad, Hercules, Calif.). The antiserum is then affinity purified by passage over a column composed of a NUSa- 109P1D4 fusion protein covalently coupled to Affigel matrix.
  • the serum is then further purified by protein G affinity chromatography to isolate the IgG fraction.
  • Sera from other His-tagged antigens and peptide immunized rabbits as well as fusion partner depleted sera are affinity purified by passage over a column matrix composed of the original protein immunogen or free peptide.
  • therapeutic mAbs to 109P1 D4 variants comprise those that react with epitopes specific for each variant protein or specific to sequences in common between the variants that would disrupt or modulate the biological function of the 109P1 D4 variants, for example those that would disrupt the interaction with ligands and binding partners.
  • Immunogens for generation of such mAbs include those designed to encode or contain the entire 109P1D4 protein variant sequence, regions predicted to contain functional motifs, and regions of the 109P1D4 protein variants predicted to be antigenic from computer analysis of the amino acid sequence (see, e.g., Figure 5, Figure 6, Figure 7, Figure 8, or Figure 9, and the Example entitled "Antigenicity Profiles and Secondary Structure").
  • Immunogens include peptides, recombinant bacterial proteins, and mammalian expressed Tag 5 proteins and human and murine IgG FC fusion proteins.
  • cells engineered to express high levels of a respective 109P1D4 variant such as 293T-109P1D4 variant 1 or 300.19- 109P1D4 variant 1 murine Pre-B cells, are used to immunize mice.
  • mice are first immunized intraperitoneally (IP) with, typically, 10-50 ⁇ g of protein immunogen or 10 7 109P1D4-expressing cells mixed in complete Freund's adjuvant. Mice are then subsequently immunized IP every 2-4 weeks with, typically, 10-50 ⁇ g of protein immunogen or 10 7 cells mixed in incomplete Freund's adjuvant.
  • IP intraperitoneally
  • MPL-TDM adjuvant is used in immunizations.
  • a DNA-based immunization protocol is employed in which a mammalian expression vector encoding a 109P1 D4 variant sequence is used to immunize mice by direct injection of the plasmid DNA.
  • amino acids 24-812 of 109P1 D4 of variant 1 is cloned into the Tag ⁇ mammalian secretion vector and the recombinant vector will then be used as immunogen.
  • the same amino acids are cloned into an Fc-fusion secretion vector in which the 109P1 D4 variant 1 sequence is fused at the amino-terminus to an IgK leader sequence and at the carboxyl- terminus to the coding sequence of the human or murine IgG Fc region.
  • This recombinant vector is then used as immunogen.
  • the plasmid immunization protocols are used in combination with purified proteins expressed from the same vector and with cells expressing the respective 109P1D4 variant.
  • mice may be immunized directly into their footpads.
  • 10-50 ⁇ g of protein immunogen or 10 7 254P1D6B-expressing cells are injected sub-cutaneously into the footpad of each hind leg.
  • the first immunization is given with Titermax (SigmaTM) as an adjuvant and subsequent injections are given with Alum-gel in conjunction with CpG oligonucleotide sequences with the exception of the final injection which is given with PBS.
  • Injections are given twice weekly (every three to four days) for a period of 4 weeks and mice are sacrificed 3-4 days after the final injection, at which point lymph nodes immediately draining from the footpad are harvested and the B-cells are collected for use as antibody producing fusion partners.
  • test bleeds are taken 7-10 days following an injection to monitor titer and specificity of the immune response. Once appropriate reactivity and specificity is obtained as determined by ELISA, Western blotting, immunoprecipitation, fluorescence microscopy, and flow cytom ⁇ fric analyses, fusion and hybridoma generation is then carried out with established procedures well known in the art (see, e.g., Harlow and Lane, 1988).
  • a Tag ⁇ antigen of variant 1 encoding amino acids 14-812 is expressed in 293T cells and purified from conditioned media.
  • Balb C mice are initially immunized intraperitoneally with 26 ⁇ g of the Tag ⁇ 109P1 D4 variant 1 protein mixed in complete Freund's adjuvant. Mice are subsequently immunized every two weeks with 25 ⁇ g of the antigen mixed in incomplete Freund's adjuvant for a total of three immunizations.
  • ELISA using the Tag ⁇ antigen determines the titer of serum from immunized mice.
  • Reactivity and specificity of serum to full length 109P1D4 variant 1 protein is monitored by Western blotting, immunoprecipitation and flow cytometry using 293T cells transfected with an expression vector encoding the 109P1D4 variant 1 cDNA (see e.g., the Example entitled "Production of Recombinant 109P1D4 in Higher Eukaryotic Systems" and Figure 21).
  • Other recombinant 109P1D4 variant 1-expressing cells or cells endogenously expressing 109P1D4 variant 1 are also used. Mice showing the strongest reactivity are rested and given a final injection of antigen in PBS and then sacrificed four days later.
  • mice The spleens of the sacrificed mice are harvested and fused to SPO/2 myeloma cells using standard procedures (Harlow and Lane, 1988). Supernatants from HAT selected growth wells are screened by ELISA, Western blot, immunoprecipitation, fluorescent microscopy, and flow cytometry to identify 109P1D4 specific antibody-producing clones.
  • immunogens are designed to encode sequences unique for each variant.
  • an antigenic peptide composed of amino acids 1-29 of 109P1D4 variant 2 is coupled to KLH to derive monoclonal antibodies specific to 109P1D4 variant 2.
  • an antigenic peptide comprised of amino acids 1 -23 of 109P1 D4 variant 6 is coupled to KLH and used as immunogen to derive varaiant 6 specific MAbs.
  • a GST-fusion protein encoding amino acids 1001-1347 of variant 3 is used as immunogen to generate antibodies that would recognize variants 3, 4, 5, and 8, and distinguish them from variants 1, 2, 6, 7and 9. Hybridoma supernatants are then screened on the respective antigen and then further screened on cells expressing the specific variant and cross-screened on cells expressing the other variants to derive variant- specific monoclonal antibodies.
  • the binding affinity of 109P1D4 variant specific monoclonal antibodies are determined using standard technologies. Affinity measurements quantify the strength of antibody to epitope binding and are used to help define which 109P1 D4 variant monoclonal antibodies preferred for diagnostic or therapeutic use, as appreciated by one of skill in the art.
  • the BIAcore system (Uppsala, Sweden) is a preferred method for determining binding affinity.
  • the BIAcore system uses surface plasmon resonance (SPR, Welford K. 1991, Opt. Quant. Elect. 23:1; Morton and Myszka, 1998, Methods in Enzymology 296: 268) to monitor biomolecular interactions in real time. BIAcore analysis conveniently generates association rate constants, dissociation rate constants, equilibrium dissociation constants, and affinity constants. Alternatively, equilibrium binding analysis of MAbs on 109P1D4-expressing cells can be used to determine affinity.
  • HLA class I and class II binding assays using purified HLA molecules are performed in accordance with disclosed protocols (e.g., PCT publications WO 94/20127 and WO 94/03205; Sidney ef al, Current Protocols in Immunology 18.3.1 (1998); Sidney, ef al, J. Immunol. 154:247 (1995); Sette, ef al, Mol. Immunol. 31:813 (1994)). Briefly, purified MHC molecules (5 to 500 nM) are incubated with various unlabeled peptide inhibitors and 1-10 nM 125 l-radiolabeled probe peptides as described.
  • MHC-peptide complexes are separated from free peptide by gel filtration and the fraction of peptide bound is determined.
  • each MHC preparation is titered in the presence of fixed amounts of radiolabeled peptides to determine the concentration of HLA molecules necessary to bind 10- 20% of the total radioactivity. All subsequent inhibition and direct binding assays are performed using these HLA concentrations.
  • Binding assays as outlined above may be used to analyze HLA supermotif and/or HLA motif-bearing peptides (see Table IV).
  • HLA vaccine compositions of the invention can include multiple epitopes.
  • the multiple epitopes can comprise multiple HLA supermotifs or motifs to achieve broad population coverage. This example illustrates the identification and confirmation of supermotif- and motif-bearing epitopes for the inclusion in such a vaccine composition. Calculation of population coverage is performed using the strategy described below.
  • Identified A2-, A3-, and DR-supermotif sequences are scored using polynomial algorithms to predict their capacity to bind to specific HLA-Class I or Class II molecules. These polynomial algorithms account for the impact of different amino acids at different positions, and are essentially based on the premise that the overall affinity (or ⁇ G) of peptide-HLA molecule interactions can be approximated as a linear polynomial function of the type: where ay/ is a coefficient which represents the effect of the presence of a given amino acid (/) at a given position () along the sequence of a peptide of n amino acids.

Abstract

A novel gene 109P1D4 and its encoded protein, and variants thereof, are described wherein 109P1D4 exhibits tissue specific expression in normal adult tissue, and is aberrantly expressed in the cancers listed in Table i. Consequently, 109P1D4 provides a diagnostic, prognostic, prophylactic and/or therapeutic target for cancer. The 109P1D4 gene or fragment thereof, or its encoded protein, or variants thereof, or a fragment thereof, can be used to elicit a humoral or cellular immune response; antibodies or T cells reactive with 109P1D4 can be used in active or passive immunization.

Description

NUCLEIC ACIDS AND CORRESPONDING PROTEINS ENTITLED 109P1D4
USEFUL IN TREATMENT AND DETECTION OF CANCER
STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH
Not applicable.
FIELD OF THE INVENTION
The invention described herein relates to genes and their encoded proteins, termed 109P1D4 and variants thereof, expressed in certain cancers, and to diagnostic and therapeutic methods and compositions useful in the management of cancers that express 109P1D4.
BACKGROUND OF THE INVENTION
Cancer is the second leading cause of human death next to coronary disease. Worldwide, millions of people die from cancer every year. In the United States alone, as reported by the American Cancer Society, cancer causes the death of well over a half-million people annually, with over 1.2 million new cases diagnosed per year. While deaths from heart disease have been declining significantly, those resulting from cancer generally are on the rise. In the early part of the next century, cancer is predicted to become the leading cause of death.
Worldwide, several cancers stand out as the leading killers. In particular, carcinomas of the lung, prostate, breast, colon, pancreas, and ovary represent the primary causes of cancer death. These and virtually all other carcinomas share a common lethal feature. With very few exceptions, metastatic disease from a carcinoma is fatal. Moreover, even for those cancer patients who initially survive their primary cancers, common experience has shown that their lives are dramatically altered. Many cancer patients experience strong anxieties driven by the awareness of the potential for recurrence or treatment failure. Many cancer patients experience physical debilitations following treatment. Furthermore, many cancer patients experience a recurrence.
Worldwide, prostate cancer is the fourth most prevalent cancer in men. In North America and Northern Europe, it is by far the most common cancer in males and is the second leading cause of cancer death in men. In the United States alone, well over 30,000 men die annually of this disease - second only to lung cancer. Despite the magnitude of these figures, there is still no effective treatment for metastatic prostate cancer. Surgical prostatectomy, radiation therapy, hormone ablation therapy, surgical castration and chemotherapy continue to be the main treatment modalities. Unfortunately, these treatments are ineffective for many and are often associated with undesirable consequences.
On the diagnostic front, the lack of a prostate tumor marker that can accurately detect early-stage, localized tumors remains a significant limitation in the diagnosis and management of this disease. Although the serum prostate specific antigen (PSA) assay has been a very useful tool, however its specificity and general utility is widely regarded as lacking in several important respects. Progress in identifying additional specific markers for prostate cancer has been improved by the generation of prostate cancer xenografts that can recapitulate different stages of the disease in mice. The LAPC (Los Angeles Prostate Cancer) xenografts are prostate cancer xenografts that have survived passage in severe combined immune deficient (SCID) mice and have exhibited the capacity to mimic the transition from androgen dependence to androgen independence (Klein ef al., 1997, Nat. Med. 3:402). More recently identified prostate cancer markers include PCTA-1 (Su ef al., 1996, Proc. Natl. Acad. Sci. USA 93: 7252), prostate-specific membrane (PSM) antigen (Pinto et al., Clin Cancer Res 1996 Sep 2 (9): 1445- 51), STEAP (Hubert, ef al., Proc Natl Acad Sci U S A. 1999 Dec 7; 96(25): 14523-8) and prostate stem cell antigen (PSCA) (Reiter ef al., 1998, Proc. Natl. Acad. Sci. USA 95: 1735).
While previously identified markers such as PSA, PSM, PCTA and PSCA have facilitated efforts to diagnose and treat prostate cancer, there is need for the identification of additional markers and therapeutic targets for prostate and related cancers in order to further improve diagnosis and therapy.
Renal cell carcinoma (RCC) accounts for approximately 3 percent of adult malignancies. Once adenomas reach a diameter of 2 to 3 cm, malignant potential exists. In the adult, the two principal malignant renal tumors are renal cell adenocarcinoma and transitional cell carcinoma of the renal pelvis or ureter. The incidence of renal cell adenocarcinoma is estimated at more than 29,000 cases in the United States, and more than 11,600 patients died of this disease in 1998. Transitional cell carcinoma is less frequent, with an incidence of approximately 500 cases per year in the United States.
Surgery has been the primary therapy for renal cell adenocarcinoma for many decades. Until recently, metastatic disease has been refractory to any systemic therapy. With recent developments in systemic therapies, particularly immunotherapies, metastatic renal cell carcinoma may be approached aggressively in appropriate patients with a possibility of durable responses. Nevertheless, there is a remaining need for effective therapies for these patients.
Of all new cases of cancer in the United States, bladder cancer represents approximately 5 percent in men (fifth most common neoplasm) and 3 percent in women (eighth most common neoplasm). The incidence is increasing slowly, concurrent with an increasing older population. In 1998, there was an estimated 54,500 cases, including 39,500 in men and 15,000 in women. The age-adjusted incidence in the United States is 32 per 100,000 for men and eight per 100,000 in women. The historic male/female ratio of 3:1 may be decreasing related to smoking patterns in women. There were an estimated 11 ,000 deaths from bladder cancer in 1998 (7,800 in men and 3,900 in women). Bladder cancer incidence and mortality strongly increase with age and will be an increasing problem as the population becomes more elderly.
Most bladder cancers recur in the bladder. Bladder cancer is managed with a combination of transurethral resection of the bladder (TUR) and intravesical chemotherapy or immunotherapy. The multifocal and recurrent nature of bladder cancer points out the limitations of TUR. Most muscle-invasive cancers are not cured by TUR alone. Radical cystectomy and urinary diversion is the most effective means to eliminate the cancer but carry an undeniable impact on urinary and sexual function. There continues to be a significant need for treatment modalities that are beneficial for bladder cancer patients.
An estimated 130,200 cases of colorectal cancer occurred in 2000 in the United States, including 93,800 cases of colon cancer and 36,400 of rectal cancer. Colorectal cancers are the third most common cancers in men and women. Incidence rates declined significantly during 1992-1996 (-2.1% per year). Research suggests that these declines have been due to increased screening and polyp removal, preventing progression of polyps to invasive cancers. There were an estimated 56,300 deaths (47,700 from colon cancer, 8,600 from rectal cancer) in 2000, accounting for about 11 % of all U.S. cancer deaths.
At present, surgery is the most common form of therapy for colorectal cancer, and for cancers that have not spread, it is frequently curative. Chemotherapy, or chemotherapy plus radiation, is given before or after surgery to most patients whose cancer has deeply perforated the bowel wall or has spread to the lymph nodes. A permanent colostomy (creation of an abdominal opening for elimination of body wastes) is occasionally needed for colon cancer and is infrequently required for rectal cancer. There continues to be a need for effective diagnostic and treatment modalities for colorectal cancer.
There were an estimated 164,100 new cases of lung and bronchial cancer in 2000, accounting for 14% of all U.S. cancer diagnoses. The incidence rate of lung and bronchial cancer is declining significantly in men, from a high of 86.5 per 100,000 in 1984 to 70.0 in 1996. In the 1990s, the rate of increase among women began to slow. In 1996, the incidence rate in women was 42.3 per 100,000.
Lung and bronchial cancer caused an estimated 156,900 deaths in 2000, accounting for 28% of all cancer deaths. During 1992-1996, mortality from lung cancer declined significantly among men (-1.7% per year) while rates for women were still significantly increasing (0.9% per year). Since 1987, more women have died each year of lung cancer than breast cancer, which, for over 40 years, was the major cause of cancer death in women. Decreasing lung cancer incidence and mortality rates most likely resulted from decreased smoking rates over the previous 30 years; however, decreasing smoking patterns among women lag behind those of men. Of concern, although the declines in adult tobacco use have slowed, tobacco use in youth is increasing again.
Treatment options for lung and bronchial cancer are determined by the type and stage of the cancer and include surgery, radiation therapy, and chemotherapy. For many localized cancers, surgery is usually the treatment of choice. Because the disease has usually spread by the time it is discovered, radiation therapy and chemotherapy are often needed in combination with surgery. Chemotherapy alone or combined with radiation is the treatment of choice for small cell lung cancer; on this regimen, a large percentage of patients experience remission, which in some cases is long lasting. There is however, an ongoing need for effective treatment and diagnostic approaches for lung and bronchial cancers.
An estimated 182,800 new invasive cases of breast cancer were expected to occur among women in the United States during 2000. Additionally, about 1 ,400 new cases of breast cancer were expected to be diagnosed in men in 2000. After increasing about 4% per year in the 1980s, breast cancer incidence rates in women have leveled off in the 1990s to about 110.6 cases per 100,000.
In the U.S. alone, there were an estimated 41,200 deaths (40,800 women, 400 men) in 2000 due to breast cancer. Breast cancer ranks second among cancer deaths in women. According to the most recent data, mortality rates declined significantly during 1992-1996 with the largest decreases in younger women, both white and black. These decreases were probably the result of earlier detection and improved treatment.
Taking into account the medical circumstances and the patient's preferences, treatment of breast cancer may involve lumpectomy (local removal of the tumor) and removal of the lymph nodes under the arm; mastectomy (surgical removal of the breast) and removal of the lymph nodes under the arm; radiation therapy; chemotherapy; or hormone therapy. Often, two or more methods are used in combination. Numerous studies have shown that, for early stage disease, long-term survival rates after lumpectomy plus radiotherapy are similar to survival rates after modified radical mastectomy. Significant advances in reconstruction techniques provide several options for breast reconstruction after mastectomy. Recently, such reconstruction has been done at the same time as the mastectomy.
Local excision of ductal carcinoma in situ (DCIS) with adequate amounts of surrounding normal breast tissue may prevent the local recurrence of the DCIS. Radiation to the breast and/or tamoxifen may reduce the chance of DCIS occurring in the remaining breast tissue. This is important because DCIS, if left untreated, may develop into invasive breast cancer. Nevertheless, there are serious side effects or sequelae to these treatments. There is, therefore, a need for efficacious breast cancer treatments. There were an estimated 23,100 new cases of ovarian cancer in the United States in 2000. It accounts for 4% of all cancers among women and ranks second among gynecologic cancers. During 1992-1996, ovarian cancer incidence rates were significantly declining. Consequent to ovarian cancer, there were an estimated 14,000 deaths in 2000. Ovarian cancer causes more deaths than any other cancer of the female reproductive system.
Surgery, radiation therapy, and chemotherapy are treatment options for ovarian cancer. Surgery usually includes the removal of one or both ovaries, the fallopian tubes (salpingo-oophorectomy), and the uterus (hysterectomy). In some very early tumors, only the involved ovary will be removed, especially in young women who wish to have children. In advanced disease, an attempt is made to remove all intra-abdominal disease to enhance the effect of chemotherapy. There continues to be an important need for effective treatment options for ovarian cancer.
There were an estimated 28,300 new cases of pancreatic cancer in the United States in 2000. Over the past 20 years, rates of pancreatic cancer have declined in men. Rates among women have remained approximately constant but may be beginning to decline. Pancreatic cancer caused an estimated 28,200 deaths in 2000 in the United States. Over the past 20 years, there has been a slight but significant decrease in mortality rates among men (about -0.9% per year) while rates have increased slightly among women.
Surgery, radiation therapy, and chemotherapy are treatment options for pancreatic cancer. These treatment options can extend survival and/or relieve symptoms in many patients but are not likely to produce a cure for most. There is a significant need for additional therapeutic and diagnostic options for pancreatic cancer.
SUMMARY OF THE INVENTION
The present invention relates to a gene, designated 109P1D4, that has now been found to be over-expressed in the cancer(s) listed in Table I. Northern blot expression analysis of 109P1D4 gene expression in normal tissues shows a restricted expression pattern in adult tissues. The nucleotide (Figure 2) and amino acid (Figure 2, and Figure 3) sequences of 109P1D4 are provided. The tissue-related profile of 109P1D4 in normal adult tissues, combined with the over-expression observed in the tissues listed in Table I, shows that 109P1 D4 is aberrantly over-expressed in at least some cancers, and thus serves as a useful diagnostic, prophylactic, prognostic, and/or therapeutic target for cancers of the tissue(s) such as those listed in Table I.
The invention provides polynucleotides corresponding or complementary to all or part of the 109P1D4 genes, mRNAs, and/or coding sequences, preferably in isolated form, including polynucleotides encoding 109P1D4-related proteins and fragments of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25 contiguous amino acids; at least 30, 35, 40, 5, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100 or more than 100 contiguous amino acids of a 109P1D4-related protein, as well as the peptides/proteins themselves; DNA, RNA, DNA/RNA hybrids, and related molecules, polynucleotides or oligonucleotides complementary or having at least a 90% homology to the 109P1 D4 genes or mRNA sequences or parts thereof, and polynucleotides or oligonucleotides that hybridize to the 109P1D4 genes, mRNAs, or to 109P1 D4-encoding polynucleotides. Also provided are means for isolating cDNAs and the genes encoding 109P1 D4. Recombinant DNA molecules containing 109P1 D4 polynucleotides, cells transformed or transduced with such molecules, and host-vector systems for the expression of 109P1 D4 gene products are also provided. The invention further provides antibodies that bind to 109P1D4 proteins and polypeptide fragments thereof, including polyclonal and monoclonal antibodies, murine and other mammalian antibodies, chimeric antibodies, humanized and fully human antibodies, and antibodies labeled with a detectable marker or therapeutic agent. In certain embodiments, there is a proviso that the entire nucleic acid sequence of Figure 2 is not encoded and/or the entire amino acid sequence of Figure 2 is not prepared. In certain embodiments, the entire nucleic acid sequence of Figure 2 is encoded and/or the entire amino acid sequence of Figure 2 is prepared, either of which are in respective human unit dose forms. The invention further provides methods for detecting the presence and status of 109P1 D4 polynucleotides and proteins in various biological samples, as well as methods for identifying cells that express 109P1 D4. A typical embodiment of this invention provides methods for monitoring 109P1 D4 gene products in a tissue or hematology sample having or suspected of having some form of growth dysregulation such as cancer.
The invention further provides various immunogenic or therapeutic compositions and strategies for treating cancers that express 109P1D4 such as cancers of tissues listed in Table I, including therapies aimed at inhibiting the transcription, translation, processing or function of 109P1 D4 as well as cancer vaccines. In one aspect, the invention provides compositions, and methods comprising them, for treating a cancer that expresses 109P1 D4 in a human subject wherein the composition comprises a carrier suitable for human use and a human unit dose of one or more than one agent that inhibits the production or function of 109P1 D4. Preferably, the carrier is a uniquely human carrier. In another aspect of the invention, the agent is a moiety that is immunoreactive with 109P1 D4 protein. Non-limiting examples of such moieties include, but are not limited to, antibodies (such as single chain, monoclonal, polyclonal, humanized, chimeric, or human antibodies), functional equivalents thereof (whether naturally occurring or synthetic), and combinations thereof. The antibodies can be conjugated to a diagnostic or therapeutic moiety. In another aspect, the agent is a small molecule as defined herein.
In another aspect, the agent comprises one or more than one peptide which comprises a cytotoxic T lymphocyte (CTL) epitope that binds an HLA class I molecule in a human to elicit a CTL response to 109P1 D4 and/or one or more than one peptide which comprises a helper T lymphocyte (HTL) epitope which binds an HLA class II molecule in a human to elicit an HTL response. The peptides of the invention may be on the same or on one or more separate polypeptide molecules. In a further aspect of the invention, the agent comprises one or more than one nucleic acid molecule that expresses one or more than one of the CTL or HTL response stimulating peptides as described above. In yet another aspect of the invention, the one or more than one nucleic acid molecule may express a moiety that is immunologically reactive with 109P1D4 as described above. The one or more than one nucleic acid molecule may also be, or encodes, a molecule that inhibits production of 109P1D4. Non-limiting examples of such molecules include, but are not limited to, those complementary to a nucleotide sequence essential for production of 109P1 D4 (e.g. antisense sequences or molecules that form a triple helix with a nucleotide double helix essential for 109P1D4 production) or a ribozyme effective to lyse 109P1D4 mRNA.
Note that to determine the starting position of any peptide set forth in Tables VIII-XXI and XXII to XLIX (collectively HLA Peptide Tables) respective to its parental protein, e.g., variant 1, variant 2, etc., reference is made to three factors: the particular variant, the length of the peptide in an HLA Peptide Table, and the Search Peptides in Table VII. Generally, a unique Search Peptide is used to obtain HLA peptides of a particular for a particular variant. The position of each Search Peptide relative to its respective parent molecule is listed in Table VII. Accordingly, if a Search Peptide begins at position "X", one must add the value "X - 1 " to each position in Tables VIII-XXI and XXII to XLIX to obtain the actual position of the HLA peptides in their parental molecule. For example, if a particular Search Peptide begins at position 150 of its parental molecule, one must add 150 - 1, i.e., 149 to each HLA peptide amino acid position to calculate the position of that amino acid in the parent molecule.
One embodiment of the invention comprises an HLA peptide, that occurs at least twice in Tables VIII-XXI and XXII to XLIX collectively, or an oligonucleotide that encodes the HLA peptide. Another embodiment of the invention comprises an HLA peptide that occurs at least once in Tables VIII-XXI and at least once in tables XXII to XLIX, or an oligonucleotide that encodes the HLA peptide.
Another embodiment of the invention is antibody epitopes, which comprise a peptide regions, or an oligonucleotide encoding the peptide region, that has one two, three, four, or five of the following characteristics: i) a peptide region of at least 5 amino acids of a particular peptide of Figure 3, in any whole number increment up to the full length of that protein in Figure 3, that includes an amino acid position having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Hydrophilicity profile of Figure 5; ii) a peptide region of at least 5 amino acids of a particular peptide of Figure 3, in any whole number increment up to the full length of that protein in Figure 3, that includes an amino acid position having a value equal to or less than 0.5, 0.4, 0.3, 0.2, 0.1, or having a value equal to 0.0, in the Hydropathicity profile of Figure 6; iii) a peptide region of at least 5 amino acids of a particular peptide of Figure 3, in any whole number increment up to the full length of that protein in Figure 3, that includes an amino acid position having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Percent Accessible Residues profile of Figure 7; iv) a peptide region of at least 5 amino acids of a particular peptide of Figure 3, in any whole number increment up to the full length of that protein in Figure 3, that includes an amino acid position having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Average Flexibility profile of Figure 8; or v) a peptide region of at least 5 amino acids of a particular peptide of Figure 3, in any whole number increment up to the full length of that protein in Figure 3, that includes an amino acid position having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Beta-turn profile of Figure 9.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1. The 109P1D4 SSH sequence of 192 nucleotides.
Figure 2. A) The cDNA and amino acid sequence of 109P1 D4 variant 1 (also called "109P1D4 v.1" or "109P1D4 variant 1") is shown in Figure 2A, The start methionine is underlined. The open reading frame extends from nucleic acid
846-3911 including the stop codon.
B) The cDNA and amino acid sequence of 109P1D4 variant 2 (also called "109P1D4 v.2") is shown in Figure 2B. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 503-3667 including the stop codon.
C) The cDNA and amino acid sequence of 109P1 D4 variant 3 (also called "109P1 D4 v.3") is shown in Figure 2C. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 846-4889 including the stop codon.
D) The cDNA and amino acid sequence of 109P1D4 variant 4 (also called "109P1D4 v.4") is shown in Figure 2D. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 846-4859 including the stop codon.
E) The cDNA and amino acid sequence of 109P1D4 variant 5 (also called "109P1D4 v.5") is shown in Figure 2E. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 846-4778 including the stop codon.
F) The cDNA and amino acid sequence of 109P1D4 variant 6 (also called "109P1D4 v.6") is shown in Figure 2F. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 614-3727 including the stop codon.
G) The cDNA and amino acid sequence of 109P1D4 variant 7 (also called "109P1D4 v.7") is shown in Figure 2G. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 735-3881 including the stop codon.
H) The cDNA and amino acid sequence of 109P1D4 variant 8 (also called "109P1D4 v.8") is shown in Figure 2H. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 735-4757 including the stop codon. I) The cDNA and amino acid sequence of 109P1D4 variant 9 (also called "109P1D4 v.9") is shown in Figure 21. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 514-3627 including the stop codon.
J) 109P1 D4 v.1, v.2 and v.3 SNP variants. Though these SNP variants are shown separately, they can also occur in any combinations and in any of the transcript variants listed above.
K) 109P1 D4 v.6, v.7 and v.S SNP variants. Though these SNP variants are shown separately, they can also occur in ai combinations and in any of the transcript variants listed above.
Figure 3.
A) The amino acid sequence of 109P1D4 v.1 is shown in Figure 3A; it has 1021 amino acids.
B) The amino acid sequence of 109P1D4 v.2 is shown in Figure 3B; it has 1054 amino acids.
C) The amino acid sequence of 109P1D4 v.S is shown in Figure 3C; it has 1347 amino acids.
D) The amino acid sequence of 109P1D4 v.4 is shown in Figure 3D; it has 1337 amino acids.
E) The amino acid sequence of 109P1D4 v.5 is shown in Figure 3E; it has 1310 amino acids.
F) The amino acid sequence of 109P1D4 v.6 is shown in Figure 3F; it has 1037 amino acids.
G) The amino acid sequence of 109P1D4 v.7 is shown in Figure 3G; it has 1048 amino acids. H) The amino acid sequence of 109P1D4 v.8 is shown in Figure 3H; it has 1340 amino acids. I) The amino acid sequence of 109P1D4 v.9 is shown in Figure 31; it has 1037 amino acids.
As used herein, a reference to 109P1D4 includes all variants thereof, including those shown in Figures 2, 3, 10, 11, and 12 unless the context clearly indicates otherwise.
Figure 4. Alignment of 109P1 D4 v.1 Protein with protocadherin-11. ,
Figure 5. Hydrophilicity amino acid profile of 109P1D4 v.1 -v.9 determined by computer algorithm sequence analysis using the method of Hopp and Woods (Hopp T.P., Woods K.R., 1981. Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828) accessed on the Protscale website located on the World Wide Web at (expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.
Figure 6. Hydropathicity amino acid profile of 109P1D4 v.l-v.9 determined by computer algorithm sequence analysis using the method of Kyte and Doolittle (Kyte J, Doolittle R.F., 1982. J. Mol. Biol. 157:105-132) accessed on the ProtScale website located on the World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.
Figure 7. Percent accessible residues amino acid profile of 109P1 D4 v.l-v.9 determined by computer algorithm sequence analysis using the method of Janin (Janin J., 1979 Nature 277:491-492) accessed on the ProtScale website located on the World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.
Figure 8. Average flexibility amino acid profile of 109P1D4 v.l-v.9 determined by computer algorithm sequence analysis using the method of Bhaskaran and Ponnuswamy (Bhaskaran R., and Ponnuswamy P.K., 1988. Int. J. Pept. Protein Res. 32:242-255) accessed on the ProtScale website located on the World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.
Figure 9. Beta-turn amino acid profile of 109P1 D4 v.1 -v.9 determined by computer algorithm sequence analysis using the method of Deleage and Roux (Deleage, G., Roux B. 1987 Protein Engineering 1 :289-294) accessed on the ProtScale website located on the World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.
Figure 10. Structure of transcript variants of 109P1D4. Variants 109P1D4 v.2 through v.9 were transcript variants of v.1. Variant v.2 shared middle portion of v.1 sequence (the 3' portion of exon 1 and 5' portion of exon 2). Variant v.6 was similar to v.2 but added an extra exon between exons 1 and 2 of v.2. V.3 shared exon 1 and 5' portion of exon 2 with v.1 with five additional exons downstream. Compared with v.3, v.4 deleted exon 4 of v.3 while v.5 deleted exons 3 and 4 of v.3. Variant v.5 lacked exons 3 and 4. This gene (called PCD11) is located in sex chromosomes X and Y. Ends of exons in the transcripts are marked above the boxes. Potential exons of this gene are shown in order as on the human genome. Poly A tails and single nucleotide differences are not shown in the figure. Lengths of introns and exons are not proportional.
Figure 11. Schematic alignment of protein variants of 109P1D4. Variants 109P1D4 v.2 through v.9 were proteins translated from the corresponding transcript variants. All these protein variants shared a common portion of the sequence, i.e., 3-1011 of v.1, except for a few amino acids different in this segment resulted from SNP in the transcripts. Variant v.6 and v.9 were the same except for two amino acids at 906 and 1001. Variant v.8 was almost the same as v.5, except for the N-terminal end, and a 2-aa deletion at 1117-8. Single amino acid difference was not shown. Numbers in parentheses corresponded to positions in variant v.3.
Figure 12. Intentionally Omitted.
Figure 13. Figures 13(a)-(i): Secondary structure and transmembrane domains prediction for 109P1D4 protein variants 1-9 (v.1 - (SEQ ID NO: 31); v.2 - (SEQ ID NO: 32); v.3 - (SEQ ID NO: 33); v.4 - (SEQ ID NO: 34); v.5 - (SEQ ID NO: 35); v.6 - (SEQ ID NO: 36); v.7 - (SEQ ID NO: 37); v.8 - (SEQ ID NO: 38); v.9 - (SEQ ID NO: 39)). The secondary structures of 109P1D4 protein variants were predicted using the HNN - Hierarchical Neural Network method (NPS@: Network Protein Sequence Analysis TIBS 2000 March Vol. 25, No 3 [291]:147-150 Combet C, Blanchet C„ Geourjon C. and Deleage G., http://pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_nn.html), accessed from the ExPasy molecular biology server located on the World Wide Web at (.expasy.ch/tools/). This method predicts the presence and location of alpha helices, extended strands, and random coils from the primary protein sequence. The percent of the protein variant in a given secondary structure is also listed. Figures 13(J)-(R) top panels: Schematic representation of the probability of existence of transmembrane regions of 109P1D4 variants based on the TMpred algorithm of Hofmann and Stoffel which utilizes TMBASE (K. Hofmann, W. Stoffel. TMBASE - A database of membrane spanning protein segments Biol. Chem. Hoppe-Seyler 374:166, 1993). Figures 13(J)-(R) bottom panels: Schematic representation of the probability of the existence of transmembrane regions of 109P1D4 variants based on the TMHMM algorithm of Sonnhammer, von Heijne, and Krogh (Erik L.L Sonnhammer, Gunnar von Heijne, and Anders Krogh: A hidden Markov model for predicting transmembrane helices in protein sequences. In Proc. of Sixth Int. Conf. on Intelligent Systems for Molecular Biology, p 175-182 Ed J. Glasgow, T. Littlejohn, F. Major, R. Lathrop, D. Sankoff, and C. Sensen Menlo Park, CA: AAAI Press, 1998). The TMpred and TMHMM algorithms are accessed from the ExPasy molecular biology server located on the World Wide Web at (.expasy.ch/tools/).
Figure 14. Expression of 109P1D4 in Lymphoma Cancer Patient Specimens. RNA was extracted from peripheral blood lymphocytes, cord blood isolated from normal individuals, and from lymphoma patient cancer specimens. Northern blots with 10μg of total RNA were probed with the 109P1D4 sequence. Size standards in kilobases are on the side. Results show expression of 109P1D4 in lymphoma patient specimens but not in the normal blood cells tested.
Figure 15. Expression of 109P1D4 by RT-PCR. First strand cDNA was prepared from vital pool 1 (liver, lung and kidney), vital pool 2 (pancreas, colon and stomach), prostate cancer pool, bladder cancer pool, kidney cancer pool, colon cancer pool, lung cancer pool, ovary cancer pool, breast cancer pool, cancer metastasis pool, and pancreas cancer pool. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 109P1 D4, was performed at 30 cycles of amplification. Results show strong expression of 109P1 D4 in all cancer pools tested. Very low expression was detected in the vital pools.
Figure 16. Expression of 109P1D4 in normal tissues. Two multiple tissue northern blots (Clontech), both with 2 μg of mRNA/lane, were probed with the 109P1D4 SSH fragment. Size standards in kilobases (kb) are indicated on the side. Results show expression of approximately 10 kb 109P1D4 transcript in ovary. Weak expression was also detected in placenta and brain, but not in the other normal tissues tested.
Figure 17. Expression of 109P1 D4 in human cancer cell lines. RNA was extracted from a number of human prostate and bone cancer cell lines. Northern blots with 10 μg of total RNA/lane were probed with the 109P1D4 SSH fragment. Size standards in kilobases (kb) are indicated on (he side. Results show expression of 109P1D4 in LAPC-9AD, LAPC-9AI, LNCaP prostate cancer cell lines, and in the bone cancer cell lines, SK-ES-1 and RD-ES.
Figure 18. Figure 18A: 109P1D4 Expression in Human Normal Tissues. An cDNA dot blot containing 76 different samples from human tissues was analyzed using a 109P1D4 SSH probe. Expression was only detected in multiple areas of the brain, placenta, ovary, and fetal brain, amongst all tissues tested. Figure 18B: Expression of 109P1D4 in patient cancer specimens, Expression of 109P1 D4 was assayed in a panel of human cancers (T) and their respective matched normal tissues (N) on RNA dot blots. Upregulated expression of 109P1D4 in tumors compared to normal tissues was observed in uterus, lung and stomach. The expression detected in normal adjacent tissues (isolated from diseased tissues) but not in normal tissues (isolated from healthy donors) may indicate that these tissues are not fully normal and that 109P1D4 may be expressed in early stage tumors.
Figure 19. 109P1D4 Expression in Lung Cancer Patient Specimens. RNA was extracted from normal lung, prostate cancer xenograft LAPC-9AD, bone cancer cell line RD-ES, and lung cancer patient tumors. Northern blots with 10 μg of total RNA were probed with 109P1D4. Size standards in kilobases are on the side. Results show strong expression of 109P1D4 in lung tumor tissues as well as the RD-ES cell line, but not in normal lung.
Figure 20. Expression of soluble secreted Tag5 109P1 D4 in 293T cells. 293T cells were transfected with either an empty vector or with the Tagδ secretion vector encoding the extracellular domain (ECD; amino acids 24-812) of 109P1 D4 variant 1 fused to a Myc/His epitope Tag. 2 days later, cells and media harvested and analyzed for expression of the recombinant Tagδ 109P1D4 protein by SDS-PAGE followed by anti-His epitope tag Western blotting. An arrow indicates the immunoreactive band corresponding to the 109P1D4 ECD present in the media and the lysate from Tagδ 109P1D4 transfected cells.
Figure 21. Expression of 109P1 D4 protein in 293T cells. 293T cells were transfected with either an empty vector or with pCDNA3.1 vector encoding the full length cDNA of 109P1 D4 variant 1 fused to a Myc/His epitope Tag. 2 days later, cells were harvested and analyzed for expression of 109P1D4 variant 1 protein by SDS-PAGE followed by anti-His epitope tag Western blotting. An arrow indicates the immunoreactive band corresponding to the full length 109P1D4 variant 1 protein expressed in cells transfected with the 109P1D4 vector but not in control cells.
Figure 22. Tyrosine phosphorylation of 109P1D4 after pervanadate treatment, 293T cells were transfected with the neomycin resistance gene alone or with 109P1 D4 in pSRμ vector. Twenty four hours after transfection, the cells were either left in 10% serum or grown in 0.1 % serum overnight. The cells were then left untreated or were treated with 200 μM pervanadate (1:1 mixture of Na3V04 and H2O2) for 30 minutes. The cells were lysed in Triton X-100, and the 109P1D4 protein was immunoprecipitated with anti-His monoclonal antibody. The immunoprecipitates were run on SDS-PAGE and then Western blotted with either anti-phosphotyrosine (upper panel) or anti-His (lower panel). The 109P1D4 protein is phosphorylated on tyrosine in response to pervanadate treatment, and a large amount of the protein moves to the insoluble fraction following pervanadate-induced activation.
Figure 23. Effect of 109P1D4 RNAi on cell proliferation. LNCaP cells were transfected with Lipofectamine 2000 alone or with siRNA oligonucleotides. The siRNA oligonucleotides included a negative control, Luc4, specific for Luciferase, a positive control, Eg5, specific for the mitotic spindle protein Eg5, or three siRNAs specific for the 109P1D4 protein, 109P1 D4.a, 109P1 D4.c and 109P1 D4.d at 20 nM concentration. Twenty four hours after transfection, the cells were pulsed with 3H-thymidine and incorporation was measured after 72 hours. All three siRNAs to 109P1D4 inhibited the proliferation of LNCaP cells, indicating that 109P1D4 expression is important for the cell growth pathway of these cancer cells.
DETAILED DESCRIPTION OF THE INVENTION Outline of Sections
I.) Definitions
II.) 109P1 D4 Polynucleotides
II A) Uses of 109P1 D4 Polynucleotides
II.A.1.) Monitoring of Genetic Abnormalities
II.A.2.) Antisense Embodiments
IIA3.) Primers and Primer Pairs
II.A.4.) Isolation of 109P1 D4-Encoding Nucleic Acid Molecules II A5.) Recombinant Nucleic Acid Molecules and Host-Vector Systems III.) 109P1D4-related Proteins
HLA.) Motif-bearing Protein Embodiments
III.B.) Expression of 109P1 D4-related Proteins
III.C.) Modifications of 109P1 D4-related Proteins
III.D.) Uses of 109P1 D4-related Proteins IV.) 109P1D4 Antibodies V.) 109P1 D4 Cellular Immune Responses VI.) 109P1D4 Transgenic Animals VII.) Methods for the Detection of 109P1 D4
VIII.) Methods for Monitoring the Status of 109P1D4-related Genes and Their Products IX.) Identification of Molecules That Interact With 109P1 D4 X.) Therapeutic Methods and Compositions
X.A.) Anti-Cancer Vaccines X.B.) 109P1 D4 as a Target for Antibody-Based Therapy X.C.) 109P1D4 as a Target for Cellular Immune Responses
X.C.1. Minigene Vaccines
X.C.2. Combinations of CTL Peptides with Helper Peptides
X.C.3. Combinations of CTL Peptides with T Cell Priming Agents
X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL and/or HTL Peptides
X.D.) Adoptive Immunotherapy X.E.) Administration of Vaccines for Therapeutic or Prophylactic Purposes XI.) Diagnostic and Prognostic Embodiments of 109P1D4. XII.) Inhibition of 109P1D4 Protein Function
XII A) Inhibition of 109P1D4 With Intracellular Antibodies
XII. B.) Inhibition of 1 9P1D4 with Recombinant Proteins
XII.C.) Inhibition of 109P1D4 Transcription or Translation
XII.D.) General Considerations for Therapeutic Strategies XIII.) Identification, Characterization and Use of Modulators of 109P1 D4 XIV.) KITS/Articles of Manufacture ) Definitions:
Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized molecular cloning methodologies described in Sambrook ef al., Molecular Cloning: A Laboratory Manual 2nd. edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer defined protocols and/or parameters unless otherwise noted.
The terms "advanced prostate cancer", "locally advanced prostate cancer", "advanced disease" and "locally advanced disease" mean prostate cancers that have extended through the prostate capsule, and are meant to include stage C disease under the American Urological Association (AUA) system, stage C1 - C2 disease under the Whitmore-Jewett system, and stage T3 - T4 and N+ disease under the TNM (tumor, node, metastasis) system. In general, surgery is not recommended for patients with locally advanced disease, and these patients have substantially less favorable outcomes compared to patients having clinically localized (organ-confined) prostate cancer. Locally advanced disease is clinically identified by palpable evidence of induration beyond the lateral border of the prostate, or asymmetry or induration above the prostate base. Locally advanced prostate cancer is presently diagnosed pathologically following radical prostatectomy if the tumor invades or penetrates the prostatic capsule, extends into the surgical margin, or invades the seminal vesicles.
"Altering the native glycosylation pattern" is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence 109P1D4 (either by removing the underlying glycosylation site or by deleting the glycosylation by chemical and/or enzymatic means), and/or adding one or more glycosylation sites that are not present in the native sequence 109P1D4. In addition, the phrase includes qualitative changes in the glycosylation of the native proteins, involving a change in the nature and proportions of the various carbohydrate moieties present.
The term "analog" refers to a molecule which is structurally similar or shares similar or corresponding attributes with another molecule (e.g. a 109P1D4-related protein). For example, an analog of a 109P1D4 protein can be specifically bound by an antibody or T cell that specifically binds to 109P1 D4.
The term "antibody" is used in the broadest sense. Therefore, an "antibody" can be naturally occurring or man-made such as monoclonal antibodies produced by conventional hybridoma technology. Anti-109P1D4 antibodies comprise monoclonal and polyclonal antibodies as well as fragments containing the antigen-binding domain and/or one or more complementarity determining regions of these antibodies.
An "antibody fragment" is defined as at least a portion of the variable region of the immunoglobulin molecule that binds to its target, i.e., the antigen-binding region. In one embodiment it specifically covers single anti-109P1D4 antibodies and clones thereof (including agonist, antagonist and neutralizing antibodies) and anti-109P1D4 antibody compositions with polyepitopic specificity.
The term "codon optimized sequences" refers to nucleotide sequences that have been optimized for a particular host species by replacing any codons having a usage frequency of less than about 20%, Nucleotide sequences that have been optimized for expression in a given host species by elimination of spurious polyadenylation sequences, elimination of exon/intron splicing signals, elimination of transposon-like repeats and/or optimization of GC content in addition to codon optimization are referred to herein as an "expression enhanced sequences."
A "combinatorial library" is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis by combining a number of chemical "building blocks" such as reagents. For example, a linear combinatorial chemical library, such as a polypeptide (e.g., mutein) library, is formed by combining a set of chemical building blocks called amino acids in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Numerous chemical compounds are synthesized through such combinatorial mixing of chemical building blocks (Gallop et al., J. Med. Chem. 37(9): 1233-1251 (1994)).
Preparation and screening of combinatorial libraries is well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Patent No. 5,010,175, Furka, Pept. Prot. Res. 37:487-493 (1991), Houghton et al., Nature, 354:84-88 (1991)), peptoids (PCT Publication No WO 91/19735), encoded peptides (PCT Publication WO 93/20242), random bio- oligomers (PCT Publication WO 92/00091), benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidal peptidomimetics with a Beta-D-Glucose scaffolding (Hirschmann et al., J. Amer. Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of small compound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbarnates (Cho, et al., Science 261:1303 (1993)), and/or peptidyl phosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)). See, generally, Gordon et al., J. Med. Chem. 37:1385 (1994), nucleic acid libraries (see, e.g., Stratagene, Corp.), peptide nucleic acid libraries (see, e.g., U.S. Patent 5,539,083), antibody libraries (see, e.g., Vaughn et al., Nature Biotechnology 14(3): 309-314 (1996), and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al., Science 274:1520-1522 (1996), and U.S. Patent No. 5,593,853), and small organic molecule libraries (see, e.g., benzodiazepines, Baum, C&EN, Jan 18, page 33 (1993); isoprenoids, U.S. Patent No. 5,569,588; thiazolidinones and metathiazanones, U.S. Patent No. 5,549,974; pyrrolidines, U.S. Patent Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. Patent No. 5,506, 337; benzodiazepines, U.S. Patent No. 5,288,514; and the like).
Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 NIPS, 390 NIPS, Advanced Chem Tech, Louisville KY; Symphony, Rainin, Woburn, MA; 433A, Applied Biosystems, Foster City, CA; 9050, Plus, Millipore, Bedford, NIA). A number of well-known robotic systems have also been developed for solution phase chemistries. These systems include automated workstations such as the automated synthesis apparatus developed by Takeda Chemical Industries, LTD. (Osaka, Japan) and many robotic systems utilizing robotic arms (Zymate H, Zymark Corporation, Hopkinton, Mass.; Orca, Hewlett-Packard, Palo Alto, Calif.), which mimic the manual synthetic operations performed by a chemist. Any of the above devices are suitable for use with the present invention. The nature and implementation of modifications to these devices (if any) so that they can operate as discussed herein will be apparent to persons skilled in the relevant art. In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, NJ; Asinex, Moscow, RU; Tripos, Inc., St. Louis, MO; ChemStar, Ltd, Moscow, RU; 3D Pharmaceuticals, Exton, PA; Martek Biosciences, Columbia, MD; etc.).
The term "cytotoxic agent" refers to a substance that inhibits or prevents the expression activity of cells, function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof. Examples of cytotoxic agents include, but are not limited to auristatins, auromycins, maytansinoids, yttrium, bismuth, ricin, ricin A-chain, combrestatin, duocarmycins, dolostatins, doxorubicin, daunorubicin, taxol, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, aipha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, Sapaonaria offioinalis inhibitor, and glucocorticoid and other chemotherapeutic agents, as well as radioisotopes such as At211, 1131, 1125, Y90, Re186, Re188, Sm153, Bi212or213, P32 and radioactive isotopes of Lu including Lu177. Antibodies may also be conjugated to an anticancer pro-drug activating enzyme capable of converting the pro-drug to its active form.
The "gene product" is sometimes referred to herein as a protein or mRNA. For example, a "gene product of the invention" is sometimes referred to herein as a "cancer amino acid sequence", "cancer protein", "protein of a cancer listed in Table I", a "cancer mRNA", "mRNA of a cancer listed in Table I", etc. In one embodiment, the cancer protein is encoded by a nucleic acid of Figure 2. The cancer protein can be a fragment, or alternatively, be the full-length protein to the fragment encoded by the nucleic acids of Figure 2. In one embodiment, a cancer amino acid sequence is used to determine sequence identity or similarity. In another embodiment, the sequences are naturally occurring allelic variants of a protein encoded by a nucleic acid of Figure 2. In another embodiment, the sequences are sequence variants as further described herein.
"High throughput screening" assays for the presence, absence, quantification, or other properties of particular nucleic acids or protein products are well known to those of skill in the art. Similarly, binding assays and reporter gene assays are similarly well known. Thus, e.g., U.S. Patent No. 5,559,410 discloses high throughput screening methods for proteins; U.S. Patent No. 5,585,639 discloses high throughput screening methods for nucleic acid binding (i.e., in arrays); while U.S. Patent Nos. 5,576,220 and 5,541 ,061 disclose high throughput methods of screening for ligand/antibody binding.
In addition, high throughput screening systems are commercially available (see, e.g., Amersham Biosciences, Piscataway, NJ; Zymark Corp., Hopkinton, MA; Air Technical Industries, Mentor, OH; Beckman Instruments, Inc. Fullerton, CA; Precision Systems, Inc., Natick, MA; etc.). These systems typically automate entire procedures, including all sample and reagent pipetting, liquid dispensing, timed incubations, and final readings of the microplate in detector(s) appropriate for the assay. These configurable systems provide high throughput and rapid start up as well as a high degree of flexibility and customization. The manufacturers of such systems provide detailed protocols for various high throughput systems. Thus, e.g., Zymark Corp. provides technical bulletins describing screening systems for detecting the modulation of gene transcription, ligand binding, and the like.
The term "homolog" refers to a molecule which exhibits homology to another molecule, by for example, having sequences of chemical residues that are the same or similar at corresponding positions.
"Human Leukocyte Antigen" or "HLA" is a human class I or class II Major Histocompatibility Complex (MHC) protein (see, e.g., Stites, ef al., IMMUNOLOGY, 8™ ED., Lange Publishing, Los Altos, CA (1994).
The terms "hybridize", "hybridizing", "hybridizes" and the like, used in the context of polynucleotides, are meant to refer to conventional hybridization conditions, preferably such as hybridization in 50% formamide/6XSSC/0.1 % SDS/100 μg/ml ssDNA, in which temperatures for hybridization are above 37 degrees C and temperatures for washing in 0.1XSSC/0.1 % SDS are above 55 degrees C.
The phrases "isolated" or "biologically pure" refer to material which is substantially or essentially free from components which normally accompany the material as it is found in its native state. Thus, isolated peptides in accordance with the invention preferably do not contain materials normally associated with the peptides in their in situ environment. For example, a polynucleotide is said to be "isolated" when it is substantially separated from contaminant polynucleotides that correspond or are complementary to genes other than the 109P1 D4 genes or that encode polypeptides other than 109P1 D4 gene product or fragments thereof. A skilled artisan can readily employ nucleic acid isolation procedures to obtain an isolated 109P1 D4 polynucleotide. A protein is said to be "isolated," for example, when physical, mechanical or chemical methods are employed to remove the 109P1D4 proteins from cellular constituents that are normally associated with the protein. A skilled artisan can readily employ standard purification methods to obtain an isolated 109P1D4 protein. Alternatively, an isolated protein can be prepared by chemical means.
The term "mammal" refers to any organism classified as a mammal, including mice, rats, rabbits, dogs, cats, cows, horses and humans. In one embodiment of the invention, the mammal is a mouse. In another embodiment of the invention, the mammal is a human.
The terms "metastatic prostate cancer" and "metastatic disease" mean prostate cancers that have spread to regional lymph nodes or to distant sites, and are meant to include stage D disease under the AUA system and stage TxNxM÷ under the TNM system. As is the case with locally advanced prostate cancer, surgery is generally not indicated for patients with metastatic disease, and hormonal (androgen ablation) therapy is a preferred treatment modality. Patients with metastatic prostate cancer eventually develop an androgen-refractory state within 12 to 18 months of treatment initiation. Approximately half of these androgen-refractory patients die within 6 months after developing that status. The most common site for prostate cancer metastasis is bone. Prostate cancer bone metastases are often osteoblastic rather than osteolytic (i.e., resulting in net bone formation). Bone metastases are found most frequently in the spine, followed by the femur, pelvis, rib cage, skull and humerus. Other common sites for metastasis include lymph nodes, lung, liver and brain. Metastatic prostate cancer is typically diagnosed by open or laparoscopic pelvic lymphadenectomy, whole body radionuclide scans, skeletal radiography, and/or bone lesion biopsy.
The term "modulator" or "test compound" or "drug candidate" or grammatical equivalents as used herein describe any molecule, e.g., protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide, etc., to be tested for the capacity to directly or indirectly alter the cancer phenotype or the expression of a cancer sequence, e.g., a nucleic acid or protein sequences, or effects of cancer sequences (e.g., signaling, gene expression, protein interaction, etc.) In one aspect, a modulator will neutralize the effect of a cancer protein of the invention. By "neutralize" is meant that an activity of a protein is inhibited or blocked, along with the consequent effect on the cell. In another aspect, a modulator will neutralize the effect of a gene, and its corresponding protein, of the invention by normalizing levels of said protein. In preferred embodiments, modulators alter expression profiles, or expression profile nucleic acids or proteins provided herein, or downstream effector pathways. In one embodiment, the modulator suppresses a cancer phenotype, e.g. to a normal tissue fingerprint. In another embodiment, a modulator induced a cancer phenotype. Generally, a plurality of assay mixtures is run in parallel with different agent concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration or below the level of detection.
Modulators, drug candidates or test compounds encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 100 and less than about 2,500 Daltons. Preferred small molecules are less than 2000, or less than 1500 or less than 1000 or less than 500 D. Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Modulators also comprise biomolecules such as peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Particularly preferred are peptides. One class of modulators are peptides, for example of from about five to about 35 amino acids, with from about five to about 20 amino acids being preferred, and from about 7 to about 15 being particularly preferred. Preferably, the cancer modulalory protein is soluble, includes a non-transmembrane region, and/or, has an N- terminal Cys to aid in solubility. In one embodiment, the C-terminus of the fragment is kept as a free acid and the N-terminus is a free amine to aid in coupling, i.e., to cysteine. In one embodiment, a cancer protein of the invention is conjugated to an immunogenic agent as discussed herein. In one embodiment, the cancer protein is conjugated to BSA. The peptides of the invention, e.g., of preferred lengths, can be linked to each other or to other amino acids to create a longer peptide/protein.
The modulatory peptides can be digests of naturally occurring proteins as is outlined above, random peptides, or "biased" random peptides. In a preferred embodiment, peptide/protein-based modulators are antibodies, and fragments thereof, as defined herein.
Modulators of cancer can also be nucleic acids. Nucleic acid modulating agents can be naturally occurring nucleic acids, random nucleic acids, or "biased" random nucleic acids. For example, digests of prokaryotic or eukaryotic genomes can be used in an approach analogous to that outlined above for proteins.
The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the antibodies comprising the population are identical except for possible naturally occurring mutations that are present in minor amounts.
A "motif, as in biological motif of a 109P1 D4-related protein, refers to any pattern of amino acids forming part of the primary sequence of a protein, that is associated with a particular function (e.g. protein-protein interaction, protein-DNA interaction, etc) or modification (e.g. that is phosphorylated, glycosylated or amidated), or localization (e.g. secretory sequence, nuclear localization sequence, etc.) or a sequence that is correlated with being immunogenic, either humorally or cellularly. A motif can be either contiguous or capable of being aligned to certain positions that are generally correlated with a certain function or property. In the context of HLA motifs, "motif refers to the pattern of residues in a peptide of defined length, usually a peptide of from about 8 to about 13 amino acids for a class I HLA motif and from about 6 to about 25 amino acids for a class II HLA motif, which is recognized by a particular HLA molecule. Peptide motifs for HLA binding are typically different for each protein encoded by each human HLA allele and differ in the pattern of the primary and secondary anchor residues.
A "pharmaceutical excipient" comprises a material such as an adjuvant, a carrier, pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservative, and the like.
"Pharmaceutically acceptable" refers to a non-toxic, inert, and/or composition that is physiologically compatible with humans or other mammals.
The term "polynucleotide" means a polymeric form of nucleotides of at least 10 bases or base pairs in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide, and is meant to include single and double stranded forms of DNA and/or RNA. In the art, this term if often used interchangeably with "oligonucleotide". A polynucleotide can comprise a nucleotide sequence disclosed herein wherein thymidine (T), as shown for example in Figure 2, can also be uracil (U); this definition pertains to the differences between the chemical structures of DNA and RNA, in particular the observation that one of the four major bases in RNA is uracil (U) instead of thymidine (T).
The term "polypeptide" means a polymer of at least about 4, 5, 6, 7, or 8 amino acids. Throughout the specification, standard three letter or single letter designations for amino acids are used. In the art, this term is often used interchangeably with "peptide" or "protein".
An HLA "primary anchor residue" is an amino acid at a specific position along a peptide sequence which is understood to provide a contact point between the immunogenic peptide and the HLA molecule. One to three, usually two, primary anchor residues within a peptide of defined length generally defines a "motif for an immunogenic peptide. These residues are understood to fit in close contact with peptide binding groove of an HLA molecule, with their side chains buried in specific pockets of the binding groove. In one embodiment, for example, the primary anchor residues for an HLA class I molecule are located at position 2 (from the amino terminal position) and at the carboxyl terminal position of a 8, 9, 10, 11, or 12 residue peptide epitope in accordance with the invention. Alternatively, in another embodiment, the primary anchor residues of a peptide binds an HLA class II molecule are spaced relative to each other, rather than to the termini of a peptide, where the peptide is generally of at least 9 amino acids in length. The primary anchor positions for each motif and supermotif are set forth in Table IV. For example, analog peptides can be created by altering the presence or absence of particular residues in the primary and/or secondary anchor positions shown in Table IV. Such analogs are used to modulate the binding affinity and/or population coverage of a peptide comprising a particular HLA motif or supermotif.
"Radioisotopes" include, but are not limited to the following (non-limiting exemplary uses are also set forth):
Examples of Medical Isotopes:
Isotope Description of use
Actinium-225
See Thorium-229 (Th-229)
(AC-225)
Actinium-227 Parent of Radium-223 (Ra-223) which is an alpha emitter used to treat metastases in the skeleton (AC-227) resulting from cancer (i.e., breast and prostate cancers), and cancer radioimmunotherapy
Bismuth-212
See Thorium-228 (Th-228) (Bi-212)
Bismuth-213 See Thorium-229 (Th-229) (Bi-213)
Cadmium-109
Cancer detection
(Cd-109)
Cobalt-60 Radiation source for radiotherapy of cancer, for food irradiators, and for sterilization of medical
(Co-60) supplies
Copper-64
A positron emitter used for cancer therapy and SPECT imaging
(Cu-64)
Copper-67' Beta/gamma emitter used in cancer radioimmunotherapy and diagnostic studies (i.e., breast and
(Cu-67) colon cancers, and lymphoma)
Dysprosium-166
Cancer radioimmunotherapy
(Dy-166)
Erbium-169
Rheumatoid arthritis treatment, particularly for the small joints associated with fingers and toes (Er-169)
Europium-152
Radiation source for food irradiation and for sterilization of medical supplies (Eu-152)
Europium-154
Radiation source for food irradiation and for sterilization of medical supplies (Eu-154)
Gadolinium-153
Osteoporosis detection and nuclear medical quality assurance devices
(Gd-153)
Gold-198
Implant and intracavity therapy of ovarian, prostate, and brain cancers
(Au-198)
Holmium-166 Multiple myeloma treatment in targeted skeletal therapy, cancer radioimmunotherapy, bone
(Ho-166) marrow ablation, and rheumatoid arthritis treatment
Osteoporosis detection, diagnostic imaging, tracer drugs, brain cancer treatment, radiolabeling, lodine-125 tumor imaging, mapping of receptors in the brain, interstitial radiation therapy, brachytherapy for (1-125) treatment of prostate cancer, determination of glomerular filtration rate (GFR), determination of plasma volume, detection of deep vein thrombosis of the legs
Thyroid function evaluation, thyroid disease detection, treatment of thyroid cancer as well as other lodine-131 non-malignant thyroid diseases (i.e., Graves disease, goiters, and hyperthyroidism), treatment of (1-131) leukemia, lymphoma, and other forms of cancer (e.g., breast cancer) using radioimmunotherapy lridium-192 Brachytherapy, brain and spinal cord tumor treatment, treatment of blocked arteries (i.e., (lr-192) arteriosclerosis and restenosis), and implants for breast and prostate tumors
Lutetium-177 Cancer radioimmunotherapy and treatment of blocked arteries (i.e., arteriosclerosis and (Lu-177) restenosis)
Parent of Technetium-99m (Tc-99m) which is used for imaging the brain, liver, lungs, heart, and
Molybdenum-99 other organs. Currently, Tc-99m is the most widely used radioisotope used for diagnostic imaging (Mo-99) of various cancers and diseases involving the brain, heart, liver, lungs; also used in detection of deep vein thrombosis of the legs
Osmium-194
Cancer radioimmunotherapy (Os-194) Palladium-103
Prostate cancer treatment (Pd-103) Platinum-195m
Studies on biodistribution and metabolism of cisplatin, a chemotherapeutic drug (Pt-195m)
Polycythemia rubra vera (blood cell disease) and leukemia treatment, bone cancer
Phosphorus-32 diagnosis/treatment; colon, pancreatic, and liver cancer treatment; radiolabeling nucleic acids for (P-32) in vitro research, diagnosis of superficial tumors, treatment of blocked arteries (i.e., arteriosclerosis and restenosis), and intracavity therapy
Phosphorus-33 Leukemia treatment, bone disease diagnosis/treatment, radiolabeling, and treatment of blocked
(P-33) arteries (i.e., arteriosclerosis and restenosis)
Radium-223
See Actinium-227 (Ac-227)
(Ra-223)
Rhenium-186 Bone cancer pain relief, rheumatoid arthritis treatment, and diagnosis and treatment of lymphoma
(Re-186) and bone, breast, colon, and liver cancers using radioimmunotherapy
Rhenium-188 Cancer diagnosis and treatment using radioimmunotherapy, bone cancer pain relief, treatment of (Re-188) rheumatoid arthritis, and treatment of prostate cancer
Rhodium-105
Cancer radioimmunotherapy
(Rh-105)
Samarium-145
Ocular cancer treatment
(Sm-145)
Samarium-153
Cancer radioimmunotherapy and bone cancer pain relief
(Sm-153)
Scandium-47
Cancer radioimmunotherapy and bone cancer pain relief
(Sc-47)
Radiotracer used in brain studies, imaging of adrenal cortex by gamma-scintigraphy, lateral
Selenium-75 locations of steroid secreting tumors, pancreatic scanning, detection of hyperactive parathyroid (Se-75) glands, measure rate of bile acid loss from the endogenous pool
Strontium-85-
Bone cancer detection and brain scans
(Sr-85)
Strontium-89
Bone cancer pain relief, multiple myeloma treatment, and osteoblastic therapy
(Sr-89)
Technetium-99m
See Molybdenum-99 (Mo-99)
(Tc-99m)
Thorium-228
Parent of Bismuth-212 (Bi-212) which is an alpha emitter used in cancer radioimmunotherapy
(Th-228)
Thorium-229 Parent of Actinium-225 (Ac-225) and grandparent of Bismuth-213 (Bi-213) which are alpha
(Th-229) emitters used in cancer radioimmunotherapy
Thulium-170
Gamma source for blood irradiators, energy source for implanted medical devices
( Tm-170)
Tin-117m
Cancer immunotherapy and bone cancer pain relief
(Sn-117m)
Parent for Rhenium-188 (Re-188) which is used for cancer diagnostics/treatment, bone cancer
Tungsten-188 pain relief, rheumatoid arthritis treatment, and treatment of blocked arteries (i.e., arteriosclerosis (W-188) and restenosis)
Xenon-127 Neuroimaging of brain disorders, high resolution SPECT studies, pulmonary function tests, and (Xe-127) cerebral blood flow studies Ytterbium-175
Cancer radioimmunotherapy (Yb-175)
Yttrium-90
Microseeds obtained from irradiating Yttrium-89 (Y-89) for liver cancer treatment (Y-90)
A gamma-emitting label for Yttrium-90 (Y-90) which is used for cancer radioimmunotherapy (i.e.,
Yttrium-91 lymphoma, breast, colon, kidney, lung, ovarian, prostate, pancreatic, and inoperable liver (Y-91) cancers) By "randomized" or grammatical equivalents as herein applied to nucleic acids and proteins is meant that each nucleic acid and peptide consists of essentially random nucleotides and amino acids, respectively. These random peptides (or nucleic acids, discussed herein) can incorporate any nucleotide or amino acid al any position. The synthetic process can be designed to generate randomized proteins or nucleic acids, to allow the formation of all or most of the possible combinations over the length of the sequence, thus forming a library of randomized candidate bioactive proteinaceous agents.
In one embodiment, a library is "fully randomized," with no sequence preferences or constants at any position. In another embodiment, the library is a "biased random" library. That is, some positions within the sequence either are held constant, or are selected from a limited number of possibilities. For example, the nucleotides or amino acid residues are randomized within a defined class, e.g., of hydrophobic amino acids, hydrophilic residues, sterically biased (either small or large) residues, towards the creation of nucleic acid binding domains, the creation of cysteines, for cross-linking, prolines for SH-3 domains, serines, threonines, tyrosines or histidines for phosphorylation sites, etc., or to purines, etc.
A "recombinant" DNA or RNA molecule is a DNA or RNA molecule that has been subjected to molecular manipulation in vitro.
Non-limiting examples of small molecules include compounds that bind or interact with 109P1 D4, ligands including hormones, neuropeptides, chemokines, odorants, phospholipids, and functional equivalents thereof that bind and preferably inhibit 109P1 D4 protein function. Such non-limiting small molecules preferably have a molecular weight of less than about 10 kDa, more preferably below about 9, about 8, about 7, about 6, about 5 or about 4 kDa. In certain embodiments, small molecules physically associate with, or bind, 109P1 D4 protein; are not found in naturally occurring metabolic pathways; and/or are more soluble in aqueous than non-aqueous solutions
"Stringency" of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured nucleic acid sequences to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature that can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel ef al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).
"Stringent conditions" or "high stringency conditions", as defined herein, are identified by, but not limited to, those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50°C; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1 % Ficoll/0.1 % polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42 °C; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCI, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1 % SDS, and 10% dextran sulfate at 42 °C, with washes at 42°C in 0.2 x SSC (sodium chloride/sodium, citrate) and 50% formamide at 55 °C, followed by a high-stringency wash consisting of 0.1 x SSC containing EDTA at 55 °C. "Moderately stringent conditions" are described by, but not limited to, those in Sambrook ef al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and %SDS) less stringent than those described above. An example of moderately stringent conditions is overnight incubation at 37°C in a solution comprising: 20% formamide, 5 x SSC (150 mM NaCI, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 37-50°C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.
An HLA "supermotif is a peptide binding specificity shared by HLA molecules encoded by two or more HLA alleles. Overall phenotypic frequencies of HLA-supertypes in different ethnic populations are set forth in Table IV (F). The non- limiting constituents of various supetypes are as follows:
A2 AO201, A*0202, A*0203, A*0204, A* 0205, A*0206, A*6802, A*6901, A*0207
A3: A3, A11 , A31 , A*3301 , A*6801 , A*0301 , A*1101 , A*3101
B7: B7, B*3501-03, B*51, B*5301, B*5401, B*5501, B*5502, B*5601, 13*6701, B7801, B*0702, B*5101, B*5602
B44: B*3701, B*4402, B*4403, B*60 (B*4001), B61 (B*4006)
All A*0102, A*2604, A*3601 , A*4301 , A*8001
A24: A*24, A*30, A*2403, A*2404, A*3002, A*3003
B27: B*1401-02, B*1503, B*1509, B*1510, B*1518, B*3801-02, B*3901, B*3902, B*3903-04, B 801-02, B*7301, B*2701-08
B58: B*1516, B*1517, B*5701, B*5702, B58
B62: B*4601. B52, B*1501 (B62), B*1502 (B7δ), B*1513 (B77) Calculated population coverage afforded by different HLA-supertype combinations are set forth in Table IV (G).
As used herein "to treat" or "therapeutic" and grammatically related terms, refer to any improvement of any consequence of disease, such as prolonged survival, less morbidity, and/or a lessening of side effects which are the byproducts of an alternative therapeutic modality; full eradication of disease is not required.
A "transgenic animal" (e.g., a mouse or rat) is an animal having cells that contain a transgene, which transgene was introduced into the animal or an ancestor of the animal at a prenatal, e.g., an embryonic stage. A "transgene" is a DNA that is integrated into the genome of a cell from which a transgenic animal develops.
As used herein, an HLA or cellular immune response "vaccine" is a composition that contains or encodes one or more peptides of the invention. There are numerous embodiments of such vaccines, such as a cocktail of one or more individual peptides; one or more peptides of the invention comprised by a polyepitopic peptide; or nucleic acids that encode such individual peptides or polypeptides, e.g., a minigene that encodes a polyepitopic peptide. The "one or more peptides" can include any whole unit integer from 1-150 or more, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 or more peptides of the invention. The peptides or polypeptides can optionally be modified, such as by lipidation, addition of targeting or other sequences. HLA class I peptides of the invention can be admixed with, or linked to, HLA class II peptides, to facilitate activation of both cytotoxic T lymphocytes and helper T lymphocytes. HLA vaccines can also comprise peptide-pulsed antigen presenting cells, e.g., dendritic cells.
The term "variant" refers to a molecule that exhibits a variation from a described type or norm, such as a protein that has one or more different amino acid residues in the corresponding position(s) of a specifically described protein (e.g. the 109P1D4 protein shown in Figure 2 or Figure 3. An analog is an example of a variant protein. Splice isoforms and single nucleotides polymorphisms (SNPs) are further examples of variants. The "109P1D4-related proteins" of the invention include those specifically identified herein, as well as allelic variants, conservative substitution variants, analogs and homologs that can be isolated/generated and characterized without undue experimentation following the methods outlined herein or readily available in the art. Fusion proteins that combine parts of different 109P1 D4 proteins or fragments thereof, as well as fusion proteins of a 109P1 D4 protein and a heterologous polypeptide are also included. Such 109P1D4 proteins are collectively referred to as the 109P1D4-related proteins, the proteins of the invention, or 109P1D4. The term "109P1D4-related protein" refers to a polypeptide fragment or a 109P1D4 protein sequence of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25 amino acids; or, at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, or 576 or more amino acids.
II.) 109P1D4 Polynucleotides
One aspect of the invention provides polynucleotides corresponding or complementary to all or part of a 109P1 D4 gene, mRNA, and/or coding sequence, preferably in isolated form, including polynucleotides encoding a 109P1 D4-related protein and fragments thereof, DNA, RNA, DNA/RNA hybrid, and related molecules, polynucleotides or oligonucleotides complementary to a 109P1D4 gene or mRNA sequence or a part thereof, and polynucleotides or oligonucleotides that hybridize to a 109P1D4 gene, mRNA, or to a 109P1D4 encoding polynucleotide (collectively, "109P1D4 polynucleotides"). In all instances when referred to in this section, T can also be U in Figure 2.
Embodiments of a 109P1D4 polynucleotide include: a 109P1D4 polynucleotide having the sequence shown in Figure 2, the nucleotide sequence of 109P1D4 as shown in Figure 2 wherein T is U; at least 10 contiguous nucleotides of a polynucleotide having the sequence as shown in Figure 2; or, at least 10 contiguous nucleotides of a polynucleotide having the sequence as shown in Figure 2 where T is U. For example, embodiments of 109P1D4 nucleotides comprise, without limitation:
(I) a polynucleotide comprising, consisting essentially of, or consisting of a sequence as shown in Figure 2, wherein T can also be U;
(II) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 2A, from nucleotide residue number 846 through nucleotide residue number 3911, including the stop codon, wherein T can also be U;
(III) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 2B, from nucleotide residue number 503 through nucleotide residue number 3667, including the stop codon, wherein T can also be U;
(IV) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 2C, from nucleotide residue number 846 through nucleotide residue number 4889, including the a stop codon, wherein T can also be U;
(V) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 2D, from nucleotide residue number 846 through nucleotide residue number 4859, including the stop codon, wherein T can also be U;
(VI) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 2E, from nucleotide residue number 846 through nucleotide residue number 4778, including the stop codon, wherein T can also be U; (VII) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 2F, from nucleotide residue number 614 through nucleotide residue number 3727, including the stop codon, wherein T can also be U;
(VIII) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 2G, from nucleotide residue number 735 through nucleotide residue number 3881, including the stop codon, wherein T can also be U;
(IX) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 2H, from nucleotide residue number 735 through nucleotide residue number 4757, including the stop codon, wherein T can also be U;
(X) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure 21, from nucleotide residue number 514 through nucleotide residue number 3627, including the stop codon, wherein T can also be U;
(XI) a polynucleotide that encodes a 109P1 D4-related protein that is at least 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100% homologous to an entire amino acid sequence shown in Figure 2A-I;
(XII) a polynucleotide that encodes a 109P1 D4-related protein that is at least 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to an entire amino acid sequence shown in Figure 2A-I;
(XIII) a polynucleotide that encodes at least one peptide set forth in Tables VIII-XXI and XXII-XLIX;
(XIV) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35 amino acids of a peptide of Figures 3A in any whole number increment up to 1021 that includes at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of Figure 5;
(XV) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35 amino acids of a peptide of Figure 3A in any whole number increment up to 1021 that includes 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of Figure 6;
(XVI) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of Figure 3A in any whole number increment up to 1021 that includes 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of Figure 7;
(XVII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35 amino acids of a peptide of Figure 3A in any whole number increment up to 1021 that includes 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of Figure 8;
(XVI II) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of Figure 3A in any whole number increment up to 1021 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of Figure 9;
(XIX) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35 amino acids of a peptide of Figure 3B, 3C, and/or 3D in any whole number increment up to 1054, 1347, and/or 1337 respectively that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of Figure 5;
(XX) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35 amino acids of a peptide of Figure 3B, 3C, and/or 3D in any whole number increment up to 1054, 1347, and/or 1337 respectively that includes 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of Figure 6;
(XXI) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35 amino acids of a peptide of Figure 3B, 3C, and or 3D in any whole number increment up to 1054, 1347, and/or 1337 respectively that includes 1, 2, 3, , 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of Figure 7;
(XXII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of Figure 3B, 3C, and/or 3D in any whole number increment up to 1054, 1347, and/or 1337 respectively that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of Figure 8;
(XXIII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11 , 12, 13, 1 , 15, 1 δ, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35 amino acids of a peptide of Figure 3B, 3C, and/or 3D in any whole number increment up to 1054, 1347, and/or 1337 respectively that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of Figure 9;
(XXIV) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of Figure 3E, 3F, 3G, 3H and/or 31 in any whole number increment up to 1310, 1037, 1048, 1340, and/or 1037 respectively that includes 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of Figure 5;
(XXV) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of Figure 3E, 3F, 3G, 3H and/or 31 in any whole number increment up to 1310, 1037, 1048, 1340, and/or 1037 respectively that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of Figure 6; (XXVI) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of Figure 3E, 3F, 3G, 3H and/or 31 in any whole number increment up to 1310, 1037, 1048, 1340, and/or 1037 respectively that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of Figure 7;
(XXVI I) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of Figure 3E, 3F, 3G, 3H and/or 31 in any whole number increment up to 1310, 1037, 1048, 1340, and/or 1037 respectively that includes 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of Figure 8;
(XXVIII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35 amino acids of a peptide of Figure 3E, 3F, 3G, 3H, and/or 31 in any whole number increment up to 1310, 1037, 1048, 1340, and/or 1037 respectively that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of Figure 9;
(XXIX) a polynucleotide that is fully complementary to a polynucleotide of any one of (l)-(XXVIII);
(XXX) a polynucleotide that is fully complementary to a polynucleotide of any one of (l)-(XXIX);
(XXXI) a peptide that is encoded by any of (I) to (XXX); and;
(XXXII) a composition comprising a polynucleotide of any of (l)-(XXX) or peptide of (XXXI) together with a pharmaceutical excipient and/or in a human unit dose form;
(XXXIII) a method of using a polynucleotide of any (l)-(XXX) or peptide of (XXXI) or a composition of (XXXII) in a method to modulate a cell expressing 109P1D4;
(XXXIV) a method of using a polynucleotide of any (l)-(XXX) or peptide of (XXXI) or a composition of (XXXII) in a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressing 109P1D4;
(XXXV) a method of using a polynucleotide of any (l)-(XXX) or peptide of (XXXI) or a composition of (XXXII) in a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressing 109P1D4, said cell from a cancer of a tissue listed in Table I;
(XXXVI) a method of using a polynucleotide of any (l)-(XXX) or peptide of (XXXI) or a composition of (XXXII) in a method to diagnose, prophylax, prognose, or treat a a cancer;
(XXXVII) a method of using a polynucleotide of any (l)-(XXX) or peptide of (XXXI) or a composition of (XXXII) in a method to diagnose, prophylax, prognose, or treat a a cancer of a tissue listed in Table I; and;
(XXXVIII) a method of using a polynucleotide of any (l)-(XXX) or peptide of (XXXI) or a composition of (XXXII) in a method to identify or characterize a modulator of a cell expressing 109P1 D4.
As used herein, a range is understood to disclose specifically all whole unit positions thereof.
Typical embodiments of the invention disclosed herein include 109P1D4 polynucleotides that encode specific portions of 109P1D4 mRNA sequences (and those which are complementary to such sequences) such as those that encode the proteins and/or fragments thereof, for example: . (a) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1010, 1020, and 1021 or more contiguous amino acids of 109P1D4 variant 1; the maximal lengths relevant for other variants are: variant 2, 1054 amino acids; variant 3, 1347 amino acids, variant 4, 1337 amino acids, variant 5, 1310 amino acids, variant 6; 1047 amino acids, variant 7; 1048 amino acids, variant 8; 1340 amino acids and variant 9; 1037 amoni acids.
For example, representative embodiments of the invention disclosed herein include: polynucleotides and their encoded peptides themselves encoding about amino acid 1 to about amino acid 10 of the 109P1D4 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 10 to about amino acid 20 of the 109P1D4 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 20 to about amino acid 30 of the 109P1D4 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 30 to about amino acid 40 of the 109P1D4 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 40 to about amino acid 50 of the 109P1D4 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 50 to about amino acid 60 of the 109P1 D4 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 60 to about amino acid 70 of the 109P1 D4 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 70 to about amino acid 80 of the 109P1D4 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 80 to about amino acid 90 of the 109P1D4 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 90 to about amino acid 100 of the 109P1D4 protein shown in Figure 2 or Figure 3, in increments of about 10 amino acids, ending at the carboxyl terminal amino acid set forth in Figure 2 or Figure 3. Accordingly, polynucleotides encoding portions of the amino acid sequence (of about 10 amino acids), of amino acids, 100 through the carboxyl terminal amino acid of the 109P1 D4 protein are embodiments of the invention. Wherein it is understood that each particular amino acid position discloses that position plus or minus five amino acid residues.
Polynucleotides encoding relatively long portions of a 109P1 D4 protein are also within the scope of the invention. For example, polynucleotides encoding from about amino acid 1 (or 20 or 30 or 40 etc.) to about amino acid 20, (or 30, or 40 or 50 etc.) of the 109P1 D4 protein "or variant" shown in Figure 2 or Figure 3 can be generated by a variety of techniques well known in the art. These polynucleotide fragments can include any portion of the 109P1D4 sequence as shown in Figure 2.
Additional illustrative embodiments of the invention disclosed herein include 109P1D4 polynucleotide fragments encoding one or more of the biological motifs contained within a 109P1D4 protein "or variant" sequence, including one or more of the motif-bearing subsequences of a 109P1 D4 protein "or variant" set forth in Tables VIII-XXI and XXII-XLIX. In another embodiment, typical polynucleotide fragments of the invention encode one or more of the regions of 109P1D4 protein or variant that exhibit homology to a known molecule. In another embodiment of the invention, typical polynucleotide fragments can encode one or more of the 109P1D4 protein or variant N-glycosylation sites, cAMP and cGMP-dependent protein kinase phosphorylation sites, casein kinase II phosphorylation sites or N-myristoylation site and amidation sites.
Note that to determine the starting position of any peptide set forth in Tables VIII-XXI and Tables XXII to XLIX (collectively HLA Peptide Tables) respective to its parental protein, e.g., variant 1 , variant 2, etc., reference is made to three factors: the particular variant, the length of the peptide in an HLA Peptide Table, and the Search Peptides listed in Table VII. Generally, a unique Search Peptide is used to obtain HLA peptides for a particular variant. The position of each Search Peptide relative to its respective parent molecule is listed in Table VII. Accordingly, if a Search Peptide begins at position "X", one must add the value "X minus 1" to each position in Tables VIII-XXI and Tables XXII-IL to obtain the actual position of the HLA peptides in their parental molecule. For example if a particular Search Peptide begins at position 150 of its parental molecule, one must add 150 - 1, i.e., 149 to each HLA peptide amino acid position to calculate the position of that amino acid in the parent molecule.
HA) Uses of 109P1 D4 Polynucleotides
II.A.1.) Monitoring of Genetic Abnormalities
The polynucleotides of the preceding paragraphs have a number of different specific uses. The human 109P1 D4 gene maps to the chromosomal location set forth in the Example entitled "Chromosomal Mapping of 109P1 D4." For example, because the 109P1 D4 gene maps to this chromosome, polynucleotides that encode different regions of the 109P1D4 proteins are used to characterize cytogenetic abnormalities of this chromosomal locale, such as abnormalities that are identified as being associated with various cancers. In certain genes, a variety of chromosomal abnormalities including rearrangements have been identified as frequent cytogenetic abnormalities in a number of different cancers (see e.g. Krajinovic ef at, Mutat. Res. 382(3-4): 81-83 (1998); Johansson ef al., Blood 86(10): 3905-3914 (1995) and Finger ef al., P.N.A.S. 85(23): 9158-9162 (1988)). Thus, polynucleotides encoding specific regions of the 109P1D4 proteins provide new tools that can be used to delineate, with greater precision than previously possible, cytogenetic abnormalities in the chromosomal region that encodes 109P1 D4 that may contribute to the malignant phenotype. In this context, these polynucleotides satisfy a need in the art for expanding the sensitivity of chromosomal screening in order to identify more subtle and less common chromosomal abnormalities (see e.g. Evans ef al., Am. J. Obstet. Gynecol 171(4): 1055-1057 (1994)).
Furthermore, as 109P1D4 was shown to be highly expressed in prostate and other cancers, 109P1D4 polynucleotides are used in methods assessing the status of 109P1D4 gene products in normal versus cancerous tissues. Typically, polynucleotides that encode specific regions of the 109P1D4 proteins are used to assess the presence of perturbations (such as deletions, insertions, point mutations, or alterations resulting in a loss of an antigen etc.) in specific regions of the 109P1D4 gene, such as regions containing one or more motifs. Exemplary assays include both RT-PCR assays as well as single-strand conformation polymorphism (SSCP) analysis (see, e.g., Marrogi et al, J. Cutan. Pathol. 26(8): 369-378 (1999), both of which utilize polynucleotides encoding specific regions of a protein to examine these regions within the protein.
II.A.2.) Antisense Embodiments
Other specifically contemplated nucleic acid related embodiments of the invention disclosed herein are genomic DNA, cDNAs, ribozymes, and antisense molecules, as well as nucleic acid molecules based on an alternative backbone, or including alternative bases, whether derived from natural sources or synthesized, and include molecules capable of inhibiting the RNA or protein expression of 109P1 D4. For example, antisense molecules can be RNAs or other molecules, including peptide nucleic acids (PNAs) or non-nucleic acid molecules such as phosphorothioate derivatives that specifically bind DNA or RNA in a base pair-dependent manner. A skilled artisan can readily obtain these classes of nucleic acid molecules using the 109P1D4 polynucleotides and polynucleotide sequences disclosed herein.
Antisense technology entails the administration of exogenous oligonucleotides that bind to a target polynucleotide located within the cells. The term "antisense" refers to the fact that such oligonucleotides are complementary to their intracellular targets, e.g., 109P1 D4. See for example, Jack Cohen, Oligodeoxynucleotides, Antisense Inhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1:1-5 (1988). The 109P1D4 antisense oligonucleotides of the present invention include derivatives such as S-oligonucleotides (phosphorothioate derivatives or S-oligos, see, Jack Cohen, supra), which exhibit enhanced cancer cell growth inhibitory action. S-oligos (nucleoside phosphorothioates) are isoelectronic analogs of an oligonucleotide (O-oligo) in which a nonbridging oxygen atom of the phosphate group is replaced by a sulfur atom. The S-oligos of the present invention can be prepared by treatment of the corresponding O-oligos with 3H-1.2- benzodithiol-3-one-1,1 -dioxide, which is a sulfur transfer reagent. See, e.g., Iyer, R. P. ef al., J. Org. Chem. 55:4693-4698 (1990); and Iyer, R. P. ef al., J. Am. Chem. Soc. 112:1253-1254 (1990). Additional 109P1D4 antisense oligonucleotides of the present invention include morpholino antisense oligonucleotides known in the art (see, e.g., Partridge ef al., 1996, Antisense & Nucleic Acid Drug Development 6: 169-175).
The 109P1 D4 antisense oligonucleotides of the present invention typically can be RNA or DNA that is complementary to and stably hybridizes with the first 100 5' codons or last 100 3' codons of a 109P1D4 genomic sequence or the corresponding mRNA. Absolute complementarity is not required, although high degrees of complementarity are preferred. Use of an oligonucleotide complementary to this region allows for the selective hybridization to 109P1 D4 mRNA and not to mRNA specifying other regulatory subunits of protein kinase. In one embodiment, 109P1 D4 antisense oligonucleotides of the present invention are 15 to 30-mer fragments of the antisense DNA molecule that have a sequence that hybridizes to 109P1 D4 mRNA. Optionally, 109P1 D4 antisense oligonucleotide is a 30-mer oligonucleotide that is complementary to a region in the first 10 5' codons or last 10 3' codons of 109P1D4. Alternatively, the antisense molecules are modified to employ ribozymes in the inhibition of 109P1 D4 expression, see, e.g., L. A. Couture & D. T. Stinchcomb; Trends Genet 12: 510-515 (1996).
II A3.) Primers and Primer Pairs
Further specific embodiments of these nucleotides of the invention include primers and primer pairs, which allow the specific amplification of polynucleotides of the invention or of any specific parts thereof, and probes that selectively or specifically hybridize to nucleic acid molecules of the invention or to any part thereof. Probes can be labeled with a detectable marker, such as, for example, a radioisotope, fluorescent compound, bioluminescent compound, a chemiluminescent compound, metal chelator or enzyme. Such probes and primers are used to detect the presence of a 109P1D4 polynucleotide in a sample and as a means for detecting a cell expressing a 109P1D4 protein.
Examples of such probes include polypeptides comprising all or part of the human 109P1 D4 cDNA sequence shown in Figure 2. Examples of primer pairs capable of specifically amplifying 109P1D4 mRNAs are also described in the Examples. As will be understood by the skilled artisan, a great many different primers and probes can be prepared based on the sequences provided herein and used effectively to amplify and/or detect a 109P1 D4 mRNA.
The 109P1 D4 polynucleotides of the invention are useful for a variety of purposes, including but not limited to their use as probes and primers for the amplification and/or detection of the 109P1D4 gene(s), mRNA(s), or fragments thereof; as reagents for the diagnosis and/or prognosis of prostate cancer and other cancers; as coding sequences capable of directing the expression of 109P1D4 polypeptides; as tools for modulating or inhibiting the expression of the 109P1D4 gene(s) and/or translation of the 109P1 D4 transcript(s); and as therapeutic agents.
The present invention includes the use of any probe as described herein to identify and isolate a 109P1 D4 or 109P1 D4 related nucleic acid sequence from a naturally occurring source, such as humans or other mammals, as well as the isolated nucleic acid sequence perse, which would comprise all or most of the sequences found in the probe used. II A4.) Isolation of 109P1D4-Encoding Nucleic Acid Molecules
The 109P1 D4 cDNA sequences described herein enable the isolation of other polynucleotides encoding 109P1 D4 gene product(s), as well as the isolation of polynucleotides encoding 109P1D4 gene product homologs, alternatively spliced isoforms, allelic variants, and mutant forms of a 109P1 D4 gene product as well as polynucleotides that encode analogs of 109P1 D4-related proteins. Various molecular cloning methods that can be employed to isolate full length cDNAs encoding a 109P1 D4 gene are well known (see, for example, Sambrook, J. ef al., Molecular Cloning: A Laboratory Manual, 2d edition, Cold Spring Harbor Press, New York, 1989; Current Protocols in Molecular Biology. Ausubel ef a/., Eds., Wiley and Sons, 1995). For example, lambda phage cloning methodologies can be conveniently employed, using commercially available cloning systems (e.g., Lambda ZAP Express, Stratagene). Phage clones containing 109P1D4 gene cDNAs can be identified by probing with a labeled 109P1D4 cDNA or a fragment thereof. For example, in one embodiment, a 109P1 D4 cDNA (e.g., Figure 2) or a portion thereof can be synthesized and used as a probe to retrieve overlapping and full-length cDNAs corresponding to a 109P1D4 gene. A 109P1D4 gene itself can be isolated by screening genomic DNA libraries, bacterial artificial chromosome libraries (BACs), yeast artificial chromosome libraries (YACs), and the like, with 109P1D4 DNA probes or primers.
II A5.) Recombinant Nucleic Acid Molecules and Host-Vector Systems
The invention also provides recombinant DNA or RNA molecules containing a 109P1D4 polynucleotide, a fragment, analog or homologue thereof, including but not limited to phages, plasmids, phagemids, cosmids, YACs, BACs, as well as various viral and non-viral vectors well known in the art, and cells transformed or transfected with such recombinant DNA or RNA molecules. Methods for generating such molecules are well known (see, for example, Sambrook ef al., 1989, supra).
The invention further provides a host-vector system comprising a recombinant DNA molecule containing a 109P1 D4 polynucleotide, fragment, analog or homologue thereof within a suitable prokaryotic or eukaryotic host cell. Examples of suitable eukaryotic host cells include a yeast cell, a plant cell, or an animal cell, such as a mammalian cell or an insect cell (e.g., a baculovirus-infectible cell such as an Sf9 or HighFive cell). Examples of suitable mammalian cells include various prostate cancer cell lines such as DU145 and TsuPrl , other transfectable or transducible prostate cancer cell lines, primary cells (PrEC), as well as a number of mammalian cells routinely used for the expression of recombinant proteins (e.g., COS, CHO, 293, 293T cells). More particularly, a polynucleotide comprising the coding sequence of 109P1D4 or a fragment, analog or homolog thereof can be used to generate 109P1 D4 proteins or fragments thereof using any number of host-vector systems routinely used and widely known in the art.
A wide range of host-vector systems suitable for the expression of 109P1 D4 proteins or fragments thereof are available, see for example, Sambrook etal., 1989, supra; Current Protocols in Molecular Biology, 1995, supra). Preferred vectors for mammalian expression include but are not limited to pcDNA 3.1 myc-His-tag (Invitrogen) and the retroviral vector pSRαtkneo (Muller ef al. , 1991 , MCB 11 : 1785). Using these expression vectors, 109P1 D4 can be expressed in several prostate cancer and non-prostate cell lines, including for example 293, 293T, rat-1, NIH 3T3 and TsuPrl. The host-vector systems of the invention are useful for the production of a 109P1 D4 protein or fragment thereof. Such host-vector systems can be employed to study the functional properties of 109P1D4 and 109P1D4 mutations or analogs.
Recombinant human 109P1 D4 protein or an analog or homolog or fragment thereof can be produced by mammalian cells transfected with a construct encoding a 109P1 D4-related nucleotide. For example, 293T cells can be transfected with an expression plasmid encoding 109P1 D4 or fragment, analog or homolog thereof, a 109P1 D4-related protein is expressed in the 293T cells, and the recombinant 109P1 D4 protein is isolated using standard purification methods (e.g., affinity purification using anti-109P1D4 antibodies). In another embodiment, a 109P1D4 coding sequence is subcloned into the retroviral vector pSRαMSVtkneo and used to infect various mammalian cell lines, such as NIH 3T3, TsuPrl, 293 and rat-1 in order to establish 109P1 D4 expressing cell lines. Various other expression systems well known in the art can also be employed. Expression constructs encoding a leader peptide joined in frame to a 109P1D4 coding sequence can be used for the generation of a secreted form of recombinant 109P1 D4 protein.
As discussed herein, redundancy in the genetic code permits variation in 109P1D4 gene sequences. In particular, it is known in the art that specific host species often have specific codon preferences, and thus one can adapt the disclosed sequence as preferred for a desired host. For example, preferred analog codon sequences typically have rare codons (i.e., codons having a usage frequency of less than about 20% in known sequences of the desired host) replaced with higher frequency codons. Codon preferences for a specific species are calculated, for example, by utilizing codon usage tables available on the INTERNET such as at URL dna.affrc.go.jp/~nakamura/codon.html.
Additional sequence modifications are known to enhance protein expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon/intron splice site signals, transposon-like repeats, and/or other such well-characterized sequences that are deleterious to gene expression. The GC content of the sequence is adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. Where possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures. Other useful modifications include the addition of a translational initiation consensus sequence at the start of the open reading frame, as described in Kozak, Mol. Cell Biol., 9:5073-5080 (1989). Skilled artisans understand that the general rule that eukaryotic ribosomes initiate translation exclusively at the 5' proximal AUG codon is abrogated only under rare conditions (see, e.g., Kozak PNAS 92(7): 2662-2666, (1995) and Kozak NAR 15(20): 8125-8148 (1987)).
III.) 109P1D4-related Proteins
Another aspect of the present invention provides 109P1 D4-related proteins. Specific embodiments of 109P1 D4 proteins comprise a polypeptide having all or part of the amino acid sequence of human 109P1 D4 as shown in Figure 2 or Figure 3. Alternatively, embodiments of 109P1D4 proteins comprise variant, homolog or analog polypeptides that have alterations in the amino acid sequence of 109P1D4 shown in Figure 2 or Figure 3.
Embodiments of a 109P1 D4 polypeptide include: a 109P1 D4 polypeptide having a sequence shown in Figure 2, a peptide sequence of a 109P1 D4 as shown in Figure 2 wherein T is U; at least 10 contiguous nucleotides of a polypeptide having the sequence as shown in Figure 2; or, at least 10 contiguous peptides of a polypeptide having the sequence as shown in Figure 2 where T is U. For example, embodiments of 109P1D4 peptides comprise, without limitation:
(I) a protein comprising, consisting essentially of, or consisting of an amino acid sequence as shown in Figure 2A-I or Figure 3A-I;
(II) a 109P1D4-related protein that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% homologous to an entire amino acid sequence shown in Figure 2A-I or 3A-I;
(III) a 109P1D4-related protein that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to an entire amino acid sequence shown in Figure 2A-1 or 3A-I; N
(IV) a protein that comprises at least one peptide set forth in Tables VIII to XLIX, optionally with a proviso that it is not an entire protein of Figure 2;
(V) a protein that comprises at least one peptide set forth in Tables VIII-XXI, collectively, which peptide is also set forth in Tables XXII to XLIX, collectively, optionally with a proviso that it is not an entire protein of Figure 2;
(VI) a protein that comprises at least two peptides selected from the peptides set forth in Tables VIII-XLIX, optionally with a proviso that it is not an entire protein of Figure 2;
(VII) a protein that comprises at least two peptides selected from the peptides set forth in Tables VIII to XLIX collectively, with a proviso that the protein is not a contiguous sequence from an amino acid sequence of Figure 2;
(VIII) a protein that comprises at least one peptide selected from the peptides set forth in Tables VIII-XXI; and at least one peptide selected from the peptides set forth in Tables XXII to XLIX, with a proviso that the protein is not a contiguous sequence from an amino acid sequence of Figure 2;
(IX) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35 amino acids of a protein of Figure 3A, 3B, 3C, 3D and/or 3E in any whole number increment up to 1021 , 1054, 1347, 1337, and/or 1310 respectively that includes at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of Figure 5;
(X) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35 amino acids of a protein of Figure 3A, 3B, 3C, 3D, and/or 3E, in any whole number increment up to 1021, 1054, 1347, 1337, and/or 1310 respectively respectively that includes at least al least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32. 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of Figure 6;
(XI) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35 amino acids of a protein of Figure 3A, 3B, 3C, 3D, and/or 3E, in any whole number increment up to 1021 , 1054, 1347, 1337, and/or 1310 respectively respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
33. 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of Figure 7;
(XI I) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of Figure 3A, 3B, 3C, 3D, and/or 3E, in any whole number increment up to 1021, 1054, 1347, 1337, and/or 1310 respectively respectively that includes at least at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of Figure 8;
(XIII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, amino acids of a protein of Figure 3A, 3B, 3C, 3D, and 3E in any whole number increment up to 1021, 1054, 1347, 1337, and/or 1310 respectively respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of Figure 9;
(XIV) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35 amino acids of a protein of Figure 3F, 3G, 3H, and/or 31, in any whole number increment up to 1037, 1048, 1340, and/or 1037 respectively that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of Figure 5;
(XV) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of Figure 3F, 3G, 3H, and/or 31 in any whole number increment up to 1037, 1048, 1340, and/or 1037 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of Figure 6;
(XVI) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35 amino acids of a protein of Figure 3F, 3G, 3H, and/or 31 in any whole number increment up to 1037, 1048, 1340, and/or 1037 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of Figure 7;
(XVII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of Figure 3F, 3G, 3H, and/or 31 in any whole number increment up to 1037, 1048, 1340, and/or 1037 respectively that includes at least at least 1, 2, 3, 4, 5, 6, , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of Figure 8;
(XVIII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, amino acids of a protein of Figure 3F, 3G, 3H, and/or 31 in any whole number increment up to 1037, 1048, 1340, and/or 1037 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of Figure 9;
(XIX) a peptide that occurs at least twice in Tables VIII-XXI and XXII to XLIX, collectively;
(XX) a peptide that occurs at least three times in Tables VIII-XXI and XXII to XLIX, collectively;
(XXI) a peptide that occurs at least four times in Tables VIII-XXI and XXII to XLIX, collectively;
(XXII) a peptide that occurs at least five times in Tables VIII-XXI and XXII to XLIX, collectively;
(XXIII) a peptide that occurs at least once in Tables VIII-XXI, and at least once in tables XXII to XLIX;
(XXIV) a peptide that occurs at least once in Tables VIII-XXI, and at least twice in tables XXII to XLIX;
(XXV) a peptide that occurs at least twice in Tables VIII-XXI, and at least once in tables XXII to XLIX;
(XXVI) a peptide that occurs at least twice in Tables VIII-XXI, and at least twice in tables XXII to XLIX;
(XXVII) a peptide which comprises one two, three, four, or five of the following characteristics, or an oligonucleotide encoding such peptide: i) a region of at least 5 amino acids of a particular peptide of Figure 3, in any whole number increment up to the full length of that protein in Figure 3, that includes an amino acid position having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Hydrophilicity profile of Figure 5; ii) a region of at least 5 amino acids of a particular peptide of Figure 3, in any whole number increment up to the full length of that protein in Figure 3, that includes an amino acid position having a value equal to or less than 0.5, 0.4, 0.3, 0.2, 0.1 , or having a value equal to 0.0, in the Hydropathicity profile of Figure 6; iii) a region of at least 5 amino acids of a particular peptide of Figure 3, in any whole number increment up to the full length of that protein in Figure 3, that includes an amino acid position having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Percent Accessible Residues profile of Figure 7; iv) a region of at least 5 amino acids of a particular peptide of Figure 3, in any whole number increment up to the full length of that protein in Figure 3, that includes an amino acid position having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Average Flexibility profile of Figure 8; or, v) a region of at least 5 amino acids of a particular peptide of Figure 3, in any whole number increment up to the full length of that protein in Figure 3, that includes an amino acid position having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Beta-turn profile of Figure 9;;
(XXVIII) a composition comprising a peptide of (l)-(XXVII) or an antibody or binding region thereof together with a pharmaceutical excipient and/or in a human unit dose form. (XXIX) a method of using a peptide of (l)-(XXVII), or an antibody or binding region thereof or a composition of (XXVIII) in a method to modulate a cell expressing 109P1D4,;
(XXX) a method of using a peptide of (l)-(XXVII) or an antibody or binding region thereof or a composition of (XXVIII) in a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressing 109P1D4;
(XXXI) a method of using a peptide of (l)-(XXVII) or an antibody or binding region thereof or a composition (XXVIII) in a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressing 109P1D4, said cell from a cancer of a tissue listed in Table I;
(XXXII) a method of using a peptide of (l)-(XXVII) or an antibody or binding region thereof or a composition of (XXVIII) in a method to diagnose, prophylax, prognose, or treat a a cancer;
(XXXIII) a method of using a peptide of (l)-(XXVII) or an antibody or binding region thereof or a composition of (XXVIII) in a method to diagnose, prophylax, prognose, or treat a a cancer of a tissue listed in Table I; and;
(XXXIV) a method of using a a peptide of (l)-(XXVII) or an antibody or binding region thereof or a composition (XXVIII) in a method to identify or characterize a modulator of a cell expressing 109P1 D4
As used herein, a range is understood to specifically disclose all whole unit positions thereof.
Typical embodiments of the invention disclosed herein include 109P1D4 polynucleotides that encode specific portions of 109P1D4 mRNA sequences (and those which are complementary to such sequences) such as those that encode the proteins and/or fragments thereof, for example:
(a) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1010, 1020, and 1021 or more contiguous amino acids of 109P1D4 variant 1; the maximal lengths relevant for other variants are: variant 2, 1054 amino acids; variant 3, 1347 amino acids, variant 4, 1337 amino acids, variant 5, 1310 amino acids, variant 6; 1037 amino acids, variant 7; 1048 amino acids, variant 8; 1340 amino acids, and variant 9; 1037 amino acids. .
In general, naturally occurring allelic variants of human 109P1 D4 share a high degree of structural identity and homology (e.g., 90% or more homology). Typically, allelic variants of a 109P1 D4 protein contain conservative amino acid substitutions within the 109P1 D4 sequences described herein or contain a substitution of an amino acid from a corresponding position in a homologue of 109P1 D4. One class of 109P1 D4 allelic variants are proteins that share a high degree of homology with at least a small region of a particular 109P1 D4 amino acid sequence, but further contain a radical departure from the sequence, such as a non-conservative substitution, truncation, insertion or frame shift. In comparisons of protein sequences, the terms, similarity, identity, and homology each have a distinct meaning as appreciated in the field of genetics. Moreover, orthology and paralogy can be important concepts describing the relationship of members of a given protein family in one organism to the members of the same family in other organisms.
Amino acid abbreviations are provided in Table II. Conservative amino acid substitutions can frequently be made in a protein without altering either the conformation or the function of the protein. Proteins of the invention can comprise 1 , 2, 3, , 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 conservative substitutions. Such changes include substituting any of isoleucine (I), valine (V), and leucine (L) for any other of these hydrophobic amino acids; aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q) for asparagine (N) and vice versa; and serine (S) for threonine (T) and vice versa. Other substitutions can also be considered conservative, depending on the environment of the particular amino acid and its role in the three- dimensional structure of the protein. For example, glycine (G) and alanine (A) can frequently be interchangeable, as can alanine (A) and valine (V). Methionine (M), which is relatively hydrophobic, can frequently be interchanged with leucine and isoleucine, and sometimes with valine. Lysine (K) and arginine (R) are frequently interchangeable in locations in which the significant feature of the amino acid residue is its charge and the differing pK's of these two amino acid residues are not significant. Still other changes can be considered "conservative" in particular environments (see, e.g. Table III herein; pages 13-15 "Biochemistry" 2"d ED. Lubert Stryer ed (Stanford University); Henikoff ef al., PNAS 1992 Vol 89 10915-10919; Lei ef al., J Biol Chem 1995 May 19; 270(20): 11882-8).
Embodiments of the invention disclosed herein include a wide variety of art-accepted variants or analogs of 109P1D4 proteins such as polypeptides having amino acid insertions, deletions and substitutions. 109P1D4 variants can be made using methods known in the art such as site-directed mutagenesis, alanine scanning, and PCR mutagenesis. Site- directed mutagenesis (Carter et al., Nucl. Acids Res., 73:4331 (1986); Zoller ef al., Nucl. Acids Res., 70:6487 (1987)), cassette mutagenesis (Wells etal., Gene, 34:315 (1985)), restriction selection mutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA, 317:415 (1986)) or other known techniques can be performed on the cloned DNA to produce the 109P1D4 variant DNA.
Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence that is involved in a specific biological activity such as a protein-protein interaction. Among the preferred scanning amino acids are relatively small, neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine. Alanine is typically a preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta- carbon and is less likely to alter the main-chain conformation of the variant. Alanine is also typically preferred because it is the most common amino acid. Further, it is frequently found in both buried and exposed positions (Creighton, 77?e Proteins, (W.H. Freeman & Co., N.Y); Chothia, J. Mol. Biol., 150:1 (1976)). If alanine substitution does not yield adequate amounts of variant, an isosteric amino acid can be used.
As defined herein, 109P1D4 variants, analogs or homologs, have the distinguishing attribute of having at least one epitope that is "cross reactive" with a 109P1D4 protein having an amino acid sequence of Figure 3. As used in this sentence, "cross reactive" means that an antibody or T cell that specifically binds to a 109P1D4 variant also specifically binds to a 109P1 D4 protein having an amino acid sequence set forth in Figure 3. A polypeptide ceases to be a variant of a protein shown in Figure 3, when it no longer contains any epitope capable of being recognized by an antibody or T cell that specifically binds to the starting 109P1D4 protein. Those skilled in the art understand that antibodies that recognize proteins bind to epitopes of varying size, and a grouping of the order of about four or five amino acids, contiguous or not, is regarded as a typical number of amino acids in a minimal epitope. See, e.g., Nair ef al., J. Immunol 2000 165(12): 6949-6955; Hebbes ef al., Mol Immunol (1989) 26(9):865-73; Schwartz ef a/., J Immunol (1985) 135(4):2598-608.
Other classes of 109P1D4-related protein variants share 70%, 75%, 80%, 85% or 90% or more similarity with an amino acid sequence of Figure 3, or a fragment thereof. Another specific class of 109P1 D4 protein variants or analogs comprises one or more of the 109P1D4 biological motifs described herein or presently known in the art. Thus, encompassed by the present invention are analogs of 109P1D4 fragments (nucleic or amino acid) that have altered functional (e.g. immunogenic) properties relative to the starting fragment. It is to be appreciated that motifs now or which become part of the art are to be applied to the nucleic or amino acid sequences of Figure 2 or Figure 3.
As discussed herein, embodiments of the claimed invention include polypeptides containing less than the full amino acid sequence of a 109P1 D4 protein shown in Figure 2 or Figure 3. For example, representative embodiments of the invention comprise peptides/proteins having any 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15 or more contiguous amino acids of a 109P1D4 protein shown in Figure 2 or Figure 3.
Moreover, representative embodiments of the invention disclosed herein include polypeptides consisting of about amino acid 1 to about amino acid 10 of a 109P1 D4 protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 10 to about amino acid 20 of a 109P1D4 prote siirn shown in Figure 2 or Figur •e 3, polypeptides consi ;ing of about amino acid 20 to about amino acid 30 of a 109P1D4 prote siiin shown in Figure 2 or Figun ■e 3. polypeptides consi :ing of about amino acid 30 to about amino acid 40 of a 109P1D4 prote siiin shown in Figure 2 or Figur polypeptides consi :ing of about amino acid 40 to about amino acid 50 of a 109P1D4 prote siiin shown in Figure 2 or Figur e 3; polypeptides consi :ing of about amino acid 50 to about amino acid 60 of a 109P1D4 prote eiirn shown in Figure 2 or Figur ■e 3, polypeptides consi :ing of about amino acid 60 to about amino acid 70 of a 109P1D4 prote siiin shown in Figure 2 or Figur •e 3. polypeptides consi :ing of about amino acid 70 to about amino acid 80 of a 109P1 D4 prote siiin shown in Figure 2 or Figur polypeptides consi :ing of about amino acid 80 to about amino acid 90 of a 109P1 D4 prote siiin shown in Figure 2 or Figur e 3 polypeptides consi ing of about amino acid 90 to about amino acid 100 of a 109P1D4 protein shown in Figure 2 or Figure 3, etc. throughout the entirety of a 109P1 D4 amino acid sequence. Moreover, polypeptides consisting of about amino acid 1 (or 20 or 30 or 40 etc.) to about amino acid 20, (or 130, or 140 or 150 etc.) of a 109P1D4 protein shown in Figure 2 or Figure 3 are embodiments of the invention. It is to be appreciated that the starting and stopping positions in this paragraph refer to the specified position as well as that position plus or minus 5 residues.
109P1D4-related proteins are generated using standard peptide synthesis technology or using chemical cleavage methods well known in the art. Alternatively, recombinant methods can be used to generate nucleic acid molecules that encode a 109P1D4-related protein. In one embodiment, nucleic acid molecules provide a means to generate defined fragments of a 109P1 D4 protein (or variants, homologs or analogs thereof). HLA.) Motif-bearing Protein Embodiments
Additional illustrative embodiments of the invention disclosed herein include 109P1D4 polypeptides comprising the amino acid residues of one or more of the biological motifs contained within a 109P1 D4 polypeptide sequence set forth in Figure 2 or Figure 3. Various motifs are known in the art, and a protein can be evaluated for the presence of such motifs by a number of publicly available Internet sites (see, e.g., URL addresses: pfam.wustl.edu/; searchlauncher.bcm.tmc.edu/seq- search/struc-predict.html; psort.ims.u-tokyo.ac.jp/; cbs.dtu.dk/; ebi.ac.uk/interpro/scan.html; expasy.cn/tools/scnpsit1.html; Epimatrix™ and Epimer™, Brown University, brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html; and BIMAS, bimas.dcrt.nih.gov/.).
Motif bearing subsequences of all 109P1 D4 variant proteins are set forth and identified in Tables VIII-XXI and XXII- XLIX.
Table V sets forth several frequently occurring motifs based on pfam searches (see URL address pfam.wustl.edu/). The columns of Table V list (1) motif name abbreviation, (2) percent identity found amongst the different member of the motif family, (3) motif name or description and (4) most common function; location information is included if the motif is relevant for location.
Polypeptides comprising one or more of the 109P1D4 motifs discussed above are useful in elucidating the specific characteristics of a malignant phenotype in view of the observation that the 109P1 D4 motifs discussed above are associated with growth dysregulation and because 109P1D4 is overexpressed in certain cancers (See, e.g., Table I). Casein kinase II, cAMP and camp-dependent protein kinase, and Protein Kinase C, for example, are enzymes known to be associated with the development of the malignant phenotype (see e.g. Chen ef al., Lab Invest., 78(2): 165-174 (1998); Gaiddon et al., Endocrinology 136(10): 4331-4338 (1995); Hall et al., Nucleic Acids Research 24(6): 1119-1126 (1996); Peterziel et al., Oncogene 18(46): 6322-6329 (1999) and O'Brian, Oncol. Rep. 5(2): 305-309 (1998)). Moreover, both glycosylation and myristoylation are protein modifications also associated with cancer and cancer progression (see e.g. Dennis ef al., Biochem. Biophys. Acta 1473(1):21-34 (1999); Raju ef al., Exp. Cell Res. 235(1): 145-154 (1997)). Amidation is another protein modification also associated with cancer and cancer progression (see e.g. Treston ef al., J. Natl. Cancer Inst. Monogr. (13): 169-175 (1992)).
In another embodiment, proteins of the invention comprise one or more of the immunoreactive epitopes identified in accordance with art-accepted methods, such as the peptides set forth in Tables VIII-XXI and XXII-XLIX. CTL epitopes can be determined using specific algorithms to identify peptides within a 109P1D4 protein that are capable of optimally binding to specified HLA alleles (e.g., Table IV; Epimatrix™ and Epimer™, Brown University, URL brown.edu/Research/TB- HIV_Lab/epimatrix/epimatrix.html; and BIMAS, URL bimas.dcrt.nih.gov/.) Moreover, processes for identifying peptides that have sufficient binding affinity for HLA molecules and which are correlated with being immunogenic epitopes, are well known in the art, and are carried out without undue experimentation. In addition, processes for identifying peptides that are immunogenic epitopes, are well known in the art, and are carried out without undue experimentation either in vitro or in vivo.
Also known in the art are principles for creating analogs of such epitopes in order to modulate immunogenicity. For example, one begins with an epitope that bears a CTL or HTL motif (see, e.g., the HLA Class I and HLA Class II motifs/supermotifs of Table IV). The epitope is analoged by substituting out an amino acid at one of the specified positions, and replacing it with another amino acid specified for that position. For example, on the basis of residues defined in Table IV, one can substitute out a deleterious residue in favor of any other residue, such as a preferred residue; substitute a less- preferred residue with a preferred residue; or substitute an originally-occurring preferred residue with another preferred residue. Substitutions can occur at primary anchor positions or at other positions in a peptide; see,, e.g., Table IV.
A variety of references reflect the art regarding the identification and generation of epitopes in a protein of interest as well as analogs thereof. See, for example, WO 97/33602 to Chesnut ef al.; Sette, Immunogenetics 1999 50(3-4): 201- 212; Sette ef a/., J. Immunol. 200 166(2): 1389-1397; Sidney etal., Hum. Immunol. 199758(1): 12-20; Kondo efa/., Immunogenetics 199745(4): 249-258; Sidney et al, J. Immunol. 1996 157(8): 3480-90; and Falk ef al., Nature 351: 290-6 (1991); Hunt ef a/., Science 255:1261-3 (1992); Parker ef a/., J. Immunol. 149:3580-7 (1992); Parker ef a/., J. Immunol. 152:163-75 (1994)); Kast ef al., 1994 152(8): 3904-12; Borras-Cuesta ef al., Hum. Immunol. 2000 61(3): 266-278; Alexander etal., J. Immunol. 2000 164(3); 164(3): 1625-1633; Alexander et al., PMID: 7895164, Ul: 95202582; O'Sullivan etal., J. Immunol. 1991 147(8): 2663-2669; Alexander ef a/., Immunity 1994 1(9): 751-761 and Alexander ef a/., Immunol. Res. 1998 18(2): 79-92.
Related embodiments of the invention include polypeptides comprising combinations of the different motifs set forth in Table VI, and/or, one or more of the predicted CTL epitopes of Tables VIII-XXI and XXII-XLIX, and/or, one or more of the predicted HTL epitopes of Tables XL I-XLIX, and/or, one or more of the T cell binding motifs known in the art. Preferred embodiments contain no insertions, deletions or substitutions either within the motifs or within the intervening sequences of the polypeptides. In addition, embodiments which include a number of either N-terminal and/or C-terminal amino acid residues on either side of these motifs may be desirable (to, for example, include a greater portion of the polypeptide architecture in which the motif is located). Typically, the number of N-terminal and/or C-terminal amino acid residues on either side of a motif is between about 1 to about 100 amino acid residues, preferably 5 to about 50 amino acid residues.
109P1 D4-related proteins are embodied in many forms, preferably in isolated form. A purified 109P1 D4 protein molecule will be substantially free of other proteins or molecules that impair the binding of 109P1 D4 to antibody, T cell or other ligand. The nature and degree of isolation and purification will depend on the intended use. Embodiments of a 109P1 D4- related proteins include purified 109P1D4-related proteins and functional, soluble 109P1D4-related proteins. In one embodiment, a functional, soluble 109P1D4 protein or fragment thereof retains the ability to be bound by antibody, T cell or other ligand.
The invention also provides 109P1D4 proteins comprising biologically active fragments of a 109P1D4 amino acid sequence shown in Figure 2 or Figure 3. Such proteins exhibit properties of the starting 109P1D4 protein, such as the ability to elicit the generation of antibodies that specifically bind an epitope associated with the starting 109P1 D4 protein; to be bound by such antibodies; to elicit the activation of HTL or CTL; and/or, to be recognized by HTL or CTL that also specifically bind to the starting protein.
109P1 D4-related polypeptides that contain particularly interesting structures can be predicted and/or identified using various analytical techniques well known in the art, including, for example, the methods of Chou-Fasman, Garnier-Robson, Kyte- Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis, or based on i munogenicity. Fragments that contain such structures are particularly useful in generating subunit-specific anti-109P1 D4 antibodies or T cells or in identifying cellular factors that bind to 109P1D4. For example, hydrophilicity profiles can be generated, and immunogenic peptide fragments identified, using the method of Hopp, T.P. and Woods, K.R., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828. Hydropathicity profiles can be generated, and immunogenic peptide fragments identified, using the method of Kyte, J. and Doolittle, R.F., 1982, J. Mol. Biol. 157:105-132. Percent (%) Accessible Residues profiles can be generated, and immunogenic peptide fragments identified, using the method of Janin J., 1979, Nature 277:491-492. Average Flexibility profiles can be generated, and immunogenic peptide fragments identified, using the method of Bhaskaran R., Ponnuswamy P.K., 1988, int. J. Pept. Protein Res. 32:242-255. Beta-turn profiles can be generated, and immunogenic peptide fragments identified, using the method of Deleage, G„ Roux B., 1987, Protein Engineering 1:289-294.
CTL epitopes can be determined using specific algorithms to identify peptides within a 109P1D4 protein that are capable of optimally binding to specified HLA alleles (e.g., by using the SYFPEITHI site at World Wide Web URL syfpeithi.bmi- heidelberg.com/; the listings in Table IV(A)-(E); Epimatrix™ and Epimer™, Brown University, URL (brown.edu/Research/TB- HIV_Lab/epimatrix/epimatrix.html); and BIMAS, URL bimas.dcrt.nih.gov ). Illustrating this, peptide epitopes from 109P1D4 that are presented in the context of human MHC Class I molecules, e.g., HLA-A1, A2, A3, A11, A24, B7 and B35 were predicted (see, e.g., Tables VIII-XXI, XXII-XLIX). Specifically, the complete amino acid sequence of the 109P1D4 protein and relevant portions of other variants, i.e., for HLA Class I predictions 9 flanking residues on either side of a point mutation or exon juction, and for HLA Class II predictions 14 flanking residues on either side of a point mutation or exon junction corresponding to that variant, were entered into the HLA Peptide Motif Search algorithm found in the Bioinformatics and Molecular Analysis Section (BIMAS) web site listed above; in addition to the site SYFPEITHI, at URL syfpeithi.bmi- heidelberg.com/.
The HLA peptide motif search algorithm was developed by Dr. Ken Parker based on binding of specific peptide sequences in the groove of HLA Class I molecules, in particular HLA-A2 (see, e.g., Falk etal., Nature 351: 290-6 (1991); Hunt ef a/., Science 255:1261-3 (1992); Parker ef al., J. Immunol. 149:3580-7 (1992); Parker ef a/., J. Immunol. 152:163-75 (1994)). This algorithm allows location and ranking of 8-mer, 9-mer, and 10-mer peptides from a complete protein sequence for predicted binding to HLA-A2 as well as numerous other HLA Class I molecules. Many HLA class I binding peptides are 8-, 9-, 10 or 11-mers. For example, for Class I HLA-A2, the epitopes preferably contain a leucine (L) or methionine (M) at position 2 and a valine (V) or leucine (L) at the C-terminus (see, e.g., Parker ef at, J. Immunol. 149:3580-7 (1992)). Selected results of 109P1D4 predicted binding peptides are shown in Tables VIII-XXI and XXII-XLIX herein. In Tables VIII- XXI and XXII-XLVII, selected candidates, 9-mers and 10-mers, for each family member are shown along with their location, the amino acid sequence of each specific peptide, and an estimated binding score. In Tables XLVI-XLIX, selected candidates, 15-mers, for each family member are shown along with their location, the amino acid sequence of each specific peptide, and an estimated binding score. The binding score corresponds to the estimated half time of dissociation of complexes containing the peptide at 37°C at pH 6.5. Peptides with the highest binding score are predicted to be the most tightly bound to HLA Class I on the cell surface for the greatest period of time and thus represent the best immunogenic targets for T-cell recognition.
Actual binding of peptides to an HLA allele can be evaluated by stabilization of HLA expression on the antigen- processing defective cell line T2 (see, e.g., Xue etal., Prostate 30:73-8 (1997) and Peshwa etal., Prostate 36:129-38 (1998)). Immunogenicity of specific peptides can be evaluated in vitro by stimulation of CD8+ cytotoxic T lymphocytes (CTL) in the presence of antigen presenting cells such as dendritic cells.
It is to be appreciated that every epitope predicted by the BIMAS site, Epimer™ and Epimatrix™ sites, or specified by the HLA class I or class II motifs available in the art or which become part of the art such as set forth in Table IV (or determined using World Wide Web site URL syfpeithi.bmi-heidelberg.com/, or BIMAS, bimas.dcrt.nih.gov/) are to be "applied" to a 109P1D4 protein in accordance with the invention. As used in this context "applied" means that a 109P1D4 protein is evaluated, e.g., visually or by computer-based patterns finding methods, as appreciated by those of skill in the relevant art. Every subsequence of a 109P1D4 protein of 8, 9, 10, or 11 amino acid residues that bears an HLA Class I motif, or a subsequence of 9 or more amino acid residues that bear an HLA Class II motif are within the scope of the invention.
III.B.) Expression of 109P1 D4-related Proteins
In an embodiment described in the examples that follow, 109P1D4 can be conveniently expressed in cells (such as 293T cells) transfected with a commercially available expression vector such as a CMV-driven expression vector encoding 109P1D4 with a C-terminal 6XHis and MYC tag (pcDNA3.1/mycHIS, Invitrogen or Tagδ, GenHunter Corporation, Nashville TN). The Tag5 vector provides an IgGK secretion signal that can be used to facilitate the production of a secreted 109P1 D4 protein in transfected cells. The secreted HIS-tagged 109P1D4 in the culture media can be purified, e.g., using a nickel column using standard techniques.
III.C.) Modifications of 109P1 D4-related Proteins
Modifications of 109P1 D4-related proteins such as covalent modifications are included within the scope of this invention. One type of covalent modification includes reacting targeted amino acid residues of a 109P1D4 polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues of a 109P1 D4 protein. Another type of covalent modification of a 109P1 D4 polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of a protein of the invention. Another type of covalent modification of 109P1D4 comprises linking a 109P1D4 polypeptide to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.
The 109P1D4-related proteins of the present invention can also be modified to form a chimeric molecule comprising 109P1D4 fused to another, heterologous polypeptide or amino acid sequence. Such a chimeric molecule can be synthesized chemically or recombinantly. A chimeric molecule can have a protein of the invention fused to another tumor- associated antigen or fragment thereof. Alternatively, a protein in accordance with the invention can comprise a fusion of fragments of a 109P1D4 sequence (amino or nucleic acid) such that a molecule is created that is not, through its length, directly homologous to the amino or nucleic acid sequences shown in Figure 2 or Figure 3. Such a chimeric molecule can comprise multiples of the same subsequence of 109P1D4. A chimeric molecule can comprise a fusion of a 109P1D4-related protein with a polyhistidine epitope tag, which provides an epitope to which immobilized nickel can selectively bind, with cytokines or with growth factors. The epitope tag is generally placed at the amino- or carboxyl- terminus of a 109P1 D4 protein. In an alternative embodiment, the chimeric molecule can comprise a fusion of a 109P1 D4-related protein with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule (also referred to as an "immunoadhesin"), such a fusion could be to the Fc region of an IgG molecule. The Ig fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of a 109P1 D4 polypeptide in place of at least one variable region within an Ig molecule. In a preferred embodiment, the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge, CHI, CH2 and CH3 regions of an IgGI molecule. For the production of immunoglobulin fusions see, e.g., U.S. Patent No. 5,428,130 issued June 27, 1995.
III.D.) Uses of 109P1 D4-related Proteins
The proteins of the invention have a number of different specific uses. As 109P1D4 is highly expressed in prostate and other cancers, 109P1D4-related proteins are used in methods that assess the status of 109P1D4 gene products in normal versus cancerous tissues, thereby elucidating the malignant phenotype. Typically, polypeptides from specific regions of a 109P1D4 protein are used to assess the presence of perturbations (such as deletions, insertions, point mutations etc.) in those regions (such as regions containing one or more motifs). Exemplary assays utilize antibodies or T cells targeting 109P1 D4-related proteins comprising the amino acid residues of one or more of the biological motifs contained within a 109P1 D4 polypeptide sequence in order to evaluate the characteristics of this region in normal versus cancerous tissues or to elicit an immune response to the epitope. Alternatively, 109P1D4-related proteins that contain the amino acid residues of one or more of the biological motifs in a 109P1 D4 protein are used to screen for factors that interact with that region of 109P1D4.
109P1D4 protein fragments/subsequences are particularly useful in generating and characterizing domain-specific antibodies (e.g., antibodies recognizing an extracellular or intracellular epitope of a 109P1 D4 protein), for identifying agents or cellular factors that bind to 109P1 D4 or a particular structural domain thereof, and in various therapeutic and diagnostic contexts, including but not limited to diagnostic assays, cancer vaccines and methods of preparing such vaccines.
Proteins encoded by the 109P1 D4 genes, or by analogs, homologs or fragments thereof, have a variety of uses, including but not limited to generating antibodies and in methods for identifying ligands and other agents and cellular constituents that bind to a 109P1D4 gene product. Antibodies raised against a 109P1D4 protein or fragment thereof are useful in diagnostic and prognostic assays, and imaging methodologies in the management of human cancers characterized by expression of 109P1D4 protein, such as those listed in Table I. Such antibodies can be expressed intracellularly and used in methods of treating patients with such cancers. 109P1D4-related nucleic acids or proteins are also used in generating HTL or CTL responses.
Various immunological assays useful for the detection of 109P1 D4 proteins are used, including but not limited to various types of radioimmunoassays, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunofluorescent assays (ELIFA), immunocytochemical methods, and the like. Antibodies can be labeled and used as immunological imaging reagents capable of detecting 109P1D4-expressing cells (e.g., in radioscintigraphic imaging methods). 109P1D4 proteins are also particularly useful in generating cancer vaccines, as further described herein.
IV.) 109P1D4 Antibodies
Another aspect of the invention provides antibodies that bind to 109P1 D4-related proteins. Preferred antibodies specifically bind to a 109P1 D4-related protein and do not bind (or bind weakly) to peptides or proteins that are not 109P1 D4- related proteins under physiological conditions. In this context, examples of physiological conditions include: 1) phosphate buffered saline; 2) Tris-buffered saline containing 25mM Tris and 150 mM NaCI; or normal saline (0.9% NaCI); 4) animal serum such as human serum; or, 5) a combination of any of 1 ) through 4); these reactions preferably taking place at pH 7.5, alternatively in a range of pH 7.0 to 8.0, or alternatively in a range of pH 6.5 to 8.5; also, these reactions taking place at a temperature between 4°C to 37°C. For example, antibodies that bind 109P1D4 can bind 109P1D4-related proteins such as the homologs or analogs thereof.
109P1 D4 antibodies of the invention are particularly useful in cancer (see, e.g., Table I) diagnostic and prognostic assays, and imaging methodologies. Similarly, such antibodies are useful in the treatment, diagnosis, and/or prognosis of other cancers, to the extent 109P1 D4 is also expressed or overexpressed in these other cancers. Moreover, intracellularly expressed antibodies (e.g., single chain antibodies) are therapeutically useful in treating cancers in which the expression of 109P1D4 is involved, such as advanced or metastatic prostate cancers.
The invention also provides various immunological assays useful for the detection and quantification of 109P1 D4 and mutant 109P1 D4-related proteins. Such assays can comprise one or more 109P1 D4 antibodies capable of recognizing and binding a 109P1D4-related protein, as appropriate. These assays are performed within various immunological assay formats well known in the art, including but not limited to various types of radioimmunoassays, enzyme-linked im unosorbent assays (ELISA), enzyme-linked immunofluorescent assays (ELIFA), and the like.
Immunological non-antibody assays of the invention also comprise T cell immunogenicity assays (inhibitory or stimulatory) as well as major histocompatibility complex (MHC) binding assays.
In addition, immunological imaging methods capable of detecting prostate cancer and other cancers expressing 109P1D4 are also provided by the invention, including but not limited to radioscintigraphic imaging methods using labeled 109P1D4 antibodies. Such assays are clinically useful in the detection, monitoring, and prognosis of 109P1D4 expressing cancers such as prostate cancer.
109P1 D4 antibodies are also used in methods for purifying a 109P1 D4-re!ated protein and for isolating 109P1 D4 homologues and related molecules. For example, a method of purifying a 109P1D4-related protein comprises incubating a 109P1 D4 antibody, which has been coupled to a solid matrix, with a lysate or other solution containing a 109P1 D4-related protein under conditions that permit the 109P1 D4 antibody to bind to the 109P1 D4-related protein; washing the solid matrix to eliminate impurities; and eluting the 109P1D4-related protein from the coupled antibody. Other uses of 109P1D4 antibodies in accordance with the invention include generating anti-idiotypic antibodies that mimic a 109P1D4 protein.
Various methods for the preparation of antibodies are well known in the art. For example, antibodies can be prepared by immunizing a suitable mammalian host using a 109P1 D4-related protein, peptide, or fragment, in isolated or immunoconjugated form (Antibodies: A Laboratory Manual, CSH Press, Eds., Harlow, and Lane (1988); Harlow, Antibodies, Cold Spring Harbor Press, NY (1989)). In addition, fusion proteins of 109P1D4 can also be used, such as a 109P1D4 GST-fusion protein. In a particular embodiment, a GST fusion protein comprising all or most of the amino acid sequence of Figure 2 or Figure 3 is produced, then used as an immunogen to generate appropriate antibodies. In another embodiment, a 109P1D4-related protein is synthesized and used as an immunogen.
In addition, naked DNA immunization techniques known in the art are used (with or without purified 109P1 D4-related protein or 109P1D4 expressing cells) to generate an immune response to the encoded immunogen (for review, see Donnelly ef a/., 1997, Ann. Rev. Immunol. 15: 617-648).
The amino acid sequence of a 109P1D4 protein as shown in Figure 2 or Figure 3 can be analyzed to select specific regions of the 109P1 D4 protein for generating antibodies. For example, hydrophobicity and hydrophilicity analyses of a 109P1 D4 amino acid sequence are used to identify hydrophilic regions in the 109P1D4 structure. Regions of a 109P1D4 protein that show immunogenic structure, as well as other regions and domains, can readily be identified using various other methods known in the art, such as Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis. Hydrophilicity profiles can be generated using the method of Hopp, T.P. and Woods, K.R., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824-
3828. Hydropathicity profiles can be generated using the method of Kyte, J. and Doolittle, R.F., 1982, J. Mol. Biol. 157:105-
132. Percent (%) Accessible Residues profiles can be generated using the method of Janin J., 1979, Nature 277:491-492.
Average Flexibility profiles can be generated using the method of Bhaskaran R., Ponnuswamy P.K., 1988, Int. J. Pept.
Protein Res. 32:242-255. Beta-turn profiles can be generated using the method of Deleage, G., Roux B., 1987, Protein
Engineering 1 :289-294. Thus, each region identified by any of these programs or methods is within the scope of the present invention. Methods for the generation of 109P1 D4 antibodies are further illustrated by way of the examples provided herein.
Methods for preparing a protein or polypeptide for use as an immunogen are well known in the art. Also well known in the art are methods for preparing immunogenic conjugates of a protein with a carrier, such as BSA, KLH or other carrier protein. In some circumstances, direct conjugation using, for example, carbodumide reagents are used; in other instances linking reagents such as those supplied by Pierce Chemical Co., Rockford, IL, are effective. Administration of a 109P1D4 immunogen is often conducted by injection over a suitable time period and with use of a suitable adjuvant, as is understood in the art. During the immunization schedule, titers of antibodies can be taken to determine adequacy of antibody formation.
109P1D4 monoclonal antibodies can be produced by various means well known in the art. For example, immortalized cell lines that secrete a desired monoclonal antibody are prepared using the standard hybridoma technology of Kohler and Milstein or modifications that immortalize antibody-producing B cells, as is generally known. Immortalized cell lines that secrete the desired antibodies are screened by immunoassay in which the antigen is a 109P1D4-related protein. When the appropriate immortalized cell culture is identified, the cells can be expanded and antibodies produced either from in vitro cultures or from ascites fluid.
The antibodies or fragments of the invention can also be produced, by recombinant means. Regions that bind specifically to the desired regions of a 109P1 D4 protein can also be produced in the context of chimeric or complementarity- determining region (CDR) grafted antibodies of multiple species origin. Humanized or human 109P1 D4 antibodies can also be produced, and are preferred for use in therapeutic contexts. Methods for humanizing murine and other non-human antibodies, by substituting one or more of the non-human antibody CDRs for corresponding human antibody sequences, are well known (see for example, Jones etal, 1986, Nature 321: 522-525; Riechmann ef al., 1988, Nature 332: 323-327; Verhoeyen ef al., 1988, Science 239: 1534-1536). See also, Carter ef al., 1993, Proc. Natl. Acad. Sci. USA 89: 4285 and Sims ef al., 1993, J. Immunol. 151: 2296.
Methods for producing fully human monoclonal antibodies include phage display and transgenic methods (for review, see Vaughan ef al, 1998, Nature Biotechnology 16: 535-539). Fully human 109P1D4 monoclonal antibodies can be generated using cloning technologies employing large human Ig gene combinatorial libraries (i.e., phage display) (Griffiths and Hoogenboom, Building an in vitro immune system: human antibodies from phage display libraries. In: Protein Engineering of Antibody Molecules for Prophylactic and Therapeutic Applications in Man, Clark, M. (Ed.), Nottingham Academic, pp 45-64 (1993); Burton and Barbas, Human Antibodies from combinatorial libraries. Id-, PP 65-82). Fully human 109P1D4 monoclonal antibodies can also be produced using transgenic mice engineered to contain human immunoglobulin gene loci as described in PCT Patent Application W098/24893, Kucherlapati and Jakobovits etal., published December 3, 1997 (see also, Jakobovits, 1998, Exp. Opin. Invest. Drugs 7(4): 607-614; U.S. patents 6,162,963 issued 19 December 2000; 6,150,584 issued 12 November 2000; and, 6,114698 issued δ September 2000). This method avoids the in vitro manipulation required with phage display technology and efficiently produces high affinity authentic human antibodies.
Reactivity of 109P1 D4 antibodies with a 109P1 D4-related protein can be established by a number of well known means, inciuding Western blot, i munoprecipitation, ELISA, and FACS analyses using, as appropriate, 109P1D4-related proteins, 109P1 D4-expressing cells or extracts thereof. A 109P1 D4 antibody or fragment thereof can be labeled with a detectable marker or conjugated to a second molecule. Suitable detectable markers include, but are not limited to, a radioisotope, a fluorescent compound, a bioluminescent compound, chemiluminescent compound, a metal chelator or an enzyme. Further, bi-specific antibodies specific for two or more 109P1 D4 epitopes are generated using methods generally known in the art. Homodimeric antibodies can also be generated by cross-linking techniques known in the art (e.g., Wolff ef al., Cancer Res. δ3: 2560-2566).
V.) 109P1 D4 Cellular Immune Responses
The mechanism by which T cells recognize antigens has been delineated. Efficacious peptide epitope vaccine compositions of the invention induce a therapeutic or prophylactic immune responses in very broad segments of the worldwide population. For an understanding of the value and efficacy of compositions of the invention that induce cellular immune responses, a brief review of immunology-related technology is provided.
A complex of an HLA molecule and a peptidic antigen acts as the ligand recognized by HLA-restricted T cells (Buus, S. et al., Cell 47:1071, 1986; Babbitt, B. P. ef al., Nature 317:359, 1985; Townsend, A. and Bodmer, H., Annu. Rev. Immunol. 7:601, 1989; Germain, R. N., Annu. Rev. Immunol. 11:403, 1993). Through the study of single amino acid substituted antigen analogs and the sequencing of endogenously bound, naturally processed peptides, critical residues that correspond to motifs required for specific binding to HLA antigen molecules have been identified and are set forth in Table IV (see also, e.g., Southwood, ef al., J. Immunol. 160:3363, 1998; Rammensee, et al., Immunogenetics 41:178, 1995; Rammensee et al., SYFPEITHI, access via World Wide Web at URL (134.2.96.221/scripts.hlaserver.dll/home.htm); Sette, A. and Sidney, J. Curr. Opin. Immunol. 10:478, 1998; Engelhard, V. H., Curr. Opin. Immunol. 6:13, 1994; Sette, A. and Grey, H. M., Curr. Opin. Immunol. 4:79, 1992; Sinigaglia, F. and Hammer, J. Curr. Biol. 6:52, 1994; Ruppert et al, Cell 74:929-937, 1993; Kondo et al., J. Immunol. 155:4307-4312, 1995; Sidney et al., J. Immunol. 157:3480-3490, 1996; Sidney et al., Human Immunol. 45:79-93, 1996; Sette, A. and Sidney, J. Immunogenetics 1999 Nov; 50(3-4) :201 -12, Review).
Furthermore, x-ray crystallographic analyses of HLA-peptide complexes have revealed pockets within the peptide binding cleft/groove of HLA molecules which accommodate, in an allele-specific mode, residues borne by peptide ligands; these residues in turn determine the HLA binding capacity of the peptides in which they are present. (See, e.g., Madden, D.R. Annu. Rev. Immunol. 13:587, 1995; Smith, ef al., Immunity 4:203, 1996; Fremont ef a/., Immunity 8:305, 1998; Stern ef al., Structure 2:245, 1994; Jones, E.Y. Curr. Opin. Immunol. 9:75, 1997; Brown, J. H. et al, Nature 364:33, 1993; Guo, H. C. et al., Proc. Natl. Acad. Sci. USA 90:8053, 1993; Guo, H. C. ef al., Nature 360:364, 1992; Silver, M. L et al., Nature 360:367, 1992; Matsumura, M. ef al., Science 257:927, 1992; Madden et al., Cell 70:1035, 1992; Fremont, D. H. et al, Science 257:919, 1992; Saper, M. A. , Bjorkman, P. J. and Wiley, D. C, J. Mol. Biol. 219:277, 1991.)
Accordingly, the definition of class I and class II allele-specific HLA binding motifs, or class I or class II supermotifs allows identification of regions within a protein that are correlated with binding to particular HLA antigen(s).
Thus, by a process of HLA motif identification, candidates for epitope-based vaccines have been identified; such candidates can be further evaluated by HLA-peptide binding assays to determine binding affinity and/or the time period of association of the epitope and its corresponding HLA molecule. Additional confirmatory work can be performed to select, amongst these vaccine candidates, epitopes with preferred characteristics in terms of population coverage, and/or immunogenicity.
Various strategies can be utilized to evaluate cellular immunogenicity, including:
1) Evaluation of primary T cell cultures from normal individuals (see, e.g., Wentworth, P. A. ef al., Mol. Immunol. 32:603, 1995; Celis, E. ef al., Proc. Natl. Acad. Sci. USA 91 :2105, 1994; Tsai, V. et al., J. Immunol. 158:1796, 1997; Kawashima, I. ef al., Human Immunol. 59:1 , 1998). This procedure involves the stimulation of peripheral blood lymphocytes (PBL) from normal subjects with a test peptide in the presence of antigen presenting cells in vitro over a period of several weeks. T cells specific for the peptide become activated during this time and are detected using, e.g., a lymphokine- or 51Cr-release assay involving peptide sensitized target cells.
2) Immunization of HLA transgenic mice (see, e.g., Wentworth, P. A. et al., J. Immunol. 26;97, 1996; Wentworth, P. A. etal., Int. Immunol. 8:651, 1996; Alexander, J. etal., J. Immunol. 159:4753, 1997). For example, in such methods peptides in incomplete Freund's adjuvant are administered subcutaneously to HLA transgenic mice. Several weeks following immunization, splenocytes are removed and cultured in vitro in the presence of test peptide for approximately one week.
Peptide-specific T cells are detected using, e.g., a 51 Cr-release assay involving peptide sensitized target cells and target cells expressing endogenously generated antigen.
3) Demonstration of recall T cell responses from immune individuals who have been either effectively vaccinated and/or from chronically ill patients (see, e.g., Rehermann, B. ef a/., J. Exp. Med. 181:1047, 1995; Doolan, D. L. ef al., Immunity 7:97, 1997; Bertoni, R. et al., J. Clin. invest 100:503, 1997; Threlkeld, S. C. et al, J. Immunol 159:1648, 1997; Diepolder, H. M. etal., J. Virol. 71 :6011, 1997). Accordingly, recall responses are detected by culturing PBL from subjects that have been exposed to the antigen due to disease and thus have generated an immune response "naturally", or from patients who were vaccinated against the antigen. PBL from subjects are cultured in vitro for 1-2 weeks in the presence of test peptide plus antigen presenting cells (APC) to allow activation of "memory" T cells, as compared to "naive" T cells. At the end of the culture period, T cell activity is detected using assays including 51 Cr release in olving peptide-sensitized targets, T cell proliferation, or lymphokine release.
VI.) 109P1 D4 Transgenic Animals
Nucleic acids that encode a 109P1D4-related protein can also be used to generate either transgenic animals or "knock out" animals that, in turn, are useful in the development and screening of therapeutically useful reagents. In accordance with established techniques, cDNA encoding 109P1 D4 can be used to clone genomic DNA that encodes 109P1D4. The cloned genomic sequences can then be used to generate transgenic animals containing cells that express DNA that encode 109P1 D4. Methods for generating transgenic animals, particularly animals such as mice or rats, have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866 issued 12 April 1988, and 4,870,009 issued 26 September 1989. Typically, particular cells would be targeted for 109P1D4 transgene incorporation with tissue-specific enhancers.
Transgenic animals that include a copy of a transgene encoding 109P1 D4 can be used to examine the effect of increased expression of DNA that encodes 109P1D4. Such animals can be used as tester animals for reagents thought to confer protection from, for example, pathological conditions associated with its overexpression. In accordance with this aspect of the invention, an animal is treated with a reagent and a reduced incidence of a pathological condition, compared to untreated animals that bear the transgene, would indicate a potential therapeutic intervention for the pathological condition.
Alternatively, non-human homologues of 109P1 D4 can be used to construct a 109P1 D4 "knock out" animal that has a defective or altered gene encoding 109P1 D4 as a result of homologous recombination between the endogenous gene encoding 109P1D4 and altered genomic DNA encoding 109P1D4 introduced into an embryonic cell of the animal. For example, cDNA that encodes 109P1 D4 can be used to clone genomic DNA encoding 109P1 D4 in accordance with established techniques. A portion of the genomic DNA encoding 109P1D4 can be deleted or replaced with another gene, such as a gene encoding a selectable marker that can be used to monitor integration. Typically, several kilobases of unaltered flanking DNA (both at the 5' and 3' ends) are included in the vector (see, e.g., Thomas and Capecchi, Cejl, δ .:503 (1987) for a description of homologous recombination vectors). The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced DNA has homologously reco bined with the endogenous DNA are selected (see, e.g., Li ef al, Cell, 69:915 (1992)). The selected cells are then injected into a blastocyst of an animal
(e.g., a mouse or rat) to form aggregation chimeras (see, e.g., Bradley, in Teratocarcinomas and Embryonic Stem Cells: A
Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152). A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal, and the embryo brought to term to create a "knock out" animal. Progeny harboring the homologously recombined DNA in their germ cells can be identified by standard techniques and used to breed animals in which all cells of the animal contain the homologously recombined DNA. Knock out animals can be characterized, for example, for their ability to defend against certain pathological conditions or for their development of pathological conditions due to absence of a 109P1D4 polypeptide.
VII.) Methods for the Detection of 109P1 4
Another aspect of the present invention relates to methods for detecting 109P1D4 polynucleotides and 109P1D4- related proteins, as well as methods for identifying a cell that expresses 109P1 D4. The expression profile of 109P1 D4 makes it a diagnostic marker for metastasized disease. Accordingly, the status of 109P1 D4 gene products provides information useful for predicting a variety of factors including susceptibility to ad anced stage disease, rate of progression, and/or tumor aggressiveness. As discussed in detail herein, the status of 109P1D4 gene products in patient samples can be analyzed by a variety protocols that are well known in the art including immunohistochemical analysis, the variety of Northern blotting techniques including in situ hybridization, RT-PCR analysis (for example on laser capture micro-dissected samples), Western blot analysis and tissue array analysis.
More particularly, the invention provides assays for the detection of 109P1D4 polynucleotides in a biological sample, such as serum, bone, prostate, and other tissues, urine, semen, cell preparations, and the like. Detectable 109P1D4 polynucleotides include, for example, a 109P1 D4 gene or fragment thereof, 109P1 D4 mRNA, alternative splice variant 109P1 D4 mRNAs, and recombinant DNA or RNA molecules that contain a 109P1 D4 polynucleotide. A number of methods for amplifying and/or detecting the presence of 109P1 D4 polynucleotides are well known in the art and can be employed in the practice of this aspect of the invention.
In one embodiment, a method for detecting a 109P1 D4 mRNA in a biological sample comprises producing cDNA from the sample by reverse transcription using at least one primer; amplifying the cDNA so produced using a 109P1 D4 polynucleotides as sense and antisense primers to amplify 109P1D4 cDNAs therein; and detecting the presence of the amplified 109P1D4 cDNA. Optionally, the sequence of the amplified 109P1D4 cDNA can be determined.
In another embodiment, a method of detecting a 109P1 D4 gene in a biological sample comprises first isolating genomic DNA from the sample; amplifying the isolated genomic DNA using 109P1 D4 polynucleotides as sense and antisense primers; and detecting the presence of the amplified 109P1 D4 gene. Any number of appropriate sense and antisense probe combinations can be designed from a 109P1D4 nucleotide sequence (see, e.g., Figure 2) and used for this purpose.
The invention also provides assays for detecting the presence of a 109P1 D4 protein in a tissue or other biological sample such as serum, semen, bone, prostate, urine, cell preparations, and the like. Methods for detecting a 109P1D4-related protein are also well known and include, for example, immunoprecipitation, immunohistochemical analysis, Western blot analysis, molecular binding assays, ELISA, ELIFA and the like. For example, a method of detecting the presence of a 109P1 D4-related protein in a biological sample comprises first contacting the sample with a 109P1 D4 antibody, a 109P1 D4-reactive fragment thereof, or a recombinant protein containing an antigen-binding region of a 109P1 D4 antibody; and then detecting the binding of 109P1D4-related protein in the sample.
Methods for identifying a cell that expresses 109P1 D4 are also within the scope of the invention. In one embodiment, an assay for identifying a cell that expresses a 109P1 D4 gene comprises detecting the presence of 109P1 D4 mRNA in the cell. Methods for the detection of particular mRNAs in cells are well known and include, for example, hybridization assays using complementary DNA probes (such as in situ hybridization using labeled 109P1D4 riboprobes, Northern blot and related techniques) and various nucleic acid amplification assays (such as RT-PCR using complementary primers specific for 109P1 D4, and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like). Alternatively, an assay for identifying a cell that expresses a 109P1 D4 gene comprises delecting the presence of 109P1 D4-related protein in the cell or secreted by the cell. Various methods for the detection of proteins are well known in the art and are employed for the detection of 109P1 D4-related proteins and cells that express 109P1D4-related proteins.
109P1 D4 expression analysis is also useful as a tool for identifying and evaluating agents that modulate 109P1 D4 gene expression. For example, 109P1 D4 expression is significantly upregulated in prostate cancer, and is expressed in cancers of the tissues listed in Table I. Identification of a molecule or biological agent that inhibits 109P1 D4 expression or over- expression in cancer cells is of therapeutic value. For example, such an agent can be identified by using a screen that quantifies 109P1D4 expression by RT-PCR, nucleic acid hybridization or antibody binding.
VIII.) Methods for Monitoring the Status of 109P1D4-related Genes and Their Products
Oncogenesis is known to be a multistep process where cellular growth becomes progressively dysregulated and cells progress from a normal physiological state to precancerous and then cancerous states (see, e.g., Alers ef al, Lab Invest. 77(5): 437-438 (1997) and Isaacs ef al, Cancer Surv. 23: 19-32 (1995)). In this context, examining a biological sample for evidence of dysregulated cell growth (such as aberrant 109P1 D4 expression in cancers) allows for early detection of such aberrant physiology, before a pathologic state such as cancer has progressed to a stage that therapeutic options are more limited and or the prognosis is worse. In such examinations, the status of 109P1 D4 in a biological sample of interest can be compared, for example, to the status of 109P1D4 in a corresponding normal sample (e.g. a sample from that individual or alternatively another individual that is not affected by a pathology). An alteration in the status of 109P1D4 in the biological sample (as compared to the normal sample) provides evidence of dysregulated cellular growth. In addition to using a biological sample that is not affected by a pathology as a normal sample, one can also use a predetermined normative value such as a predetermined normal level of mRNA expression (see, e.g., Grever ef al., J. Comp. Neurol. 1996 Dec 9; 376(2): 306-14 and U.S. Patent No. 5,837,601) to compare 109P1D4 status in a sample.
The term "status" in this context is used according to its art accepted meaning and refers to the condition or state of a gene and its products. Typically, skilled artisans use a number of parameters to evaluate the condition or state of a gene and its products. These include, but are not limited to the location of expressed gene products (including the location of 109P1 D4 expressing cells) as well as the level, and biological activity of expressed gene products (such as 109P1D4 mRNA, polynucleotides and polypeptides). Typically, an alteration in the status of 109P1D4 comprises a change in the location of 109P1D4 and/or 109P1D4 expressing cells and/or an increase in 109P1D4 mRNA and/or protein expression.
109P1 D4 status in a sample can be analyzed by a number of means well known in the art, including without limitation, immunohistochemical analysis, in situ hybridization, RT-PCR analysis on laser capture micro-dissected samples, Western blot analysis, and tissue array analysis. Typical protocols for evaluating the status of a 109P1 D4 gene and gene products are found, for example in Ausubel et al. eds., 1996, Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4 (Southern Blotting), 1δ (Immunoblotting) and 18 (PCR Analysis). Thus, the status of 109P1D4 in a biological sample is evaluated by various methods utilized by skilled artisans including, but not limited to genomic Southern analysis (to examine, for example perturbations in a 109P1D4 gene), Northern analysis and/or PCR analysis of 109P1D4 mRNA (to examine, for example alterations in the polynucleotide sequences or expression levels of 109P1D4 mRNAs), and, Western and/or immunohistochemical analysis (to examine, for example alterations in polypeptide sequences, alterations in polypeptide localization within a sample, alterations in expression levels of 109P1D4 proteins and/or associations of 109P1D4 proteins with polypeptide binding partners). Detectable 109P1D4 polynucleotides include, for example, a 109P1D4 gene or fragment thereof, 109P1 D4 mRNA, alternative splice variants, 109P1 D4 mRNAs, and recombinant DNA or RNA molecules containing a 109P1D4 polynucleotide.
The expression profile of 109P1 D4 makes it a diagnostic marker for local and/or melastasized disease, and provides information on the growth or oncogenic potential of a biological sample. In particular, the status of 109P1 D4 provides information useful for predicting susceptibility to particular disease stages, progression, and/or tumor aggressiveness. The invention provides methods and assays for determining 109P1 D4 status and diagnosing cancers that express 109P1 D4, such as cancers of the tissues listed in Table I. For example, because 109P1 D4 mRNA is so highly expressed in prostate and other cancers relative to normal prostate tissue, assays that evaluate the levels of 109P1D4 mRNA transcripts or proteins in a biological sample can be used to diagnose a disease associated with 109P1D4 dysregulation, and can provide prognostic information useful in defining appropriate therapeutic options.
The expression status of 109P1D4 provides information including the presence, stage and location of dysplastic, precancerous and cancerous cells, predicting susceptibility to various stages of disease, and/or for gauging tumor aggressiveness. Moreover, the expression profile makes it useful as an imaging reagent for metastasized disease. Consequently, an aspect of the invention is directed to the various molecular prognostic and diagnostic methods for examining the status of 109P1 D4 in biological samples such as those from individuals suffering from, or suspected of suffering from a pathology characterized by dysregulated cellular growth, such as cancer.
As described above, the status of 109P1D4 in a biological sample can be examined by a number of well-known procedures in the art. For example, the status of 109P1D4 in a biological sample taken from a specific location in the body can be examined by evaluating the sample for the presence or absence of 109P1 D4 expressing cells (e.g. those that express 109P1 D4 mRNAs or proteins). This examination can provide evidence of dysregulated cellular growth, for example, when 109P1D4-expressing cells are found in a biological sample that does not normally contain such cells (such as a lymph node), because such alterations in the status of 109P1D4 in a biological sample are often associated with dysregulated cellular growth. Specifically, one indicator of dysregulated cellular growth is the metastases of cancer cells from an organ of origin (such as the prostate) to a different area of the body (such as a lymph node). In this context, evidence of dysregulated cellular growth is important for example because occult lymph node metastases can be detected in a substantial proportion of patients with prostate cancer, and such metastases are associated with known predictors of disease progression (see, e.g., Murphy ef a/., Prostate 42(4): 316-317 (2000);Su ef al, Semin. Surg. Oncol. 18(1): 17-28 (2000) and Freeman et al, J Urol 1995 Aug 154(2 Pt 1):474-8).
In one aspect, the invention provides methods for monitoring 109P1D4 gene products by determining the status of 109P1D4 gene products expressed by cells from an individual suspected of having a disease associated with dysregulated cell growth (such as hyperplasia or cancer) and then comparing the status so determined to the status of 109P1D4 gene products in a corresponding normal sample. The presence of aberrant 109P1 D4 gene products in the test sample relative to the normal sample provides an indication of the presence of dysregulated cell growth within the cells of the individual.
In another aspect, the invention provides assays useful in determining the presence of cancer in an individual, comprising detecting a significant increase in 109P1D4 mRNA or protein expression in a test cell or tissue sample relative to expression levels in the corresponding normal cell or tissue. The presence of 109P1 D4 mRNA can, for example, be evaluated in tissues including but not limited to those listed in Table I. The presence of significant 109P1D4 expression in any of these tissues is useful to indicate the emergence, presence and/or severity of a cancer, since the corresponding normal tissues do not express 109P1 D4 mRNA or express it at lower levels.
In a related embodiment, 109P1 D4 status is determined at the protein level rather than at the nucleic acid level. For example, such a method comprises determining the level of 109P1 D4 protein expressed by cells in a test tissue sample and comparing the level so determined to the level of 109P1D4 expressed in a corresponding normal sample. In one embodiment, the presence of 109P1D4 protein is evaluated, for example, using immunohistochemical methods. 109P1D4 antibodies or binding partners capable of detecting 109P1 D4 protein expression are used in a variety of assay formats well known in the art for this purpose.
In a further embodiment, one can evaluate the status of 109P1 D4 nucleotide and amino acid sequences in a biological sample in order to identify perturbations in the structure of these molecules. These perturbations can include insertions, deletions, substitutions and the like. Such evaluations are useful because perturbations in the nucleotide and amino acid sequences are observed in a large number of proteins associated with a growth dysregulated phenotype (see, e.g., Marrogi ef al, 1999, J. Cutan. Pathol. 26(8):369-378). For example, a mutation in the sequence of 109P1D4 may be indicative of the presence or promotion of a tumor. Such assays therefore have diagnostic and predictive value where a mutation in 109P1 D4 indicates a potential loss of function or increase in tumor growth.
A wide variety of assays for observing perturbations in nucleotide and amino acid sequences are well known in the art. For example, the size and structure of nucleic acid or amino acid sequences of 109P1 D4 gene products are observed by the Northern, Southern, Western, PCR and DNA sequencing protocols discussed herein. In addition, other methods for observing perturbations in nucleotide and amino acid sequences such as single strand conformation polymorphism analysis are well known in the art (see, e.g., U.S. Patent Nos. 5,382,610 issued 7 September 1999, and 6,962,170 issued 17 January 1995).
Additionally, one can examine the methylation status of a 109P1D4 gene in a biological sample. Aberrant demethylation and/or hypermethylation of CpG islands in gene 5' regulatory regions frequently occurs in immortalized and transformed cells, and can result in altered expression of various genes. For example, promoter hypermethylation of the pi-class glutathione S-transferase (a protein expressed in normal prostate but not expressed in >90% of prostate carcinomas) appears to permanently silence transcription of this gene and is the most frequently detected genomic alteration in prostate carcinomas (De Marzo et al, Am. J. Pathol. 155(6): 1985-1992 (1999)). In addition, this alteration is present in at least 70% of cases of high-grade prostatic intraepithelial neoplasia (PIN) (Brooks et al, Cancer Epidemiol. Biomarkers Prev., 1998, 7:631-636). In another example, expression of the LAGE-I tumor specific gene (which is not expressed in normal prostate but is expressed in 2δ-δ0% of prostate cancers) is induced by deoxy-azacytidine in lymphoblastoid cells, suggesting that tumoral expression is due to demethylation (Lethe ef al, Int. J. Cancer 76(6): 903-908 (1998)). A variety of assays for examining methylation status of a gene are well known in the art. For example, one can utilize, in Southern hybridization approaches, methylation-sensitive restriction enzymes that cannot cleave sequences that contain methylated CpG sites to assess the methylation status of CpG islands. In addition, MSP (methylation specific PCR) can rapidly profile the methylation status of all the CpG sites present in a CpG island of a given gene. This procedure involves initial modification of DNA by sodium bisulfite (which will convert all unmethylated cytosines to uracil) followed by amplification using primers specific for methylated versus unmethylated DNA, Protocols involving methylation interference can also be found for example in Current Protocols In Molecular Biology, Unit 12, Frederick M. Ausubel ef al. eds., 1996.
Gene amplification is an additional method for assessing the status of 109P1D4. Gene amplification is measured in a sample directly, for example, by conventional Southern blotting or Northern blotting to quantitate the transcription of mRNA (Thomas, 1980, Proc. Natl. Acad. Sci. USA, 77:5201-5205), dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, antibodies are employed that recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn are labeled and the assay carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.
Biopsied tissue or peripheral blood can be conveniently assayed for the presence of cancer cells using for example, Northern, dot blot or RT-PCR analysis to detect 109P1 D4 expression. The presence of RT-PCR amplifiable 109P1 D4 mRNA provides an indication of the presence of cancer. RT-PCR assays are well known in the art. RT-PCR detection assays for tumor cells in peripheral blood are currently being evaluated for use in the diagnosis and management of a number of human solid tumors, in the prostate cancer field, these include RT-PCR assays for the detection of cells expressing PSA and PSM (Verkaik ef al, 1997, Urol. Res.25:373-384; Ghossein ef al, 1995, J. Clin. Oncol. 13:1195-2000; Heston ef al, 1995, Clin. Chem.41:1687- 1688).
A further aspect of the invention is an assessment of the susceptibility that an individual has for developing cancer. In one embodiment, a method for predicting susceptibility to cancer comprises detecting 109P1 D4 mRNA or 109P1 D4 protein in a tissue sample, its presence indicating susceptibility to cancer, wherein the degree of 109P1 D4 mRNA expression correlates to the degree of susceptibility, in a specific embodiment, the presence of 109P1 D4 in prostate or other tissue is examined, with the presence of 109P1 D4 in the sample providing an indication of prostate cancer susceptibility (or the emergence or existence of a prostate tumor). Similarly, one can evaluate the integrity 109P1 D4 nucleotide and amino acid sequences in a biological sample, in order to identify perturbations in the structure of these molecules such as insertions, deletions, substitutions and the like. The presence of one or more perturbations in 109P1 D4 gene products in the sample is an indication of cancer susceptibility (or the emergence or existence of a tumor).
The invention also comprises methods for gauging tumor aggressiveness. In one embodiment, a method for gauging aggressiveness of a tumor comprises determining the level of 109P1 D4 mRNA or 109P1 D4 protein expressed by tumor cells, comparing the level so determined to the level of 109P1 D4 mRNA or 109P1 D4 protein expressed in a corresponding normal tissue taken from the same individual or a normal tissue reference sample, wherein the degree of 109P1D4 mRNA or 109P1D4 protein expression in the tumor sample relative to the normal sample indicates the degree of aggressiveness. In a specific embodiment, aggressiveness of a tumor is evaluated by determining the extent to which 109P1 D4 is expressed in the tumor cells, with higher expression levels indicating more aggressive tumors. Another embodiment is the evaluation of the integrity of 109P1 D4 nucleotide and amino acid sequences in a biological sample, in order to identify perturbations in the structure of these molecules such as insertions, deletions, substitutions and the like. The presence of one or more perturbations indicates more aggressive tumors.
Another embodiment of the invention is directed to methods for observing the progression of a malignancy in an individual over time. In one embodiment, methods for observing the progression of a malignancy in an individual over time comprise determining the level of 109P1D4 mRNA or 109P1D4 protein expressed by cells in a sample of the tumor, comparing the level so determined to the level of 109P1 D4 mRNA or 109P1 D4 protein expressed in an equivalent tissue sample taken from the same individual at a different time, wherein the degree of 109P1 D4 mRNA or 109P1 D4 protein expression in the tumor sample over time provides information on the progression of the cancer. In a specific embodiment, the progression of a cancer is evaluated by determining 109P1 D4 expression in the tumor cells over time, where increased expression over time indicates a progression of the cancer. Also, one can evaluate the integrity 109P1D4 nucleotide and amino acid sequences in a biological sample in order to identify perturbations in the structure of these molecules such as insertions, deletions, substitutions and the like, where the presence of one or more perturbations indicates a progression of the cancer.
The above diagnostic approaches can be combined with any one of a wide variety of prognostic and diagnostic protocols known in the art. For example, another embodiment of the invention is directed to methods for observing a coincidence between the expression of 109P1 D4 gene and 109P1 D4 gene products (or perturbations in 109P1 D4 gene and 109P1 D4 gene products) and a factor that is associated with malignancy, as a means for diagnosing and prognosticating the status of a tissue sample. A wide variety of factors associated with malignancy can be utilized, such as the expression of genes associated with malignancy (e.g. PSA, PSCA and PSM expression for prostate cancer etc.) as well as gross cytological observations (see, e.g., Booking etal, 1984, Anal. Quant. Cytol. 6(2):74-88; Epstein, 1995, Hum. Pathol. 26(2):223-9; Thorson et al, 1998, Mod. Pathol. 11(6):543-δ1; Baisden et al, 1999, Am. J. Surg. Pathol. 23(8):918-24). Methods for observing a coincidence between the expression of 109P1D4 gene and 109P1D4 gene products (or perturbations in 109P1D4 gene and 109P1D4 gene products) and another factor that is associated with malignancy are useful, for example, because the presence of a set of specific factors that coincide with disease provides information crucial for diagnosing and prognosticating the status of a tissue sample.
In one embodiment, methods for observing a coincidence between the expression of 109P1 D4 gene and 109P1 D4 gene products (or perturbations in 109P1D4 gene and 109P1D4 gene products) and another factor associated with malignancy entails detecting the overexpression of 109P1D4 mRNA or protein in a tissue sample, detecting the overexpression of PSA mRNA or protein in a tissue sample (or PSCA or PSM expression), and observing a coincidence of 109P1 D4 mRNA or protein and PSA mRNA or protein overexpression (or PSCA or PSM expression). In a specific embodiment, the expression of 109P1 D4 and PSA mRNA in prostate tissue is examined, where the coincidence of 109P1D4 and PSA mRNA overexpression in the sample indicates the existence of prostate cancer, prostate cancer susceptibility or the emergence or status of a prostate tumor.
Methods for detecting and quantifying the expression of 109P1 D4 mRNA or protein are described herein, and standard nucleic acid and protein detection and quantification technologies are well known in the art. Standard methods for the detection and quantification of 109P1 D4 mRNA include in situ hybridization using labeled 109P1 D4 riboprobes, Northern blot and related techniques using 109P1D4 polynucleotide probes, RT-PCR analysis using primers specific for 109P1D4, and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like. In a specific embodiment, semi- quantitative RT-PCR is used to detect and quantify 109P1 D4 mRNA expression. Any number of primers capable of amplifying 109P1 D4 can be used for this purpose, including but not limited to the various primer sets specifically described herein. In a specific embodiment, polyclonal or monoclonal antibodies specifically reactive with the wild-type 109P1 D4 protein can be used in an immunohistochemical assay of biopsied tissue.
IX.) Identification of Molecules That Interact With 109P1 D4
The 109P1D4 protein and nucleic acid sequences disclosed herein allow a skilled artisan to identify proteins, small molecules and other agents that interact with 109P1 D4, as well as pathways activated by 109P1 D4 via any one of a variety of art accepted protocols. For example, one can utilize one of the so-called interaction trap systems (also referred to as the "two-hybrid assay"). In such systems, molecules interact and reconstitute a transcription factor which directs expression of a reporter gene, whereupon the expression of the reporter gene is assayed. Other systems identify protein-protein interactions in vivo through reconstitution of a eukaryotic transcriptional activator, see, e.g., U.S. Patent Nos. 5,955,280 issued 21 September 1999, 5,926,623 issued 20 July 1999, 5,846,722 issued 8 December 1998 and 6,004,746 issued 21 December 1999. Algorithms are also available in the art for genome-based predictions of protein function (see, e.g., Marcotte, ef al, Nature 402: 4 November 1999, 83-86).
Alternatively one can screen peptide libraries to identify molecules that interact with 109P1 D4 protein sequences. In such methods, peptides that bind to 109P1D4 are identified by screening libraries that encode a random or controlled collection of amino acids. Peptides encoded by the libraries are expressed as fusion proteins of bacteriophage coat proteins, the bacteriophage particles are then screened against the 109P1D4 protein(s).
Accordingly, peptides having a wide variety of uses, such as therapeutic, prognostic or diagnostic reagents, are thus identified without any prior information on the structure of the expected ligand or receptor molecule. Typical peptide libraries and screening methods that can be used to identify molecules that interact with 109P1 D4 protein sequences are disclosed for example in U.S. Patent Nos. 5,723,286 issued 3 March 1998 and 5,733,731 issued 31 March 1998.
Alternatively, cell lines that express 109P1D4 are used to identify protein-protein interactions mediated by 109P1D4. Such interactions can be examined using immunoprecipitation techniques (see, e.g., Hamilton B.J., et al. Biochem. Biophys. Res. Commun. 1999, 261 :646-51). 109P1D4 protein can be immunoprecipitated from 109P1D4- expressing cell lines using anti-109P1D4 antibodies. Alternatively, antibodies against His-tag can be used in a cell line engineered to express fusions of 109P1D4 and a His-tag (vectors mentioned above). The immunoprecipitated complex can be examined for protein association by procedures such as Western blotting, 35S-methionine labeling of proteins, protein microsequencing, silver staining and two-dimensional gel electrophoresis.
Small molecules and ligands that interact with 109P1D4 can be identified through related embodiments of such screening assays. For example, small molecules can be identified that interfere with protein function, including molecules that interfere with 109P1D4's ability to mediate phosphorylation and de-phosphorylation, interaction with DNA or RNA molecules as an indication of regulation of cell cycles, second messenger signaling or tumorigenesis. Similarly, small molecules that modulate 109P1D4-related ion channel, protein pump, or cell communication functions are identified and used to treat patients that have a cancer that expresses 109P1 D4 (see, e.g., Hille, B., Ionic Channels of Excitable Membranes 2nd Ed., Sinauer Assoc, Sunderland, MA, 1992). Moreover, ligands that regulate 109P1D4 function can be identified based on their ability to bind 109P1D4 and activate a reporter construct. Typical methods are discussed for example in U.S. Patent No. 5,928,868 issued 27 July 1999, and include methods for forming hybrid ligands in which at least one ligand is a small molecule. In an illustrative embodiment, cells engineered to express a fusion protein of 109P1D4 and a DNA-binding protein are used to co-express a fusion protein of a hybrid ligand/small molecule and a cDNA library transcriptional activator protein. The cells further contain a reporter gene, the expression of which is conditioned on the proximity of the first and second fusion proteins to each other, an event that occurs only if the hybrid ligand binds to target sites on both hybrid proteins. Those cells that express the reporter gene are selected and the unknown small molecule or the unknown ligand is identified. This method provides a means of identifying modulators, which activate or inhibit 109P1D4.
An embodiment of this invention comprises a method of screening for a molecule that interacts with a 109P1 D4 amino acid sequence shown in Figure 2 or Figure 3, comprising the steps of contacting a population of molecules with a 109P1D4 amino acid sequence, allowing the population of molecules and the 109P1D4 amino acid sequence to interact under conditions that facilitate an interaction, determining the presence of a molecule that interacts with the 109P1 D4 amino acid sequence, and then separating molecules that do not interact with the 109P1 D4 amino acid sequence from molecules that do. In a specific embodiment, the method further comprises purifying, characterizing and identifying a molecule that interacts with the 109P1D4 amino acid sequence. The identified molecule can be used to modulate a function performed by 109P1 D4. In a preferred embodiment, the 109P1 D4 amino acid sequence is contacted with a library of peptides.
>Q Therapeutic Methods and Compositions
The identification of 109P1 D4 as a protein that is normally expressed in a restricted set of tissues, but which is also expressed in cancers such as those listed in Table I, opens a number of therapeutic approaches to the treatment of such cancers.
Of note, targeted antitumor therapies have been useful even when the targeted protein is expressed on normal tissues, even vital normal organ tissues. A vital organ is one that is necessary to sustain life, such as the heart or colon. A non-vital organ is one that can be removed whereupon the individual is still able to survive. Examples of non-vital organs are ovary, breast, and prostate. For example, Herceptin® is an FDA approved pharmaceutical that has as its active ingredient an antibody which is immunoreactive with the protein variously known as HER2, HER2/neu, and erb-b-2. It is marketed by Genentech and has been a commercially successful antitumor agent. Herceptin sales reached almost $400 million in 2002. Herceptin is a treatment for HER2 positive metastatic breast cancer. However, the expression of HER2 is not limited to such tumors. The same protein is expressed in a number of normal tissues. In particular, it is known that HER2/neu is present in normal kidney and heart, thus these tissues are present in all human recipients of Herceptin. The presence of HER2/neu in normal kidney is also confirmed by Latif, Z., et al., B.J.U. International (2002) 89:5-9. As shown in this article (which evaluated whether renal cell carcinoma should be a preferred indication for anti-HER2 antibodies such as Herceptin) both protein and mRNA are produced in benign renal tissues. Notably, HER2/neu protein was strongly overexpressed in benign renal tissue. Despite the fact that HER2/neu is expressed in such vital tissues as heart and kidney, Herceptin is a very useful, FDA approved, and commercially successful drug. The effect of Herceptin on cardiac tissue, i.e., "cardiotoxicity," has merely been a side effect to treatment. When patients were treated with Herceptin alone, significant cardiotoxicity occurred in a very low percentage of patients.
Of particular note, although kidney tissue is indicated to exhibit normal expression, possibly even higher expression than cardiac tissue, kidney has no appreciable Herceptin side effect whatsoever. Moreover, of the diverse array of normal tissues in which HER2 is expressed, there is very little occurrence of any side effect. Only cardiac tissue has manifested any appreciable side effect at all. A tissue such as kidney, where HER2/neu expression is especially notable, has not been the basis for any side effect.
Furthermore, favorable therapeutic effects have been found for antitumor therapies that target epidermal growth factor receptor (EGFR). EGFR is also expressed in numerous normal tissues. There have been very limited side effects in normal tissues following use of anti-EGFR therapeutics.
Thus, expression of a target protein in normal tissue, even vital normal tissue, does not defeat the utility of a targeting agent for the protein as a therapeutic for certain tumors in which the protein is also overexpressed.
Accordingly, therapeutic approaches that inhibit the activity of a 109P1 D4 protein are useful for patients suffering from a cancer that expresses 109P1D4. These therapeutic approaches generally fall into two classes. One class comprises various methods for inhibiting the binding or association of a 109P1 D4 protein with its binding partner or with other proteins. Another class comprises a variety of methods for inhibiting the transcription of a 109P1D4 gene or translation of 109P1D4 mRNA.
X.A.) Anti-Cancer Vaccines
The invention provides cancer vaccines comprising a 109P1D4-related protein or 109P1D4-related nucleic acid. In view of the expression of 109P1 D4, cancer vaccines prevent and/or treat 109P1 D4-expressing cancers with minimal or no effects on non-target tissues. The use of a tumor antigen in a vaccine that generates humoral and/or cell-mediated immune responses as anti-cancer therapy is well known in the art and has been employed in prostate cancer using human PSMA and rodent PAP immunogens (Hodge etal, 1995, Int. J. Cancer 63:231-237; Fong etal, 1997, J. Immunol. 159:3113-3117).
Such methods can be readily practiced by employing a 109P1D4-related protein, or a 109P1D4-encoding nucleic acid molecule and recombinant vectors capable of expressing and presenting the 109P1 D4 immunogen (which typically comprises a number of antibody or T cell epitopes). Skilled artisans understand that a wide variety of vaccine systems for delivery of immunoreactive epitopes are known in the art (see, e.g., Heryln ef a/., Ann Med 1999 Feb 31(1):66-78; Maruyama ef al, Cancer Immunol Immunother 2000 Jun 49(3):123-32) Briefly, such methods of generating an immune response (e.g. humoral and/or cell-mediated) in a mammal, comprise the steps of: exposing the mammal's immune system to an immunoreactive epitope (e.g. an epitope present in a 109P1D4 protein shown in Figure 3 or analog or homolog thereof) so that the mammal generates an immune response that is specific for that epitope (e.g. generates antibodies that specifically recognize that epitope). In a preferred method, a 109P1D4 immunogen contains a biological motif, see e.g., Tables VIII-XXI and XXII-XLIX, or a peptide of a size range from 109P1 D4 indicated in Figure 5, Figure 6, Figure 7, Figure 8, and Figure 9.
The entire 109P1D4 protein, immunogenic regions or epitopes thereof can be combined and delivered by various means. Such vaccine compositions can include, for example, lipopeptides (e.g.,Viliello, A. ef al, J. Clin. Invest. 95:341 , 1996), peptide compositions encapsulated in poly(DL-lactide-co-glycolide) ("PLG") microspheres (see, e.g., Eldridge, ef a/., Molec. Immunol. 28:287-294, 1991: Alonso ef al., Vaccine 12:299-306, 1994; Jones et al, Vaccine 13:676-681, 1996), peptide compositions contained in immune stimulating complexes (ISCOMS) (see, e.g., Takahashi ef al, Nature 344:873- 875, 1990; Hu ef al, Clin Exp Immunol 113:236-243, 1998), multiple antigen peptide systems (MAPs) (see e.g., Tarn, J. P., Proc. Natl. Acad. Sci. U.S.A. 85:5409-6413, 1988; Tarn, J.P., J. Immunol. Methods 196:17-32, 1996), peptides formulated as multivalent peptides; peptides for use in ballistic delivery systems, typically crystallized peptides, viral delivery vectors (Perkus, M. E. et al, In: Concepts in vaccine development, Kaufmann, S. H. E., ed., p. 379, 1996; Chakrabarti, S. ef al, Nature 320:535, 1986; Hu, S. L. ef al, Nature 320:637, 1986; Kieny, M.-P. ef al, AIDS Bio/Technology 4:790, 1986; Top, F. H. ef al, J. Infect. Dis. 124:148, 1971; Chanda, P. K. et al, Virology 175:536, 1990), particles of viral or synthetic origin (e.g., Kofler, N. ef al, J. Immunol Methods. 192:26, 1996; Eldridge, J. H. et al, Sem. HematoL 30:16, 1993; Falo, L. D, Jr. ef al, Nature Med. 7:649, 1996), adjuvants (Warren, H. S., Vogel, F. R., and Chedid, L. A. Annu. Rev. Immunol. 4:369, 1986; Gupta, R. K. et al, Vaccine 11:293, 1993), liposomes (Reddy, R. et al, J. Immunol. 148:1686, 1992; Rock, K. L, Immunol. Today 17:131, 1996), or, naked or particle absorbed cDNA (Ulmer, J. B. ef al, Science 259:1745, 1993; Robinson, H. L, Hunt, L. A., and Webster, R. G., Vaccine 11 :957, 1993; Shiver, J. W. et al, In: Concepts in vaccine development, Kaufmann, S. H. E., ed., p. 423, 1996; Cease, K. B., and Berzofsky, J. A., Annu. Rev. Immunol. 12:923, 1994 and Eldridge, J. H. etal, Sem. HematoL 30:16, 1993). Toxin-targeted delivery technologies, also known as receptor mediated targeting, such as those of Avant Immunotherapeutics, Inc. (Needham, Massachusetts) may also be used.
In patients with 109P1D4-associated cancer, the vaccine compositions of the invention can also be used in conjunction with other treatments used for cancer, e.g., surgery, chemotherapy, drug therapies, radiation therapies, efc. including use in combination with immune adjuvants such as IL-2, IL-12, GM-CSF, and the like.
Cellular Vaccines:
CTL epitopes can be determined using specific algorithms to identify peptides within 109P1 D4 protein that bind corresponding HLA alleles (see e.g., Table IV; Epimer™ and Epimatrix™, Brown University (URL brown.edu/Research/TB- HIV_Lab/epimatrix/epimatrix.html); and, BIMAS, (URL bimas.dcrt.nih.gov/; SYFPEITHI at URL syfpeithi.bmi-heidelberg.com/). In a preferred embodiment, a 109P1D4 immunogen contains one or more amino acid sequences identified using techniques well known in the art, such as the sequences shown in Tables VIII-XXI and XXII-XLIX or a peptide of 8, 9, 10 or 11 amino acids specified by an HLA Class I motif/supermotif (e.g., Table IV (A), Table IV (D), or Table IV (E)) and/or a peptide of at least 9 amino acids that comprises an HLA Class II motif/supermotif (e.g., Table IV (B) or Table IV (C)). As is appreciated in the art, the HLA Class I binding groove is essentially closed ended so that peptides of only a particular size range can fit into the groove and be bound, generally HLA Class I epitopes are 8, 9, 10, or 11 amino acids long. In contrast, the HLA Class II binding groove is essentially open ended; therefore a peptide of about 9 or more amino acids can be bound by an HLA Class II molecule. Due to the binding groove differences between HLA Class I and II, HLA Class I motifs are length specific, i.e., position two of a Class I motif is the second amino acid in an amino to carboxyl direction of the peptide. The amino acid positions in a Class II motif are relative only to each other, not the overall peptide, i.e., additional amino acids can be attached to the amino and/or carboxyl termini of a motif-bearing sequence. HLA Class II epitopes are often 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25 amino acids long, or longer than 25 amino acids.
Antibody-based Vaccines
A wide variety of methods for generating an immune response in a mammal are known in the art (for example as the first step in the generation of hybridomas). Methods of generating an immune response in a mammal comprise exposing the mammal's immune system to an immunogenic epitope on a protein (e.g. a 109P1D4 protein) so that an immune response is generated. A typical embodiment consists of a method for generating an immune response to 109P1D4 in a host, by contacting the host with a sufficient amount of al least one 109P1 D4 B cell or cytotoxic T-cell epitope or analog thereof; and at least one periodic interval thereafter re-contacting the host with the 109P1 D4 B cell or cytotoxic T-cell epitope or analog thereof. A specific embodiment consists of a method of generating an immune response against a 109P1 D4- related protein or a man-made multiepitopic peptide comprising: administering 109P1D4 immunogen (e.g. a 109P1D4 protein or a peptide fragment thereof, a 109P1D4 fusion protein or analog etc.) in a vaccine preparation to a human or another mammal. Typically, such vaccine preparations further contain a suitable adjuvant (see, e.g., U.S. Patent No. 6,146,635) or a universal helper epitope such as a PADRE™ peptide (Epimmune Inc., San Diego, CA; see, e.g., Alexander ef al, J. Immunol. 2000 164(3); 164(3): 1625-1633; Alexander ef al, Immunity 1994 1(9): 751-761 and Alexander ef al, Immunol. Res. 1998 18(2): 79-92). An alternative method comprises generating an immune response in an individual against a 109P1D4 immunogen by: administering in vivo to muscle or skin of the individual's body a DNA molecule that comprises a DNA sequence that encodes a 109P1D4 immunogen, the DNA sequence operatively linked to regulatory sequences which control the expression of the DNA sequence; wherein the DNA molecule is taken up by cells, the DNA sequence is expressed in the cells and an immune response is generated against the immunogen (see, e.g., U.S. Patent No. 5,962,428). Optionally a genetic vaccine facilitator such as anionic lipids; saponins; lectins; estrogenic compounds; hydroxylated lower alkyls; dimethyl sulfoxide; and urea is also administered. In addition, an antiidiotypic antibody can be administered that mimics 109P1D4, in order to generate a response to the target antigen.
Nucleic Acid Vaccines:
Vaccine compositions of the invention include nucleic acid-mediated modalities. DNA or RNA that encode protein(s) of the invention can be administered to a patient. Genetic immunization methods can be employed to generate prophylactic or therapeutic humoral and cellular immune responses directed against cancer cells expressing 109P1D4. Constructs comprising DNA encoding a 109P1D4-related protein/immunogen and appropriate regulatory sequences can be injected directly into muscle or skin of an individual, such that the cells of the muscle or skin take-up the construct and express the encoded 109P1 D4 protein/immunogen. Alternatively, a vaccine comprises a 109P1D4-related protein. Expression of the 109P1 D4-related protein immunogen results in the generation of prophylactic or therapeutic humoral and cellular immunity against cells that bear a 109P1D4 protein. Various prophylactic and therapeutic genetic immunization techniques known in the art can be used (for review, see information and references published at Internet address genweb.com). Nucleic acid-based delivery is described, for instance, in Wolff ef. a/., Science 247:1466 (1990) as well as U.S. Patent Nos. 6,680,859; 5,589,466; 5,804,666; 5,739,118; 5,736,524; 5,679,647; WO 98/04720. Examples of DNA- based delivery technologies include "naked DNA", facilitated (bupivicaine, polymers, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated ("gene gun") or pressure-mediated delivery (see, e.g., U.S. Patent No. 5,922,687).
For therapeutic or prophylactic immunization purposes, proteins of the invention can be expressed via viral or bacterial vectors. Various viral gene delivery systems that can be used in the practice of the invention include, but are not limited to, vaccinia, fowlpox, canarypox, adenovirus, influenza, poliovirus, adeno-associated virus, lentivirus, and sindbis virus (see, e.g., Restifo, 1996, Curr. Opin. Immunol. 8:658-663; Tsang ef al. J. Natl. Cancer Inst. 87:982-990 (1995)). Non-viral delivery systems can also be employed by introducing naked DNA encoding a 109P1D4-related protein into the patient (e.g., intramuscularly or intradermally) to induce an anti-tumor response.
Vaccinia virus is used, for example, as a vector to express nucleotide sequences that encode the peptides of the invention. Upon introduction into a host, the recombinant vaccinia virus expresses the protein immunogenic peptide, and thereby elicits a host immune response. Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Patent No. 4,722,848. Another vector is BCG (Bacille Cal ette Guerin). BCG vectors are described in Stover ef al, Nature 351:456-460 (1991). A wide variety of other vectors useful for therapeutic administration or immunization of the peptides of the invention, e.g. adeno and adeno-associated virus vectors, retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, and the like, will be apparent to those skilled in the art from the description herein.
Thus, gene delivery systems are used to deliver a 109P1D4-related nucleic acid molecule. In one embodiment, the full- length human 109P1 D4 cDNA is employed. In another embodiment, 109P1 D4 nucleic acid molecules encoding specific cytotoxic T lymphocyte (CTL) and/or antibody epitopes are employed.
Ex Vivo Vaccines
Various ex vivo strategies can also be employed to generate an immune response. One approach involves the use of antigen presenting cells (APCs) such as dendritic cells (DC) to present 109P1 D4 antigen to a patient's immune system. Dendritic cells express MHC class I and II molecules, B7 co-stimulator, and IL-12, and are thus highly specialized antigen presenting cells. In prostate cancer, autologous dendritic cells pulsed with peptides of the prostate-specific membrane antigen (PSMA) are being used in a Phase I clinical trial to stimulate prostate cancer patients' immune systems (Tjoa ef a/., 1996, Prostate 28:65- 69; Murphy etal, 1996, Prostate 29:371-380). Thus, dendritic cells can be used to present 109P1D4 peptides to T cells in the context of MHC class I or II molecules. In one embodiment, autologous dendritic cells are pulsed with 109P1 D4 peptides capable of binding to MHC class I and/or class II molecules. In another embodiment, dendritic cells are pulsed with the complete 109P1D4 protein. Yet another embodiment involves engineering the overexpression of a 109P1D4 gene in dendritic cells using various implementing vectors known in the art, such as adenovirus (Arthur ef a/., 1997, Cancer Gene Ther. 4:17-25), retrovirus (Henderson et al, 1996, Cancer Res. 66:3763-3770), lentivirus, adeno-associated virus, DNA transfection (Ribas ef a/., 1997, Cancer Res. 67:2865-2869), or tumor-derived RNA transfection (Ashley et al, 1997, J. Exp. Med. 186:1177-1182). Cells that express 109P1D4 can also be engineered to express immune modulators, such as GM- CSF, and used as immunizing agents.
X.B.) 109P1 D4 as a Target for Antibody-based Therapy
109P1 D4 is an attractive target for antibody-based therapeutic strategies. A number of antibody strategies are known in the art for targeting both extracellular and intracellular molecules (see, e.g., complement and ADCC mediated killing as well as the use of intrabodies). Because 109P1D4 is expressed by cancer cells of various lineages relative to corresponding normal cells, systemic administration of 109P1 D4-immunoreactive compositions are prepared that exhibit excellent sensitivity without toxic, non-specific and/or non-target effects caused by binding of the immunoreactive composition to non-target organs and tissues. Antibodies specifically reactive with domains of 109P1D4 are useful to treat 109P1D4-expressing cancers systemically, either as conjugates with a toxin or therapeutic agent, or as naked antibodies capable of inhibiting cell proliferation or function.
109P1D4 antibodies can be introduced into a patient such" that the antibody binds to 109P1D4 and modulates a function, such as an interaction with a binding partner, and consequently mediates destruction of the tumor cells and/or inhibits the growth of the tumor cells. Mechanisms by which such antibodies exert a therapeutic effect can include complement-mediated cytolysis, antibody-dependent cellular cytotoxicity, modulation of the physiological function of 109P1 D4, inhibition of ligand binding or signal transduction pathways, modulation of tumor cell differentiation, alteration of tumor angiogenesis factor profiles, and/or apoptosis.
Those skilled in the art understand that antibodies can be used to specifically target and bind immunogenic molecules such as an immunogenic region of a 109P1 D4 sequence shown in Figure 2 or Figure 3. In addition, skilled artisans understand that it is routine to conjugate antibodies to cytotoxic agents (see, e.g., Slevers ef al. Blood 93:11 3678- 3684 (June 1, 1999)). When cytotoxic and/or therapeutic agents are delivered directly to cells, such as by conjugating them to antibodies specific for a molecule expressed by that cell (e.g. 109P1 D4), the cytotoxic agent will exert its known biological effect (i.e. cytotoxicity) on those cells.
A wide variety of compositions and methods for using antibody-cytotoxic agent conjugates to kill cells are known in the art. In the context of cancers, typical methods entail administering to an animal having a tumor a biologically effective amount of a conjugate comprising a selected cytotoxic and/or therapeutic agent linked to a targeting agent (e.g. an anti- 109P1D4 antibody) that binds to a marker (e.g. 109P1D4) expressed, accessible to binding or localized on the cell surfaces. A typical embodiment is a method of delivering a cytotoxic and/or therapeutic agent to a cell expressing 109P1D4, comprising conjugating the cytotoxic agent to an antibody that immunospecifically binds to a 109P1 D4 epitope, and, exposing the cell to the antibody-agent conjugate. Another illustrative embodiment is a method of treating an individual suspected of suffering from metastasized cancer, comprising a step of administering parenterally to said individual a pharmaceutical composition comprising a therapeutically effective amount of an antibody conjugated to a cytotoxic and/or therapeutic agent.
Cancer immunotherapy using anti-109P1D4 antibodies can be done in accordance with various approaches that have been successfully employed in the treatment of other types of cancer, including but not limited to colon cancer (Arlen ef a/., 1998, Crit. Rev. Immunol. 18:133-138), multiple myeloma (Ozaki ef al, 1997, Blood 90:3179-3186, Tsunenari et al, 1997, Blood 90:2437-2444), gastric cancer (Kasprzyk ef a/., 1992, Cancer Res. 52:2771-2776), B-cell lymphoma (Funakoshi et al, 1996, J. Immunother. Emphasis Tumor Immunol. 19:93-101), leukemia (Zhong et al., 1996, Leuk. Res. 20:681-689), colorectal cancer (Moun ef al, 1994, Cancer Res. 64:6160-6166; Velders ef al, 1995, Cancer Res. 56:4398-4403), and breast cancer (Shepard ef a/., 1991, J. Clin. Immunol. 11:117-127). Some therapeutic approaches involve conjugation of naked antibody to a toxin or radioisotope, such as the conjugation of Y91 or I131 to anti-CD20 antibodies (e.g., Zevalin™, IDEC Pharmaceuticals Corp. or Bexxar™, Coulter Pharmaceuticals), while others involve co-administration of antibodies and other therapeutic agents, such as Herceptin™ (trastuzumab) with paclitaxel (Genentech, Inc.). The antibodies can be conjugated to a therapeutic agent. To treat prostate cancer, for example, 109P1D4 antibodies can be administered in conjunction with radiation, chemotherapy or hormone ablation. Also, antibodies can be conjugated to a toxin such as calicheamicin (e.g., Mylotarg™, Wyeth-Ayerst, Madison, NJ, a recombinant humanized lgG kappa antibody conjugated to antitumor antibiotic calicheamicin) or a maytansinoid (e.g., taxane-based Tumor-Activated Prodrug, TAP, platform, ImmunoGen, Cambridge, MA, also see e.g., US Patent 5,416,064).
Although 109P1 D4 antibody therapy is useful for all stages of cancer, antibody therapy can be particularly appropriate in advanced or metastatic cancers. Treatment with the antibody therapy of the invention is indicated for patients who have received one or more rounds of chemotherapy. Alternatively, antibody therapy of the invention is combined with a chemotherapeutic or radiation regimen for patients who have not received chemotherapeutic treatment. Additionally, antibody therapy can enable the use of reduced dosages of concomitant chemotherapy, particularly for patients who do not tolerate the toxicity of the chemotherapeutic agent very well. Fan et al. (Cancer Res. 53:4637-4642, 1993), Prewett et al. (International J. of Onco. 9:217-224, 1996), and Hancock et al. (Cancer Res. 51 :4576-4680, 1991) describe the use of various antibodies together with chemotherapeutic agents.
Although 109P1D4 antibody therapy is useful for all stages of cancer, antibody therapy can be particularly appropriate in advanced or metastatic cancers. Treatment with the antibody therapy of the invention is indicated for patients who have received one or more rounds of chemotherapy. Alternatively, antibody therapy of the invention is combined with a chemotherapeutic or radiation regimen for patients who have not received chemotherapeutic treatment. Additionally, antibody therapy can enable the use of reduced dosages of concomitant chemotherapy, particularly for patients who do not tolerate the toxicity of the chemotherapeutic agent very well.
Cancer patients can be evaluated for the presence and level of 109P1D4 expression, preferably using immunohistochemical assessments of tumor tissue, quantitative 109P1 D4 imaging, or other techniques that reliably indicate the presence and degree of 109P1 D4 expression. Immunohistochemical analysis of tumor biopsies or surgical specimens is preferred for this purpose. Methods for immunohistochemical analysis of tumor tissues are well known in the art.
Anti-109P1D4 monoclonal antibodies that treat prostate and other cancers include those that initiate a potent immune response against the tumor or those that are directly cytotoxic. In this regard, anti-109P1D4 monoclonal antibodies (mAbs) can elicit tumor cell lysis by either complement-mediated or antibody-dependent cell cytotoxicity (ADCC) mechanisms, both of which require an intact Fc portion of the immunoglobulin molecule for interaction with effector cell Fc receptor sites on complement proteins. In addition, anti-109P1D4 mAbs that exert a direct biological effect on tumor growth are useful to treat cancers that express 109P1 D4. Mechanisms by which directly cytotoxic mAbs act include: inhibition of cell growth, modulation of cellular differentiation, modulation of tumor angiogenesis factor profiles, and the induction of apoptosis. The mechanism(s) by which a particular anti-109P1 D4 mAb exerts an anti-tumor effect is evaluated using any number of in vitro assays that evaluate cell death such as ADCC, ADMMC, complement-mediated cell lysis, and so forth, as is generally known in the art.
In some patients, the use of murine or other non-human monoclonal antibodies, or human/mouse chimeric mAbs can induce moderate to strong immune responses against the non-human antibody. This can result in clearance of the antibody from circulation and reduced efficacy. In the most severe cases, such an immune response can lead to the extensive formation of immune complexes which, potentially, can cause renal failure. Accordingly, preferred monoclonal antibodies used in the therapeutic methods of the invention are those that are either fully human or humanized and that bind specifically to the target 109P1D4 antigen with high affinity but exhibit low or no antigenicity in the patient.
Therapeutic methods of the invention contemplate the administration of single anti-109P1 D4 mAbs as well as combinations, or cocktails, of different mAbs. Such mAb cocktails can have certain advantages inasmuch as they contain mAbs that target different epitopes, exploit different effector mechanisms or combine directly cytotoxic mAbs with mAbs that rely on immune effector functionality. Such mAbs in combination can exhibit synergistic therapeutic effects. In addition, anti- 109P1 D4 mAbs can be administered concomitantly with other therapeutic modalities, including but not limited to various chemotherapeutic agents, androgen-blockers, immune modulators (e.g., lL-2, GM-CSF), surgery or radiation. The anti- 109P1 D4 mAbs are administered in their "naked" or unconjugated form, or can have a therapeutic agent(s) conjugated to them.
Anti-109P1 D4 antibody formulations are administered via any route capable of delivering the antibodies to a tumor cell. Routes of administration include, but are not limited to, intravenous, intraperitoneal, intramuscular, intratumor, intradermal, and the like. Treatment generally involves repeated administration of the anti-109P1D4 antibody preparation, via an acceptable route of administration such as intravenous injection (IV), typically at a dose in (he range of about 0.1, .2, .3, .4, .5, .6, .7, .8, .9., 1, 2, 3, 4, δ, 6, 7, 8, 9, 10, 15, 20, or 25 mg/kg body weight. In general, doses in the range of 10-1000 mg mAb per week are effective and well tolerated.
Based on clinical experience with the Herceptin™ mAb in the treatment of metastatic breast cancer, an initial loading dose of approximately 4 mg/kg patient body weight IV, followed by weekly doses of about 2 mg/kg IV of the anti- 109P1D4 mAb preparation represents an acceptable dosing regimen. Preferably, the initial loading dose is administered as a 90-minute or longer infusion. The periodic maintenance dose is administered as a 30 minute or longer infusion, provided the initial dose was well tolerated. As appreciated by those of skill in the art, various factors can influence the ideal dose regimen in a particular case. Such factors include, for example, the binding affinity and half life of the Ab or mAbs used, the degree of 109P1 D4 expression in the patient, the extent of circulating shed 109P1 D4 antigen, the desired steady-state antibody concentration level, frequency of treatment, and the influence of chemotherapeutic or other agents used in combination with the treatment method of the invention, as well as the health status of a particular patient.
Optionally, patients should be evaluated for the levels of 109P1D4 in a given sample (e.g. the levels of circulating 109P1D4 antigen and/or 109P1D4 expressing cells) in order to assist in the determination of the most effective dosing regimen, etc. Such evaluations are also used for monitoring purposes throughout therapy, and are useful to gauge therapeutic success in combination with the evaluation of other parameters (for example, urine cytology and/or ImmunoCyt levels in bladder cancer therapy, or by analogy, serum PSA levels in prostate cancer therapy).
Anti-idiotypic anti-109P1D4 antibodies can also be used in anti-cancer therapy as a vaccine for inducing an immune response to cells expressing a 109P1D4-related protein. In particular, the generation of anti-idiotypic antibodies is well known in the art; this methodology can readily be adapted to generate anti-idiotypic anti-109P1D4 antibodies that mimic an epitope on a 109P1D4-related protein (see, for example, Wagner ef al, 1997, Hybridoma 16: 33-40; Foon ef al, 1995, J. Clin. Invest. 96:334-342; Herlyn etal, 1996, Cancer Immunol. Immunother. 43:65-76). Such an anti-idiotypic antibody can be used in cancer vaccine strategies.
X.C.) 109P1D4 as a Target for Cellular Immune Responses
Vaccines and methods of preparing vaccines that contain an immunogenically effective amount of one or more HLA-binding peptides as described herein are further embodiments of the invention. Furthermore, vaccines in accordance with the invention encompass compositions of one or more of the claimed peptides. A peptide can be present in a vaccine individually. Alternatively, the peptide can exist as a homopolymer comprising multiple copies of the same peptide, or as a heteropolymer of various peptides. Polymers have the advantage of increased immunological reaction and, where different peptide epitopes are used to make up the polymer, the additional ability to induce antibodies and/or CTLs that react with different antigenic determinants of the pathogenic organism or tumor-related peptide targeted for an immune response. The composition can be a naturally occurring region of an antigen or can be prepared, e.g., recombinantly or by chemical synthesis.
Carriers that can be used with vaccines of the invention are well known in the art, and include, e.g., thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly L-lysine, poly L-glutamic acid, influenza, hepatitis B virus core protein, and the like. The vaccines can contain a physiologically tolerable (i.e., acceptable) diluent such as water, or saline, preferably phosphate buffered saline. The vaccines also typically include an adjuvant. Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum are examples of materials well known in the art. Additionally, as disclosed herein, CTL responses can be primed by conjugating peptides of the invention to lipids, such as tripalmitoyl-S-glycerylcysteinlyseryl- serine (P3CSS). Moreover, an adjuvant such as a synthetic cytosine-phosphorothiolated-guanine-containing (CpG) oligonucleotides has been found to increase CTL responses 10- to 100-fold. (see, e.g. Davila and Celis, J. Immunol. 165:539-547 (2000))
Upon immunization with a peptide composition in accordance with the invention, via injection, aerosol, oral, transdermal, transmucosal, infrapleural, intrathecal, or other suitable routes, the immune system of the host responds to the vaccine by producing large amounts of CTLs and/or HTLs specific for the desired antigen. Consequently, the host becomes at least partially immune to later development of cells that express or overexpress 109P1D4 antigen, or derives at least some therapeutic benefit when the antigen was tumor-associated.
In some embodiments, it may be desirable to combine the class I peptide components with components that induce or facilitate neutralizing antibody and or helper T cell responses directed to the target antigen. A preferred embodiment of such a composition comprises class I and class II epitopes in accordance with the invention. An alternative embodiment of such a composition comprises a class I and/or class II epitope in accordance with the invention, along with a cross reactive HTL epitope such as PADRE™ (Epimmune, San Diego, CA) molecule (described e.g., in U.S. Patent Number 5,736,142).
A vaccine of the invention can also include antigen-presenting cells (APC), such as dendritic cells (DC), as a vehicle to present peptides of the invention. Vaccine compositions can be created in vitro, following dendritic cell mobilization and harvesting, whereby loading of dendritic cells occurs in vitro. For example, dendritic cells are transfected, e.g., with a miπigene in accordance with the invention, or are pulsed with peptides. The dendritic cell can then be administered to a patient to elicit immune responses in vivo. Vaccine compositions, either DNA- or peptide-based, can also be administered in vivo in combination with dendritic cell mobilization whereby loading of dendritic cells occurs in vivo.
Preferably, the following principles are utilized when selecting an array of epitopes for inclusion in a polyepitopic composition for use in a vaccine, or for selecting discrete epitopes to be included in a vaccine and/or to be encoded by nucleic acids such as a minigene. It is preferred that each of the following principles be balanced in order to make the selection. The multiple epitopes to be incorporated in a given vaccine composition may be, but need not be, contiguous in sequence in the native antigen from which the epitopes are derived.
1.) Epitopes are selected which, upon administration, mimic immune responses that have been observed to be correlated with tumor clearance. For HLA Class I this includes 3-4 epitopes that come from at least one tumor associated antigen (TAA). For HLA Class II a similar rationale is employed; again 3-4 epitopes are selected from at least one TAA (see, e.g., Rosenberg ef a/., Science 278:1447-1450). Epitopes from one TAA may be used in combination with epitopes from one or more additional TAAs to produce a vaccine that targets tumors with varying expression patterns of frequently-expressed TAAs.
2.) Epitopes are selected that have the requisite binding affinity established to be correlated with immunogenicity: for HLA Class I an ICso of 500 nM or less, often 200 nM or less; and for Class II an ICso of 1000 nM or less.
3.) Sufficient supermotif bearing-peptides, or a sufficient array of allele-specific motif-bearing peptides, are selected to give broad population coverage. For example, it is preferable to have at least 80% population coverage. A Monte Carlo analysis, a statistical evaluation known in the art, can be employed to assess the breadth, or redundancy of, population coverage.
4.) When selecting epitopes from cancer-related antigens it is often useful to select analogs because the patient may have developed tolerance to the native epitope.
5.) Of particular relevance are epitopes referred to as "nested epitopes." Nested epitopes occur where at least two epitopes overlap in a given peptide sequence. A nested peptide sequence can comprise B cell, HLA class I and/or HLA class II epitopes. When providing nested epitopes, a general objective is to provide the greatest number of epitopes per sequence. Thus, an aspect is to avoid providing a peptide that is any longer than the amino terminus of the amino terminal epitope and the carboxyl terminus of the carboxyl terminal epitope in the peptide. When providing a multi-epitopic sequence, such as a sequence comprising nested epitopes, it is generally important to screen the sequence in order to insure that it does not have pathological or other deleterious biological properties.
6.) If a polyepitopic protein is created, or when creating a minigene, an objective is to generate the smallest peptide that encompasses the epitopes of interest. This principle is similar, if not the same as that employed when selecting a peptide comprising nested epitopes. However, with an artificial polyepitopic peptide, the size minimization objective is balanced against the need to integrate any spacer sequences between epitopes in the polyepitopic protein. Spacer amino acid residues can, for example, be introduced to avoid junctional epitopes (an epitope recognized by the immune system, not present in the target antigen, and only created by the man-made juxtaposition of epitopes), or to facilitate cleavage between epitopes and thereby enhance epitope presentation. Junctional epitopes are generally to be avoided because the recipient may generate an immune response to that non-native epitope. Of particular concern is a junctional epitope that is a "dominant epitope." A dominant epitope may lead to such a zealous response that immune responses to other epitopes are diminished or suppressed.
7.) Where the sequences of multiple variants of the same target protein are present, potential peptide epitopes can also be selected on the basis of their conservancy. For example, a criterion for conservancy may define that the entire sequence of an HLA class I binding peptide or the entire 9-mer core of a class II binding peptide be conserved in a designated percentage of the sequences evaluated for a specific protein antigen. X.C.1. Minigene Vaccines
A number of different approaches are available which allow simultaneous delivery of multiple epitopes. Nucleic acids encoding the peptides of the invention are a particularly useful embodiment of the invention. Epitopes for inclusion in a minigene are preferably selected according to the guidelines set forth in the previous section. A preferred means of administering nucleic acids encoding the peptides of the invention uses minigene constructs encoding a peptide comprising one or multiple epitopes of the invention.
The use of muiti-epitope inigenes is described below and in, Ishioka ef al, J. Immunol 162:3915-3925, 1999; An, L. and Whitton, J. L, J. Virol. 71:2292, 1997; Thomson, S. A. et al, J. Immunol. 157:822, 1996; Whitton, J. L. et al, J. Virol. 67:348, 1993; Hanke, R. ef al, Vaccine 16:426, 1998. For example, a multi-epitope DNA plasmid encoding supermotif- and/or motif-bearing epitopes derived 109P1D4, the PADRE® universal helper T cell epitope or multiple HTL epitopes from 109P1D4 (see e.g., Tables VIII-XXI and XXII to XLIX), and an endoplasmic reticulum-translocating signal sequence can be engineered. A vaccine may also comprise epitopes that are derived from other TAAs.
The immunogenicity of a multi-epitopic minigene can be confirmed in transgenic mice to evaluate the magnitude of CTL induction responses against the epitopes tested. Further, the immunogenicity of DNA-encoded epitopes in vivo can be correlated with the in vitro responses of specific CTL lines against target cells transfected with the DNA plasmid. Thus, these experiments can show that the minigene serves to both: 1.) generate a CTL response and 2.) that the induced CTLs recognized cells expressing the encoded epitopes.
For example, to create a DNA sequence encoding the selected epitopes (minigene) for expression in human cells, the amino acid sequences of the epitopes may be reverse translated. A human codon usage table can be used to guide the codon choice for each amino acid. These epitope-encoding DNA sequences may be directly adjoined, so that when translated, a continuous polypeptide sequence is created. To optimize expression and/or immunogenicity, additional elements can be incorporated into the minigene design. Examples of amino acid sequences that can be reverse translated and included in the minigene sequence include: HLA class I epitopes, HLA class II epitopes, antibody epitopes, a ubiquitination signal sequence, and/or an endoplasmic reticulum targeting signal. In addition, HLA presentation of CTL and HTL epitopes may be improved by including synthetic (e.g. poly-alanine) or naturally-occurring flanking sequences adjacent to the CTL or HTL epitopes; these larger peptides comprising the epitope(s) are within the scope of the invention.
The minigene sequence may be converted to DNA by assembling oligonucleotides that encode the plus and minus strands of the minigene. Overlapping oligonucleotides (30-100 bases long) may be synthesized, phosphorylated, purified and annealed under appropriate conditions using well known techniques. The ends of the oligonucleotides can be joined, for example, using T4 DNA ligase. This synthetic minigene, encoding the epitope polypeptide, can then be cloned into a desired expression vector.
Standard regulatory sequences well known to those of skill in the art are preferably included in the vector to ensure expression in the target cells. Several vector elements are desirable: a promoter with a down-stream cloning site for minigene insertion; a polyadenylation signal for efficient transcription termination; an E. coli origin of replication; and an £ coli selectable marker (e.g. ampicillin or kanamycin resistance). Numerous promoters can be used for this purpose, e.g., the human cytomegalovirus (hCMV) promoter. See, e.g., U.S. Patent Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.
Additional vector modifications may be desired to optimize minigene expression and immunogenicity. In some cases, introns are required for efficient gene expression, and one or more synthetic or naturally-occurring introns could be incorporated into the transcribed region of the minigene. The inclusion of mRNA stabilization sequences and sequences for replication in mammalian cells may also be considered for increasing minigene expression.
Once an expression vector is selected, the minigene is cloned into the polylinker region downstream of the promoter. This plasmid is transformed into an appropriate £ coli strain, and DNA is prepared using standard techniques. The orientation and DNA sequence of the minigene, as well as all other elements included in the vector, are confirmed using restriction mapping and DNA sequence analysis. Bacterial cells harboring the correct plasmid can be stored as a master cell bank and a working cell bank.
In addition, immunostimulatory sequences (ISSs or CpGs) appear to play a role in the immunogenicity of DNA vaccines. These sequences may be included in the vector, outside the minigene coding sequence, if desired to enhance immunogenicity.
In some embodiments, a bi-cistronic expression vector which allows production of both the minigene-encoded epitopes and a second protein (included to enhance or decrease immunogenicity) can be used. Examples of proteins or polypeptides that could beneficially enhance the immune response if co-expressed include cytokines (e.g., IL-2, IL-12, GM- CSF), cytokine-inducing molecules (e.g., LelF), costimulatory molecules, or for HTL responses, pan-DR binding proteins (PADRE™, Epimmune, San Diego, CA). Helper (HTL) epitopes can be joined to intracellular targeting signals and expressed separately from expressed CTL epitopes; this allows direction of the HTL epitopes to a cell compartment different than that of the CTL epitopes. If required, this could facilitate more efficient entry of HTL epitopes into the HLA class II pathway, thereby improving HTL induction. In contrast to HTL or CTL induction, specifically decreasing the immune response by co-expression of immunosuppressive molecules (e.g. TGF-β) may be beneficial in certain diseases.
Therapeutic quantities of plasmid DNA can be produced for example, by fermentation in £ coli, followed by purification. Aliquots from the working cell bank are used to inoculate growth medium, and grown to saturation in shaker flasks or a bioreactor according to well-known techniques. Plasmid DNA can be purified using standard bioseparation technologies such as solid phase anion-exchange resins supplied by QIAGEN, Inc. (Valencia, California). If required, supercoiled DNA can be isolated from the open circular and linear forms using gel electrophoresis or other methods.
Purified plasmid DNA can be prepared for injection using a variety of formulations. The simplest of these is reconstitution of lyophilized DNA in sterile phosphate-buffer saline (PBS). This approach, known as "naked DNA," is currently being used for intramuscular (IM) administration in clinical trials. To maximize the immunotherapeutic effects of minigene DNA vaccines, an alternative method for formulating purified plasmid DNA may be desirable. A variety of methods have been described, and new techniques may become available. Cationic lipids, glycolipids, and fusogenic liposomes can also be used in the formulation (see, e.g., as described by WO 93/24640; Mannino & Gould-Fogerite, BioTechniques 6(7): 682 (1988); U.S. Pat No. 5,279,833; WO 91/06309; and Feigner, ef al, Proc. Nat'IAcad. Sci. USA 84:7413 (1987). In addition, peptides and compounds referred to collectively as protective, interactive, non-condensing compounds (PINC) could also be complexed to purified plasmid DNA to influence variables such as stability, intramuscular dispersion, or trafficking to specific organs or cell types.
Target cell sensitization can be used as a functional assay for expression and HLA class I presentation of minigene-encoded CTL epitopes. For example, the plasmid DNA is introduced into a mammalian cell line that is suitable as a target for standard CTL chromium release assays. The transfection method used will be dependent on the final formulation. Electroporation can be used for "naked" DNA, whereas cationic lipids allow direct in vitro transfection. A plasmid expressing green fluorescent protein (GFP) can be co-transfected to allow enrichment of transfected cells using fluorescence activated cell sorting (FACS). These cells are then chromium-51 (51Cr) labeled and used as target cells for epitope-specific CTL lines; cytolysis, detected by 51Cr release, indicates both production of, and HLA presentation of, minigene-encoded CTL epitopes. Expression of HTL epitopes may be evaluated in an analogous manner using assays to assess HTL activity.
In vivo immunogenicity is a second approach for functional testing of minigene DNA formulations. Transgenic mice expressing appropriate human HLA proteins are immunized with the DNA product. The dose and route of administration are formulation dependent (e.g., IM for DNA in PBS, intraperitoneal (i.p.) for lipid-complexed DNA). Twenty-one days after immunization, splenocytes are harvested and restimulated for one week in the presence of peptides encoding each epitope being tested. Thereafter, for CTL effector cells, assays are conducted for cytolysis of peptide-loaded, 51Cr-labeled target cells using standard techniques. Lysis of target cells that were sensitized by HLA loaded with peptide epitopes, corresponding to minigene-encoded epitopes, demonstrates DNA vaccine function for in vivo induction of CTLs. Immunogenicity of HTL epitopes is confirmed in transgenic mice in an analogous manner.
Alternatively, the nucleic acids can be administered using ballistic delivery as described, for instance, in U.S. Patent No. 5,204,253. Using this technique, particles comprised solely of DNA are administered. In a further alternative embodiment, DNA can be adhered to particles, such as gold particles.
Minigenes can also be delivered using other bacterial or viral delivery systems well known in the art, e.g., an expression construct encoding epitopes of the invention can be incorporated into a viral vector such as vaccinia.
X.C.2. Combinations of CTL Peptides with Helper Peptides
Vaccine compositions comprising CTL peptides of the invention can be modified, e.g., analoged, to provide desired attributes, such as improved serum half life, broadened population coverage or enhanced immunogenicity.
For instance, the ability of a peptide to induce CTL activity can be enhanced by linking the peptide to a sequence which contains at least one epitope that is capable of inducing a T helper cell response. Although a CTL peptide can be directly linked to a T helper peptide, often CTL epitope/HTL epitope conjugates are linked by a spacer molecule. The spacer is typically comprised of relatively small, neutral molecules, such as amino acids or amino acid mimetics, which are substantially uncharged under physiological conditions. The spacers are typically selected from, e.g., Ala, Gly, or other neutral spacers of nonpolar amino acids or neutral polar amino acids, it will be understood that the optionally present spacer need not be comprised of the same residues and thus may be a hetero- or homo-oligomer. When present, the spacer will usually be at least one or two residues, more usually three to six residues and sometimes 10 or more residues. The CTL peptide epitope can be linked to the T helper peptide epitope either directly or via a spacer either at the amino or carboxy terminus of the CTL peptide. The amino terminus of either the immunogenic peptide or the T helper peptide may be acylated.
In certain embodiments, the T helper peptide is one that is recognized by T helper cells present in a majority of a genetically diverse population. This can be accomplished by selecting peptides that bind to many, most, or all of the HLA class II molecules. Examples of such amino acid bind many HLA Class II molecules include sequences from antigens such as fefamvs toxoid at positions 830-843 QYIKANSKFIGITE; (SEQ ID NO: 40), Plasmodium falciparum circumsporozoite (CS) protein at positions 378-398 DIEKKIAKMEKASSVFNWNS; (SEQ ID NO: 41), and Streptococcus 18kD protein at positions 116-131 GAVDSILGGVATYGAA; (SEQ ID NO: 42). Other examples include peptides bearing a DR 1-4-7 supermotif, or either of the DR3 motifs.
Alternatively, it is possible to prepare synthetic peptides capable of stimulating T helper lymphocytes, in a loosely HLA-restricted fashion, using amino acid sequences not found in nature (see, e.g., PCT publication WO 95/07707). These synthetic compounds called Pan-DR-binding epitopes (e.g., PADRE™, Epimmune, Inc., San Diego, CA) are designed, most preferably, to bind most HLA-DR (human HLA class II) molecules. For instance, a pan-DR-binding epitope peptide having the formula: xKXVAAWTLKAAx (SEQ ID NO: 43), where "X" is either cyclohexylalanine, phenylalanine, or tyrosine, and a is either D-alanine or L-alanine, has been found to bind to most HLA-DR alleles, and to stimulate the response of T helper lymphocytes from most individuals, regardless of their HLA type. An alternative of a pan-DR binding epitope comprises all "L" natural amino acids and can be provided in the form of nucleic acids that encode the epitope.
HTL peptide epitopes can also be modified to alter their biological properties. For example, they can be modified to include D-amino acids to increase their resistance to proteases and thus extend their serum half life, or they can be conjugated to other molecules such as lipids, proteins, carbohydrates, and the like to increase their biological activity. For example, a T helper peptide can be conjugated to one or more palmitic acid chains at either the amino or carboxyl termini.
X.C.3. Combinations of CTL Peptides with T Cell Priming Agents
In some embodiments it may be desirable to include in the pharmaceutical compositions of the invention at least one component which primes B lymphocytes or T lymphocytes. Lipids have been identified as agents capable of priming CTL in vivo. For example, palmitic acid residues can be attached to the ε-and α- amino groups of a lysine residue and then linked, e.g., via one or more linking residues such as Gly, Gly-Gly-, Ser, Ser-Ser, or the like, to an immunogenic peptide. The lipidated peptide can then be administered either directly in a micelle or particle, incorporated into a liposome, or emulsified in an adjuvant, e.g., incomplete Freund's adjuvant. In a preferred embodiment, a particularly effective immunogenic composition comprises palmitic acid attached to ε- and α- amino groups of Lys, which is attached via linkage, e.g., Ser-Ser, to the amino terminus of the immunogenic peptide.
As another example of lipid priming of CTL responses, £ coli lipoproteins, such as tripalmitoyl-S- glycerylcysteinlyseryl- serine (P3CSS) can be used to prime virus specific CTL when covalently attached to an appropriate peptide (see, e.g., Deres, etal, Nature 342:561, 1989). Peptides of the invention can be coupled to P3CSS, for example, and the lipopeptide administered to an individual to prime specifically an immune response to the target antigen. Moreover, because the induction of neutralizing antibodies can also be primed with p3CSS-conjugated epitopes, two such compositions can be combined to more effectively elicit both humoral and cell-mediated responses.
X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL and/or HTL Peptides
An embodiment of a vaccine composition in accordance with the invention comprises ex vivo administration of a cocktail of epitope-bearing peptides to PBMC, or isolated DC therefrom, from the patient's blood. A pharmaceutical to facilitate harvesting of DC can be used, such as Progenipoielin™ (Pharmacia-Monsanto, St. Louis, MO) or GM-CSF/IL-4. After pulsing the DC with peptides and prior to reinfusion into patients, the DC are washed to remove unbound peptides. In this embodiment, a vaccine comprises peptide-pulsed DCs which present the puised peptide epitopes complexed with HLA molecules on their surfaces.
The DC can be pulsed ex vivo with a cocktail of peptides, some of which stimulate CTL responses to 109P1D4. Optionally, a helper T cell (HTL) peptide, such as a natural or artificial loosely restricted HLA Class II peptide, can be included to facilitate the CTL response. Thus, a vaccine in accordance with the invention is used to treat a cancer which expresses or overexpresses 109P1D4.
X.D. Adoptive Immunotherapy
Antigenic 109P1 D4-related peptides are used to elicit a CTL and/or HTL response ex vivo, as well. The resulting CTL or HTL cells, can be used to treat tumors in patients that do not respond to other conventional forms of therapy, or will not respond to a therapeutic vaccine peptide or nucleic acid in accordance with the invention. Ex vivo CTL or HTL responses to a particular antigen are induced by incubating in tissue culture the patient's, or genetically compatible, CTL or HTL precursor cells together with a source of antigen-presenting cells (APC), such as dendritic cells, and the appropriate immunogenic peptide. After an appropriate incubation time (typically about 7-28 days), in which the precursor cells are activated and expanded into effector cells, the cells are infused back into the patient, where they will destroy (CTL) or facilitate destruction (HTL) of their specific target cell (e.g., a tumor cell). Transfected dendritic cells may also be used as antigen presenting cells.
X.E. Administration of Vaccines for Therapeutic or Prophylactic Purposes
Pharmaceutical and vaccine compositions of the invention are typically used to treat and/or prevent a cancer that expresses or overexpresses 109P1D4. In therapeutic applications, peptide and/or nucleic acid compositions are administered to a patient in an amount sufficient to elicit an effective B cell, CTL and/or HTL response to the antigen and to cure or at least partially arrest or slow symptoms and/or complications. An amount adequate to accomplish this is defined as "therapeutically effective dose." Amounts effective for this use will depend on, e.g., the particular composition administered, the manner of administration, the stage and severity of the disease being treated, the weight and general state of health of the patient, and the judgment of the prescribing physician.
For pharmaceutical compositions, the immunogenic peptides of the invention, or DNA encoding them, are generally administered to an individual already bearing a tumor that expresses 109P1D4. The peptides or DNA encoding them can be administered individually or as fusions of one or more peptide sequences. Patients can be treated with the immunogenic peptides separately or in conjunction with other treatments, such as surgery, as appropriate.
For therapeutic use, administration should generally begin at the first diagnosis of 109P1 D4-associated cancer. This is followed by boosting doses until at least symptoms are substantially abated and for a period thereafter. The embodiment of the vaccine composition (i.e., including, but not limited to embodiments such as peptide cocktails, polyepitopic polypeptides, minigenes, or TAA-specific CTLs or pulsed dendritic cells) delivered to the patient may vary according to the stage of the disease or the patient's health status. For example, in a patient with a tumor that expresses 109P1 D4, a vaccine comprising 109P1 D4-specific CTL may be more efficacious in killing tumor cells in patient with advanced disease than alternative embodiments.
It is generally important to provide an amount of the peptide epitope delivered by a mode of administration sufficient to stimulate effectively a cytotoxic T cell response; compositions which stimulate helper T cell responses can also be given in accordance with this embodiment of the invention.
The dosage for an initial therapeutic immunization generally occurs in a unit dosage range where the lower value is about 1, 5, 50, 500, or 1,000 μg and the higher value is about 10,000; 20,000; 30,000; or 50,000 μg. Dosage values for a human typically range from about 500 μg to about 50,000 μg per 70 kilogram patient. Boosting dosages of between about 1.0 μg to about 60,000 μg of peptide pursuant to a boosting regimen over weeks to months may be administered depending upon the patient's response and condition as determined by measuring the specific activity of CTL and HTL obtained from the patient's blood. Administration should continue until at least clinical symptoms or laboratory tests indicate that the neoplasia, has been eliminated or reduced and for a period thereafter. The dosages, routes of administration, and dose schedules are adjusted in accordance with methodologies known in the art.
In certain embodiments, the peptides and compositions of the present invention are employed in serious disease states, that is, life-threatening or potentially life threatening situations. In such cases, as a result of the minimal amounts of extraneous substances and the relative nontoxic nature of the peptides in preferred compositions of the invention, it is possible and may be felt desirable by the treating physician to administer substantial excesses of these peptide compositions relative to these stated dosage amounts.
The vaccine compositions of the invention can also be used purely as prophylactic agents. Generally the dosage for an initial prophylactic immunization generally occurs in a unit dosage range where the lower value is about 1, 5, 60, 600, or 1000 μg and the higher value is about 10,000; 20,000; 30,000; or 60,000 μg. Dosage values for a human typically range from about 500 μg to about 50,000 μg per 70 kilogram patient. This is followed by boosting dosages of between about 1.0 μg to about 50,000 μg of peptide administered at defined intervals from about four weeks to six months after the initial administration of vaccine. The immunogenicity of the vaccine can be assessed by measuring the specific activity of CTL and HTL obtained from a sample of the patient's blood.
The pharmaceutical compositions for therapeutic treatment are intended for parenteral, topical, oral, nasal, intrathecal, or local (e.g. as a cream or topical ointment) administration. Preferably, the pharmaceutical compositions are administered parentally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly. Thus, the invention provides compositions for parenteral administration which comprise a solution of the immunogenic peptides dissolved or suspended in an acceptable carrier, preferably an aqueous carrier.
A variety of aqueous carriers may be used, e.g., water, buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid and the like. These compositions may be sterilized by conventional, well-known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration.
The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservatives, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanola ine oleate, etc.
The concentration of peptides of the invention in the pharmaceutical formulations can vary widely, i.e., from less than about 0.1 %, usually at or at least about 2% to as much as 20% to 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, efc, in accordance with the particular mode of administration selected.
A human unit dose form of a composition is typically included in a pharmaceutical composition that comprises a human unit dose of an acceptable carrier, in one embodiment an aqueous carrier, and is administered in a volume/quantity that is known by those of skill in the art to be used for administration of such compositions to humans (see, e.g., Remington's Pharmaceutical Sciences, 17th Edition, A. Gennaro, Editor, Mack Publishing Co., Easton, Pennsylvania, 1985). For example a peptide dose for initial immunization can be from about 1 to about 50,000 μg, generally 100-5,000 μg, for a 70 kg patient. For example, for nucleic acids an initial immunization may be performed using an expression vector in the form of naked nucleic acid administered IM (or SC or ID) in the amounts of 0.5-5 mg at multiple sites. The nucleic acid (0.1 to 1000 μg) can also be administered using a gene gun. Following an incubation period of 3-4 weeks, a booster dose is then administered. The booster can be recombinant fowlpox virus administered at a dose of 5-107 to 5x109 pfu. For antibodies, a treatment generally involves repeated administration of the anti-109P1D4 antibody preparation, via an acceptable route of administration such as intravenous injection (IV), typically at a dose in the range of about 0.1 to about 10 mg/kg body weight. In general, doses in the range of 10-500 mg mAb per week are effective and well tolerated. Moreover, an initial loading dose of approximately 4 mg/kg patient body weight IV, followed by weekly doses of about 2 mg/kg IV of the anti- 109P1 D4 mAb preparation represents an acceptable dosing regimen. As appreciated by those of skill in the art, various factors can influence the ideal dose in a particular case. Such factors include, for example, half life of a composition, the binding affinity of an Ab, the immunogenicity of a substance, the degree of 109P1 D4 expression in the patient, the extent of circulating shed 109P1D4 antigen, the desired steady-state concentration level, frequency of treatment, and the influence of chemotherapeutic or other agents used in combination with the treatment method of the invention, as well as the health status of a particular patient. Non-limiting preferred human unit doses are, for example, δOOμg - 1 mg, 1 mg - 50mg, 50mg - 100mg, 100mg - 200mg, 200mg - 300mg, 400mg - δOOmg, 500mg - 600mg, 600mg - 700mg, 700mg - 800mg, 800mg - 900mg, 900mg - 1g, or 1mg - 700mg. In certain embodiments, the dose is in a range of 2-5 mg/kg body weight, e.g., with follow on weekly doses of 1-3 mg/kg; 0.5mg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10mg/kg body weight followed, e.g., in two, three or four weeks by weekly doses; 0.5 - 10mg/kg body weight, e.g., followed in two, three or four weeks by weekly doses; 225, 250, 275, 300, 325, 350, 375, 400mg m2 of body area weekly; 1-600mg mz of body area weekly; 225-400mg m2 of body area weekly; these does can be followed by weekly doses for 2, 3, 4, δ, 6, 7, 8, 9, 19, 11 , 12 or more weeks.
In one embodiment, human unit dose forms of polynucleotides comprise a suitable dosage range or effective amount that provides any therapeutic effect. As appreciated by one of ordinary skill in the art a therapeutic effect depends on a number of factors, including the sequence of the polynucleotide, molecular weight of the polynucleotide and route of administration. Dosages are generally selected by the physician or other health care professional in accordance with a variety of parameters known in the art, such as severity of symptoms, history of the patient and the like. Generally, for a polynucleotide of about 20 bases, a dosage range may be selected from, for example, an independently selected lower limit such as about 0.1, 0.25, 0.5, 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 mg/kg up to an independently selected upper limit, greater than the lower limit, of about 60, 80, 100, 200, 300, 400, 500, 750, 1000, 1600, 2000, 3000, 4000, 6000, 6000, 7000, 8000, 9000 or 10,000 mg/kg. For example, a dose may be about any of the following: 0.1 to 100 mg/kg, 0.1 to 60 mg/kg, 0.1 to 25 mg/kg, 0.1 to 10 mg/kg, 1 to 500 mg/kg, 100 to 400 mg/kg, 200 to 300 mg/kg, 1 to 100 mg/kg, 100 to 200 mg/kg, 300 to 400 mg/kg, 400 to 500 mg/kg, 500 to 1000 mg/kg, 500 to 5000 mg/kg, or 500 to 10,000 mg/kg. Generally, parenteral routes of administration may require higher doses of polynucleotide compared to more direct application to the nucleotide to diseased tissue, as do polynucleotides of increasing length.
In one embodiment, human unit dose forms of T-cells comprise a suitable dosage range or effective amount that provides any therapeutic effect. As appreciated by one of ordinary skill in the art, a therapeutic effect depends on a number of factors. Dosages are generally selected by the physician or other health care professional in accordance with a variety of parameters known in the art, such as severity of symptoms, history of the patient and the like. A dose may be about 104 cells to about 106 cells, about 106 cells to about 108 cells, about 103 to about 1011 cells, or about 10B to about 5 x 10™ cells. A dose may also about 106 cells/m2 to about 1010 cells/m2, or about 106 cells/m2 to about 108 cells/m2.
Proteins(s) of the invention, and/or nucleic acids encoding the protein(s), can also be administered via liposomes, which may also serve to: 1) target the proteins(s) to a particular tissue, such as lymphoid tissue; 2) to target selectively to diseases cells; or, 3) to increase the half-life of the peptide composition. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. In these preparations, the peptide to be delivered is incorporated as part of a liposome, alone or in conjunction with a molecule which binds to a receptor prevalent among lymphoid cells, such as monoclonal antibodies which bind to the CD45 antigen, or with other therapeutic or immunogenic compositions. Thus, liposomes either filled or decorated with a desired peptide of the invention can be directed to the site of lymphoid cells, where the liposomes then deliver the peptide compositions. Liposomes for use in accordance with the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of, e.g., liposome size, acid lability and stability of the liposomes in the blood stream. A variety of methods are available for preparing liposomes, as described in, e.g., Szoka, ef a/., Ann. Rev. Biophys. Bioeng. 9:467 (1980), and U.S. Patent Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.
For targeting cells of the immune system, a ligand to be incorporated into the liposome can include, e.g., antibodies or fragments thereof specific for cell surface determinants of the desired immune system cells. A liposome suspension containing a peptide may be administered intravenously, locally, topically, efc. in a dose which varies according to, infer a//a, the manner of administration, the peptide being delivered, and the stage of the disease being treated.
For solid compositions, conventional nontoxic solid carriers may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10- 95% of active ingredient, that is, one or more peptides of the invention, and more preferably at a concentration of 25%-7δ%.
For aerosol administration, immunogenic peptides are preferably supplied in finely divided form along with a surfactant and propellant. Typical percentages of peptides are about 0.01 %-20% by weight, preferably about 1%-10%. The surfactant must, of course, be nontoxic, and preferably soluble in the propellant. Representative of such agents are the esters or partial esters of fatty acids containing from about 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides may be employed. The surfactant may constitute about 0.1 %-20% by weight of the composition, preferably about 0.25-6%. The balance of the composition is ordinarily propellant. A carrier can also be included, as desired, as with, e.g., lecithin for intranasal delivery.
XI.) Diagnostic and Prognostic Embodiments of 109P1 D4.
As disclosed herein, 109P1 D4 polynucleotides, polypeptides, reactive cytotoxic T cells (CTL), reactive helper T cells (HTL) and anti-polypeptide antibodies are used in well known diagnostic, prognostic and therapeutic assays that examine conditions associated with dysregulated cell growth such as cancer, in particular the cancers listed in Table I (see, e.g., both its specific pattern of tissue expression as well as its overexpression in certain cancers as described for example in the Example entitled "Expression analysis of 109P1 D4 in normal tissues, and patient specimens").
109P1D4 can be analogized to a prostate associated antigen PSA, the archetypal marker that has been used by medical practitioners for years to identify and monitor the presence of prostate cancer (see, e.g., Merrill ef al, J. Urol. 163(2): 503-5120 (2000); Polascik ef al, J. Urol. Aug; 162(2):293-306 (1999) and Fortier ef al., J. Nat. Cancer Inst. 91(19): 1635- 1640(1999)). A variety of other diagnostic markers are also used in similar contexts including p53 and K-ras (see, e.g., Tulchinsky ef al, Int J Mol Med 1999 Jul 4(1):99-102 and Mini oto ef a/., Cancer Detect Prev 2000;24(1):1-12). Therefore, this disclosure of 109P1D4 polynucleotides and polypeptides (as well as 109P1D4 polynucleotide probes and anti-109P1D4 antibodies used to identify the presence of these molecules) and their properties allows skilled artisans to utilize these molecules in methods that are analogous to those used, for example, in a variety of diagnostic assays directed to examining conditions associated with cancer.
Typical embodiments of diagnostic methods which utilize the 109P1D4 polynucleotides, polypeptides, reactive T cells and antibodies are analogous to those methods from well-established diagnostic assays, which employ, e.g., PSA polynucleotides, polypeptides, reactive T cells and antibodies. For example, just as PSA polynucleotides are used as probes (for example in Northern analysis, see, e.g., Sharief ef al, Biochem. Mol. Biol. Int. 33(3) :567-74(1994)) and primers (for example in PCR analysis, see, e.g., Okegawa etal, J. Urol. 163(4): 1189-1190 (2000)) to observe the presence and/or the level of PSA mRNAs in methods of monitoring PSA overexpression or the metastasis of prostate cancers, the 109P1 D4 polynucleotides described herein can be utilized in the same way to detect 109P1D4 overexpression or the metastasis of prostate and other cancers expressing this gene. Alternatively, just as PSA polypeptides are used to generate antibodies specific for PSA which can then be used to observe the presence and/or the level of PSA proteins in methods to monitor PSA protein overexpression (see, e.g., Stephan et al, Urology 65(4):560-3 (2000)) or the metastasis of prostate cells (see, e.g., Alanen et al, Pathol. Res. Pract. 192(3):233-7 (1996)), the 109P1D4 polypeptides described herein can be utilized to generate antibodies for use in detecting 109P1 D4 overexpression or the metastasis of prostate cells and cells of other cancers expressing this gene.
Specifically, because metastases involves the movement of cancer cells from an organ of origin (such as the lung or prostate gland etc.) to a different area of the body (such as a lymph node), assays which examine a biological sample for the presence of cells expressing 109P1D4 polynucleotides and/or polypeptides can be used to provide evidence of metastasis. For example, when a biological sample from tissue that does not normally contain 109P1D4-expressing cells (lymph node) is found to contain 109P1D4-expressing cells such as the 109P1D4 expression seen in LAPC4 and LAPC9, xenografts isolated from lymph node and bone metastasis, respectively, this finding is indicative of metastasis.
Alternatively 109P1D4 polynucleotides and/or polypeptides can be used to provide evidence of cancer, for example, when cells in a biological sample that do not normally express 109P1D4 or express 109P1D4 at a different level are found to express 109P1D4 or have an increased expression of 109P1D4 (see, e.g., the 109P1D4 expression in the cancers listed in Table I and in patient samples etc. shown in the accompanying Figures). In such assays, artisans may further wish to generate supplementary evidence of metastasis by testing the biological sample for the presence of a second tissue restricted marker (in addition to 109P1D4) such as PSA, PSCA etc. (see, e.g., Alanen ef al, Pathol. Res. Pract. 192(3): 233- 237 (1996)).
The use of immunohistochemistry to identify the presence of a 109P1 D4 polypeptide within a tissue section can indicate an altered state of certain cells within that tissue. It is well understood in the art that the ability of an antibody to localize to a polypeptide that is expressed in cancer cells is a way of diagnosing presence of disease, disease stage, progression and/or tumor aggressiveness. Such an antibody can also detect an altered distribution of the polypeptide within the cancer cells, as compared to corresponding non-malignant tissue.
The 109P1 D4 polypeptide and immunogenic compositions are also useful in view of the phenomena of altered subcellular protein localization in disease states. Alteration of cells from normal to diseased state causes changes in cellular morphology and is often associated with changes in subcellular protein localization/distribution. For example, cell membrane proteins that are expressed in a polarized manner in normal cells can be altered in disease, resulting in distribution of the protein in a non-polar manner over the whole cell surface.
The phenomenon of altered subcellular protein localization in a disease state has been demonstrated with MUC1 and Her2 protein expression by use of immunohistochemical means. Normal epithelial cells have a typical apical distribution of MUC1, in addition to some supranuclear localization of the glycoprotein, whereas malignant lesions often demonstrate an apolar staining pattern (Diaz et al, The Breast Journal, 7; 40-45 (2001 ); Zhang ef al, Clinical Cancer Research, 4; 2669-2676 (1998): Cao, ef al, The Journal of Histochemistry and Cytochemistry, 45: 1547-1657 (1997)). In addition, normal breast epithelium is either negative for Her2 protein or exhibits only a basolateral distribution whereas malignant cells can express the protein over the whole cell surface (De Potter, et al, International Journal of Cancer, 44; 969-974 (1989): McCormick, et al, 117; 935-943 (2002)). Alternatively, distribution of the protein may be altered from a surface only localization to include diffuse cytoplasmic expression in the diseased state. Such an example can be seen with MUC1 (Diaz, etal, The Breast Journal, 7: 40-45 (2001)).
Alteration in the localization/distribution of a protein in the cell, as detected by immunohistochemical methods, can also provide valuable information concerning the favorability of certain treatment modalities. This last point is illustrated by a situation where a protein may be intracellular in normal tissue, but cell surface in malignant cells; the cell surface location makes the cells favorably amenable to antibody-based diagnostic and treatment regimens. When such an alteration of protein localization occurs for 109P1D4, the 109P1D4 protein and immune responses related thereto are very useful. Accordingly, the ability to determine whether alteration of subcellular protein localization occurred for 24P4C12 make the 109P1D4 protein and immune responses related thereto very useful. Use of the 109P1D4 compositions allows those skilled in the art to make important diagnostic and therapeutic decisions.
Immunohistochemical reagents specific to 109P1D4 are also useful to detect metastases of tumors expressing 109P1D4 when the polypeptide appears in tissues where 109P1D4 is not normally produced.
Thus, 109P1D4 polypeptides and antibodies resulting from immune responses thereto are useful in a variety of important contexts such as diagnostic, prognostic, preventative and/or therapeutic purposes known to those skilled in the art.
Just as PSA polynucleotide fragments and polynucleotide variants are employed by skilled artisans for use in methods of monitoring PSA, 109P1D4 polynucleotide fragments and polynucleotide variants are used in an analogous manner. In particular, typical PSA polynucleotides used in methods of monitoring PSA are probes or primers which consist of fragments of the PSA cDNA sequence. Illustrating this, primers used to PCR amplify a PSA polynucleotide must include less than the whole PSA sequence to function in the polymerase chain reaction. In the context of such PCR reactions, skilled artisans generally create a variety of different polynucleotide fragments that can be used as primers in order to amplify different portions of a polynucleotide of interest or to optimize amplification reactions (see, e.g., Caetano-Anolles, G. Biotechniques 25(3): 472-476, 478-480 (1998); Robertson etal, Methods Mol. Biol. 98:121-154 (1998)). An additional illustration of the use of such fragments is provided in the Example entitled "Expression analysis of 109P1 D4 in normal tissues, and patient specimens," where a 109P1D4 polynucleotide fragment is used as a probe to show the expression of 109P1D4 RNAs in cancer cells. In addition, variant polynucleotide sequences are typically used as primers and probes for the corresponding mRNAs in PCR and Northern analyses (see, e.g., Sawai ef a/., Fetal Diagn. Ther. 1996 Nov-Dec 11(6):407-13 and Current Protocols In Molecular Biology, Volume 2, Unit 2, Frederick M. Ausubel ef a/, eds., 1995)). Polynucleotide fragments and variants are useful in this context where they are capable of binding to a target polynucleotide sequence (e.g., a 109P1 D4 polynucleotide shown in Figure 2 or variant thereof) under conditions of high stringency.
Furthermore, PSA polypeptides which contain an epitope that can be recognized by an antibody or T cell that specifically binds to that epitope are used in methods of monitoring PSA. 109P1 D4 polypeptide fragments and polypeptide analogs or variants can also be used in an analogous manner. This practice of using polypeptide fragments or polypeptide variants to generate antibodies (such as anti-PSA antibodies or T cells) is typical in the art with a wide variety of systems such as fusion proteins being used by practitioners (see, e.g., Current Protocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubel etal. eds., 1995). In this context, each epitope(s) functions to provide the architecture with which an antibody or T cell is reactive. Typically, skilled artisans create a variety of different polypeptide fragments that can be used in order to generate immune responses specific for different portions of a polypeptide of interest (see, e.g., U.S. Patent No. 5,840,501 and U.S. Patent No. 5,939,533). For example it may be preferable to utilize a polypeptide comprising one of the 109P1D4 biological motifs discussed herein or a motif-bearing subsequence which is readily identified by one of skill in the art based on motifs available in the art. Polypeptide fragments, variants or analogs are typically useful in this context as long as they comprise an epitope capable of generating an antibody or T cell specific for a target polypeptide sequence (e.g. a 109P1D4 polypeptide shown in Figure 3).
As shown herein, the 109P1D4 polynucleotides and polypeptides (as well as the 109P1D4 polynucleotide probes and anti-109P1 D4 antibodies or T cells used to identify the presence of these molecules) exhibit specific properties that make them useful in diagnosing cancers such as those listed in Table I. Diagnostic assays that measure the presence of 109P1 D4 gene products, in order to evaluate the presence or onset of a disease condition described herein, such as prostate cancer, are used to identify patients for preventive measures or further monitoring, as has been done so successfully with PSA. Moreover, these materials satisfy a need in the art for molecules having similar or complementary characteristics to PSA in situations where, for example, a definite diagnosis of metastasis of prostatic origin cannot be made on the basis of a test for PSA alone (see, e.g., Alanen ef al, Pathol. Res. Pract. 192(3): 233-237 (1996)), and consequently, materials such as 109P1D4 polynucleotides and polypeptides (as well as the 109P1D4 polynucleotide probes and anti- 109P1D4 antibodies used to identify the presence of these molecules) need to be employed to confirm a metastases of prostatic origin.
Finally, in addition to their use in diagnostic assays, the 109P1D4 polynucleotides disclosed herein have a number of other utilities such as their use in the identification of oncogenetic associated chromosomal abnormalities in the chromosomal region to which the 109P1D4 gene maps (see the Example entitled "Chromosomal Mapping of 109P1D4" below). Moreover, in addition to their use in diagnostic assays, the 109P1D4-related proteins and polynucleotides disclosed herein have other utilities such as their use in the forensic analysis of tissues of unknown origin (see, e.g., Takahama K Forensic Sci Int 1996 Jun 28;80(1-2): 63-9).
Additionally, 109P1D4-related proteins or polynucleotides of the invention can be used to treat a pathologic condition characterized by the over-expression of 109P1 D4. For example, the amino acid or nucleic acid sequence of Figure 2 or Figure 3, or fragments of either, can be used to generate an immune response to a 109P1D4 antigen. Antibodies or other molecules that react with 109P1 D4 can be used to modulate the function of this molecule, and thereby provide a therapeutic benefit.
XII.) Inhibition of 109P1 D4 Protein Function
The invention includes various methods and compositions for inhibiting the binding of 109P1 D4 to its binding partner or its association with other protein(s) as well as methods for inhibiting 109P1D4 function.
XII.A.) Inhibition of 109P1 D4 With Intracellular Antibodies
In one approach, a recombinant vector that encodes single chain antibodies that specifically bind to 109P1 D4 are introduced into 109P1D4 expressing cells via gene transfer technologies. Accordingly, the encoded single chain anti- 109P1D4 antibody is expressed intracellularly, binds to 109P1D4 protein, and thereby inhibits its function. Methods for engineering such intracellular single chain antibodies are well known. Such intracellular antibodies, also known as "intrabodies", are specifically targeted to a particular compartment within the cell, providing control over where the inhibitory activity of the treatment is focused. This technology has been successfully applied in the art (for review, see Richardson and Marasco, 1995, TIBTECH vol. 13). Intrabodies have been shown to virtually eliminate the expression of otherwise abundant cell surface receptors (see, e.g., Richardson ef al, 1995, Proc. Natl. Acad. Sci. USA 92: 3137-3141; Beerli ef al, 1994, J. Biol. Chem. 289: 23931-23936; Deshane ef al, 1994, Gene Ther. 1: 332-337).
Single chain antibodies comprise the variable domains of the heavy and light chain joined by a flexible linker polypeptide, and are expressed as a single polypeptide. Optionally, single chain antibodies are expressed as a single chain variable region fragment joined to the light chain constant region. Well-known intracellular trafficking signals are engineered into recombinant polynucleotide vectors encoding such single chain antibodies in order to target precisely the intrabody to the desired intracellular compartment. For example, intrabodies targeted to the endoplasmic reticulum (ER) are engineered to incorporate a leader peptide and, optionally, a C-terminal ER retention signal, such as the KDEL amino acid motif. Intrabodies intended to exert activity in the nucleus are engineered to include a nuclear localization signal. Lipid moieties are joined to intrabodies in order to tether the intrabody to the cytosolic side of the plasma membrane. Intrabodies can also be targeted to exert function in the cytosol. For example, cytosolic intrabodies are used to sequester factors within the cytosol, thereby preventing them from being transported to their natural cellular destination.
In one embodiment, intrabodies are used to capture 109P1D4 in the nucleus, thereby preventing its activity within the nucleus. Nuclear targeting signals are engineered into such 109P1 D4 intrabodies in order to achieve the desired targeting. Such 109P1D4 intrabodies are designed to bind specifically to a particular 109P1D4 domain. In another embodiment, cytosolic intrabodies that specifically bind to a 109P1D4 protein are used to prevent 109P1D4 from gaining access to the nucleus, thereby preventing it from exerting any biological activity within the nucleus (e.g., preventing 109P1 D4 from forming transcription complexes with other factors).
In order to specifically direct the expression of such intrabodies to particular cells, the transcription of the intrabody is placed under the regulatory control of an appropriate tumor-specific promoter and/or enhancer. In order to target intrabody expression specifically to prostate, for example, the PSA promoter and/or promoter/enhancer can be utilized (See, for example, U.S. Patent No. 5,919,652 issued 6 July 1999).
XII.B.) Inhibition of 109P1 D4 with Recombinant Proteins
In another approach, recombinant molecules bind to 109P1D4 and thereby inhibit 109P1D4 function. For example, these recombinant molecules prevent or inhibit 109P1D4 from accessing/binding to its binding partner(s) or associating with other protein(s). Such recombinant molecules can, for example, contain the reactive partis) of a 109P1 D4 specific antibody molecule. In a particular embodiment, the 109P1 D4 binding domain of a 109P1 D4 binding partner is engineered into a dimeric fusion protein, whereby the fusion protein comprises two 109P1D4 ligand binding domains linked to the Fc portion of a human IgG, such as human lgG1. Such IgG portion can contain, for example, the CH2 and CH3 domains and the hinge region, but not the CH1 domain. Such dimeric fusion proteins are administered in soluble form to patients suffering from a cancer associated with the expression of 109P1 D4, whereby the dimeric fusion protein specifically binds to 109P1 D4 and blocks 109P1 D4 interaction with a binding partner. Such dimeric fusion proteins are further combined into multimeric proteins using known antibody linking technologies.
XII.C) Inhibition of 109P1D4 Transcription or Translation
The present invention also comprises various methods and compositions for inhibiting the transcription of the 109P1 D4 gene. Similarly, the invention also provides methods and compositions for inhibiting the translation of 109P1 D4 mRNA into protein.
In one approach, a method of inhibiting the transcription of the 109P1D4 gene comprises contacting the 109P1D4 gene with a 109P1 D4 antisense polynucleotide. In another approach, a method of inhibiting 109P1 D4 mRNA translation comprises contacting a 109P1D4 mRNA with an antisense polynucleotide. In another approach, a 109P1 D4 specific ribozyme is used to cleave a 109P1D4 message, thereby inhibiting translation. Such antisense and ribozyme based methods can also be directed to the regulatory regions of the 109P1D4 gene, such as 109P1D4 promoter and/or enhancer elements. Similarly, proteins capable of inhibiting a 109P1 D4 gene transcription factor are used to inhibit 109P1 D4 mRNA transcription. The various polynucleotides and compositions useful in the aforementioned methods have been described above. The use of antisense and ribozyme molecules to inhibit transcription and translation is well known in the art.
Other factors that inhibit the transcription of 109P1 D4 by interfering with 109P1 D4 transcriptional activation are also useful to treat cancers expressing 109P1D4. Similarly, factors that interfere with 109P1D4 processing are useful to treat cancers that express 109P1 D4. Cancer treatment methods utilizing such factors are also within the scope of the invention.
XII.D.) General Considerations for Therapeutic Strategies
Gene transfer and gene therapy technologies can be used to deliver therapeutic polynucleotide molecules to tumor cells synthesizing 109P1D4 (i.e., antisense, ribozyme, polynucleotides encoding intrabodies and other 109P1D4 inhibitory molecules). A number of gene therapy approaches are known in the art. Recombinant vectors encoding 109P1 D4 antisense polynucleotides, ribozymes, factors capable of interfering with 109P1 D4 transcription, and so forth, can be delivered to target tumor cells using such gene therapy approaches.
The above therapeutic approaches can be combined with any one of a wide variety of surgical, chemotherapy or radiation therapy regimens. The therapeutic approaches of the invention can enable the use of reduced dosages of chemotherapy (or other therapies) and/or less frequent administration, an advantage for all patients and particularly for those that do not tolerate the toxicity of the chemotherapeutic agent well.
The anti-tumor activity of a particular composition (e.g., antisense, ribozyme, intrabody), or a combination of such compositions, can be evaluated using various in vitro and in vivo assay systems. In vitro assays that evaluate therapeutic activity include cell growth assays, soft agar assays and other assays indicative of tumor promoting activity, binding assays capable of determining the extent to which a therapeutic composition will inhibit the binding of 109P1 D4 to a binding partner, etc.
In vivo, the effect of a 109P1 D4 therapeutic composition can be evaluated in a suitable animal model. For example, xenogenic prostate cancer models can be used, wherein human prostate cancer explants or passaged xenograft tissues are introduced into immune compromised animals, such as nude or SCID mice (Klein et al, 1997, Nature Medicine 3: 402-408). For example, PCT Patent Application W098/16628 and U.S. Patent 6,107,540 describe various xenograft models of human prostate cancer capable of recapitulating the development of primary tumors, micrometastasis, and the formation of osteoblastic metastases characteristic of late stage disease. Efficacy can be predicted using assays that measure inhibition of tumor formation, tumor regression or metastasis, and the like.
In vivo assays that evaluate the promotion of apoptosis are useful in evaluating therapeutic compositions. In one embodiment, xenografts from tumor bearing mice treated with the therapeutic composition can be examined for the presence of apoptotic foci and compared to untreated control xenograft-bearing mice. The extent to which apoptotic foci are found in the tumors of the treated mice provides an indication of the therapeutic efficacy of the composition.
The therapeutic compositions used in the practice of the foregoing methods can be formulated into pharmaceutical compositions comprising a carrier suitable for the desired delivery method. Suitable carriers include any material that when combined with the therapeutic composition retains the anti-tumor function of the therapeutic composition and is generally non-reactive with the patient's immune system. Examples include, but are not limited to, any of a number of standard pharmaceutical carriers such as sterile phosphate buffered saline solutions, bacteriostatic water, and the like (see, generally, Remington's Pharmaceutical Sciences 16th Edition, A. Osal., Ed., 1980).
Therapeutic formulations can be solubilized and administered via any route capable of delivering the therapeutic composition to the tumor site. Potentially effective routes of administration include, but are not limited to, intravenous, parenteral, intraperitoneal, intramuscular, intratumor, intradermal, intraorgan, orthotopic, and the like. A preferred formulation for intravenous injection comprises the therapeutic composition in a solution of preserved bacteriostatic water, sterile unpreserved water, and/or diluted in polyvinylchloride or polyethylene bags containing 0.9% sterile Sodium Chloride for Injection, USP. Therapeutic protein preparations can be lyophilized and stored as sterile powders, preferably under vacuum, and then reconstituted in bacteriostatic water (containing for example, benzyl alcohol preservative) or in sterile water prior to injection.
Dosages and administration protocols for the treatment of cancers using the foregoing methods will vary with the method and the target cancer, and will generally depend on a number of other factors appreciated in the art.
XIII.) Identification. Characterization and Use of Modulators of 109P1D4
Methods to Identify and Use Modulators
In one embodiment, screening is performed to identify modulators that induce or suppress a particular expression profile, suppress or induce specific pathways, preferably generating the associated phenotype thereby. In another embodiment, having identified differentially expressed genes important in a particular state; screens are performed to identify modulators that alter expression of individual genes, either increase or decrease. In another embodiment, screening is performed to identify modulators that alter a biological function of the expression product of a differentially expressed gene. Again, having identified the importance of a gene in a particular state, screens are performed to identify agents that bind and/or modulate the biological activity of the gene product.
In addition, screens are done for genes that are induced in response to a candidate agent. After identifying a modulator (one that suppresses a cancer expression pattern leading to a normal expression pattern, or a modulator of a cancer gene that leads to expression of the gene as in normal tissue) a screen is performed to identify genes that are specifically modulated in response to the agent. Comparing expression profiles between normal tissue and agent-treated cancer tissue reveals genes that are not expressed in normal tissue or cancer tissue, but are expressed in agent treated tissue, and vice versa. These agent-specific sequences are identified and used by methods described herein for cancer genes or proteins. In particular these sequences and the proteins they encode are used in marking or identifying agent- treated cells. In addition, antibodies are raised against the agent-induced proteins and used to target novel therapeutics to the treated cancer tissue sample.
Modulator-related Identification and Screening Assays:
Gene Expression-related Assays
Proteins, nucleic acids, and antibodies of the invention are used in screening assays. The cancer-associated proteins, antibodies, nucleic acids, modified proteins and cells containing these sequences are used in screening assays, such as evaluating the effect of drug candidates on a "gene expression profile," expression profile of polypeptides or alteration of biological function. In one embodiment, the expression profiles are used, preferably in conjunction with high throughput screening techniques to allow monitoring for expression profile genes after treatment with a candidate agent (e.g., Davis, GF, et al, J Biol Screen 7:69 (2002); Zlokamik, et al., Science 279:84-8 (1998); Heid, Genome Res 6:986- 94,1996).
The cancer proteins, antibodies, nucleic acids, modified proteins and cells containing the native or modified cancer proteins or genes are used in screening assays. That is, the present invention comprises methods for screening for compositions which modulate the cancer phenotype or a physiological function of a cancer protein of the invention. This is done on a gene itself or by evaluating the effect of drug candidates on a "gene expression profile" or biological function. In one embodiment, expression profiles are used, preferably in conjunction with high throughput screening techniques to allow monitoring after treatment with a candidate agent, see Zlokamik, supra.
A variety of assays are executed directed to the genes and proteins of the invention. Assays are run on an individual nucleic acid or protein level. That is, having identified a particular gene as up regulated in cancer, test compounds are screened for the ability to modulate gene expression or for binding to the cancer protein of the invention. "Modulation" in this context includes an increase or a decrease in gene expression. The preferred amount of modulation will depend on the original change of the gene expression in normal versus tissue undergoing cancer, with changes of at least 10%, preferably 50%, more preferably 100-300%, and in some embodiments 300-1000% or greater. Thus, if a gene exhibits a 4-fold increase in cancer tissue compared to normal tissue, a decrease of about four-fold is often desired; similarly, a 10-fold decrease in cancer tissue compared to normal tissue a target value of a 10-fold increase in expression by the test compound is often desired. Modulators that exacerbate the type of gene expression seen in cancer are also useful, e.g., as an upregulated target in further analyses.
The amount of gene expression is monitored using nucleic acid probes and the quantification of gene expression levels, or, alternatively, a gene product itself is monitored, e.g., through the use of antibodies to the cancer protein and standard immunoassays. Proteomics and separation techniques also allow for quantification of expression.
Expression Monitoring to Identify Compounds that Modify Gene Expression
In one embodiment, gene expression monitoring, i.e., an expression profile, is monitored simultaneously for a number of entities. Such profiles will typically involve one or more of the genes of Figure 2. In this embodiment, e.g., cancer nucleic acid probes are attached to biochips to detect and quantify cancer sequences in a particular cell. Alternatively, PCR can be used. Thus, a series, e.g., wells of a microtiter plate, can be used with dispensed primers in desired wells. A PCR reaction can then be performed and analyzed for each well.
Expression monitoring is performed to identify compounds that modify the expression of one or more cancer- associated sequences, e.g., a polynucleotide sequence set out in Figure 2. Generally, a test modulator is added to the cells prior to analysis. Moreover, screens are also provided to identify agents that modulate cancer, modulate cancer proteins of the invention, bind to a cancer protein of the invention, or interfere with the binding of a cancer protein of the invention and an antibody or other binding partner.
In one embodiment, high throughput screening methods involve providing a library containing a large number of potential therapeutic compounds (candidate compounds). Such "combinatorial chemical libraries" are then screened in one or more assays to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional "lead compounds," as compounds for screening, or as therapeutics.
In certain embodiments, combinatorial libraries of potential modulators are screened for an ability to bind to a cancer polypeptide or to modulate activity. Conventionally, new chemical entities with useful properties are generated by identifying a chemical compound (called a "lead compound") with some desirable property or activity, e.g., inhibiting activity, creating variants of the lead compound, and evaluating the property and activity of those variant compounds. Often, high throughput screening (HTS) methods are employed for such an analysis.
As noted above, gene expression monitoring is conveniently used to test candidate modulators (e.g., protein, nucleic acid or small molecule). After the candidate agent has been added and the cells allowed to incubate for a period, the sample containing a target sequence to be analyzed is, e.g., added to a biochip.
If required, the target sequence is prepared using known techniques. For example, a sample is treated to lyse the cells, using known lysis buffers, electroporation, etc., with purification and/or amplification such as PCR performed as appropriate. For example, an in vitro transcription with labels covalently attached to the nucleotides is performed. Generally, the nucleic acids are labeled with biotin-FITC or PE, or with cy3 or cy5.
The target sequence can be labeled with, e.g., a fluorescent, a chemiluminescent, a chemical, or a radioactive signal, to provide a means of detecting the target sequence's specific binding to a probe. The label also can be an enzyme, such as alkaline phosphatase or horseradish peroxidase, which when provided with an appropriate substrate produces a product that is detected. Alternatively, the label is a labeled compound or small molecule, such as an enzyme inhibitor, that binds but is not catalyzed or altered by the enzyme. The label also can be a moiety or compound, such as, an epitope tag or biotin which specifically binds to streptavidin. For the example of biotin, the streptavidin is labeled as described above, thereby, providing a detectable signal for the bound target sequence. Unbound labeled streptavidin is typically removed prior to analysis.
As will be appreciated by those in the art, these assays can be direct hybridization assays or can comprise "sandwich assays", which include the use of multiple probes, as is generally outlined in U.S. Patent Nos. 5, 681,702; 5,597,909; 5,545,730; 5,594,117; 6,691,584; 5,571,670; 5,580,731; 5,571,670; 5,691,584; 5,624,802; 5,635,352; 5,594,118; 5,359,100; 5,124, 246; and 5,681,697. In this embodiment, in general, the target nucleic acid is prepared as outlined above, and then added to the biochip comprising a plurality of nucleic acid probes, under conditions that allow the formation of a hybridization complex.
A variety of hybridization conditions are used in the present invention, including high, moderate and low stringency conditions as outlined above. The assays are generally run under stringency conditions which allow formation of the label probe hybridization complex only in the presence of target. Stringency can be controlled by altering a step parameter that is a thermodynamic variable, including, but not limited to, temperature, formamide concentration, salt concentration, chaotropic salt concentration pH, organic solvent concentration, etc. These parameters may also be used to control non-specific binding, as is generally outlined in U.S. Patent No. 5,681,697. Thus, it can be desirable to perform certain steps at higher stringency conditions to reduce non-specific binding.
The reactions outlined herein can be accomplished in a variety of ways. Components of the reaction can be added simultaneously, or sequentially, in different orders, with preferred embodiments outlined below. In addition, the reaction may include a variety of other reagents. These include salts, buffers, neutral proteins, e.g. albumin, detergents, etc. which can be used to facilitate optimal hybridization and detection, and/or reduce nonspecific or background interactions. Reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti- icrobial agents, etc., may also be used as appropriate, depending on the sample preparation methods and purity of the target. The assay data are analyzed to determine the expression levels of individual genes, and changes in expression levels as between states, forming a gene expression profile.
Biological Activity-related Assays
The invention provides methods identify or screen for a compound that modulates the activity of a cancer-related gene or protein of the invention. The methods comprise adding a test compound, as defined above, to a cell comprising a cancer protein of the invention. The cells contain a recombinant nucleic acid that encodes a cancer protein of the invention. In another embodiment, a library of candidate agents is tested on a plurality of cells.
In one aspect, the assays are evaluated in the presence or absence or previous or subsequent exposure of physiological signals, e.g. hormones, antibodies, peptides, antigens, cytokines, growth factors, action potentials, pharmacological agents including chemotherapeutics, radiation, carcinogenics, or other cells (i.e., cell-cell contacts). In another example, the determinations are made at different stages of the cell cycle process. In this way, compounds that modulate genes or proteins of the invention are identified. Compounds with pharmacological activity are able to enhance or interfere with the activity of the cancer protein of the invention. Once identified, similar structures are evaluated to identify critical structural features of the compound.
In one embodiment, a method of modulating ( e.g., inhibiting) cancer cell division is provided; the method comprises administration of a cancer modulator. In another embodiment, a method of modulating ( e.g., inhibiting) cancer is provided; the method comprises administration of a cancer modulator. In a further embodiment, methods of treating cells or individuals with cancer are provided; the method comprises administration of a cancer modulator.
In one embodiment, a method for modulating the status of a cell that expresses a gene of the invention is provided. As used herein status comprises such art-accepted parameters such as growth, proliferation, survival, function, apoptosis, senescence, location, enzymatic activity, signal transduction, etc. of a cell. In one embodiment, a cancer inhibitor is an antibody as discussed above. In another embodiment, the cancer inhibitor is an antisense molecule. A variety of cell growth, proliferation, and metastasis assays are known to those of skill in the art, as described herein.
High Throughput Screening to Identify Modulators
The assays to identify suitable modulators are amenable to high throughput screening. Preferred assays thus detect enhancement or inhibition of cancer gene transcription, inhibition or enhancement of polypeptide expression, and inhibition or enhancement of polypeptide activity.
In one embodiment, modulators evaluated in high throughput screening methods are proteins, often naturally occurring proteins or fragments of naturally occurring proteins. Thus, e.g., cellular extracts containing proteins, or random or directed digests of proteinaceous cellular extracts, are used. In this way, libraries of proteins are made for screening in the methods of the invention. Particularly preferred in this embodiment are libraries of bacterial, fungal, viral, and mammalian proteins, with the latter being preferred, and human proteins being especially preferred. Particularly useful test compound will be directed to the class of proteins to which the target belongs, e.g., substrates for enzymes, or ligands and receptors.
Use of Soft Agar Growth and Colony Formation to Identify and Characterize Modulators
Normal cells require a solid substrate to attach and grow. When cells are transformed, they lose this phenotype and grow detached from the substrate. For example, transformed cells can grow in stirred suspension culture or suspended in semi-solid media, such as semi-solid or soft agar. The transformed cells, when transfected with tumor suppressor genes, can regenerate normal phenotype and once again require a solid substrate to attach to and grow. Soft agar growth or colony formation in assays are used to identify modulators of cancer sequences, which when expressed in host cells, inhibit abnormal cellular proliferation and transformation. A modulator reduces or eliminates the host cells' ability to grow suspended in solid or semisolid media, such as agar.
Techniques for soft agar growth or colony formation in suspension assays are described in Freshney, Culture of Animal Cells a Manual of Basic Technique (3rd ed., 1994). See also, the methods section of Garkavtsev et al. (1996), supra.
Evaluation of Contact Inhibition and Growth Density Limitation to Identify and Characterize Modulators
Normal cells typically grow in a flat and organized pattern in cell culture until they touch other cells. When the cells touch one another, they are contact inhibited and stop growing. Transformed cells, however, are not contact inhibited and continue to grow to high densities in disorganized foci. Thus, transformed cells grow to a higher saturation density than corresponding normal cells. This is detected morphologically by the formation of a disoriented monolayer of cells or cells in foci. Alternatively, labeling index with (3H)-thymidine at saturation density is used to measure density limitation of growth, similarly an MTT or Alamar blue assay will reveal proliferation capacity of cells and the the ability of modulators to affect same. See Freshney (1994), supra. Transformed cells, when transfected with tumor suppressor genes, can regenerate a normal phenotype and become contact inhibited and would grow to a lower density. In this assay, labeling index with 3H)-thymidine at saturation density is a preferred method of measuring density limitation of growth. Transformed host cells are transfected with a cancer-associated sequence and are grown for 24 hours at saturation density in non-limiting medium conditions. The percentage of cells labeling with (3H)-thymidine is determined by incorporated cpm.
Contact independent growth is used to identify modulators of cancer sequences, which had led to abnormal cellular proliferation and transformation. A modulator reduces or eliminates contact independent growth, and returns the cells to a normal phenotype.
Evaluation of Growth Factor or Serum Dependence to Identify and Characterize Modulators
Transformed cells have lower serum dependence than their normal counterparts (see, e.g., Temin, J. Natl. Cancer Inst. 37:167-175 (1966); Eagle et al., J. Exp. Med 131:836-879 (1970)); Freshney, supra. This is in part due to release of various growth factors by the transformed cells. The degree of growth factor or serum dependence of transformed host cells can be compared with that of control. For example, growth factor or serum dependence of a cell is monitored in methods to identify and characterize compounds that modulate cancer-associated sequences of the invention.
Use of Tumor-specific Marker Levels to Identify and Characterize Modulators Tumor cells release an increased amount of certain factors (hereinafter "tumor specific markers") than their normal counterparts. For example, plasminogen activator (PA) is released from human glioma at a higher level than from normal brain cells (see, e.g., Gullino, Angiogenesis, Tumor Vascularization, and Potential Interference with Tumor Growth, in Biological Responses in Cancer, pp. 178-184 (Mihich (ed.) 1985)). Similarly, Tumor Angiogenesis Factor (TAF) is released at a higher level in tumor cells than their normal counterparts. See, e.g., Folkman, Angiogenesis and Cancer, Sem Cancer Biol. (1992)), while bFGF is released from endothelial tumors (Ensoli, B et al).
Various techniques which measure the release of these factors are described in Freshney (1994), supra. Also, see, Unkless et al., J. Biol. Chem. 249:4296-4306 (1974); Strickland & Beers, J. Biol. Chem. 261:6694-6702 (1976); Whur et al., Br. J. Cancer 42:305312 (1980); Gullino, Angiogenesis, Tumor Vascularization, and Potential Interference with Tumor Growth, in Biological Responses in Cancer, pp. 178-184 (Mihich (ed.) 1985); Freshney, Anticancer Res. 5:111-130 (1986). For example, tumor specific marker levels are monitored in methods to identify and characterize compounds that modulate cancer-associated sequences of the invention.
Invasiveness into Matriqel to Identify and Characterize Modulators
The degree of invasiveness into Matrigel or an extracellular matrix constituent can be used as an assay to identify and characterize compounds that modulate cancer associated sequences. Tumor cells exhibit a positive correlation between malignancy and invasiveness of cells into Matrigel or some other extracellular matrix constituent. In this assay, tumorigenic cells are typically used as host cells. Expression of a tumor suppressor gene in these host cells would decrease invasiveness of the host cells. Techniques described in Cancer Res. 1999; 59:6010; Freshney (1994), supra, can be used. Briefly, the level of invasion of host cells is measured by using filters coated with Matrigel or some other extracellular matrix constituent. Penetration into the gel, or through to the distal side of the filter, is rated as invasiveness, and rated histologically by number of cells and distance moved, or by prelabeling the cells with 1251 and counting the radioactivity on the distal side of the filter or bottom of the dish. See, e.g., Freshney (1984), supra.
Evaluation of Tumor Growth In Vivo to Identify and Characterize Modulators
Effects of cancer-associated sequences on cell growth are tested in transgenic or immune-suppressed organisms. Transgenic organisms are prepared in a variety of art-accepted ways. For example, knock-out transgenic organisms, e.g., mammals such as mice, are made, in which a cancer gene is disrupted or in which a cancer gene is inserted. Knock-out transgenic mice are made by insertion of a marker gene or other heterologous gene into the endogenous cancer gene site in the mouse genome via homologous recombination. Such mice can also be made by substituting the endogenous cancer gene with a mutated version of the cancer gene, or by mutating the endogenous cancer gene, e.g., by exposure to carcinogens.
To prepare transgenic chimeric animals, e.g., mice, a DNA construct is introduced into the nuclei of embryonic stem cells. Cells containing the newly engineered genetic lesion are injected into a host mouse embryo, which is re- implanted into a recipient female. Some of these embryos develop into chimeric mice that possess germ cells some of which are derived from the mutant cell line. Therefore, by breeding the chimeric mice it is possible to obtain a new line of mice containing the introduced genetic lesion (see, e.g., Capecchi et al., Science 244:1288 (1989)). Chimeric mice can be derived according to US Patent 6,365,797, issued 2 April 2002; US Patent 6,107,540 issued 22 August 2000; Hogan et al., Manipulating the Mouse Embryo: A laboratory Manual, Cold Spring Harbor Laboratory (1988) and Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, Robertson, ed., IRL Press, Washington, D.C., (1987).
Alternatively, various immune-suppressed or immune-deficient host animals can be used. For example, a genetically athymic "nude" mouse (see, e.g., Giovanella et al., J. Natl. Cancer Inst. 52:921 (1974)), a SCID mouse, a thymectornized mouse, or an irradiated mouse (see, e.g., Bradley et al., Br. J. Cancer 38:263 (1978); Selby et al., Br. J. Cancer 41:52 (1980)) can be used as a host. Transplantable tumor cells (typically about 106 cells) injected into isogenic hosts produce invasive tumors in a high proportion of cases, while normal cells of similar origin will not. In hosts which developed invasive tumors, cells expressing cancer-associated sequences are injected subcutaneously or orthotopically. Mice are then separated into groups, including control groups and treated experimental groups) e.g. treated with a modulator). After a suitable length of time, preferably 4-8 weeks, tumor growth is measured (e.g., by volume or by its two largest dimensions, or weight) and compared to the control. Tumors that have statistically significant reduction (using, e.g., Student's T test) are said to have inhibited growth.
In Vitro Assays to Identify and Characterize Modulators
Assays to identify compounds with modulating activity can be performed in vitro. For example, a cancer polypeptide is first contacted with a potential modulator and incubated for a suitable amount of time, e.g., from 0.5 to 48 hours. In one embodiment, the cancer polypeptide levels are determined in vitro by measuring the level of protein or mRNA. The level of protein is measured using immunoassays such as Western blotting, ELISA and the like with an antibody that selectively binds to the cancer polypeptide or a fragment thereof. For measurement of mRNA, amplification, e.g., using PCR, LCR, or hybridization assays, e. g., Northern hybridization, RNAse protection, dot blotting, are preferred. The level of protein or mRNA is detected using directly or indirectly labeled detection agents, e.g., fluorescently or radioactively labeled nucleic acids, radioactively or enzymatically labeled antibodies, and the like, as described herein.
Alternatively, a reporter gene system can be devised using a cancer protein promoter operably linked to a reporter gene such as luciferase, green fluorescent protein, CAT, or P-gal. The reporter construct is typically transfected into a cell. After treatment with a potential modulator, the amount of reporter gene transcription, translation, or activity is measured according to standard techniques known to those of skill in the art (Davis GF, supra; Gonzalez, J. & Negulescu, P. Curr. Opin. Biotechnol. 1998: 9:624).
As outlined above, in vitro screens are done on individual genes and gene products. That is, having identified a particular differentially expressed gene as important in a particular state, screening of modulators of the expression of the gene or the gene product itself is performed.
In one embodiment, screening for modulators of expression of specific gene(s) is performed. Typically, the expression of only one or a few genes is evaluated. In another embodiment, screens are designed to first find compounds that bind to differentially expressed proteins. These compounds are then evaluated for the ability to modulate differentially expressed activity. Moreover, once initial candidate compounds are identified, variants can be further screened to better evaluate structure activity relationships.
Binding Assays to Identify and Characterize Modulators
In binding assays in accordance with the invention, a purified or isolated gene product of the invention is generally used. For example, antibodies are generated to a protein of the invention, and immunoassays are run to determine the amount and/or location of protein. Alternatively, cells comprising the cancer proteins are used in the assays.
Thus, the methods comprise combining a cancer protein of the invention and a candidate compound such as a ligand, and determining the binding of the compound to the cancer protein of the invention. Preferred embodiments utilize the human cancer protein; animal models of human disease of can also be developed and used. Also, other analogous mammalian proteins also can be used as appreciated by those of skill in the art. Moreover, in some embodiments variant or derivative cancer proteins are used.
Generally, the cancer protein of the invention, or the ligand, is non-diffusibly bound to an insoluble support. The support can, e.g., be one having isolated sample receiving areas (a microtiter plate, an array, etc.). The insoluble supports can be made of any composition to which the compositions can be bound, is readily separated from soluble material, and is otherwise compatible with the overall method of screening. The surface of such supports can be solid or porous and of any convenient shape.
Examples of suitable insoluble supports include microtiter plates, arrays, membranes and beads. These are typically made of glass, plastic (e.g., polystyrene), polysaccharide, nylon, nitrocellulose, or Teflon™, etc. Microtiter plates and arrays are especially convenient because a large number of assays can be carried out simultaneously, using small amounts of reagents and samples. The particular manner of binding of the composition to the support is not crucial so long as it is compatible with the reagents and overall methods of the invention, maintains the activity of the composition and is nondiffusable. Preferred methods of binding include the use of antibodies which do not sterically block either the ligand binding site or activation sequence when attaching the protein to the support, direct binding to "sticky" or ionic supports, chemical crosslinking, the synthesis of the protein or agent on the surface, etc. Following binding of the protein or ligand/binding agent to the support, excess unbound material is removed by washing. The sample receiving areas may then be blocked through incubation with bovine serum albumin (BSA), casein or other innocuous protein or other moiety.
Once a cancer protein of the invention is bound to the support, and a test compound is added to the assay. Alternatively, the candidate binding agent is bound to the support and the cancer protein of the invention is then added. Binding agents include specific antibodies, non-natural binding agents identified in screens of chemical libraries, peptide analogs, etc.
Of particular interest are assays to identify agents that have a low toxicity for human cells. A wide variety of assays can be used for this purpose, including proliferation assays, cAMP assays, labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, functional assays (phosphorylation assays, etc.) and the like.
A determination of binding of the test compound (ligand, binding agent, modulator, etc.) to a cancer protein of the invention can be done in a number of ways. The test compound can be labeled, and binding determined directly, e.g., by attaching all or a portion of the cancer protein of the invention to a solid support, adding a labeled candidate compound (e.g., a fluorescent label), washing off excess reagent, and determining whether the label is present on the solid support. Various blocking and washing steps can be utilized as appropriate. In certain embodiments, only one of the components is labeled, e.g., a protein of the invention or ligands labeled. Alternatively, more than one component is labeled with different labels, e.g., I125, for the proteins and a fluorophor for the compound. Proximity reagents, e.g., quenching or energy transfer reagents are also useful.
Competitive Binding to Identify and Characterize Modulators
In one embodiment, the binding of the "test compound" is determined by competitive binding assay with a "competitor." The competitor is a binding moiety that binds to the target molecule (e.g., a cancer protein of the invention). Competitors include compounds such as antibodies, peptides, binding partners, ligands, etc. Under certain circumstances, the competitive binding between the test compound and the competitor displaces the test compound. In one embodiment, the test compound is labeled. Either the test compound, the competitor, or both, is added to the protein for a time sufficient to allow binding. Incubations are performed at a temperature that facilitates optimal activity, typically between four and 40°C. Incubation periods are typically optimized, e.g., to facilitate rapid high throughput screening; typically between zero and one hour will be sufficient. Excess reagent is generally removed or washed away. The second component is then added, and the presence or absence of the labeled component is followed, to indicate binding.
In one embodiment, the competitor is added first, followed by the test compound. Displacement of the competitor is an indication that the test compound is binding to the cancer protein and thus is capable of binding to, and potentially modulating, the activity of the cancer protein, in this embodiment, either component can be labeled. Thus, e.g., if the competitor is labeled, the presence of label in the post-test compound wash solution indicates displacement by the test compound. Alternatively, if the test compound is labeled, the presence of the label on the support indicates displacement.
In an alternative embodiment, the test compound is added first, with incubation and washing, followed by the competitor. The absence of binding by the competitor indicates that the test compound binds to the cancer protein with higher affinity than the competitor. Thus, if the test compound is labeled, the presence of the label on the support, coupled with a lack of competitor binding, indicates that the test compound binds to and thus potentially modulates the cancer protein of the invention.
Accordingly, the competitive binding methods comprise differential screening to identity agents that are capable of modulating the activity of the cancer proteins of the invention. In this embodiment, the methods comprise combining a cancer protein and a competitor in a first sample. A second sample comprises a test compound, the cancer protein, and a competitor. The binding of the competitor is determined for both samples, and a change, or difference in binding between the two samples indicates the presence of an agent capable of binding to the cancer protein and potentially modulating its activity. That is, if the binding of the competitor is different in the second sample relative to the first sample, the agent is capable of binding to the cancer protein.
Alternatively, differential screening is used to identify drug candidates that bind to the native cancer protein, but cannot bind to modified cancer proteins. For example the structure of the cancer protein is modeled and used in rational drug design to synthesize agents that interact with that site, agents which generally do not bind to site-modified proteins. Moreover, such drug candidates that affect the activity of a native cancer protein are also identified by screening drugs for the ability to either enhance or reduce the activity of such proteins.
Positive controls and negative controls can be used in the assays. Preferably control and test samples are performed in at least triplicate to obtain statistically significant results. Incubation of all samples occurs for a time sufficient to allow for the binding of the agent to the protein. Following incubation, samples are washed free of non-specifically bound material and the amount of bound, generally labeled agent determined. For example, where a radiolabel is employed, the samples can be counted in a scintillation counter to determine the amount of bound compound. A variety of other reagents can be included in the screening assays. These include reagents like salts, neutral proteins, e.g. albumin, detergents, etc. which are used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., can be used. The mixture of components is added in an order that provides for the requisite binding.
Use of Polynucleotides to Down-reoulate or Inhibit a Protein of the Invention.
Polynucleotide modulators of cancer can be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand-binding molecule, as described in WO 91/04753. Suitable ligand-binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or block entry of the sense or antisense oligonucleotide or its conjugated version into the cell. Alternatively, a polynucleotide modulator of cancer can be introduced into a cell containing the target nucleic acid sequence, e.g., by formation of a polynucleotide-lipid complex, as described in WO 90/10448. It is understood that the use of antisense molecules or knock out and knock in models may also be used in screening assays as discussed above, in addition to methods of treatment.
Inhibitory and Antisense Nucleotides
In certain embodiments, the activity of a cancer-associated protein is down-regulated, or entirely inhibited, by the use of antisense polynucleotide or inhibitory small nuclear RNA (snRNA), i.e., a nucleic acid complementary to, and which can preferably hybridize specifically to, a coding mRNA nucleic acid sequence, e.g., a cancer protein of the invention, mRNA, or a subsequence thereof. Binding of the antisense polynucleotide to the mRNA reduces the translation and/or stability of the mRNA.
In the context of this invention, antisense polynucleotides can comprise naturally occurring nucleotides, or synthetic species formed from naturally occurring subunits or their close homologs. Antisense polynucleotides may also have altered sugar moieties or inter-sugar linkages. Exemplary among these are the phosphorothioate and other sulfur containing species which are known for use in the art. Analogs are comprised by this invention so long as they function effectively to hybridize with nucleotides of the invention. See, e.g., Isis Pharmaceuticals, Carlsbad, CA; Sequitor, Inc., Natick, MA.
Such antisense polynucleotides can readily be synthesized using recombinant means, or can be synthesized in vitro. Equipment for such synthesis is sold by several vendors, including Applied Biosystems. The preparation of other oligonucleotides such as phosphorothioates and alkylated derivatives is also well known to those of skill in the art.
Antisense molecules as used herein include antisense or sense oligonucleotides. Sense oligonucleotides can, e.g., be employed to block transcription by binding to the anti-sense strand. The antisense and sense oligonucleotide comprise a single stranded nucleic acid sequence (either RNA or DNA) capable of binding to target mRNA (sense) or DNA (antisense) sequences for cancer molecules. Antisense or sense oligonucleotides, according to the present invention, comprise a fragment generally at least about 12 nucleotides, preferably from about 12 to 30 nucleotides. The ability to derive an antisense or a sense oligonucleotide, based upon a cDNA sequence encoding a given protein is described in, e.g., Stein &Cohen (Cancer Res. 48:2659 (1988 and van der Krol et al. (BioTechniques 6:958 (1988)).
Ribozymes
In addition to antisense polynucleotides, ribozymes can be used to target and inhibit transcription of cancer- associated nucleotide sequences. A ribozyme is an RNA molecule that catalytically cleaves other RNA molecules. Different kinds of ribozymes have been described, including group I ribozymes, hammerhead ribozymes, hairpin ribozymes, RNase P, and axhead ribozymes (see, e.g., Castanotto et al., Adv. in Pharmacology 25: 289-317 (1994) for a general review of the properties of different ribozymes).
The general features of hairpin ribozymes are described, e.g., in Hampel et al., Nucl. Acids Res. 18:299-304 (1990); European Patent Publication No. 0360257; U.S. Patent No. 5,254,678. Methods of preparing are well known to those of skill in the art (see, e.g., WO 94/26877; Ojwang et al., Proc. Natl. Acad. Sci. USA 90:6340-6344 (1993); Yamada et al., Human Gene Therapy 1:39-45 (1994); Leavitt et al., Proc. Natl. Acad Sci. USA 92:699- 703 (1995); Leavitt et al., Human Gene Therapy 5: 1151-120 (1994); and Yamada et al., Virology 205: 121-126 (1994)).
Use of Modulators in Phenotypic Screening
In one embodiment, a test compound is administered to a population of cancer cells, which have an associated cancer expression profile. By "administration" or "contacting" herein is meant that the modulator is added to the cells in such a manner as to allow the modulator to act upon the cell, whether by uptake and intracellular action, or by action at the cell surface. In some embodiments, a nucleic acid encoding a proteinaceous agent (i.e., a peptide) is put into a viral construct such as an adenoviral or retroviral construct, and added to the cell, such that expression of the peptide agent is accomplished, e.g., PCT US97/01019. Regulatable gene therapy systems can also be used. Once the modulator has been administered to the cells, the cells are washed if desired and are allowed to incubate under preferably physiological conditions for some period. The cells are then harvested and a new gene expression profile is generated. Thus, e.g., cancer tissue is screened for agents that modulate, e.g., induce or suppress, the cancer phenotype. A change in at least one gene, preferably many, of the expression profile indicates that the agent has an effect on cancer activity. Similarly, altering a biological function or a signaling pathway is indicative of modulator activity. By defining such a signature for the cancer phenotype, screens for new drugs that alter the phenotype are devised. With this approach, the drug target need not be known and need not be represented in the original gene/protein expression screening platform, nor does the level of transcript for the target protein need to change. The modulator inhibiting function will serve as a surrogate marker
As outlined above, screens are done to assess genes or gene products. That is, having identified a particular differentially expressed gene as important in a particular state, screening of modulators of either the expression of the gene or the gene product itself is performed.
Use of Modulators to Affect Peptides of the Invention
Measurements of cancer polypeptide activity, or of the cancer phenotype are performed using a variety of assays. For example, the effects of modulators upon the function of a cancer polypeptide(s) are measured by examining parameters described above. A physiological change that affects activity is used to assess the influence of a test compound on the polypeptides of this invention. When the functional outcomes are determined using intact cells or animals, a variety of effects can be assesses such as, in the case of a cancer associated with solid tumors, tumor growth, tumor metastasis, neovascularization, hormone release, transcriptional changes to both known and uncharacterized genetic markers (e.g., by Northern blots), changes in cell metabolism such as cell growth or pH changes, and changes in intracellular second messengers such as cGNIP.
Methods of Identifying Characterizing Cancer-associated Sequences
Expression of various gene sequences is correlated with cancer. Accordingly, disorders based on mutant or variant cancer genes are determined. In one embodiment, the invention provides methods for identifying cells containing variant cancer genes, e.g., determining the presence of, all or part, the sequence of at least one endogenous cancer gene in a cell. This is accomplished using any number of sequencing techniques. The invention comprises methods of identifying the cancer genotype of an individual, e.g., determining all or part of the sequence of at least one gene of the invention in the individual. This is generally done in at least one tissue of the individual, e.g., a tissue set forth in Table I, and may include the evaluation of a number of tissues or different samples of the same tissue. The method may include comparing the sequence of the sequenced gene to a known cancer gene, i.e., a wild-type gene to determine the presence of family members, homologies, mutations or variants. The sequence of all or part of the gene can then be compared to the sequence of a known cancer gene to determine if any differences exist. This is done using any number of known homology programs, such as BLAST, Bestftt, etc. The presence of a difference in the sequence between the cancer gene of the patient and the known cancer gene correlates with a disease state or a propensity for a disease state, as outlined herein.
In a preferred embodiment, the cancer genes are used as probes to determine the number of copies of the cancer gene in the genome. The cancer genes are used as probes to determine the chromosomal localization of the cancer genes. Information such as chromosomal localization finds use in providing a diagnosis or prognosis in particular when chromosomal abnormalities such as translocations, and the like are identified in the cancer gene locus.
XIV.) Kits/Articles of Manufacture
For use in the laboratory, prognostic, prophylactic, diagnostic and therapeutic applications described herein, kits are within the scope of the invention. Such kits can comprise a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in the method, along with a label or insert comprising instructions for use, such as a use described herein. For example, the container(s) can comprise a probe that is or can be detectably labeled. Such probe can be an antibody or polynucleotide specific for a protein or a gene or message of the invention, respectively. Where the method utilizes nucleic acid hybridization to detect the target nucleic acid, the kit can also have containers containing nucleotide(s) for amplification of the target nucleic acid sequence. Kits can comprise a container comprising a reporter, such as a biotin- binding protein, such as avidin or streptavidin, bound to a reporter molecule, such as an enzymatic, fluorescent, or radioisotope label; such a reporter can be used with, e.g., a nucleic acid or antibody. The kit can include all or part of the amino acid sequences in Figure 2 or Figure 3 or analogs thereof, or a nucleic acid molecule that encodes such amino acid sequences.
The kit of the invention will typically comprise the container described above and one or more other containers associated therewith that comprise materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes; carrier, package, container, vial and/or tube labels listing contents and/or instructions for use, and package inserts with instructions for use.
A label can be present on or with the container to indicate that the composition is used for a specific therapy or non- therapeutic application, such as a prognostic, prophylactic, diagnostic or laboratory application, and can also indicate directions for either in vivo or in vitro use, such as those described herein. Directions and or other information can also be included on an insert(s) or label(s) which is included with or on the kit. The label can be on or associated with the container. A label a can be on a container when letters, numbers or other characters forming the label are molded or etched into the container itself; a label can be associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert. The label can indicate that the composition is used for diagnosing, treating, prophylaxing or prognosing a condition, such as a neoplasia of a tissue set forth in Table I.
The terms "kit" and "article of manufacture" can be used as synonyms. In another embodiment of the invention, an article(s) of manufacture containing compositions, such as amino acid sequence(s), small molecule(s), nucleic acid sequence(s), and/or antibody(s), e.g., materials useful for the diagnosis, prognosis, prophylaxis and/or treatment of neoplasias of tissues such as those set forth in Table I is provided. The article of manufacture typically comprises at least one container and at least one label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers can be formed from a variety of materials such as glass, metal or plastic. The container can hold amino acid sequence(s), small molβcule(s), nucleic acid sequence(s), cell population(s) and/or antibody(s). In one embodiment, the container holds a polynucleotide for use in examining the mRNA expression profile of a cell, together with reagents used for this purpose. In another embodiment a container comprises an antibody, binding fragment thereof or specific binding protein for use in evaluating protein expression of) 09P1 D4 in cells and tissues, or for relevant laboratory, prognostic, diagnostic, prophylactic and therapeutic purposes; indications and/or directions for such uses can be included on or with such container, as can reagents and other compositions or tools used for these purposes. In another embodiment, a container comprises materials for eliciting a cellular or humoral immune response, together with associated indications and/or directions. In another embodiment, a container comprises materials for adoptive immunotherapy, such as cytotoxic T cells (CTL) or helper T cells (HTL), together with associated indications and/or directions; reagents and other compositions or tools used for such purpose can also be included.
The container can alternatively hold a composition that is effective for treating, diagnosis, prognosing or prophylaxing a condition and can have a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The active agents in the composition can be an antibody capable of specifically binding 109P1D4 and modulating the function of 109P1D4.
The article of manufacture can further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution and/or dextrose solution. It can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, stirrers, needles, syringes, and/or package inserts with indications and/or instructions for use.
EXAMPLES:
Various aspects of the invention are further described and illustrated by way of the several examples that follow, none of which is intended to limit the scope of the invention.
Example 1 : SSH-Generated Isolation of cDNA Fragment of the 109P1 D4 Gene
To isolate genes that are over-expressed in prostate cancer we used the Suppression Subtractive Hybridization (SSH) procedure using cDNA derived from prostate cancer tissues. The 109P1 D4 SSH cDNA sequence was from an experiment where cDNA derived from LNCaP cells that was androgen-deprived (by growing in the presence of charcoal-stripped serum) was subtracted from cDNA derived from LNCaP cells that were stimulated with mibolerone for 9 hours.
Materials and Methods
Human Tissues:
The patient cancer and normal tissues were purchased from different sources such as the NDRI (Philadelphia, PA). mRNA for some normal tissues were purchased from different companies such as Clontech, Palo Alto, CA.
RNA Isolation:
Tissues were homogenized in Trizol reagent (Life Technologies, Gibco BRL) using 10 ml/ g tissue to isolate total RNA. Poly A RNA was purified from total RNA using Qiagen's Oiigotex mRNA Mini and Midi kits. Total and mRNA were quantified by spectrophotometric analysis (O.D. 260/280 nm) and analyzed by gel electrophoresis. Oligonucleotides:
The following HPLC purified oligonucleotides were used.
DPNCDN (cDNA synthesis primer):
5'TTTTGATCAAGCTT3o3' (SEQ ID NO: 44)
Adaptor 1:
5OTMTACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3' (SEQ ID NO: 45)
3'GGCCCGTCCTAG5' (SEQ ID NO: 46)
Adaptor 2:
5'GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3' (SEQ ID NO: 47)
3'CGGCTCCTAG5' (SEQ ID NO: 48)
PCR primer 1:
5OTAATACGACTCACTATAGGGC3' (SEQ ID NO: 49)
Nested primer (NP)1:
5 CGAGCGGCCGCCCGGGCAGGA3' (SEQ ID NO: 50)
Nested primer (NP)2:
5ΑGCGTGGTCGCGGCCGAGGA3' (SEQ ID NO: 51)
Suppression Subtractive Hybridization:
Suppression Subtractive Hybridization (SSH) was used to identify cDNAs corresponding to genes that may be differentially expressed in prostate cancer. The SSH reaction utilized cDNA from LNCaP prostate cancer cells.
The 109P1D4 SSH sequence was derived from cDNA subtraction of LNCaP stimulated with mibolerone minus LNCaP in the absence of androgen. The SSH DNA sequence (Figure 1) was identified.
The cDNA derived from androgen-deprived LNCaP cells was used as the source of the "driver" cDNA, while the cDNA from androgen-stimulated LNCaP cells was used as the source of the "tester" cDNA. Double stranded cDNAs corresponding to tester and driver cDNAs were synthesized from 2 μg of poly(A)+ RNA isolated from the relevant xenograft tissue, as described above, using CLONTECH's PCR-Select cDNA Subtraction Kit and 1 μg of oligonucleotide DPNCDN as primer. First- and second- strand synthesis were carried out as described in the Kit's user manual protocol (CLONTECH Protocol No. PT1117-1, Catalog No. K1804-1). The resulting cDNA was digested with Dpn II for 3 hrs at 37°C. Digested cDNA was extracted with phenol/chloroform (1:1) and ethanol precipitated.
Tester cDNA was generated by diluting 1 μl of Dpn II digested cDNA from the relevant tissue source (see above) (400 ng) in 5 μl of water. The diluted cDNA (2 μl, 160 ng) was then ligated to 2 μl of Adaptor 1 and Adaptor 2 (10 μM), in separate ligation reactions, in a total volume of 10 μl at 16°C overnight, using 400 μl of T4 DNA ligase (CLONTECH). Ligation was terminated with 1 μl of 0.2 M EDTA and heating at 72°C for 5 min.
The first hybridization was performed by adding 1.5 μl (600 ng) of driver cDNA to each of two tubes containing 1.5 μl (20 ng) Adaptor 1- and Adaptor 2- ligated tester cDNA. In a final volume of 4 μl, the samples were overlaid with mineral oil, denatured in an MJ Research thermal cycler at 98°C for 1.5 minutes, and then were allowed to hybridize for 8 hrs at 68°C. The two hybridizations were then mixed together with an additional 1 μl of fresh denatured driver cDNA and were allowed to hybridize overnight at 68°C. The second hybridization was then diluted in 200 μl of 20 mM Hepes, pH 8.3, 50 mM NaCI, 0.2 mM EDTA, heated at 70°C for 7 min. and stored at -20°C.
PCR Amplification, Cloning and Sequencing of Gene Fragments Generated from SSH: To amplify gene fragments resulting from SSH reactions, two PCR amplifications were performed. In the primary PCR reaction 1 μl of the diluted final hybridization mix was added to 1 μl of PCR primer 1 (10 μM), 0.5 μl dNTP mix (10 μM), 2.5 μl 10 x reaction buffer (CLONTECH) and 0.5 μl 50 x Advantage cDNA polymerase Mix (CLONTECH) in a final volume of 25 μl. PCR 1 was conducted using the following conditions: 75°C for 5 min., 94°C for 25 sec, then 27 cycles of 94°C for 10 sec, 66°C for 30 sec, 72°C for 1.5 min. Five separate primary PCR reactions were performed for each experiment. The products were pooled and diluted 1:10 with water. For the secondary PCR reaction, 1 μl from the pooled and diluted primary PCR reaction was added to the same reaction mix as used for PCR 1, except that primers NP1 and NP2 (10 μM) were used instead of PCR primer 1. PCR 2 was performed using 10-12 cycles of 94°C for 10 sec, 68°C for 30 sec, and 72°C for 1.5 minutes. The PCR products were analyzed using 2% agarose gel electrophoresis.
The PCR products were inserted into pCR2.1 using the T/A vector cloning kit (Invitrogen). Transformed £ coli were subjected to blue/white and ampicillin selection. White colonies were picked and arrayed into 96 well plates and were grown in liquid culture overnight. To identify inserts, PCR amplification was performed on 1 μl of bacterial culture using the conditions of PCR1 and NP1 and NP2 as primers. PCR products were analyzed using 2% agarose gel electrophoresis.
Bacterial clones were stored in 20% glycerol in a 96 well format. Plasmid DNA was prepared, sequenced, and subjected to nucleic acid homology searches of the GenBank, dBest, and NCI-CGAP databases.
RT-PCR Expression Analysis:
First strand cDNAs can be generated from 1 μg of mRNA with oligo (dT)12-18 priming using the Gibco-BRL Superscript Preamplification system. The manufacturer's protocol was used which included an incubation for 50 min at 42°C with reverse transcriptase followed by RNAse H treatment at 37°C for 20 min. After completing the reaction, the volume can be increased to 200 μl with water prior to normalization. First strand cDNAs from 16 different normal human tissues can be obtained from Clontech.
Normalization of the first strand cDNAs from multiple tissues was performed by using the primers 5ΑTATCGCCGCGCTCGTCGTCGACAA3' (SEQ ID NO: 52) and 5'AGCCACACGCAGCTCATTGTAGAAGG 3' (SEQ ID NO: 53) to amplify β-actin. First strand cDNAs (5 μl) were amplified in a total volume of 50 μl containing 0.4 μM primers, 0.2 μM each dNTPs, 1X PCR buffer (Clontech, 10 mM Tris-HCL, 1.5 mM MgC , 50 mM KCl, pH8.3) and 1X Klentaq DNA polymerase (Clontech). Five μl of the PCR reaction can be removed at 18, 20, and 22 cycles and used for agarose gel electrophoresis. PCR was performed using an MJ Research thermal cycler under the following conditions: Initial denaturation can be at 94°C for 15 sec, followed by a 18, 20, and 22 cycles of 94°C for 15, 65°C for 2 min, 72°C for 5 sec. A final extension at 72°C was carried out for 2 min. After agarose gel electrophoresis, the band intensities of the 283 base pair β-actin bands from multiple tissues were compared by visual inspection. Dilution factors for the first strand cDNAs were calculated to result in equal β-actin band intensities in all tissues after 22 cycles of PCR. Three rounds of normalization can be required to achieve equal band intensities in all tissues after 22 cycles of PCR.
To determine expression levels of the 109P1 D4 gene, 5 μl of normalized first strand cDNA were analyzed by PCR using 26, and 30 cycles of amplification. Semi-quantitative expression analysis can be achieved by comparing the PCR products at cycle numbers that give light band intensities. The primers used for RT-PCR were designed using the 109P1D4 SSH sequence and are listed below:
109P1D4.1
5'- TGGTCTTTCAGGTAATTGCTGTTG - 3' (SEQ ID NO: 54)
109P1D4.2
5'- CTCCATCAATGTTATGTTGCCTGT - 3' (SEQ ID NO: 55) A typical RT-PCR expression analysis is shown in Figure 15.
Example 2: Isolation of Full Length 109P1D4 encoding DNA
The 109P1D4 SSH sequence of 192 bp (Figure 1) exhibited homology to protocadherin 11 (PCDH11), a cell adhesion molecule related to the calcium dependent cadherins. The human cDNA sequence encodes a 1021 amino acid protein with an N- terminal leader sequence and a transmembrane domain. 109P1D4 v.1 of 4603bp was cloned from human prostate cancer xenograft LAPC-9AD cDNA library, revealing an ORF of 1021 amino acids (Figure 2 and Figure 3). Other variants (Transcript and SNP) of 109P1D4 were also identified and these are listed sequentially in Figure 2 and Figure 3.
Example 3: Chromosomal Mapping of 109P1D4
Chromosomal localization can implicate genes in disease pathogenesis. Several chromosome mapping approaches are available including fluorescent in situ hybridization (FISH), human/hamster radiation hybrid (RH) panels (Walter et al., 1994; Nature Genetics 7:22; Research Genetics, Huntsville Al), human-rodent somatic cell hybrid panels such as is available from the Coriell Institute (Camden, New Jersey), and genomic viewers utilizing BLAST homologies to sequenced and mapped genomic clones (NCBI, Bethesda, Maryland).
109P1D4 maps to chromosome Xq21.3 using 109P1D4 sequence and the NCBI BLAST tool: located on the World Wide Web at: (.ncbi.nlm.nih.gov/genome/seq/page.cgi?F=HsBlast.html&&ORG=Hs). 109P1D4 was also identified on chromosome Yp11.2, a region of 99% identity to Xq21.
Example 4: Expression Analysis of 109P1D4 in Normal Tissues and Patient Specimens
Expression analysis by RT-PCR and Northern analysis demonstrated that normal tissue expression of a gene of Figure 2 is restricted predominantly to the tissues set forth in Table I.
Therapeutic applications for a gene of Figure 2 include use as a small molecule therapy and/or a vaccine (T cell or antibody) target. Diagnostic applications for a gene of Figure 2 include use as a diagnostic marker for local and/or metastasized disease. The restricted expression of a gene of Figure 2 in normal tissues makes it useful as a tumor target for diagnosis and therapy. Expression analysis of a gene of Figure 2 provides information useful for predicting susceptibility to advanced stage disease, rate of progression, and/or tumor aggressiveness. Expression status of a gene of Figure 2 in patient samples, tissue arrays and/or cell lines may be analyzed by: (i) immunohistochemical analysis; (ii) in situ hybridization; (iii) RT-PCR analysis on laser capture micro-dissected samples; (iv) Western blot analysis; and (v) Northern analysis.
RT-PCR analysis and Northern blotting were used to evaluate gene expression in a selection of normal and cancerous urological tissues. The results are summarized in Figures 15-19.
Figure 14 shows expression of 109P1 D4 in lymphoma cancer patient specimens. RNA was extracted from peripheral blood lymphocytes, cord blood isolated from normal individuals, and from lymphoma patient cancer specimens. Northern blots with 10μg of total RNA were probed with the 109P1 D4 sequence. Size standards in kilobases are on the side. Results show expression of 109P1D4 in lymphoma patient specimens but not in the normal blood cells tested.
Figure 15 shows expression of 109P1D4 by RT-PCR. First strand cDNA was prepared from vital pool 1 (liver, lung and kidney), vital pool 2 (pancreas, colon and stomach), prostate cancer pool, bladder cancer pool, kidney cancer pool, colon cancer pool, lung cancer pool, ovary cancer pool, breast cancer pool, cancer metastasis pool, and pancreas cancer pool. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 109P1 D4, was performed at 30 cycles of amplification. Results show strong expression of 109P1 D4 in all cancer pools tested. Very low expression was detected in the vital pools.
Figure 16 shows expression of 109P1D4 in normal tissues. Two multiple tissue northern blots (Clontech), both with 2 μg of mRNA/lane, were probed with the 109P1D4 SSH fragment. Size standards in kilobases (kb) are indicated on the side. Results show expression of approximately 10 kb 109P1D4 transcript in ovary. Weak expression was also detected in placenta and brain, but not in the other normal tissues tested.
Figure 17 shows expression of 109P1D4 in human cancer cell lines. RNA was extracted from a number of human prostate and bone cancer cell lines. Northern blots with 10 μg of total RNA/lane were probed with the 109P1 D4 SSH fragment. Size standards in kilobases (kb) are indicated on the side. Results show expression of 109P1D4 in LAPC-9AD, LAPC-9AI, LNCaP prostate cancer cell lines, and in the bone cancer cell lines, SK-ES-1 and RD-ES.
Extensive expression of 109P1 D4 in normal tissues is shown in Figure 18A. A cDNA dot blot containing 76 different samples from human tissues was analyzed using a 109P1 D4 SSH probe. Expression was only detected in multiple areas of the brain, placenta, ovary, and fetal brain, amongst all tissues tested.
Figure 18B shows expression of 109P1D4 in patient cancer specimens. Expression of 109P1D4 was assayed in a panel of human cancers (T) and their respective matched normal tissues (N) on RNA dot blots. Upregulated expression of 109P1D4 in tumors compared to normal tissues was observed in uterus, lung and stomach. The expression detected in normal adjacent tissues (isolated from diseased tissues) but not in normal tissues (isolated from healthy donors) may indicate that these tissues are not fully normal and that 109P1D4 may be expressed in early stage tumors.
Figure 19 shows 109P1D4 expression in lung cancer patient specimens. RNA was extracted from normal fung, prostate cancer xenograft LAPC-9AD, bone cancer cell line RD-ES, and lung cancer patient tumors. Northern blots with 10 μg of total RNA were probed with 109P1D4. Size standards in kilobases are on the side. Results show strong expression of 109P1D4 in lung tumor tissues as well as the RD-ES cell line, but not in normal lung.
The restricted expression of 109P1D4 in normal tissues and the expression detected in cancer patient specimens suggest that 109P1 D4 is a potential therapeutic target and a diagnostic marker for human cancers.
Example 5: Splice Variants of 109P1 D4
Transcript variants are variants of mature mRNA from the same gene which arise by alternative transcription or alternative splicing. Alternative transcripts are transcripts from the same gene but start transcription at different points. Splice variants are mRNA variants spliced differently from the same transcript, in eukaryotes, when a multi-exon gene is transcribed from genomic DNA, the initial RNA is spliced to produce functional mRNA, which has only exons and is used for translation into an amino acid sequence. Accordingly, a given gene can have zero to many alternative transcripts and each transcript can have zero to many splice variants. Each transcript variant has a unique exon makeup, and can have different coding and/or non-coding (5' or 3' end) portions, from the original transcript. Transcript variants can code for similar or different proteins with the same or a similar function or can encode proteins with different functions, and can be expressed in the same tissue at the same time, or in different tissues at the same time, or in the same tissue at different times, or in different tissues at different times. Proteins encoded by transcript variants can have similar or different cellular or extracellular localizations, e.g., secreted versus intracellular.
Transcript variants are identified by a variety of art-accepted methods. For example, alternative transcripts and splice variants are identified by full-length cloning experiment, or by use of full-length transcript and EST sequences. First, all human ESTs were grouped into clusters which show direct or indirect identity with each other. Second, ESTs in the same cluster were further grouped into sub-clusters and assembled into a consensus sequence. The original gene sequence is compared to the consensus sequence(s) or other full-length sequences. Each consensus sequence is a potential splice variant for that gene. Even when a variant is identified that is not a full-length clone, that portion of the variant is very useful for antigen generation and for further cloning of the full-length splice variant, using techniques known in the art.
Moreover, computer programs are available in the art that identify transcript variants based on genomic sequences. Genomic-based transcript variant identification programs include FgenesH (A. Salamov and V. Solovyev, "Ab initio gene finding in Drosophila genomic DNA," Genome Research. 2000 April; 10(4):516-22); Grail (URL compbio.ornl.gov/Grail-bin/EmptyGrailForm) and GenScan (URL genes.mit.edu/GENSCAN.html). For a general discussion of splice variant identification protocols see., e.g., Southan, C, A genomic perspective on human proteases, FEBS Lett. 2001 Jun 8; 98(2-3):214-8; de Souza, S.J., ef al, identification of human chromosome 22 transcribed sequences with ORF expressed sequence tags, Proc. Natl Acad Sci U S A. 2000 Nov 7; 97(23): 12690-3.
To further confirm the parameters of a transcript variant, a variety of techniques are available in the art, such as full-length cloning, proteomic validation, PCR-based validation, and 5' RACE validation, etc. (see e.g., Proteomic Validation: Brennan, S.O., ef al, Albumin banks peninsula: a new termination variant characterized by electrospray mass spectrometry, Biochem Biophys Acta. 1999 Aug 17; 1433( 1 -2) :321 -6; Ferranti P, et al, Differential splicing of pre-messenger RNA produces multiple forms of mature caprine alpha(s1)-casein, Eur J Biochem. 1997 Oct 1;249(1):1-7. For PCR-based Validation: Wellmann S, ef al, Specific reverse transcription-PCR quantification of vascular endothelial growth factor (VEGF) splice variants by LightCycler technology, Clin Chem. 2001 Apr;47(4):654-60; Jia, H.P., ef al, Discovery of new human beta- defensins using a geno ics-based approach, Gene. 2001 Jan 24; 263(1-2):211-8. For PCR-based and 5' RACE Validation: Brigle, K.E., ef al, Organization of the murine reduced folate carrier gene and identification of variant splice forms, Biochem Biophys Acta. 1997 Aug 7; 1353(2): 191-8).
It is known in the art that genomic regions are modulated in cancers. When the genomic region to which a gene maps is modulated in a particular cancer, the alternative transcripts or splice variants of the gene are modulated as well. Disclosed herein is that 109P1D4 has a particular expression profile related to cancer. Alternative transcripts and splice variants of 109P1D4 may also be involved in cancers in the same or different tissues, thus serving as tumor-associated markers/antigens.
Using the full-length gene and EST sequences, 8 transcript variants were identified, designated as 109P1D4 v.2, v.3, v.4, v.5, v.6, v.7, v.8 and v.9. The boundaries of the exon in the original transcript, 109P1D4 v.1, were shown in Table LI. Compared with 109P1D4 v.1, transcript variant 109P1D4 v.3 has spliced out 2069-2395 from variant 109P1D4 v.1, as shown in Figure 12. Variant 109P1D4 v.4 spliced out 1162-2096 of variant 109P1D4 v.1. Variant 109P1D4 v.5 added one exon to the 5' and extended 2 bp to the 5' end and 288 bp to the 3' end of variant 109P1D4 v.1. Theoretically, each different combination of exons in spatial order, e.g. exon 1 of v.5 and exons 1 and 2 of v.3 or v.4, is a potential splice variant.
Tables Lll through LV are set forth on a variant-by-variant basis. Tables Lll(a)-(h) show nucleotide sequence of the transcript variants. Tables Llll(a)-(h) show the alignment of the transcript variants with nucleic acid sequence of 109P1 D4 v.1. Tables LIV(a)-(h) lay out amino acid translation of the transcript variants for the identified reading frame orientation. Tables LV(a)-(h) displays alignments of the amino acid sequence encoded by the splice variants with that of 109P1D4 V.1.
Example 6: Single Nucleotide Polymorphisms of 109P1D4
A Single Nucleotide Polymorphism (SNP) is a single base pair variation in a nucleotide sequence at a specific location. At any given point of the genome, there are four possible nucleotide base pairs: A/T, C/G, G/C and T/A. Genotype refers to the specific base pair sequence of one or more locations in the genome of an individual. Haplotype refers to the base pair sequence of more than one location on the same DNA molecule (or the same chromosome in higher organisms), often in the context of one gene or in the context of several tightly linked genes. SNP that occurs on a cDNA is called cSNP. This cSNP may change amino acids of the protein encoded by the gene and thus change the functions of the protein. Some SNP cause inherited diseases; others contribute to quantitative variations in phenotype and reactions to environmental factors including diet and drugs among individuals. Therefore, SNP and/or combinations of alleles (called haplotypes) have many applications, including diagnosis of inherited diseases, determination of drug reactions and dosage, identification of genes responsible for diseases, and analysis of the genetic relationship between individuals (P. Nowotny, J. M. Kwon and A. M. Goate, " SNP analysis to dissect human traits," Curr. Opin. Neurobiol. 2001 Oct; 11(5):637-641; M. Pirmohamed and B. K. Park, "Genetic susceptibility to adverse drug reactions," Trends Pharmacol. Sci. 2001 Jun; 22(6):298-305; J. H. Riley, C. J. Allan, E. Lai and A. Roses, "The use of single nucleotide polymorphisms in the isolation of common disease genes," Pharmacogenomics. 2000 Feb; 1(1):39-47; R. Judson, J. C. Stephens and A. Winde uth, "The predictive power of haplotypes in clinical response," Pharmacogenomics. 2000 Feb; 1(1):15-26).
SNP are identified by a variety of art-accepted methods (P. Bean, "The promising voyage of SNP target discovery," Am. Clin. Lab. 2001 Oct-Nov; 20(9):18-20; K. M. Weiss, "In search of human variation," Genome Res. 1998 Jul; 8(7):691- 697; M. M. She, "Enabling large-scale pharmacogenetic studies by high-throughput mutation detection and genotyping technologies," Clin. Chem. 2001 Feb; 47(2):164-172). For example, SNP can be identified by sequencing DNA fragments that show polymorphism by gel-based methods such as restriction fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE). They can also be discovered by direct sequencing of DNA samples pooled from different individuals or by comparing sequences from different DNA samples. With the rapid accumulation of sequence data in public and private databases, one can discover SNP by comparing sequences using computer programs (Z. Gu, L. Hillier and P. Y. Kwok, "Single nucleotide polymorphism hunting in cyberspace," Hum. Mutat. 1998; 12(4):221-225). SNP can be verified and genotype or haplotype of an individual can be determined by a variety of methods including direct sequencing and high throughput microarrays (P. Y. Kwok, "Methods for genotyping single nucleotide polymorphisms," Annu. Rev. Genomics Hum. Genet. 2001; 2:235-258; M. Kokoris, K. Dix, K. Moynihan, J. Mathis, B. Erwin, P. Grass, B. Hines and A. Duesterhoeft, "High-throughput SNP genotyping with the Masscode system," Mol. Diagn. 2000 Dec; 5(4):329-340).
Using the methods described above, SNP were identified in the original transcript, 109P4D4 v.1, and its variants (see Figure 2J and Figure 2K). These alleles of the SNP, though shown separately here, can occur in different combinations (haplotypes) and in any one of the transcript variants (such as 109P4D4 v.4 or v.5) that contains the site of the SNP. Transcript variants v.4 and v.5 contained those SNP in the exons shared with variant v.3, and transcript variant v.9 contained all the SNP occurred in variant v.6 (see Figure 10).
Example 7: Production of Recombinant 109P1D4 in Prokaryotic Systems
To express recombinant 109P1D4 and 109P1D4 variants in prokaryotic cells, the full or partial length 109P1D4 and 109P1D4 variant cDNA sequences are cloned into any one of a variety of expression vectors known in the art. One or more of the following regions of 109P1D4 variants are expressed: the full length sequence presented in Figures 2 and 3, or any 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from 109P1D4, variants, or analogs thereof.
A. In vitro transcription and translation constructs: pCRIl: To generate 109P1D4 sense and anti-sense RNA probes for RNA in situ investigations, pCRII constructs (Invitrogen, Carlsbad CA) are generated encoding either all or fragments of the 109P1 D4 cDNA. The pCRIl vector has Sp6 and T7 promoters flanking the insert to drive the transcription of 109P1 D4 RNA for use as probes in RNA in situ hybridization experiments. These probes are used to analyze the cell and tissue expression of 109P1 D4 at the RNA level. Transcribed 109P1D4 RNA representing the cDNA amino acid coding region of the 109P1D4 gene is used in in vitro translation systems such as the TnT™ Coupled Reticulolysate System (Promega, Corp., Madison, Wl) to synthesize 109P1D4 protein.
B. Bacterial Constructs: pGEX Constructs: To generate recombinant 109P1D4 proteins in bacteria that are fused to the Glutathione S- transferase (GST) protein, all or parts of the 109P1D4 cDNA protein coding sequence are cloned into the pGEX family of GST-fusion vectors (Amersham Pharmacia Biotech, Piscataway, NJ). These constructs allow controlled expression of recombinant 109P1 D4 protein sequences with GST fused at the amino-terminus and a six histidine epitope (6X His) at the carboxyl-terminus. The GST and 6X His tags permit purification of the recombinant fusion protein from induced bacteria with the appropriate affinity matrix and allow recognition of the fusion protein with anti-GST and anti-His antibodies. The 6X His tag is generated by adding 6 histidine codons to the cloning primer at the 3' end, e.g., of the open reading frame (ORF). A proteolytic cleavage site, such as the PreScission™ recognition site in pGEX-6P-1, may be employed such that it permits cleavage of the GST tag from 109P1D4-related protein. The ampicillin resistance gene and pBR322 origin permits selection and maintenance of the pGEX plasmids in £ coli. pMAL Constructs: To generate, in bacteria, recombinant 109P1D4 proteins that are fused to maltose-binding protein (MBP), all or parts of the 109P1D4 cDNA protein coding sequence are fused to the MBP gene by cloning into the pMAL-c2X and pMAL-p2X vectors (New England Biolabs, Beverly, MA). These constructs allow controlled expression of recombinant 109P1D4 protein sequences with MBP fused at the amino-terminus and a 6X His epitope tag at the carboxyl- terminus. The MBP and 6X His tags permit purification of the recombinant protein from induced bacteria with the appropriate affinity matrix and allow recognition of the fusion protein with anti-MBP and anti-His antibodies. The 6X His epitope tag is generated by adding 6 histidine codons to the 3' cloning primer. A Factor Xa recognition site permits cleavage of the pMAL tag from 109P1D4. The pMAL-c2X and pMAL-p2X vectors are optimized to express the recombinant protein in the cytoplasm or periplasm respectively. Periplasm expression enhances folding of proteins with disulfide bonds. In one embodiment, amino acids 24-419 of 109P1D4 variant 1 was cloned into the pMAL-c2X vector and was used to express the fusion protein. pET Constructs: To express 109P1D4 in bacterial cells, all or parts of the 109P1D4 cDNA protein coding sequence are cloned into the pET family of vectors (Novagen, Madison, Wl). These vectors allow tightly controlled expression of recombinant 109P1D4 protein in bacteria with and without fusion to proteins that enhance solubility, such as NusA and thioredoxin (Trx), and epitope tags, such as 6X His and S-Tag ™ that aid purification and detection of the recombinant protein. For example, constructs are made utilizing pET NusA fusion system 43.1 such that regions of the 109P1D4 protein are expressed as amino-terminal fusions to NusA. In 2 embodiments, amino acids 24-419 and 24-815 were cloned into pET43.1 vector and used to express the fusion protein.
C. Yeast Constructs: pESC Constructs: To express 109P1 D4 in the yeast species Saccharomyces cerevisiae for generation of recombinant protein and functional studies, all or parts of the 109P1 D4 cDNA protein coding sequence are cloned into the pESC family of vectors each of which contain 1 of 4 selectable markers, HIS3, TRP1, LEU2, and URA3 (Stratagene, La Jolla, CA). These vectors allow controlled expression from the same plasmid of up to 2 different genes or cloned sequences containing either Flag™ or Myc epitope tags in the same yeast cell. This system is useful to confirm protein-protein interactions of 109P1 D4. In addition, expression in yeast yields similar post-translational modifications, such as glycosylations and phosphorylations, that are found when expressed in eukaryotic cells. pESP Constructs: To express 109P1 D4 in the yeast species Saccharomyces pombe, all or parts of the 109P1 D4 cDNA protein coding sequence are cloned into the pESP family of vectors. These vectors allow controlled high level of expression of a 109P1D4 protein sequence that is fused at either the amino terminus or at the carboxyl terminus to GST which aids purification of the recombinant protein. A Flag™ epitope tag allows detection of the recombinant protein with anti- Flag™ antibody.
Example 8: Production of Recombinant 109P1D4 in Higher Eukaryotic Systems
A. Mammalian Constructs:
To express recombinant 109P1D4 in eukaryotic cells, the full or partial length 109P1D4 cDNA sequences were cloned into any one of a variety of expression vectors known in the art. One or more of the following regions of 109P1 D4 were expressed in these constructs, amino acids 1 to 1021 or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from 109P1D4 v.1; amino acids 1 to 1054, 1 to 1347, 1 to 1337, 1 to 1310, 1 to 1037, 1 to 1048, 1 to 1340 of v.2, v.3, v.4, v.5, v.6, v.7, and v.8 respectively; or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from 109P1D4 variants, or analogs thereof.
The constructs can be transfected into any one of a wide variety of mammalian cells such as 293T cells. Transfected 293T cell lysates can be probed with the anti-109P1D4 polyclonal serum, described herein.
pcDNA4/HisMax Constructs: To express 109P1D4 in mammalian cells, a 109P1D4 ORF, or portions thereof, of 109P1D4 are cloned into pcDNA4/HisMax Version A (Invitrogen, Carlsbad, CA). Protein expression is driven from the cytomegalovirus (CMV) promoter and the SP16 translational enhancer. The recombinant protein has Xpress™ and six histidine (6X His) epitopes fused to the amino-terminus. The pcDNA4/HisMax vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen. The Zeocin resistance gene allows for selection of mammalian cells expressing the protein and the ampicillin resistance gene and ColE1 origin permits selection and maintenance of the plasmid in £ coli. pcDNA3.1/MycHis Constructs: To express 109P1D4 in mammalian cells, a 109P1 D4 ORF, or portions thereof, of 109P1D4 with a consensus Kozak translation initiation site was cloned into pcDNA3.1/MycHis Version A (Invitrogen, Carlsbad, CA). Protein expression is driven from the cytomegalovirus (CMV) promoter. The recombinant proteins have the myc epitope and 6X His epitope fused to the carboxyl-terminus. The pcDNA3.1/MycHis vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability, along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen. The Neomycin resistance gene can be used, as it allows for selection of mammalian cells expressing the protein and the ampicillin resistance gene and ColE1 origin permits selection and maintenance of the plasmid in £ coli.
The complete ORF of 109P1D4 v.1 was cloned into the pcDNA3.1/MycHis construct to generate 109P1D4.pcDNA3.1/MycHis. pcDNA3.1/CT-GFP-TOPO Construct: To express 109P1D4 in mammalian cells and to allow detection of the recombinant proteins using fluorescence, a 109P1D4 ORF, or portions thereof, with a consensus Kozak translation initiation site are cloned into pcDNA3.1/CT-GFP-TOPO (Invitrogen, CA). Protein expression is driven from the cytomegalovirus (CMV) promoter. The recombinant proteins have the Green Fluorescent Protein (GFP) fused to the carboxyl-terminus facilitating non-invasive, in vivo detection and cell biology studies. The pcDNA3.1CT-GFP-TOPO vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen. The Neomycin resistance gene allows for selection of mammalian cells that express the protein, and the ampicillin resistance gene and ColE1 origin permits selection and maintenance of the plasmid in £ coli. Additional constructs with an amino- terminal GFP fusion are made in pcDNA3.1/NT-GFP-TOPO spanning the entire length of a 109P1D4 protein.
PAPtag: A 109P1D4 ORF, or portions thereof, is cloned into pAPtag-5 (GenHunter Corp. Nashville, TN). This construct generates an alkaline phosphatase fusion at the carboxyl-terminus of a 109P1 D4 protein while fusing the lgGκ signal sequence to the amino-terminus. Constructs are also generated in which alkaline phosphatase with an amino- terminal lgG signal sequence is fused to the amino-terminus of a 109P1D4 protein. The resulting recombinant 109P1D4 proteins are optimized for secretion into the media of transfected mammalian cells and can be used to identify proteins such as ligands or receptors that interact with 109P1 D4 proteins. Protein expression is driven from the CMV promoter and the recombinant proteins also contain myc and 6X His epitopes fused at the carboxyl-terminus that facilitates detection and purification. The Zeocin resistance gene present in the vector allows for selection of mammalian cells expressing the recombinant protein and the ampicillin resistance gene permits selection of the plasmid in £ coli. pTagδ: A 109P1D4 ORF, or portions thereof, were cloned into pTag-5. This vector is similar to pAPtag but without the alkaline phosphatase fusion. This construct generated 109P1D4 protein with an amino-terminal lgGκ signal sequence and myc and 6X His epitope tags at the carboxyl-terminus that facilitate detection and affinity purification. The resulting recombinant 109P1D4 protein was optimized for secretion into the media of transfected mammalian cells, and was used as immunogen or ligand to identify proteins such as ligands or receptors that interact with the 109P1D4 proteins. Protein expression is driven from the CMV promoter. The Zeocin resistance gene present in the vector allows for selection of mammalian cells expressing the protein, and the ampicillin resistance gene permits selection of the plasmid in £ coli.
PsecFc: A 109P1D4 ORF, or portions thereof, is also cloned into psecFc. The psecFc vector was assembled by cloning the human immunoglobulin G1 (IgG) Fc (hinge, CH2, CH3 regions) into pSecTag2 (Invitrogen, California). This construct generates an lgG1 Fc fusion at the carboxyl-terminus of the 109P1 D4 proteins, while fusing the IgGK signal sequence to N-terminus. 109P1D4 fusions utilizing the murine lgG1 Fc region are also used. The resulting recombinant 109P1D4 proteins are optimized for secretion into the media of transfected mammalian cells, and can be used as immunogens or to identify proteins such as ligands or receptors that interact with 109P1 D4 protein. Protein expression is driven from the CMV promoter. The hygromycin resistance gene present in the vector allows for selection of mammalian cells that express the recombinant protein, and the ampicillin resistance gene permits selection of the plasmid in £ coli. pSRα Constructs: To generate mammalian cell lines that express 109P1D4 constitutively, 109P1D4 ORF, or portions thereof, were cloned into pSRα constructs. Amphotropic and ecotropic retroviruses were generated by transfection of pSRα constructs into the 293T-10A1 packaging line or co-transfection of pSRα and a helper plasmid (containing deleted packaging sequences) into the 293 cells, respectively. The retrovirus is used to infect a variety of mammalian cell lines, resulting in the integration of the cloned gene, 109P1D4, into the host cell-lines. Protein expression is driven from a long terminal repeat (LTR). The Neomycin resistance gene present in the vector allows for selection of mammalian cells that express the protein, and the ampicillin resistance gene and ColE1 origin permit selection and maintenance of the plasmid in £ coli. The retroviral vectors can thereafter be used for infection and generation of various ceil lines using, for example, PC3, NIH 3T3, TsuPrl, 293 or rat-1 cells.
Additional pSRα constructs are made that fuse an epitope tag such as the FLAG™ tag to the carboxyl-terminus of 109P1D4 sequences to allow detection using anti-Flag antibodies. For example, the FLAG™ sequence 5' GAT TAG AAG GAT GAC GAC GAT AAG 3' (SEQ ID NO: 56) is added to cloning primer at the 3' end of the ORF. Additional pSRα constructs are made to produce both amino-terminal and carboxyl-terminal GFP and myc/6X His fusion proteins of the full- length 109P1D4 proteins. Additional Viral Vectors: Additional constructs are made for viral-mediated delivery and expression of 109P1D4. High virus titer leading to high level expression of 109P1D4 is achieved in viral delivery systems such as adenoviral vectors and herpes amplicon vectors. A 109P1D4 coding sequence or fragments thereof are amplified by PCR and subcloned into the AdEasy shuttle vector (Stratagene). Recombination and virus packaging are performed according to the manufacturer's instructions to generate adenoviral vectors. Alternatively, 109P1 D4 coding sequences or fragments thereof are cloned into the HSV-1 vector (Imgenex) to generate herpes viral vectors. The viral vectors are thereafter used for infection of various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.
Regulated Expression Systems: To control expression of 109P1D4 in mammalian cells, coding sequences of 109P1D4, or portions thereof, are cloned into regulated mammalian expression systems such as the T-Rex System (Invitrogen), the GeneSwitch System (Invitrogen) and the tightly-regulated Ecdysone System (Stratagene). These systems allow the study of the temporal and concentration dependent effects of recombinant 109P1 D4. These vectors are thereafter used to control expression of 109P1D4 in various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.
B. Baculovirus Expression Systems
To generate recombinant 109P1D4 proteins in a baculovirus expression system, 109P1D4 ORF, or portions thereof, are cloned into the baculovirus transfer vector pBlueBac 4.5 (Invitrogen), which provides a His-tag at the N-terminus. Specifically, pBlueBac-109P1D4 is co-transfected with helper plasmid pBac-N-Blue (Invitrogen) into SF9 (Spodoptera frugiperda) insect cells to generate recombinant baculovirus (see Invitrogen instruction manual for details). Baculovirus is then collected from cell supernatant and purified by plaque assay.
Recombinant 109P1 D4 protein is then generated by infection of HighFive insect cells (Invitrogen) with purified baculovirus. Recombinant 109P1D4 protein can be detected using anti-109P1D4 or anti-His-tag antibody. 109P1D4 protein can be purified and used in various cell-based assays or as immunogen to generate polyclonal and monoclonal antibodies specific for 109P1D4.
Example 9: Antiαenicitv Profiles and Secondary Structure
Figure(s) 5A-I, Figure 6A-I, Figure 7A-1, Figure 8A-I, and Figure 9A-I depict graphically five amino acid profiles of 109P1D4 variants 1 through 9, each assessment available by accessing the ProtScale website located on the World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) on the ExPasy molecular biology server.
These profiles: Figure 5, Hydrophilicity, (Hopp T.P., Woods K.R., 1981. Proc. Natl. Acad. Sci. U.S.A. 78:3824- 3828); Figure 6, Hydropathicity, (Kyte J., Doolittle R.F., 1982. J. Mol. Biol. 157:105-132); Figure 7, Percentage Accessible Residues (Janin J., 1979 Nature 277:491-492); Figure 8, Average Flexibility, (Bhaskaran R„ and Ponnuswamy P.K., 1988. Int. J. Pept. Protein Res. 32:242-255); Figure 9, Beta-turn (Deleage, G., Roux B. 1987 Protein Engineering 1:289-294); and optionally others available in the art, such as on the ProtScale website, were used to identify antigenic regions of each of the 109P1D4 variant proteins. Each of the above amino acid profiles of 109P1D4 variants were generated using the following ProtScale parameters for analysis: 1) A window size of 9; 2) 100% weight of the window edges compared to the window center; and, 3) amino acid profile values normalized to lie between 0 and 1.
Hydrophilicity (Figure 5), Hydropathicity (Figure 6) and Percentage Accessible Residues (Figure 7) profiles were used to determine stretches of hydrophilic amino acids (i.e., values greater than 0.5 on the Hydrophilicity and Percentage Accessible Residues profile, and values less than 0.5 on the Hydropathicity profile). Such regions are likely to be exposed to the aqueous environment, be present on the surface of the protein, and thus available for immune recognition, such as by antibodies.
Average Flexibility (Figure 8) and Beta-turn (Figure 9) profiles determine stretches of amino acids (i.e., values greater than 0.5 on the Beta-turn profile and the Average Flexibility profile) that are not constrained in secondary structures such as beta sheets and alpha helices. Such regions are also more likely to be exposed on the protein and thus accessible to immune recognition, such as by antibodies.
Antigenic sequences of the 109P1 D4 variant proteins indicated, e.g., by the profiles set forth in Figure 5, Figure 6, Figure 7, Figure 8, and/or Figure 9 are used to prepare immunogens, either peptides or nucleic acids that encode them, to generate therapeutic and diagnostic anti-109P1D4 antibodies. The immunogen can be any 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more than 50 contiguous amino acids, or the corresponding nucleic acids that encode them, from the 109P1D4 protein variants listed in Figures 2 and 3. In particular, peptide immunogens of the invention can comprise, a peptide region of at least 5 amino acids of Figures 2 and 3 in any whole number increment that includes an amino acid position having a value greater than 0.5 in the Hydrophilicity profiles of Figure 5; a peptide region of at least 5 amino acids of Figures 2 and 3 in any whole number increment that includes an amino acid position having a value less than 0.5 in the Hydropathicity profile of Figure 6 ; a peptide region of at least 5 amino acids of Figures 2 and 3 in any whole number increment that includes an amino acid position having a value greater than 0.5 in the Percent Accessible Residues profiles of Figure 7; a peptide region of at least 5 amino acids of Figures 2 and 3 in any whole number increment that includes an amino acid position having a value greater than 0.5 in the Average Flexibility profiles on Figure 8 ; and, a peptide region of at least 5 amino acids of Figures 2 and 3 in any whole number increment that includes an amino acid position having a value greater than 0.5 in the Beta-turn profile of Figures 9 . Peptide immunogens of the invention can also comprise nucleic acids that encode any of the forgoing.
All immunogens of the invention, peptide or nucleic acid, can be embodied in human unit dose form, or comprised by a composition that includes a pharmaceutical excipient compatible with human physiology.
The secondary structure of 109P1D4 protein variants, namely the predicted presence and location of alpha helices, extended strands, and random coils, are predicted from the primary amino acid sequence using the HNN - Hierarchical Neural Network method (NPS@: Network Protein Sequence Analysis TIBS 2000 March Vol. 25, No 3 [291]:147-150 Combet C, Blanchet C, Geourjon C. and Deleage G., http://pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_nn.html), accessed from the ExPasy molecular biology server located on the World Wide Web at (www.expasy.ch/tools/). This analysis for protein variants 1 through 9 are shown in Figure 13A through 131 respectively. The percent of structure for each variant comprised of alpha helix, extended strand, and random coil is also indicated.
Analysis for the potential presence of transmembrane domains in 109P1 D4 variant proteins was carried out using a variety of transmembrane prediction algorithms accessed from the ExPasy molecular biology server located on the World Wide Web at (www.expasy.ch/tools/). Shown graphically in figures 13J-R are the results of analyses using the TMpred program (top panels) and the TMHMM program (bottom panels) of 109P1D4 protein variants 1 through 9 respectively. Analyses of the variants using other structural prediction programs are summarized in Table VI and Table L.
Example 10: Generation of 109P1D4 Polyclonal Antibodies
Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. In addition to immunizing with a full length 109P1D4 protein variant, computer algorithms are employed in design of immunogens that, based on amino acid sequence analysis contain characteristics of being antigenic and available for recognition by the immune system of the immunized host (see the Example entitled "Antigenicity Profiles and Secondary Structure"). Such regions would be predicted to be hydrophilic, flexible, in beta-turn conformations, and be exposed on the surface of the protein (see, e.g., Figure 5, Figure 6, Figure 7, Figure 8, or Figure 9 for amino acid profiles that indicate such regions of 109P1D4 protein variant 1).
For example, recombinant bacterial fusion proteins or peptides containing hydrophilic, flexible, beta-turn regions of 109P1D4 protein variants are used as antigens to generate polyclonal antibodies in New Zealand White rabbits or monoclonal antibodies as described in the example entitled "Generation of 109P1D4 Monoclonal Antibodies (mAbs)". For example, in 109P1D4 variant 1, such regions include, but are not limited to, amino acids 22-39, amino acids 67-108, amino acids 200-232, amino acids 454-499, amino acids 525-537, amino acids 640-660, amino acids 834-880, and amino acids 929-942. It is useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include, but are not limited to, keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. In 2 embodiments, peptides encoding amino acids 77-90 and amino acids 929-942 of 109P1D4 variant 1 were synthesized, conjugated to KLH, and used to immunize separate rabbits. Alternatively the immunizing agent may include all or portions of the 109P1 D4 variant proteins, analogs or fusion proteins thereof. For example, the 109P1D4 variant 1 amino acid sequence can be fused using recombinant DNA techniques to any one of a variety of fusion protein partners that are well known in the art, such as glutathione-S-transferase (GST) and HIS tagged fusion proteins. In 1 embodiment, amino acids 24-419 of 109P1D4 variant 1 was fused to NUSa using recombinant techniques and the pET43.1 expression vector, expressed, purified and used to immunize a rabbit. Such fusion proteins are purified from induced bacteria using the appropriate affinity matrix.
Other recombinant bacterial fusion proteins that may be employed include maltose binding protein, LacZ, thioredoxin, NusA, or an immunoglobulin constant region (see the section entitled "Production of 109P1D4 in Prokaryotic Systems" and Current Protocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubul et al. eds., 1995; Linsley, P.S., Brady, W., Urnes, M., Grosmaire, L, Damle, N„ and Ledbetter, J.(1991) J.Exp. Med. 174, 561-566).
In addition to bacterial derived fusion proteins, mammalian expressed protein antigens are also used. These antigens are expressed from mammalian expression vectors such as the Tag5 and Fc-fusion vectors (see the section entitled "Production of Recombinant 109P1D4 in Eukaryotic Systems"), and retain post-translational modifications such as glycosylations found in native protein. In one embodiment, amino acids 24-812 of 109P1 D4 variant 1 was cloned into the Tag5 mammalian secretion vector, and expressed in 293T cells (See Figure 20). The recombinant protein is purified by metal chelate chromatography from tissue culture supernatants of 293T cells stably expressing the recombinant vector. The purified Tagδ 109P1D4 protein is then used as immunogen.
During the immunization protocol, it is useful to mix or emulsify the antigen in adjuvants that enhance the immune response of the host animal. Examples of adjuvants include, but are not limited to, complete Freund's adjuvant (CFA) and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
In a typical protocol, rabbits are initially immunized subcutaneously with up to 200 μg, typically 100-200 μg, of fusion protein or peptide conjugated to KLH mixed in complete Freund's adjuvant (CFA). Rabbits are then injected subcutaneously every two weeks with up to 200 μg, typically 100-200 μg, of the immunogen in incomplete Freund's adjuvant (IFA). Test bleeds are taken approximately 7-10 days following each immunization and used to monitor the titer of the antiserum by ELISA.
To test reactivity and specificity of immune serum, such as the rabbit serum derived from immunization with the NUSa-fusion of 109P1D4 variant 1 protein, the full-length 109P1D4 variant 1 cDNA is cloned into pCDNA 3.1 myc-his expression vector (Invitrogen, see the Example entitied "Production of Recombinant 109P1D4 in Eukaryotic Systems"). After transfection of the constructs into 293T cells, cell lysates are probed with the anti-109P1D4 serum to determine specific reactivity to denatured 109P1D4 protein using the Western blot technique. Probing with anti-His antibody serves as a positive control for expression of 109P1D4 in the transfected cells (See Figure 21). In addition, the immune serum is tested by fluorescence microscopy, flow cytometry and immunoprecipitatioπ against 293T and other recombinant 109P1 D4- expressing cells to determine specific recognition of native protein. Western blot, immunoprecipitation, fluorescent microscopy, and flow cytometric techniques using cells that endogenously express 109P1 D4 are also carried out to test reactivity and specificity.
Anti-serum from rabbits immunized with 109P1D4 variant fusion proteins, such as GST and MBP fusion proteins, are purified by depletion of antibodies reactive to the fusion partner sequence by passage over an affinity column containing the fusion partner either alone or in the context of an irrelevant fusion protein. For example, antiserum derived from a NUSa- 109P1D4 variant 1 fusion protein is first purified by passage over a column of MBP protein covalently coupled to AffiGel matrix (BioRad, Hercules, Calif.). The antiserum is then affinity purified by passage over a column composed of a NUSa- 109P1D4 fusion protein covalently coupled to Affigel matrix. The serum is then further purified by protein G affinity chromatography to isolate the IgG fraction. Sera from other His-tagged antigens and peptide immunized rabbits as well as fusion partner depleted sera are affinity purified by passage over a column matrix composed of the original protein immunogen or free peptide.
Example 11: Generation of 109P1D4 Monoclonal Antibodies (mAbs)
In one embodiment, therapeutic mAbs to 109P1 D4 variants comprise those that react with epitopes specific for each variant protein or specific to sequences in common between the variants that would disrupt or modulate the biological function of the 109P1 D4 variants, for example those that would disrupt the interaction with ligands and binding partners. Immunogens for generation of such mAbs include those designed to encode or contain the entire 109P1D4 protein variant sequence, regions predicted to contain functional motifs, and regions of the 109P1D4 protein variants predicted to be antigenic from computer analysis of the amino acid sequence (see, e.g., Figure 5, Figure 6, Figure 7, Figure 8, or Figure 9, and the Example entitled "Antigenicity Profiles and Secondary Structure"). Immunogens include peptides, recombinant bacterial proteins, and mammalian expressed Tag 5 proteins and human and murine IgG FC fusion proteins. In addition, cells engineered to express high levels of a respective 109P1D4 variant, such as 293T-109P1D4 variant 1 or 300.19- 109P1D4 variant 1 murine Pre-B cells, are used to immunize mice.
To generate mAbs to a 109P1D4 variant, mice are first immunized intraperitoneally (IP) with, typically, 10-50 μg of protein immunogen or 107 109P1D4-expressing cells mixed in complete Freund's adjuvant. Mice are then subsequently immunized IP every 2-4 weeks with, typically, 10-50 μg of protein immunogen or 107 cells mixed in incomplete Freund's adjuvant. Alternatively, MPL-TDM adjuvant is used in immunizations. In addition to the above protein and cell-based immunization strategies, a DNA-based immunization protocol is employed in which a mammalian expression vector encoding a 109P1 D4 variant sequence is used to immunize mice by direct injection of the plasmid DNA. For example, amino acids 24-812 of 109P1 D4 of variant 1 is cloned into the Tagδ mammalian secretion vector and the recombinant vector will then be used as immunogen. In another example the same amino acids are cloned into an Fc-fusion secretion vector in which the 109P1 D4 variant 1 sequence is fused at the amino-terminus to an IgK leader sequence and at the carboxyl- terminus to the coding sequence of the human or murine IgG Fc region. This recombinant vector is then used as immunogen. The plasmid immunization protocols are used in combination with purified proteins expressed from the same vector and with cells expressing the respective 109P1D4 variant.
Alternatively, mice may be immunized directly into their footpads. In this case, 10-50 μg of protein immunogen or 107 254P1D6B-expressing cells are injected sub-cutaneously into the footpad of each hind leg. The first immunization is given with Titermax (Sigma™) as an adjuvant and subsequent injections are given with Alum-gel in conjunction with CpG oligonucleotide sequences with the exception of the final injection which is given with PBS. Injections are given twice weekly (every three to four days) for a period of 4 weeks and mice are sacrificed 3-4 days after the final injection, at which point lymph nodes immediately draining from the footpad are harvested and the B-cells are collected for use as antibody producing fusion partners.
During the immunization protocol, test bleeds are taken 7-10 days following an injection to monitor titer and specificity of the immune response. Once appropriate reactivity and specificity is obtained as determined by ELISA, Western blotting, immunoprecipitation, fluorescence microscopy, and flow cytomβfric analyses, fusion and hybridoma generation is then carried out with established procedures well known in the art (see, e.g., Harlow and Lane, 1988).
In one embodiment for generating 109P1 D4 monoclonal antibodies, a Tagδ antigen of variant 1 encoding amino acids 14-812 is expressed in 293T cells and purified from conditioned media. Balb C mice are initially immunized intraperitoneally with 26 μg of the Tagδ 109P1 D4 variant 1 protein mixed in complete Freund's adjuvant. Mice are subsequently immunized every two weeks with 25 μg of the antigen mixed in incomplete Freund's adjuvant for a total of three immunizations. ELISA using the Tagδ antigen determines the titer of serum from immunized mice. Reactivity and specificity of serum to full length 109P1D4 variant 1 protein is monitored by Western blotting, immunoprecipitation and flow cytometry using 293T cells transfected with an expression vector encoding the 109P1D4 variant 1 cDNA (see e.g., the Example entitled "Production of Recombinant 109P1D4 in Higher Eukaryotic Systems" and Figure 21). Other recombinant 109P1D4 variant 1-expressing cells or cells endogenously expressing 109P1D4 variant 1 are also used. Mice showing the strongest reactivity are rested and given a final injection of antigen in PBS and then sacrificed four days later. The spleens of the sacrificed mice are harvested and fused to SPO/2 myeloma cells using standard procedures (Harlow and Lane, 1988). Supernatants from HAT selected growth wells are screened by ELISA, Western blot, immunoprecipitation, fluorescent microscopy, and flow cytometry to identify 109P1D4 specific antibody-producing clones.
To generate monoclonal antibodies that are specific for a 109P1D4 variant protein, immunogens are designed to encode sequences unique for each variant. In one embodiment, an antigenic peptide composed of amino acids 1-29 of 109P1D4 variant 2 is coupled to KLH to derive monoclonal antibodies specific to 109P1D4 variant 2. In another embodiment, an antigenic peptide comprised of amino acids 1 -23 of 109P1 D4 variant 6 is coupled to KLH and used as immunogen to derive varaiant 6 specific MAbs. In another example, a GST-fusion protein encoding amino acids 1001-1347 of variant 3 is used as immunogen to generate antibodies that would recognize variants 3, 4, 5, and 8, and distinguish them from variants 1, 2, 6, 7and 9. Hybridoma supernatants are then screened on the respective antigen and then further screened on cells expressing the specific variant and cross-screened on cells expressing the other variants to derive variant- specific monoclonal antibodies.
The binding affinity of 109P1D4 variant specific monoclonal antibodies are determined using standard technologies. Affinity measurements quantify the strength of antibody to epitope binding and are used to help define which 109P1 D4 variant monoclonal antibodies preferred for diagnostic or therapeutic use, as appreciated by one of skill in the art. The BIAcore system (Uppsala, Sweden) is a preferred method for determining binding affinity. The BIAcore system uses surface plasmon resonance (SPR, Welford K. 1991, Opt. Quant. Elect. 23:1; Morton and Myszka, 1998, Methods in Enzymology 296: 268) to monitor biomolecular interactions in real time. BIAcore analysis conveniently generates association rate constants, dissociation rate constants, equilibrium dissociation constants, and affinity constants. Alternatively, equilibrium binding analysis of MAbs on 109P1D4-expressing cells can be used to determine affinity.
Example 12: HLA Class I and Class II Binding Assays
HLA class I and class II binding assays using purified HLA molecules are performed in accordance with disclosed protocols (e.g., PCT publications WO 94/20127 and WO 94/03205; Sidney ef al, Current Protocols in Immunology 18.3.1 (1998); Sidney, ef al, J. Immunol. 154:247 (1995); Sette, ef al, Mol. Immunol. 31:813 (1994)). Briefly, purified MHC molecules (5 to 500 nM) are incubated with various unlabeled peptide inhibitors and 1-10 nM 125l-radiolabeled probe peptides as described. Following incubation, MHC-peptide complexes are separated from free peptide by gel filtration and the fraction of peptide bound is determined. Typically, in preliminary experiments, each MHC preparation is titered in the presence of fixed amounts of radiolabeled peptides to determine the concentration of HLA molecules necessary to bind 10- 20% of the total radioactivity. All subsequent inhibition and direct binding assays are performed using these HLA concentrations.
Since under these conditions [label]<[HLA] and ICso>[HLA], the measured ICso values are reasonable approximations of the true KD values. Peptide inhibitors are typically tested at concentrations ranging from 120 μg/ml to 1.2 ng/ml, and are tested in two to four completely independent experiments. To allow comparison of the data obtained in different experiments, a relative binding figure is calculated for each peptide by dividing the ICso of a positive control for inhibition by the ICso for each tested peptide (typically unlabeled versions of the radiolabeled probe peptide). For database purposes, and inter-experiment comparisons, relative binding values are compiled. These values can subsequently be converted back into ICso nM values by dividing the ICso nM of the positive controls for inhibition by the relative binding of the peptide of interest. This method of data compilation is accurate and consistent for comparing peptides that have been tested on different days, or with different lots of purified MHC.
Binding assays as outlined above may be used to analyze HLA supermotif and/or HLA motif-bearing peptides (see Table IV).
Example 13: Identification of HLA Supermotif- and Motif-Bearing CTL Candidate Epitopes
HLA vaccine compositions of the invention can include multiple epitopes. The multiple epitopes can comprise multiple HLA supermotifs or motifs to achieve broad population coverage. This example illustrates the identification and confirmation of supermotif- and motif-bearing epitopes for the inclusion in such a vaccine composition. Calculation of population coverage is performed using the strategy described below.
Computer searches and algorithms for identification of supermotif and/or motif-bearing epitopes
The searches performed to identify the motif-bearing peptide sequences in the Example entitled "Antigenicity Profiles" and Tables VIII-XXI and XXII-XLIX employ the protein sequence data from the gene product of 109P1 D4 set forth in Figures 2 and 3, the specific search peptides used to generate the tables are listed in Table VII.
Computer searches for epitopes bearing HLA Class I or Class II supermotifs or motifs are performed as follows. All translated 109P1D4 protein sequences are analyzed using a text string search software program to identify potential peptide sequences containing appropriate HLA binding motifs; such programs are readily produced in accordance with information in the art in view of known motif/supermotif disclosures. Furthermore, such calculations can be made mentally.
Identified A2-, A3-, and DR-supermotif sequences are scored using polynomial algorithms to predict their capacity to bind to specific HLA-Class I or Class II molecules. These polynomial algorithms account for the impact of different amino acids at different positions, and are essentially based on the premise that the overall affinity (or ΔG) of peptide-HLA molecule interactions can be approximated as a linear polynomial function of the type:
Figure imgf000097_0001
where ay/ is a coefficient which represents the effect of the presence of a given amino acid (/) at a given position () along the sequence of a peptide of n amino acids. The crucial assumption of this method is that the effects at each position are essentially independent of each other (i.e., independent binding of individual side-chains). When residue j occurs at position / in the peptide, it is assumed to contribute a constant amount • to the free energy of binding of the peptide irrespective of the sequence of the rest of the peptide.
The method of derivation of specific algorithm coefficients has been described in Gulukota ef al, J. Mol. Biol. 267:1258-126, 1997; (see also Sidney ef al, Human Immunol. 45:79-93, 1996; and Southwood ef al, J. Immunol. 160:3363- 3373, 1998). Briefly, for all / positions, anchor and non-anchor alike, the geometric mean of the average relative binding (ARB) of all peptides carrying j is calculated relative to the remainder of the group, and used as the estimate of j/. For Class II peptides, if multiple alignments are possible, only the highest scoring alignment is utilized, following an iterative procedure. To calculate an algorithm score of a given peptide in a test set, the ARB values corresponding to the sequence of the peptide are multiplied. If this product exceeds a chosen threshold, the peptide is predicted to bind. Appropriate thresholds are chosen as a function of the degree of stringency of prediction desired.
Selection of HLA-A2 supertype cross-reactive peptides
Protein sequences from 109P1D4 are scanned utilizing motif identification software, to identify 8-, 9- 10- and 11- mer sequences containing the HLA-A2-supermotif main anchor specificity. Typically, these sequences are then scored using the protocol described above and the peptides corresponding to the positive-scoring sequences are synthesized and tested for their capacity to bind purified HLA-A*0201 molecules in vitro (HLA-A*0201 is considered a prototype A2 supertype molecule).
These peptides are then tested for the capacity to bind to additional A2-supertype molecules (A*0202, A*0203, A*0206, and A*6802). Peptides that bind to at least three of the five A2-supertype alleles tested are typically deemed A2- supertype cross-reactive binders. Preferred peptides bind at an affinity equal to or less than 500 nM to three or more HLA- A2 supertype molecules.
Selection of HLA-A3 supermotif-bearing epitopes
The 109P1 D4 protein sequence(s) scanned above is also examined for the presence of peptides with the HLA-A3- supermotif primary anchors. Peptides corresponding to the HLA A3 supermotif-bearing sequences are then synthesized and tested for binding to HLA-A*0301 and HLA-A*1101 molecules, the molecules encoded by the two most prevalent A3- supertype alleles. The peptides that bind at least one of the two alleles with binding affinities of <500 nM, often < 200 nM, are then tested for binding cross-reactivity to the other common A3-supertype alleles (e.g., A*3101, A*3301, and A*6801) to identify those that can bind at least three of the five HLA-A3-supertype molecules tested.
Selection of HLA-B7 supermotif bearing epitopes
The 109P1D4 protein(s) scanned above is also analyzed for the presence of 8-, 9- 10-, or 11-mer peptides with the HLA-B7-supermotif. Corresponding peptides are synthesized and tested for binding to HLA-B 02, the molecule encoded by the most common B7-supertype allele (i.e., the prototype B7 supertype allele). Peptides binding B 02 with ICso of <500 nM are identified using standard methods. These peptides are then tested for binding to other common B7-supertype molecules (e.g., B*3501, B*5101, B*5301, and B*5401). Peptides capable of binding to three or more of the five B7- supertype alleles tested are thereby identified.
Selection of A1 and A24 motif-bearing epitopes
To further increase population coverage, HLA-A1 and -A24 epitopes can also be incorporated into vaccine compositions. An analysis of the 109P1D4 protein can also be performed to identify HLA-A1- and A24-motif-containing sequences. High affinity and/or cross-reactive binding epitopes that bear other motif and/or supermotifs are identified using analogous methodology.
Example 14: Confirmation of Immunogenicity
Cross-reactive candidate CTL A2-supermotif-bearing peptides that are identified as described herein are selected to confirm in vitro immunogenicity. Confirmation is performed using the following methodology:
Target Cell Lines for Cellular Screening:
The .221A2.1 cell line, produced by transferring the HLA-A2.1 gene into the HLA-A, -B, -C null mutant human B- lymphoblastoid cell line 721.221, is used as the peptide-loaded target to measure activity of HLA-A2.1 -restricted CTL. This cell line is grown in RPMI-1640 medium supplemented with antibiotics, sodium pyruvate, nonessential amino acids and 10% (v/v) heat inactivated FCS. Cells that express an antigen of interest, or transfectants comprising the gene encoding the antigen of interest, can be used as target cells to confirm the ability of peptide-specific CTLs to recognize endogenous antigen.
Primary CTL Induction Cultures:
Generation of Dendritic Cells (DC): PBMCs are thawed in RPMI with 30 μg/ml DNAse, washed twice and resuspended in complete medium (RPMI-1640 plus 5% AB human serum, non-essential amino acids, sodium pyruvate, L- glutamine and penicillin/streptomycin). The monocytes are purified by plating 10 x 106 PBMC/well in a 6-well plate. After 2 hours at 37°C, the non-adherent cells are removed by gently shaking the plates and aspirating the supernatants. The wells are washed a total of three times with 3 ml RPMI to remove most of the non-adherent and loosely adherent cells. Three ml of complete medium containing 60 ng/ml of GM-CSF and 1,000 U/ml of IL-4 are then added to each well. TNFα is added to the DCs on day 6 at 75 ng/ml and the cells are used for CTL induction cultures on day 7.
Induction of CTL with DC and Peptide: CD8+ T-cells are isolated by positive selection with Dynal immunomagnetic beads (Dynabeads® M-450) and the detacha-bead® reagent. Typically about 200-250x106 PBMC are processed to obtain 24x106 CD8+ T-cells (enough for a 48-well plate culture). Briefly, the PBMCs are thawed in RPMI with 30μg/ml DNAse, washed once with PBS containing 1% human AB serum and resuspended in PBS/1 % AB serum at a concentration of 20x106cells/ml. The magnetic beads are washed 3 times with PBS/AB serum, added to the cells (140μl beads/20x106 cells) and incubated for 1 hour at 4°C with continuous mixing. The beads and cells are washed 4x with PBS/AB serum to remove the nonadherent cells and resuspended at 100x106 cells/ml (based on the original cell number) in PBS/AB serum containing 100μl/ml detacha-bead® reagent and 30 μg/ml DNAse. The mixture is incubated for 1 hour at room temperature with continuous mixing. The beads are washed again with PBS/AB/DNAse to collect the CD8+ T-cells. The DC are collected and centrifuged at 1300 rpm for 5-7 minutes, washed once with PBS with 1% BSA, counted and pulsed with 40μg/ml of peptide at a cell concentration of 1-2x106/ml in the presence of 3μg/ml Ik- microglobulin for 4 hours at 20°C. The DC are then irradiated (4,200 rads), washed 1 time with medium and counted again.
Setting up induction cultures: 0.25 ml cytokine-generated DC (at 1x105 cells/ml) are co-cultured with 0.25ml of CD8+ T-cells (at 2x106 cell/ml) in each well of a 48-well plate in the presence of 10 ng/ml of IL-7. Recombinant human IL-10 is added the next day at a final concentration of 10 ng/ml and rhuman IL-2 is added 48 hours later at 10 lU/ml.
Restimulation of the induction cultures with peptide-pulsed adherent cells: Seven and fourteen days after the primary induction, the cells are restimulated with peptide-pulsed adherent cells. The PBMCs are thawed and washed twice with RPMI and DNAse. The cells are resuspended at 5x10s cells/ml and irradiated at ~4200 rads. The PBMCs are plated at 2x106 in 0.5 ml complete medium per well and incubated for 2 hours at 37CC. The plates are washed twice with RPMI by tapping the plate gently to remove the nonadherent cells and the adherent cells pulsed with 10μg/ml of peptide in the presence of 3 μg/ml β2 microglobulin in 0.25ml RPMI/5%AB per well for 2 hours at 37°C. Peptide solution from each well is aspirated and the wells are washed once with RPMI. Most of the media is aspirated from the induction cultures (CD8+ cells) and brought to 0.5 ml with fresh media. The cells are then transferred to the wells containing the peptide-pulsed adherent cells. Twenty four hours later recombinant human IL-10 is added at a final concentration of 10 ng/ml and recombinant human 1L2 is added the next day and again 2-3 days later at 50IU/ml (Tsai ef al, Critical Reviews in Immunology 18(1-2):65-75, 1998). Seven days later, the cultures are assayed for CTL activity in a 51Cr release assay. In some experiments the cultures are assayed for peptide-specific recognition in the in situ IFNγ ELISA at the time of the second restimulation followed by assay of endogenous recognition 7 days later. After expansion, activity is measured in both assays for a side-by-side comparison.
Measurement of CTL lytic activity by 51Cr release.
Seven days after the second restimulation, cytotoxicity is determined in a standard (5 hr) 51Cr release assay by assaying individual wells at a single E:T. Peptide-pulsed targets are prepared by incubating the cells with 10μg/ml peptide overnight at 37°C.
Adherent target cells are removed from culture flasks with trypsin-EDTA. Target cells are labeled with 200μCi of 51Cr sodium chromate (Dupont, Wilmington, DE) for 1 hour at 37°C. Labeled target cells are resuspended at 106 per ml and diluted 1:10 with K562 cells at a concentration of 3.3x106/ml (an NK-sensitive erythroblastoma cell line used to reduce nonspecific lysis). Target cells (100 μl) and effectors (100μl) are plated in 96 well round-bottom plates and incubated for 5 hours at 37°C. At that time, 100 μl of supernatant are collected from each well and percent lysis is determined according to the formula:
[(cpm of the test sample- cpm of the spontaneous 5 Cr release sample)/(cpm of the maximal 51Cr release sample- cpm of the spontaneous 51Cr release sample)] x 100.
Maximum and spontaneous release are determined by incubating the labeled targets with 1% Triton X-100 and media alone, respectively. A positive culture is defined as one in which the specific lysis (sample- background) is 10% or higher in the case of individual wells and is 16% or more at the two highest E:T ratios when expanded cultures are assayed.
In situ Measurement of Human IFNγ Production as an Indicator of Peptide-specific and Endogenous Recognition
Immulon 2 plates are coated with mouse anti-human IFNy. monoclonal antibody (4 μg/ml 0.1M NaHCθ3, pH8.2) overnight at 4°C. The plates are washed with Ca2+, Mg2+-free PBS/0.05% Tween 20 and blocked with PBS/10% FCS for two hours, after which the CTLs (100 μl/well) and targets (100 μl/well) are added to each well, leaving empty wells for the standards and blanks (which received media only). The target cells, either peptide-pulsed or endogenous targets, are used at a concentration of 1x106 cells/ml. The plates are incubated for 48 hours at 37°C with 5% C02.
Recombinant human IFN-gamma is added to the standard wells starting at 400 pg or 1200pg/100 microliter/well and the plate incubated for two hours at 37°C. The plates are washed and 100 μl of biotinylated mouse anti-human IFN- gamma monoclonal antibody (2 microgram/ml in PBS/3%FCS/0.05% Tween 20) are added and incubated for 2 hours at room temperature. After washing again, 100 microliter HRP-streptavidin (1:4000) are added and the plates incubated for one hour at room temperature. The plates are then washed 6x with wash buffer, 100 microliter/well developing solution (TMB 1:1) are added, and the plates allowed to develop for 5-15 minutes. The reaction is stopped with 50 microliter/well 1M H3PO4 and read at OD460. A culture is considered positive if it measured at least 60 pg of IFN-gamma/well above background and is twice the background level of expression.
CTL Expansion.
Those cultures that demonstrate specific lytic activity against peptide-pulsed targets and/or tumor targets are expanded over a two week period with anti-CD3. Briefly, 5x104 CD8+ cells are added to a T25 flask containing the following: 1x106 irradiated (4,200 rad) PBMC (autologous or allogeneic) per ml, 2x105 irradiated (8,000 rad) EBV- transformed cells per ml, and OKT3 (anti-CD3) at 30ng per ml in RPMI-1640 containing 10% (v/v) human AB serum, non-essential amino acids, sodium pyruvate, 25μM 2-mercaptoethanol, L-glutamine and penicillin/streptomycin. Recombinant human IL2 is added 24 hours later at a final concentration of 200IU/ml and every three days thereafter with fresh media at 50IU/ml. The cells are split if the cell concentration exceeds 1x106/ml and the cultures are assayed between days 13 and 15 at E:T ratios of 30, 10, 3 and 1:1 in the 51Cr release assay or at 1x106/ml in the in situ IFNγ assay using the same targets as before the expansion.
Cultures are expanded in the absence of anti-CD3+ as follows. Those cultures that demonstrate specific lytic activity against peptide and endogenous targets are selected and 5x104 CD8+ cells are added to a T25 flask containing the following: 1x106 autologous PBMC per ml which have been peptide-pulsed with 10 μg/ml peptide for two hours at 37°C and irradiated (4,200 rad); 2x105 irradiated (8,000 rad) EBV-transformed cells per ml RPMI-1640 containing 10%(v/v) human AB serum, non-essential AA, sodium pyruvate, 2δmM 2-ME, L-glutamine and gentamicin.
Immunogenicity of A2 supermotif-bearing peptides
A2-supermotif cross-reactive binding peptides are tested in the cellular assay for the ability to induce peptide- specific CTL in normal individuals. In this analysis, a peptide is typically considered to be an epitope if it induces peptide- specific CTLs in at least individuals, and preferably, also recognizes the endogenously expressed peptide.
Immunogenicity can also be confirmed using PBMCs isolated from patients bearing a tumor that expresses 109P1D4. Briefly, PBMCs are isolated from patients, re-stimulated with peptide-pulsed monocytes and assayed for the ability to recognize peptide-pulsed target cells as well as transfected cells endogenously expressing the antigen.
Evaluation of A*03/A11 immunogenicity
HLA-A3 supermotif-bearing cross-reactive binding peptides are also evaluated for immunogenicity using methodology analogous for that used to evaluate the immunogenicity of the HLA-A2 supermotif peptides.
Evaluation of B7 immunogenicity
Immunogenicity screening of the B7-supertype cross-reactive binding peptides identified as set forth herein are confirmed in a manner analogous to the confirmation of A2-and A3-supermotif-bearing peptides.
Peptides bearing other supermotifs/motifs, e.g., HLA-A1, HLA-A24 etc. are also confirmed using similar methodology
Example 15: Implementation of the Extended Supermotif to Improve the Binding Capacity of Native Epitopes by Creating Analogs
HLA motifs and supermotifs (comprising primary and/or secondary residues) are useful in the identification and preparation of highly cross-reactive native peptides, as demonstrated herein. Moreover, the definition of HLA motifs and supermotifs also allows one to engineer highly cross-reactive epitopes by identifying residues within a native peptide sequence which can be analoged to confer upon the peptide certain characteristics, e.g. greater cross-reactivity within the group of HLA molecules that comprise a supertype, and/or greater binding affinity for some or all of those HLA molecules. Examples of analoging peptides to exhibit modulated binding affinity are set forth in this example.
Analoging at Primary Anchor Residues
Peptide engineering strategies are implemented to further increase the cross-reactivity of the epitopes. For example, the main anchors of A2-supermotif-bearing peptides are altered, for example, to introduce a preferred L, I, V, or M at position 2, and I or V at the C-terminus.
To analyze the cross-reactivity of the analog peptides, each engineered analog is initially tested for binding to the prototype A2 supertype allele A*0201, then, if A*0201 binding capacity is maintained, for A2-supertype cross-reactivity. Alternatively, a peptide is confirmed as binding one or all supertype members and then analoged to modulate binding affinity to any one (or more) of the supertype members to add population coverage.
The selection of analogs for immunogenicity in a cellular screening analysis is typically further restricted by the capacity of the parent wild type (WT) peptide to bind at least weakly, i.e., bind at an ICso of 5000nM or less, to three of more A2 supertype alleles. The rationale for this requirement is that the WT peptides must be present endogenously in sufficient quantity to be biologically relevant. Analoged peptides have been shown to have increased immunogenicity and cross- reactivity by T cells specific for the parent epitope (see, e.g., Parkhurst ef al, J. Immunol. 157:2539, 1996; and Pogue ef al., Proc. Natl. Acad. Sci. USA 92:8166, 1995).
In the cellular screening of these peptide analogs, it is important to confirm that analog-specific CTLs are also able to recognize the wild-type peptide and, when possible, target cells that endogenously express the epitope.
Analoging of HLA-A3 and B7-supermotif-bearing peptides
Analogs of HLA-A3 supermotif-bearing epitopes are generated using strategies similar to those employed in analoging HLA-A2 supermotif-bearing peptides. For example, peptides binding to 3/5 of the A3-supertype molecules are engineered at primary anchor residues to possess a preferred residue (V, S, M, or A) at position 2.
The analog peptides are then tested for the ability to bind A*03 and A*11 (prototype A3 supertype alleles). Those peptides that demonstrate < 500 nM binding capacity are then confirmed as having A3-supertype cross-reactivity.
Similarly to the A2- and A3- motif bearing peptides, peptides binding 3 or more B7-supertype alleles can be improved, where possible, to achieve increased cross-reactive binding or greater binding affinity or binding half life. B7 supermotif-bearing peptides are, for example, engineered to possess a preferred residue (V, I, L, or F) at the C-terminal primary anchor position, as demonstrated by Sidney et al. (J. Immunol. 157:3480-3490, 1996).
Analoging at primary anchor residues of other motif and/or supermotif-bearing epitopes is performed in a like manner.
The analog peptides are then be confirmed for immunogenicity, typically in a cellular screening assay. Again, it is generally important to demonstrate that analog-specific CTLs are also able to recognize the wild-type peptide and, when possible, targets that endogenously express the epitope.
Analoging at Secondary Anchor Residues
Moreover, HLA supermotifs are of value in engineering highly cross-reactive peptides and/or peptides that bind HLA molecules with increased affinity by identifying particular residues at secondary anchor positions that are associated with such properties. For example, the binding capacity of a B7 supermotif-bearing peptide with an F residue at position 1 is analyzed. The peptide is then analoged to, for example, substitute L for F at position 1. The analoged peptide is evaluated for increased binding affinity, binding half life and/or increased cross-reactivity. Such a procedure identifies analoged peptides with enhanced properties.
Engineered analogs with sufficiently improved binding capacity or cross-reactivity can also be tested for immunogenicity in HLA-B7-transgenic mice, following for example, IFA immunization or lipopeptide immunization. Analoged peptides are additionally tested for the ability to stimulate a recall response using PBMC from patients with 109P1 D4- expressing tumors.
Other analoging strategies
Another form of peptide analoging, unrelated to anchor positions, involves the substitution of a cysteine with α- amino butyric acid. Due to its chemical nature, cysteine has the propensity to form disulfide bridges and sufficiently alter the peptide structurally so as to reduce binding capacity. Substitution of α-amino butyric acid for cysteine not only alleviates this problem, but has been shown to improve binding and crossbinding capabilities in some instances (see, e.g., the review by Sette etal, In: Persistent Viral Infections, Eds. R. Ahmed and I. Chen, John Wiley & Sons, England, 1999).
Thus, by the use of single amino acid substitutions, the binding properties and/or cross-reactivity of peptide ligands for HLA supertype molecules can be modulated.
Example 18: Identification and confirmation of 109P1D4-derived sequences with HLA-DR binding motifs
Peptide epitopes bearing an HLA class II supermotif or motif are identified and confirmed as outlined below using methodology similar to that described for HLA Class I peptides.
Selection of HLA-DR-supermotif-bearing epitopes.
To identify 109P1D4-derived, HLA class II HTL epitopes, a 109P1D4 antigen is analyzed for the presence of sequences bearing an HLA-DR-motif or supermotif. Specifically, 15-mer sequences are selected comprising a DR- supermotif, comprising a 9-mer core, and three-residue N- and C-terminal flanking regions (16 amino acids total).
Protocols for predicting peptide binding to DR molecules have been developed (Southwood ef a/., J. Immunol. 160:3363-3373, 1998). These protocols, specific for individual DR molecules, allow the scoring, and ranking, of 9-mer core regions. Each protocol not only scores peptide sequences for the presence of DR-supermotif primary anchors (i.e., at position 1 and position 6) within a 9-mer core, but additionally evaluates sequences for the presence of secondary anchors. Using allele-specific selection tables (see, e.g., Southwood ef al, ibid.), it has been found that these protocols efficiently select peptide sequences with a high probability of binding a particular DR molecule. Additionally, it has been found that performing these protocols in tandem, specifically those for DR1, DR4w4, and DR7, can efficiently select DR cross-reactive peptides.
The 109P1D4-derived peptides identified above are tested for their binding capacity for various common HLA-DR molecules. All peptides are initially tested for binding to the DR molecules in the primary panel: DR1, DR4w4, and DR7. Peptides binding at least two of these three DR molecules are then tested for binding to DR2w2 β1, DR2w2 β2, DR6w19, and DR9 molecules in secondary assays. Finally, peptides binding at least two of the four secondary panel DR molecules, and thus cumulatively at least four of seven different DR molecules, are screened for binding to DR4w15, DRδwl 1 , and DR8w2 molecules in tertiary assays. Peptides binding at least seven of the ten DR molecules comprising the primary, secondary, and tertiary screening assays are considered cross-reactive DR binders. 109P1 D4-derived peptides found to bind common HLA-DR alleles are of particular interest.
Selection of DR3 motif peptides
Because HLA-DR3 is an allele that is prevalent in Caucasian, Black, and Hispanic populations, DR3 binding capacity is a relevant criterion in the selection of HTL epitopes. Thus, peptides shown to be candidates may also be assayed for their DR3 binding capacity. However, in view of the binding specificity of the DR3 motif, peptides binding only to DR3 can also be considered as candidates for inclusion in a vaccine formulation.
To efficiently identify peptides that bind DR3, target 109P1D4 antigens are analyzed for sequences carrying one of the two DR3-specific binding motifs reported by Geluk ef al. (J. Immunol. 152:5742-5748, 1994). The corresponding peptides are then synthesized and confirmed as having the ability to bind DR3 with an affinity of 1 μM or better, i.e., less than 1 μM. Peptides are found that meet this binding criterion and qualify as HLA class II high affinity binders.
DR3 binding epitopes identified in this manner are included in vaccine compositions with DR supermotif-bearing peptide epitopes.
Similarly to the case of HLA class I motif-bearing peptides, the class II motif-bearing peptides are analoged to improve affinity or cross-reactivity. For example, aspartic acid at position 4 of the 9-mer core sequence is an optimal residue for DR3 binding, and substitution for that residue often improves DR 3 binding.
Example 17: Immunogenicity of 109P1D4-derived HTL epitopes
This example determines immunogenic DR supermotif- and DR3 motif-bearing epitopes among those identified using the methodology set forth herein.
Immunogenicity of HTL epitopes are confirmed in a manner analogous to the determination of immunogenicity of CTL epitopes, by assessing the ability to stimulate HTL responses and/or by using appropriate transgenic mouse models. Immunogenicity is determined by screening for: 1.) in vitro primary induction using normal PBMC or 2.) recall responses from patients who have 109P1D4-expressing tumors.
Example 18: Calculation of phenotypic frequencies of HLA-supertypes in various ethnic backgrounds to determine breadth of population coverage
This example illustrates the assessment of the breadth of population coverage of a vaccine composition comprised of multiple epitopes comprising multiple supermotifs and/or motifs.
In order to analyze population coverage, gene frequencies of HLA alleles are determined. Gene frequencies for each HLA allele are calculated from antigen or allele frequencies utilizing the binomial distribution formulae gf=1-(SQRT(1- af)) (see, e.g., Sidney ef al, Human Immunol. 45:79-93, 1996). To obtain overall phenotypic frequencies, cumulative gene frequencies are calculated, and the cumulative antigen frequencies derived by the use of the inverse formula [af=1-(1-Cgf)2j.
Where frequency data is not available at the level of DNA typing, correspondence to the serologically defined antigen frequencies is assumed. To obtain total potential supertype population coverage no linkage disequilibrium is assumed, and only alleles confirmed to belong to each of the supertypes are included (minimal estimates). Estimates of total potential coverage achieved by inter-loci combinations are made by adding to the A coverage the proportion of the non-A covered population that could be expected to be covered by the B alleles considered (e.g., total=A+B*(1-A)). Confirmed members of the A3-like supertype are A3, A11 , A31 , A*3301 , and A*6801. Although the A3-like supertype may also include A34, A66, and A*7401, these alleles were not included in overall frequency calculations. Likewise, confirmed members of the A2-like supertype family are A*0201, A*0202, A*0203, A*0204, A*0205, A*0206, A*0207, A*6802, and A*6901. Finally, the B7-like supertype-confirmed alleles are: B7, B*3501-03, B51, B*5301, B*5401, B*5501-2, B*5601, B*6701, and B*7801 (potentially also B*1401, B*3504-06, B*4201, and B*δ602).
Population coverage achieved by combining the A2-, A3- and B7-supertypes is approximately 86% in five major ethnic groups. Coverage may be extended by including peptides bearing the A1 and A24 motifs. On average, A1 is present in 12% and A24 in 29% of the population across five different major ethnic groups (Caucasian, North American Black, Chinese, Japanese, and Hispanic). Together, these alleles are represented with an average frequency of 39% in these same ethnic populations. The total coverage across the major ethnicities when A1 and A24 are combined with the coverage of the A2-, A3- and B7-supertype alleles is >95%, see, e.g., Table IV (G). An analogous approach can be used to estimate population coverage achieved with combinations of class II motif-bearing epitopes.
Immunogenicity studies in humans (e.g., Bertoni ef al, J. Clin. Invest. 100:503, 1997; Doolan ef al, Immunity 7:97, 1997; and Threlkeld ef al, J. Immunol. 169:1648, 1997) have shown that highly cross-reactive binding peptides are almost always recognized as epitopes. The use of highly cross-reactive binding peptides is an important selection criterion in identifying candidate epitopes for inclusion in a vaccine that is immunogenic in a diverse population.
With a sufficient number of epitopes (as disclosed herein and from the art), an average population coverage is predicted to be greater than 9δ% in each of five major ethnic populations. The game theory Monte Carlo simulation analysis, which is known in the art (see e.g., Osborne, M.J. and Rubinstein, A. "A course in game theory" MIT Press, 1994), can be used to estimate what percentage of the individuals in a population comprised of the Caucasian, North American Black, Japanese, Chinese, and Hispanic ethnic groups would recognize the vaccine epitopes described herein. A preferred percentage is 90%. A more preferred percentage is 96%.
Example 19: CTL Recognition Of Endogenously Processed Antigens After Priming
This example confirms that CTL induced by native or analoged peptide epitopes identified and selected as described herein recognize endogenously synthesized, i.e., native antigens.
Effector cells isolated from transgenic mice that are immunized with peptide epitopes, for example HLA-A2 supermotif-bearing epitopes, are re-stimulated in vitro using peptide-coated stimulator cells. Six days later, effector cells are assayed for cytotoxicity and the cell lines that contain peptide-specific cytotoxic activity are further re-stimulated. An additional six days later, these cell lines are tested for cytotoxic activity on 51Cr labeled Jurkat-A2.1/Kb target cells in the absence or presence of peptide, and also tested on 6 Cr labeled target cells bearing the endogenously synthesized antigen, i.e. cells that are stably transfected with 109P1D4 expression vectors.
The results demonstrate that CTL lines obtained from animals primed with peptide epitope recognize endogenously synthesized 109P1 D4 antigen. The choice of transgenic mouse model to be used for such an analysis depends upon the epitope(s) that are being evaluated. In addition to HLA-A*0201/Kb transgenic mice, several other transgenic mouse models including mice with human A11 , which may also be used to evaluate A3 epitopes, and B7 alleles have been characterized and others (e.g., transgenic mice for HLA-A1 and A24) are being developed. HLA-DR1 and HLA- DR3 mouse models have also been developed, which may be used to evaluate HTL epitopes.
Example 20: Activity Of CTL-HTL Conjugated Epitopes In Transgenic Mice
This example illustrates the induction of CTLs and HTLs in transgenic mice, by use of a 109P1D4-derived CTL and HTL peptide vaccine compositions. The vaccine composition used herein comprise peptides to be administered to a patient with a 109P1D4-expressing tumor. The peptide composition can comprise multiple CTL and/or HTL epitopes. The epitopes are identified using methodology as described herein. This example also illustrates that enhanced immunogenicity can be achieved by inclusion of one or more HTL epitopes in a CTL vaccine composition; such a peptide composition can comprise an HTL epitope conjugated to a CTL epitope. The CTL epitope can be one that binds to multiple HLA family members at an affinity of 600 nM or less, or analogs of that epitope. The peptides may be lipidated, if desired.
Immunization procedures: Immunization of transgenic mice is performed as described (Alexander ef a/., J. Immunol. 169:4753-4761, 1997). For example, A2/Kb mice, which are transgenic for the human HLA A2.1 allele and are used to confirm the immunogenicity of HLA-A*0201 motif- or HLA-A2 supermotif-bearing epitopes, and are primed subcutaneously (base of the tail) with a 0.1 ml of peptide in Incomplete Freund's Adjuvant, or if the peptide composition is a lipidated CTL/HTL conjugate, in DMSO/saline, or if the peptide composition is a polypeptide, in PBS or Incomplete Freund's Adjuvant. Seven days after priming, splenocytes obtained from these animals are restimulated with syngenic irradiated LPS- activated lymphoblasts coated with peptide.
Cell lines: Target cells for peptide-specific cytotoxicity assays are Jurkat cells transfected with the HLA-A2.1/Kb chimeric gene (e.g., Vitiello etal, J. Exp. Med. 173:1007, 1991)
In vitro CTL activation: One week after priming, spleen cells (30x10δ cells/flask) are co-cultured at 37°C with syngeneic, irradiated (3000 rads), peptide coated lymphoblasts (10x106 cells/flask) in 10 ml of culture medium/T25 flask. After six days, effector cells are harvested and assayed for cytotoxic activity.
Assay for cytotoxic activity: Target cells (1.0 to 1.6x106) are incubated at 37°C in the presence of 200 μl of 51Cr. After 60 minutes, cells are washed three times and resuspended in R10 medium. Peptide is added where required at a concentration of 1 μg/ml. For the assay, 104 51Cr-labeled target cells are added to different concentrations of effector cells (final volume of 200 μl) in U-bottom 96-well plates. After a six hour incubation period at 37°C, a 0.1 ml aliquot of supernatant is removed from each well and radioactivity is determined in a Micromedic automatic gamma counter. The percent specific lysis is determined by the formula: percent specific release = 100 x (experimental release - spontaneous rβ!ease)/(maximum release - spontaneous release). To facilitate comparison between separate CTL assays run under the same conditions, % 51Cr release data is expressed as lytic units/106 cells. One lytic unit is arbitrarily defined as the number of effector cells required to achieve 30% lysis of 10,000 target cells in a six hour 51Cr release assay. To obtain specific lytic units/106, the lytic units/106 obtained in the absence of peptide is subtracted from the lytic units/106 obtained in the presence of peptide. For example, if 30% 51Cr release is obtained at the effector (E): target (T) ratio of 60:1 (i.e., 6x105 effector cells for 10,000 targets) in the absence of peptide and 6:1 (i.e., δx104 effector cells for 10,000 targets) in the presence of peptide, the specific lytic units would be: [(1/50,000)-(1/500,000)j x 106 = 18 LU.
The results are analyzed to assess the magnitude of the CTL responses of animals injected with the immunogenic CTL/HTL conjugate vaccine preparation and are compared to the magnitude of the CTL response achieved using, for example, CTL epitopes as outlined above in the Example entitled "Confirmation of Immunogenicity." Analyses similar to this may be performed to confirm the immunogenicity of peptide conjugates containing multiple CTL epitopes and/or multiple HTL epitopes. In accordance with these procedures, it is found that a CTL response is induced, and concomitantly that an HTL response is induced upon administration of such compositions.
Example 21: Selection of CTL and HTL epitopes for inclusion in a 109P1D4-specific vaccine.
This example illustrates a procedure for selecting peptide epitopes for vaccine compositions of the invention. The peptides in the composition can be in the form of a nucleic acid sequence, either single or one or more sequences (i.e., minigene) that encodes peptide(s), or can be single and/or polyepitopic peptides.
The following principles are utilized when selecting a plurality of epitopes for inclusion in a vaccine composition. Each of the following principles is balanced in order to make the selection.
Epitopes are selected which, upon administration, mimic immune responses that are correlated with 109P1D4 clearance. The number of epitopes used depends on observations of patients who spontaneously clear 109P1 D4. For example, if it has been observed that patients who spontaneously clear 109P1D4-expressing cells generate an immune response to at least three (3) epitopes from 109P1 D4 antigen, then at least three epitopes should be included for HLA class I. A similar rationale is used to determine HLA class II epitopes.
Epitopes are often selected that have a binding affinity of an ICso of 500 nM or less for an HLA class I molecule, or for class II, an ICso of 1000 nM or less; or HLA Class I peptides with high binding scores from the BIMAS web site, at URL bimas.dcrt.nih.gov/.
In order to achieve broad coverage of the vaccine through out a diverse population, sufficient supermotif bearing peptides, or a sufficient array of allele-specific motif bearing peptides, are selected to give broad population coverage. In one embodiment, epitopes are selected to provide at least 80% population coverage. A Monte Carlo analysis, a statistical evaluation known in the art, can be employed to assess breadth, or redundancy, of population coverage.
When creating polyepitopic compositions, or a minigene that encodes same, it is typically desirable to generate the smallest peptide possible that encompasses the epitopes of interest. The principles employed are similar, if not the same, as those employed when selecting a peptide comprising nested epitopes. For example, a protein sequence for the vaccine composition is selected because it has maximal number of epitopes contained within the sequence, i.e., it has a high concentration of epitopes. Epitopes may be nested or overlapping (i.e., frame shifted relative to one another). For example, with overlapping epitopes, two 9-mer epitopes and one 10-mer epitope can be present in a 10 amino acid peptide. Each epitope can be exposed and bound by an HLA molecule upon administration of such a peptide. A multi-epitopic, peptide can be generated synthetically, recombinantly, or via cleavage from the native source. Alternatively, an analog can be made of this native sequence, whereby one or more of the epitopes comprise substitutions that alter the cross-reactivity and/or binding affinity properties of the polyepitopic peptide. Such a vaccine composition is administered for therapeutic or prophylactic purposes. This embodiment provides for the possibility that an as yet undiscovered aspect of immune system processing will apply to the native nested sequence and thereby facilitate the production of therapeutic or prophylactic immune response-inducing vaccine compositions. Additionally such an embodiment provides for the possibility of motif- bearing epitopes for an HLA makeup that is presently unknown. Furthermore, this embodiment (absent the creating of any analogs) directs the immune response to multiple peptide sequences that are actually present in 109P1D4, thus avoiding the need to evaluate any junctional epitopes. Lastly, the embodiment provides an economy of scale when producing nucleic acid vaccine compositions. Related to this embodiment, computer programs can be derived in accordance with principles in the art, which identify in a target sequence, the greatest number of epitopes per sequence length.
A vaccine composition comprised of selected peptides, when administered, is safe, efficacious, and elicits an immune response similar in magnitude to an immune response that controls or clears cells that bear or overexpress 109P1D4.
Example 22: Construction of "Minigene" Multi-Epitope DNA Plasmids
This example discusses the construction of a minigene expression plasmid. Minigene plasmids may, of course, contain various configurations of B cell, CTL and/or HTL epitopes or epitope analogs as described herein.
A minigene expression plasmid typically includes multiple CTL and HTL peptide epitopes. In the present example, HLA-A2, -A3, -B7 supermotif-bearing peptide epitopes and HLA-A1 and -A24 motif-bearing peptide epitopes are used in conjunction with DR supermotif-bearing epitopes and/or DR3 epitopes. HLA class I supermotif or motif-bearing peptide epitopes derived 109P1D4, are selected such that multiple supermotifs/motifs are represented to ensure broad population coverage. Similarly, HLA class II epitopes are selected from 109P1D4 to provide broad population coverage, i.e. both HLA DR-1-4-7 supermotif-bearing epitopes and HLA DR-3 motif-bearing epitopes are selected for inclusion in the minigene construct. The selected CTL and HTL epitopes are then incorporated into a minigene for expression in an expression vector.
Such a construct may additionally include sequences that direct the HTL epitopes to the endoplasmic reticulum. For example, the Ii protein may be fused to one or more HTL epitopes as described in the art, wherein the CLIP sequence of the Ii protein is removed and replaced with an HLA class II epitope sequence so that HLA class II epitope is directed to the endoplasmic reticulum, where the epitope binds to an HLA class II molecules.
This example illustrates the methods to be used for construction of a minigene-bearing expression plasmid. Other expression vectors that may be used for minigene compositions are available and known to those of skill in the art.
The minigene DNA plasmid of this example contains a consensus Kozak sequence and a consensus murine kappa Ig-light chain signal sequence followed by CTL and/or HTL epitopes selected in accordance with principles disclosed herein. The sequence encodes an open reading frame fused to the Myc and His antibody epitope tag coded for by the pcDNA 3.1 Myc-His vector.
Overlapping oligonucleotides that can, for example, average about 70 nucleotides in length with 15 nucleotide overlaps, are synthesized and HPLC-purified. The oligonucleotides encode the selected peptide epitopes as well as appropriate linker nucleotides, Kozak sequence, and signal sequence. The final multiepitope minigene is assembled by extending the overlapping oligonucleotides in three sets of reactions using PCR. A Perkin/Elmer 9600 PCR machine is used and a total of 30 cycles are performed using the following conditions: 95°C for 15 sec, annealing temperature (5° below the lowest calculated Tm of each primer pair) for 30 sec, and 72°C for 1 min.
For example, a minigene is prepared as follows. For a first PCR reaction, δ μg of each of two oligonucleotides are annealed and extended: In an example using eight oligonucleotides, i.e., four pairs of primers, oligonucleotides 1+2, 3+4, δ+6, and 7+8 are combined in 100 μl reactions containing Pfu polymerase buffer (1x= 10 mM KCL, 10 mM (NH4)2S04, 20 mM Tris-chloride, pH 8.76, 2 mM MgS04, 0.1% Triton X-100, 100 μg/ml BSA), 0.26 mM each dNTP, and 2.6 U of Pfu polymerase. The full-length dimer products are gel-purified, and two reactions containing the product of 1 +2 and 3+4, and the product of δ+6 and 7+8 are mixed, annealed, and extended for 10 cycles. Half of the two reactions are then mixed, and δ cycles of annealing and extension carried out before flanking primers are added to amplify the full length product. The full- length product is gel-purified and cloned into pCR-blunt (Invitrogen) and individual clones are screened by sequencing.
Example 23: The Plasmid Construct and the Degree to Which It Induces Immunogenicity.
The degree to which a plasmid construct, for example a plasmid constructed in accordance with the previous Example, is able to induce immunogenicity is confirmed in vitro by determining epitope presentation by APC following transduction or transfection of the APC with an epitope-expressing nucleic acid construct. Such a study determines "antigenicity" and allows the use of human APC. The assay determines the ability of the epitope to be presented by the APC in a context that is recognized by a T cell by quantifying the density of epitope-HLA class I complexes on the cell surface. Quantitation can be performed by directly measuring the amount of peptide eluted from the APC (see, e.g., Sijts ef al, J. Immunol. 166:683-692, 1996; Demotz ef al, Nature 342:682-684, 1989); or the number of peptide-HLA class I complexes can be estimated by measuring the amount of lysis or lymphokine release induced by diseased or transfected target cells, and then determining the concentration of peptide necessary to obtain equivalent levels of lysis or lymphokine release (see, e.g., Kageyama et al, J. Immunol. 154:667-676, 1995).
Alternatively, immunogenicity is confirmed through in vivo injections into mice and subsequent in vitro assessment of CTL and HTL activity, which are analyzed using cytotoxicity and proliferation assays, respectively, as detailed e.g., in Alexander ef al, Immunity 1 :751-761, 1994.
For example, to confirm the capacity of a DNA minigene construct containing at least one HLA-A2 supermotif peptide to induce CTLs in vivo, HLA-A2.1/Kb transgenic mice, for example, are immunized intramuscularly with 100 μg of naked cDNA. As a means of comparing the level of CTLs induced by cDNA immunization, a control group of animals is also immunized with an actual peptide composition that comprises multiple epitopes synthesized as a single polypeptide as they would be encoded by the minigene.
Splenocytes from immunized animals are stimulated twice with each of the respective compositions (peptide epitopes encoded in the minigene or the polyepitopic peptide), then assayed for peptide-specific cytotoxic activity in a 51Cr release assay. The results indicate the magnitude of the CTL response directed against the A2-restricted epitope, thus indicating the in vivo immunogenicity of the minigene vaccine and polyepitopic vaccine.
It is, therefore, found that the minigene elicits immune responses directed toward the HLA-A2 supermotif peptide epitopes as does the polyepitopic peptide vaccine. A similar analysis is also performed using other HLA-A3 and HLA-B7 transgenic mouse models to assess CTL induction by HLA-A3 and HLA-B7 motif or supermotif epitopes, whereby it is also found that the minigene elicits appropriate immune responses directed toward the provided epitopes. To confirm the capacity of a class II epitope-encoding minigene to induce HTLs in vivo, DR transgenic mice, or for those epitopes that cross react with the appropriate mouse MHC molecule, l-Ab-restricted mice, for example, are immunized intramuscularly with 100 μg of plasmid DNA. As a means of comparing the level of HTLs induced by DNA immunization, a group of control animals is also immunized with an actual peptide composition emulsified in complete Freund's adjuvant. CD4+ T cells, i.e. HTLs, are purified from splenocytes of immunized animals and stimulated with each of the respective compositions (peptides encoded in the minigene). The HTL response is measured using a 3H-thymidine incorporation proliferation assay, (see, e.g., Alexander ef al. Immunity 1:751-761, 1994). The results indicate the magnitude of the HTL response, thus demonstrating the in vivo immunogenicity of the minigene.
DNA minigenes, constructed as described in the previous Example, can also be confirmed as a vaccine in combination with a boosting agent using a prime boost protocol. The boosting agent can consist of recombinant protein (e.g., Barnett ef al., Aids Res. and Human Retroviruses 14, Supplement 3:S299-S309, 1998) or recombinant vaccinia, for example, expressing a minigene or DNA encoding the complete protein of interest (see, e.g., Hanke ef al, Vaccine 16:439- 446, 1998; Sedegah ef al, Proc. Nail. Acad. Sci USA 96:7648-53, 1998; Hanke and McMichael, Immunol Letters 66:177- 181, 1999; and Robinson ef a/., Nature Med. 6:526-34, 1999).
For example, the efficacy of the DNA minigene used in a prime boost protocol is initially evaluated in transgenic mice. In this example, A2.1/Kb transgenic mice are immunized IM with 100 μg of a DNA minigene encoding the immunogenic peptides including at least one HLA-A2 supermotif-bearing peptide. After an incubation period (ranging from 3- 9 weeks), the mice are boosted IP with 107 pfu/mouse of a recombinant vaccinia virus expressing the same sequence encoded by the DNA minigene. Control mice are immunized with 100 μg of DNA or recombinant vaccinia without the minigene sequence, or with DNA encoding the minigene, but without the vaccinia boost. After an additional incubation period of two weeks, splenocytes from the mice are immediately assayed for peptide-specific activity in an ELISPOT assay. Additionally, splenocytes are stimulated in vitro with the A2-restricted peptide epitopes encoded in the minigene and recombinant vaccinia, then assayed for peptide-specific activity in an alpha, beta and/or gamma IFN ELISA.
It is found that the minigene utilized in a prime-boost protocol elicits greater immune responses toward the HLA-A2 supermotif peptides than with DNA alone. Such an analysis can also be performed using HLA-A11 or HLA-B7 transgenic mouse models to assess CTL induction by HLA-A3 or HLA-B7 motif or supermotif epitopes. The use of prime boost protocols in humans is described below in the Example entitled "Induction of CTL Responses Using a Prime Boost Protocol."
Example 24: Peptide Compositions for Prophylactic Uses
Vaccine compositions of the present invention can be used to prevent 109P1D4 expression in persons who are at risk for tumors that bear this antigen. For example, a polyepitopic peptide epitope composition (or a nucleic acid comprising the same) containing multiple CTL and HTL epitopes such as those selected in the above Examples, which are also selected to target greater than 80% of the population, is administered to individuals at risk for a 109P1 D4-associated tumor.
For example, a peptide-based composition is provided as a single polypeptide that encompasses multiple epitopes. The vaccine is typically administered in a physiological solution that comprises an adjuvant, such as Incomplete Freunds Adjuvant. The dose of peptide for the initial immunization is from about 1 to about 50,000 μg, generally 100-6,000 μg, for a 70 kg patient. The initial administration of vaccine is followed by booster dosages at 4 weeks followed by evaluation of the magnitude of the immune response in the patient, by techniques that determine the presence of epitope- specific CTL populations in a PBMC sample. Additional booster doses are administered as required. The composition is found to be both safe and efficacious as a prophylaxis against 109P1D4-associated disease.
Alternatively, a composition typically comprising transfecting agents is used for the administration of a nucleic acid- based vaccine in accordance with methodologies known in the art and disclosed herein.
Example 25: Polyepitopic Vaccine Compositions Derived from Native 109P1D4 Sequences
A native 109P1 D4 polyprotein sequence is analyzed, preferably using computer algorithms defined for each class I and/or class II supermotif or motif, to identify "relatively short" regions of the polyprotein that comprise multiple epitopes. The "relatively short" regions are preferably less in length than an entire native antigen. This relatively short sequence that contains multiple distinct or overlapping, "nested" epitopes can be used to generate a minigene construct. The construct is engineered to express the peptide, which corresponds to the native protein sequence. The "relatively short" peptide is generally less than 250 amino acids in length, often less than 100 amino acids in length, preferably less than 75 amino acids in length, and more preferably less than 50 amino acids in length. The protein sequence of the vaccine composition is selected because it has maximal number of epitopes contained within the sequence, i.e., it has a high concentration of epitopes. As noted herein, epitope motifs may be nested or overlapping (i.e., frame shifted relative to one another). For example, with overlapping epitopes, two 9-mer epitopes and one 10-mer epitope can be present in a 10 amino acid peptide. Such a vaccine composition is administered for therapeutic or prophylactic purposes.
The vaccine composition will include, for example, multiple CTL epitopes from 109P1 D4 antigen and at least one HTL epitope. This polyepitopic native sequence is administered either as a peptide or as a nucleic acid sequence which encodes the peptide. Alternatively, an analog can be made of this native sequence, whereby one or more of the epitopes comprise substitutions that alter the cross-reactivity and/or binding affinity properties of the polyepitopic peptide.
The embodiment of this example provides for the possibility that an as yet undiscovered aspect of immune system processing will apply to the native nested sequence and thereby facilitate the production of therapeutic or prophylactic immune response-inducing vaccine compositions. Additionally, such an embodiment provides for the possibility of motif- bearing epitopes for an HLA makeup(s) that is presently unknown. Furthermore, this embodiment (excluding an analoged embodiment) directs the immune response to multiple peptide sequences that are actually present in native 109P1D4, thus avoiding the need to evaluate any junctional epitopes. Lastly, the embodiment provides an economy of scale when producing peptide or nucleic acid vaccine compositions.
Related to this embodiment, computer programs are available in the art which can be used to identify in a target sequence, the greatest number of epitopes per sequence length.
Example 26: Polyepitopic Vaccine Compositions from Multiple Antigens
The 109P1 D4 peptide epitopes of the present invention are used in conjunction with epitopes from other target tumor-associated antigens, to create a vaccine composition that is useful for the prevention or treatment of cancer that expresses 109P1D4 and such other antigens. For example, a vaccine composition can be provided as a single polypeptide that incorporates multiple epitopes from 109P1D4 as well as tumor-associated antigens that are often expressed with a target cancer associated with 109P1 D4 expression, or can be administered as a composition comprising a cocktail of one or more discrete epitopes. Alternatively, the vaccine can be administered as a minigene construct or as dendritic cells which have been loaded with the peptide epitopes in vitro.
Example 27: Use of peptides to evaluate an immune response
Peptides of the invention may be used to analyze an immune response for the presence of specific antibodies, CTL or HTL directed to 109P1 D4. Such an analysis can be performed in a manner described by Ogg ef al, Science 279:2103-2106, 1998. In this Example, peptides in accordance with the invention are used as a reagent for diagnostic or prognostic purposes, not as an immunogen.
In this example highly sensitive human leukocyte antigen tetrameric complexes ("tetramers") are used for a cross- sectional analysis of, for example, 109P1D4 HLA-A*0201 -specific CTL frequencies from HLA A*0201 -positive individuals at different stages of disease or following immunization comprising a 109P1D4 peptide containing an A*0201 motif. Tetrameric complexes are synthesized as described (Musey ef al, N. Engl. J. Med. 337:1267, 1997). Briefly, purified HLA heavy chain (A*0201 in this example) and β2-microglobulin are synthesized by means of a prokaryotic expression system. The heavy chain is modified by deletion of the transme brane-cytosolic tail and COOH-terminal addition of a sequence containing a BirA enzymatic biotinylation site. The heavy chain, β2-microglobulin, and peptide are refolded by dilution. The 45-kD refolded product is isolated by fast protein liquid chromatography and then biotinylated by BirA in the presence of biotin (Sigma, St. Louis, Missouri), adenosine 5' triphosphate and magnesium. Slreptavidin-phycoerythrin conjugate is added in a 1:4 molar ratio, and the tetrameric product is concentrated to 1 mg/ml. The resulting product is referred to as tetramer- phycoerythrin.
For the analysis of patient blood samples, approximately one million PBMCs are centrifuged at 300g for 5 minutes and resuspended in 50 μl of cold phosphate-buffered saline. Tri-color analysis is performed with the tetramer-phycoerythrin, along with anti-CD8-Tricolor, and anti-CD38. The PBMCs are incubated with tetramer and antibodies on ice for 30 to 60 min and then washed twice before formaldehyde fixation. Gates are applied to contain >99.98% of control samples. Controls for the tetramers include both A*0201 -negative individuals and A*0201 -positive non-diseased donors. The percentage of cells stained with the tetramer is then determined by flow cytometry. The results indicate the number of cells in the PBMC sample that contain epitope-restricted CTLs, thereby readily indicating the extent of immune response to the 109P1 D4 epitope, and thus the status of exposure to 109P1 D4, or exposure to a vaccine that elicits a protective or therapeutic response.
Example 28: Use of Peptide Epitopes to Evaluate Recall Responses
The peptide epitopes of the invention are used as reagents to evaluate T cell responses, such as acute or recall responses, in patients. Such an analysis may be performed on patients who have recovered from 109P1D4-associated disease or who have been vaccinated with a 109P1D4 vaccine.
For example, the class I restricted CTL response of persons who have been vaccinated may be analyzed. The vaccine may be any 109P1D4 vaccine. PBMC are collected from vaccinated individuals and HLA typed. Appropriate peptide epitopes of the invention that, optimally, bear supermotifs to provide cross-reactivity with multiple HLA supertype family members, are then used for analysis of samples derived from individuals who bear that HLA type.
PBMC from vaccinated individuals are separated on Ficoll-Histopaque density gradients (Sigma Chemical Co., St. Louis, MO), washed three times in HBSS (GIBCO Laboratories), resuspended in RPMI-1640 (GIBCO Laboratories) supplemented with L-glutamine (2mM), penicillin (50U/ml), streptomycin (50 μg/ml), and Hepes (10mM) containing 10% heat-inactivated human AB serum (complete RPMI) and plated using microculture formats. A synthetic peptide comprising an epitope of the invention is added at 10 μg/ml to each well and HBV core 128-140 epitope is added at 1 μg/ml to each well as a source of T cell help during the first week of stimulation.
In the microculture format, 4 x 105 PBMC are stimulated with peptide in 8 replicate cultures in 96-well round bottom plate in 100 μl/well of complete RPMI. On days 3 and 10, 100 μi of complete RPMI and 20 U/ml final concentration of rlL-2 are added to each well. On day 7 the cultures are transferred into a 96-well flat-bottom plate and restimulated with peptide, rlL-2 and 105 irradiated (3,000 rad) autologous feeder cells. The cultures are tested for cytotoxic activity on day 14. A positive CTL response requires two or more of the eight replicate cultures to display greater than 10% specific 51Cr release, based on comparison with non-diseased control subjects as previously described (Rehermann, ef al, Nature Med. 2:1104,1108, 1996; Rehermann ef al., J. Clin. invest. 97:1655-1665, 1996; and Rehermann et al J. Clin. Invest. 98:1432- 1440, 1996).
Target cell lines are autologous and allogeneic EBV-transformed B-LCL that are either purchased from the American Society for Histocompatibility and Immunogenetics (ASHI, Boston, MA) or established from the pool of patients as described (Guilhot, et al. J. Virol. 66:2670-2678, 1992).
Cytotoxicity assays are performed in the following manner. Target cells consist of either allogeneic HLA-matched or autologous EBV-transformed B lymphoblastoid cell line that are incubated overnight with the synthetic peptide epitope of the invention at 10 μM, and labeled with 100 μCi of 51Cr (Amersham Corp., Arlington Heights, IL) for 1 hour after which they are washed four times with HBSS.
Cytolytic activity is determined in a standard 4-h, split well 51Cr release assay using U-bottomed 96 well plates containing 3,000 targets/well. Stimulated PBMC are tested at effector/target (E/T) ratios of 20-50:1 on day 14. Percent cytotoxicity is determined from the formula: 100 x [(experimental release-spontaneous releaseVmaximum release- spontaneous release)]. Maximum release is determined by lysis of targets by detergent (2% Triton X-100; Sigma Chemical Co., St. Louis, MO). Spontaneous release is <2δ% of maximum release for all experiments.
The results of such an analysis indicate the extent to which HLA-restricted CTL populations have been stimulated by previous exposure to 109P1D4 or a 109P1D4 vaccine.
Similarly, Class II restricted HTL responses may also be analyzed. Purified PBMC are cultured in a 96-well flat bottom plate at a density of 1.5x105 cells/well and are stimulated with 10 μg/ml synthetic peptide of the invention, whole 109P1D4 antigen, or PHA. Cells are routinely plated in replicates of 4-6 wells for each condition. After seven days of culture, the medium is removed and replaced with fresh medium containing 10U/ml lL-2. Two days later, 1 μCi 3H-thymidine is added to each well and incubation is continued for an additional 18 hours. Cellular DNA is then harvested on glass fiber mats and analyzed for 3H-thymidine incorporation. Antigen-specific T cell proliferation is calculated as the ratio of 3H- thy idine incorporation in the presence of antigen divided by the 3H-thymidine incorporation in the absence of antigen.
Example 29: Induction Of Specific CTL Response In Humans
A human clinical trial for an immunogenic composition comprising CTL and HTL epitopes of the invention is set up as an IND Phase I, dose escalation study and carried out as a randomized, double-blind, placebo-controlled trial. Such a trial is designed, for example, as follows:
A total of about 27 individuals are enrolled and divided into 3 groups: . Group I: 3 subjects are injected with placebo and 6 subjects are injected with 5 μg of peptide composition;
Group II: 3 subjects are injected with placebo and 6 subjects are injected with 50 μg peptide composition;
Group III: 3 subjects are injected with placebo and 6 subjects are injected with 500 μg of peptide composition.
After 4 weeks following the first injection, all subjects receive a booster inoculation at the same dosage.
The endpoints measured in this study relate to the safety and tolerability of the peptide composition as well as its immunogenicity. Cellular immune responses to the peptide composition are an index of the intrinsic activity of this the peptide composition, and can therefore be viewed as a measure of biological efficacy. The following summarize the clinical and laboratory data that relate to safety and efficacy endpoints.
Safety: The incidence of adverse events is monitored in the placebo and drug treatment group and assessed in terms of degree and reversibility.
Evaluation of Vaccine Efficacy: For evaluation of vaccine efficacy, subjects are bled before and after injection. Peripheral blood mononuclear cells are isolated from fresh heparinized blood by Ficoll-Hypaque density gradient centrifugation, aliquoted in freezing media and stored frozen. Samples are assayed for CTL and HTL activity. The vaccine is found to be both safe and efficacious.
Example 30: Phase II Trials In Patients Expressing 109P1D4
Phase II trials are performed to study the effect of administering the CTL-HTL peptide compositions to patients having cancer that expresses 109P1 D4. The main objectives of the trial are to determine an effective dose and regimen for inducing CTLs in cancer patients that express 109P1 D4, to establish the safety of inducing a CTL and HTL response in these patients, and to see to what extent activation of CTLs improves the clinical picture of these patients, as manifested, e.g., by the reduction and/or shrinking of lesions. Such a study is designed, for example, as follows:
The studies are performed in multiple centers. The trial design is an open-label, uncontrolled, dose escalation protocol wherein the peptide composition is administered as a single dose followed six weeks later by a single booster shot of the same dose. The dosages are 50, 500 and 5,000 micrograms per injection. Drug-associated adverse effects (severity and reversibility) are recorded.
There are three patient groupings. The first group is injected with 50 micrograms of the peptide composition and the second and third groups with 500 and 5,000 micrograms of peptide composition, respectively. The patients within each group range in age from 21-65 and represent diverse ethnic backgrounds. All of them have a tumor that expresses 109P1D4.
Clinical manifestations or antigen-specific T-cell responses are monitored to assess the effects of administering the peptide compositions. The vaccine composition is found to be both safe and efficacious in the treatment of 109P1D4- associated disease.
Example 31 : Induction of CTL Responses Using a Prime Boost Protocol
A prime boost protocol similar in its underlying principle to that used to confirm the efficacy of a DNA vaccine in transgenic mice, such as described above in the Example entitled "The Plasmid Construct and the Degree to Which It Induces Immunogenicity," can also be used for the administration of the vaccine to humans. Such a vaccine regimen can include an initial administration of, for example, naked DNA followed by a boost using recombinant virus encoding the vaccine, or recombinant protein/polypeptide or a peptide mixture administered in an adjuvant.
For example, the initial immunization may be performed using an expression vector, such as that constructed in the Example entitled "Construction of "Minigene" Multi-Epitope DNA Plasmids" in the form of naked nucleic acid administered IM (or SC or ID) in the amounts of 0.5-5 mg at multiple sites. The nucleic acid (0.1 to 1000 μg) can also be administered using a gene gun. Following an incubation period of 3-4 weeks, a booster dose is then administered. The booster can be recombinant fowlpox virus administered at a dose of 5-107 to 5x109 pfu. An alternative recombinant virus, such as an MVA, canarypox, adenovirus, or adeno-associated virus, can also be used for the booster, or the polyepitopic protein or a mixture of the peptides can be administered. For evaluation of vaccine efficacy, patient blood samples are obtained before immunization as well as at intervals following administration of the initial vaccine and booster doses of the vaccine. Peripheral blood mononuclear cells are isolated from fresh heparinized blood by Ficoll-Hypaque density gradient centrifugation, aliquoted in freezing media and stored frozen. Samples are assayed for CTL and HTL activity.
Analysis of the results indicates that a magnitude of response sufficient to achieve a therapeutic or protective immunity against 109P1 D4 is generated.
Example 32: Administration of Vaccine Compositions Using Dendritic Cells (DC) Vaccines comprising peptide epitopes of the invention can be administered using APCs, or "professional" APCs such as DC. In this example, peptide-pulsed DC are administered to a patient to stimulate a CTL response in vivo. In this method, dendritic cells are isolated, expanded, and pulsed with a vaccine comprising peptide CTL and HTL epitopes of the invention. The dendritic cells are infused back into the patient to elicit CTL and HTL responses in vivo. The induced CTL and HTL then destroy or facilitate destruction, respectively, of the target cells that bear the 109P1 D4 protein from which the epitopes in the vaccine are derived.
For example, a cocktail of epitope-comprising peptides is administered ex vivo to PBMC, or isolated DC therefrom. A pharmaceutical to facilitate harvesting of DC can be used, such as Progenipoietin™ (Monsanto, St. Louis, MO) or GM- CSF/IL-4. After pulsing the DC with peptides, and prior to reinfusion into patients, the DC are washed to remove unbound peptides.
As appreciated clinically, and readily determined by one of skill based on clinical outcomes, the number of DC reinfused into the patient can vary (see, e.g., Nature Med. 4:328, 1998; Nature Med. 2:62, 1996 and Prosfafe 32:272, 1997). Although 2-50 x 106 DC per patient are typically administered, larger number of DC, such as 107 or 108 can also be provided. Such cell populations typically contain between 50-90% DC.
In some embodiments, peptide-loaded PBMC are injected into patients without purification of the DC. For example, PBMC generated after treatment with an agent such as Progenipoietin™ are injected into patients without purification of the DC. The total number of PBMC that are administered often ranges from 108 to 1010. Generally, the cell doses injected into patients is based on the percentage of DC in the blood of each patient, as determined, for example, by immunofluorescence analysis with specific anti-DC antibodies. Thus, for example, if Progenipoietin™ mobilizes 2% DC in the peripheral blood of a given patient, and that patient is to receive 5 x 106 DC, then the patient will be injected with a total of 2.6 x 108 peptide-loaded PBMC. The percent DC mobilized by an agent such as Progenipoietin™ is typically estimated to be between 2-10%, but can vary as appreciated by one of skill in the art.
Ex vivo activation of CTL/HTL responses
Alternatively, ex vivo CTL or HTL responses to 109P1D4 antigens can be induced by incubating, in tissue culture, the patient's, or genetically compatible, CTL or HTL precursor cells together with a source of APC, such as DC, and immunogenic peptides. After an appropriate incubation time (typically about 7-28 days), in which the precursor cells are activated and expanded into effector cells, the cells are infused into the patient, where they will destroy (CTL) or facilitate destruction (HTL) of their specific target cells, i.e., tumor cells.
Example 33: An Alternative Method of Identifying and Confirming Motif-Bearing Peptides
Another method of identifying and confirming motif-bearing peptides is to elute them from cells bearing defined MHC molecules. For example, EBV transformed B cell lines used for tissue typing have been extensively characterized to determine which HLA molecules they express. In certain cases these cells express only a single type of HLA molecule. These cells can be transfected with nucleic acids that express the antigen of interest, e.g. 109P1D4. Peptides produced by endogenous antigen processing of peptides produced as a result of transfection will then bind to HLA molecules within the cell and be transported and displayed on the cell's surface. Peptides are then eluted from the HLA molecules by exposure to mild acid conditions and their amino acid sequence determined, e.g., by mass spectral analysis (e.g., Kubo ef al, J. Immunol. 162:3913, 1994). Because the majority of peptides that bind a particular HLA molecule are motif-bearing, this is an alternative modality for obtaining the motif-bearing peptides correlated with the particular HLA molecule expressed on the cell.
Alternatively, cell lines that do not express endogenous HLA molecules can be transfected with an expression construct encoding a single HLA allele. These cells can then be used as described, i.e., they can then be transfected with nucleic acids that encode 109P1 D4 to isolate peptides corresponding to 109P1 D4 that have been presented on the cell surface. Peptides obtained from such an analysis will bear motif(s) that correspond to binding to the single HLA allele that is expressed in the cell.
As appreciated by one in the art, one can perform a similar analysis on a cell bearing more than one HLA allele and subsequently determine peptides specific for each HLA allele expressed. Moreover, one of skill would also recognize that means other than transfection, such as loading with a protein antigen, can be used to provide a source of antigen to the cell.
Example 34: Complementary Polynucleotides
Sequences complementary to the 109P1D4-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring 109P1 D4. Although use of oligonucleotides comprising from about 15 to 30 base pairs is described, essentially the same procedure is used with smaller or with larger sequence fragments. Appropriate oligonucleotides are designed using, e.g., OLIGO 4.06 software (National Biosciences) and the coding sequence of 109P1 D4. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence. To inhibit translation, a complementary oligonucleotide is designed to prevent ribosomal binding to a 109P1D4-encoding transcript.
Example 35: Purification of Naturally-occurring or Recombinant 109P1D4 Using 109P1D4-Specific Antibodies
Naturally occurring or recombinant 109P1D4 is substantially purified by immunoaffinity chromatography using antibodies specific for 109P1 D4. An immunoaffinity column is constructed by covalently coupling anti-109P1 D4 antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.
Media containing 109P1 D4 are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of 109P1 D4 (e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/109P1D4 binding (e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion), and GCR.P is collected.
Example 36: Identi ication of Molecules Which Interact with 109P1 D4
109P1D4, or biologically active fragments thereof, are labeled with 121 1 Bolton-Hunter reagent. (See, e.g., Bolton et al. (1973) Biochem. J. 133:529.) Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled 109P1 D4, washed, and any wells with labeled 109P1 D4 complex are assayed. Data obtained using different concentrations of 109P1 D4 are used to calculate values for the number, affinity, and association of 109P1 D4 with the candidate molecules.
Example 37: In Vivo Assay for 109P1D4 Tumor Growth Promotion
The effect of a 109P1 D4 protein on tumor cell growth is evaluated in vivo by gene overexpression in tumor-bearing mice. For example, SCID mice are injected subcutaneously on each flank with 1 x 106 of either PC3, DU146 or 3T3 cells containing tkNeo empty vector or a nucleic acid sequence of the invention. At least two strategies can be used: (1) Constitutive expression under regulation of a promoter such as a constitutive promoter obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), or from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, provided such promoters are compatible with the host cell systems, and (2) Regulated expression under control of an inducible vector system, such as ecdysone, tet, etc., provided such promoters are compatible with the host cell systems. Tumor volume is then monitored at the appearance of palpable tumors and followed over time to determine if the cells expressing a gene of the invention grow at a faster rate and whether tumors of a 109P1 D4 protein-expressing cells demonstrate characteristics of altered aggressiveness (e.g. enhanced metastasis, vascularization, reduced responsiveness to chemotherapeutic drugs).
Additionally, mice can be implanted with 1 x 105 of the same cells orthotopically to determine if a protein of the invention has an effect on local growth in the prostate or on the ability of the cells to metastasize, specifically to lungs, lymph nodes, and bone marrow.
The assay is also useful to determine the inhibitory effect of candidate therapeutic compositions, such as for example, 109P1D4 protein-related intrabodies, 109P1D4 gene-related antisense molecules and ribozymes.
Example 38: 109P1D4 Monoclonal Antibody-mediated Inhibition of Tumors In Vivo
The significant expression of 109P1 D4 proteins in the cancer tissues of Table I and its restrictive expression in normal tissues, together with its expected cell surface expression, makes 109P1 D4 proteins excellent targets for antibody therapy. Similarly, 109P1 D4 proteins are a target for T cell-based immunotherapy. Thus, for 109P1 D4 genes expressed, e.g., in prostate cancer, the therapeutic efficacy of anti-109P1D4 protein mAbs in human prostate cancer xenograft mouse models is evaluated by using androgen-independent LAPC-4 and LAPC-9 xenografts (Craft, N., ef al.,. Cancer Res, 1999. 59(19): p. 5030-6) and the androgen independent recombinant cell line PC3-of 109P1D4 (see, e.g., Kaighn, M.E., ef al, Invest Urol, 1979. 17(1): p. 16-23); analogous models are used for other cancers.
Antibody efficacy on tumor growth and metastasis formation is studied, e.g., in a mouse orthotopic prostate cancer xenograft models and mouse kidney xenograft models. The antibodies can be unconjugated, as discussed in this Example, or can be conjugated to a therapeutic modality, as appreciated in the art. Anti-109P1D4 protein mAbs inhibit formation of both the androgen-dependent LAPC-9 and androgen-independent PC3-109P1 D4 protein tumor xenografts. Anti-109P1 D4 protein mAbs also retard the growth of established orthotopic tumors and prolonged survival of tumor-bearing mice. These results indicate the utility of anti-109P1 D4 protein mAbs in the treatment of local and advanced stages of prostate cancer. (See, e.g., (Saffran, D., et al., PNAS 10:1073-1078 or World Wide Web URL ww.pnas.org/cgi/doi/10.1073/pnas.051624698).
Administration of the anti-109P1D4 protein mAbs lead to retardation of established orthotopic tumor growth and inhibition of metastasis to distant sites, resulting in a significant prolongation in the survival of tumor-bearing mice. These studies indicate that proteins of the invention are attractive targets for immunotherapy and demonstrate the therapeutic potential of anti-109P1 D4 protein mAbs for the treatment of local and metastatic cancer. This example demonstrates that unconjugated 109P1D4 protein-related monoclonal antibodies are effective to inhibit the growth of human prostate tumor xenografts and human kidney xenografts grown in SCID mice; accordingly a combination of such efficacious monoclonal antibodies is also effective.
Tumor inhibition using multiple unconjugated mAbs Materials and Methods
109P1D4 Protein-related Monoclonal Antibodies:
Monoclonal antibodies are raised against proteins of the invention as described in the Example entitled "Generation of 109P1D4 Monoclonal Antibodies". The antibodies are characterized by ELISA, Western blot, FACS, and immunoprecipitation for their capacity to bind to the respective protein of the invention. Epitope mapping data for, e.g., the anti-109P1D4 protein mAbs, as determined by ELISA and Western analysis, indicate that the antibodies recognize epitopes on the respective 109P1D4 protein. Immunohistochemical analysis of prostate cancer tissues and cells with these antibodies is performed.
The monoclonal antibodies are purified from ascites or hybridoma tissue culture supernatants by Protein-G Sepharose chromatography, dialyzed against PBS, filter sterilized, and stored at -20°C. Protein determinations are performed by a Bradford assay (Bio-Rad, Hercules, CA). A therapeutic monoclonal antibody or a cocktail comprising a mixture of individual monoclonal antibodies is prepared and used for the treatment of mice receiving subcutaneous or orthotopic injections of LAPC-9 prostate tumor xenografts.
Cancer Xenografts and Cell Lines
The LAPC-9 xenograft, which expresses a wild-type androgen receptor and produces prostate-specific antigen (PSA), is passaged in 6- to 8-week-old male ICR-severe combined immunodeficient (SCID) mice (Taconic Farms) by s.c. trocar implant (Craft, N., ef al., supra). The prostate carcinoma cell line PC3 (American Type Culture Collection) is maintained in RPMI supplemented with L-glutamine and 10% FBS.
Recombinant PC3 and 3T3- cell populations expressing a protein of the invention are generated by retroviral gene transfer as described in Hubert, R.S., et al., STEAP: a prostate-specific cell-surface antigen highly expressed in human prostate tumors. Proc Natl Acad Sci U S A, 1999. 96(26): p. 14623-8. Anti-protein of the invention staining is detected by using an FITC-conjugated goat anti-mouse antibody (Southern Biotechnology Associates) followed by analysis on a Coulter Epics-XL flow cytometer.
Xenograft Mouse Models.
Subcutaneous (s.c.) tumors are generated by injection of 1 x 106 LAPC-9, PC3, recombinant PC3-protein of the invention, 3T3 or recombinant 3T3-protein of the invention cells mixed at a 1:1 dilution with Matrigel (Collaborative Research) in the right flank of male SCID mice. To test antibody efficacy on tumor formation, i.p. antibody injections are started on the same day as tumor-cell injections. As a control, mice are injected with either purified mouse IgG (ICN) or PBS; or a purified monoclonal antibody that recognizes an irrelevant antigen not expressed in human cells. In preliminary studies, no difference is found between mouse IgG or PBS on tumor growth. Tumor sizes are determined by vernier caliper measurements, and the tumor volume is calculated as length x width x height. Mice with s.c. tumors greater than 1.5 cm in diameter are sacrificed. PSA levels are determined by using a PSA ELISA kit (Anogen, Mississauga, Ontario). Circulating levels of, e.g., anti-109P1D4 protein mAbs are determined by a capture ELISA kit (Bethyl Laboratories, Montgomery, TX). (See, e.g., Saffran, D., et al., PNAS 10:1073-1078 or www.pnas.org/cgi/ doi/10.1073/pnas.051624698)
Orthotopic injections are performed under anesthesia by using ketamine/xylazine. For prostate orthotopic studies, an incision is made through the abdominal muscles to expose the bladder and seminal vesicles, which then are delivered through the incision to expose the dorsal prostate. LAPC-9 or PC3 cells (5 x 105 ) mixed with Matrigel are injected into each dorsal lobe in a 10-μl volume. To monitor tumor growth, mice are bled on a weekly basis for determination of PSA levels. The mice are segregated into groups for the appropriate treatments, with anti-protein of the invention or control mAbs being injected i.p.
Anti-109P1 D4 Protein mAbs Inhibit Growth of Respective 109P1 D4 Protein-Expressing Xenograft-Cancer Tumors
The effect of anti-109P1 D4 protein mAbs on tumor formation is tested by using LAPC-9 and recombinant PC3- protein of the invention orthotopic models. As compared with the s.c. tumor model, the orthotopic model, which requires injection of tumor cells directly in the mouse prostate or kidney, respectively, results in a local tumor growth, development of metastasis in distal sites, deterioration of mouse health, and subsequent death (Saffran, D., et al., PNAS supra; Fu, X., et al., Int J Cancer, 1992. 52(6): p. 987-90; Kubota, T., J Cell Biochem, 1994. 56(1): p.4-8). The features make the orthotopic model more representative of human disease progression and allowed us to follow the therapeutic effect of mAbs on clinically relevant end points.
Accordingly, tumor cells are injected into the mouse prostate or kidney, and 2 days later, the mice are segregated into two groups and treated with either: a) 200-500μg, of anti-109P1 D4 protein Ab, or b) PBS three times per week for two to five weeks.
A major advantage of the orthotopic prostate-cancer model is the ability to study the development of metastases. Formation of metastasis in mice bearing established orthotopic tumors is studied by IHC analysis on lung sections using an antibody against a prostate-specific cell-surface protein STEAP expressed at high levels in LAPC-9 xenografts (Hubert, R.S., ef al, Proc Natl Acad Sci U S A, 1999. 96(25): p. 14523-8).
Mice bearing established orthotopic LAPC-9 or recombinant PC3-109P1D4 protein tumors are administered 1000μg injections of either anti-109P1D4 protein mAbs or PBS over a 4-week period. Mice in both groups are allowed to establish a high tumor burden (PSA levels greater than 300 ng/ml for IAPC-9), to ensure a high frequency of metastasis formation in mouse lungs. Mice then are killed and their prostate and lungs are analyzed for the presence of tumor cells by IHC analysis.
These studies demonstrate a broad anti-tumor efficacy of anti-109P1 D4 protein antibodies on initiation and progression of prostate cancer in xenograft mouse models. Anti-109P1D4 protein antibodies inhibit tumor formation of both androgen-dependent and androgen-independent tumors, retard the growth of already established tumors, and prolong the survival of treated mice. Moreover, anti-109P1D4 protein mAbs demonstrate a dramatic inhibitory effect on the spread of local prostate tumor to distal sites, even in the presence of a large tumor burden. Thus, anti-109P1 D4 protein mAbs are efficacious on major clinically relevant end points (tumor growth), prolongation of survival, and health.
Example 39: Therapeutic and Diagnostic use of Anti-109P1D4 Antibodies in Humans.
Anti-109P1D4 monoclonal antibodies are safely and effectively used for diagnostic, prophylactic, prognostic and/or therapeutic purposes in humans. Western blot and immunohistochemical analysis of cancer tissues and cancer xenografts with anti-109P1 D4 mAb show strong extensive staining in carcinoma but significantly lower or undetectable levels in normal tissues. Detection of 109P1D4 in carcinoma and in metastatic disease demonstrates the usefulness of the mAb as a diagnostic and/or prognostic indicator. Anti-109P1D4 antibodies are therefore used in diagnostic applications such as immunohistochemistry of kidney biopsy specimens to detect cancer from suspect patients.
As determined by flow cytometry, anti-109P1D4 mAb specifically binds to carcinoma cells. Thus, anti-109P1D4 antibodies are used in diagnostic whole body imaging applications, such as radioimmunoscintigraphy and radioimmunotherapy, (see, e.g., Potamianos S., et. al. Anticancer Res 20(2A):925-948 (2000)) for the detection of localized and metastatic cancers that exhibit expression of 109P1D4. Shedding or release of an extracellular domain of 109P1D4 into the extracellular milieu, such as that seen for alkaline phosphodiesterase B10 (Meerson, N. R., Hepatology 27:563-568 (1998)), allows diagnostic detection of 109P1D4 by anti-109P1D4 antibodies in serum and/or urine samples from suspect patients.
Anti-109P1 D4 antibodies that specifically bind 109P1 D4 are used in therapeutic applications for the treatment of cancers that express 109P1D4. Anti-109P1D4 antibodies are used as an unconjugated modality and as conjugated form in which the antibodies are attached to one of various therapeutic or imaging modalities well known in the art, such as a prodrugs, enzymes or radioisotopes. In preclinical studies, unconjugated and conjugated anti-109P1D4 antibodies are tested for efficacy of tumor prevention and growth inhibition in the SCID mouse cancer xenograft models, e.g., kidney cancer models AGS-K3 and AGS-K6, (see, e.g., the Example entitled "109P1D4 Monoclonal Antibody-mediated Inhibition of Bladder and Lung Tumors In Vivo'). Either conjugated and unconjugated anti-109P1D4 antibodies are used as a therapeutic modality in human clinical trials either alone or in combination with other treatments as described in following Examples.
Example 40: Human Clinical Trials for the Treatment and Diagnosis of Human Carcinomas through use of Human Anti-109P1D4 Antibodies In vivo
Antibodies are used in accordance with the present invention which recognize an epitope on 109P1D4, and are used in the treatment of certain tumors such as those listed in Table I. Based upon a number of factors, including 109P1D4 expression levels, tumors such as those listed in Table I are presently preferred indications. In connection with each of these indications, three clinical approaches are successfully pursued.
I.) Adjunclive therapy: In adjunctive therapy, patients are treated with anti-109P1 D4 antibodies in combination with a chemotherapeutic or antineoplastic agent and/or radiation therapy. Primary cancer targets, such as those listed in Table I, are treated under standard protocols by the addition anti-109P1D4 antibodies to standard first and second line therapy. Protocol designs address effectiveness as assessed by reduction in tumor mass as well as the ability to reduce usual doses of standard chemotherapy. These dosage reductions allow additional and/or prolonged therapy by reducing dose-related toxicity of the chemotherapeutic agent. Anti-109P1D4 antibodies are utilized in several adjunctive clinical trials in combination with the chemotherapeutic or antineoplastic agents adriamycin (advanced prostrate carcinoma), cisplatin (advanced head and neck and lung carcinomas), taxol (breast cancer), and doxorubicin (preclinical).
II.) Monotherapy: In connection with the use of the anti-109P1D4 antibodies in monotherapy of tumors, the antibodies are administered to patients without a chemotherapeutic or antineoplastic agent. In one embodiment, monotherapy is conducted clinically in end stage cancer patients with extensive metastatic disease. Patients show some disease stabilization. Trials demonstrate an effect in refractory patients with cancerous tumors.
111.) Imaging Agent: Through binding a radionuclide (e.g., iodine or yttrium (I131, Y90) to anti-109P1D4 antibodies, the radiolabeled antibodies are utilized as a diagnostic and/or imaging agent. In such a role, the labeled antibodies localize to both solid tumors, as well as, metastatic lesions of cells expressing 109P1D4. In connection with the use of the anti-109P1 D4 antibodies as imaging agents, the antibodies are used as an adjunct to surgical treatment of solid tumors, as both a pre-surgical screen as well as a post-operative follow-up to determine what tumor remains and/or returns. In one embodiment, a ( 11 ln)-109P1D4 antibody is used as an imaging agent in a Phase I human clinical trial in patients having a carcinoma that expresses 109P1D4 (by analogy see, e.g., Divgi ef al. J. Natl. Cancerlnst. 83:97-104 (1991)). Patients are followed with standard anterior and posterior gamma camera. The results indicate that primary lesions and metastatic lesions are identified.
Dose and Route of Administration
As appreciated by those of ordinary skill in the art, dosing considerations can be determined through comparison with the analogous products that are in the clinic. Thus, anti-109P1D4 antibodies can be administered with doses in the range of 5 to 400 mg/m 2 , with the lower doses used, e.g., in connection with safety studies. The affinity of anti-109P1 D4 antibodies relative to the affinity of a known antibody for its target is one parameter used by those of skill in the art for determining analogous dose regimens. Further, anti-109P1D4 antibodies that are fully human antibodies, as compared to the chimeric antibody, have slower clearance; accordingly, dosing in patients with such fully human anti-109P1D4 antibodies can be lower, perhaps in the range of 50 to 300 mg/m2, and still remain efficacious. Dosing in mg/m2, as opposed to the conventional measurement of dose in mg/kg, is a measurement based on surface area and is a convenient dosing measurement that is designed to include patients of all sizes from infants to adults. Three distinct delivery approaches are useful for delivery of anti-109P1 D4 antibodies. Conventional intravenous delivery is one standard delivery technique for many tumors. However, in connection with tumors in the peritoneal cavity, such as tumors of the ovaries, biliary duct, other ducts, and the like, intraperitoneal administration may prove favorable for obtaining high dose of antibody at the tumor and to also minimize antibody clearance. In a similar manner, certain solid tumors possess vasculature that is appropriate for regional perfusion. Regional perfusion allows for a high dose of antibody al the site of a tumor and minimizes short term clearance of the antibody.
Clinical Development Plan (CDP)
Overview: The CDP follows and develops treatments of anti-109P1D4 antibodies in connection with adjunctive therapy, monotherapy, and as an imaging agent. Trials initially demonstrate safety and thereafter confirm efficacy in repeat doses. Trails are open label comparing standard chemotherapy with standard therapy plus anti-109P1D4 antibodies. As will be appreciated, one criteria that can be utilized in connection with enrollment of patients is 109P1 D4 expression levels in their tumors as determined by biopsy.
As with any protein or antibody infusion-based therapeutic, safety concerns are related primarily to (i) cytokine release syndrome, i.e., hypotension, fever, shaking, chills; (ii) the development of an immunogenic response to the material (i.e., development of human antibodies by the patient to the antibody therapeutic, or HAHA response); and, (iii) toxicity to normal cells that express 109P1D4. Standard tests and follow-up are utilized to monitor each of these safety concerns. Anti-109P1D4 antibodies are found to be safe upon human administration.
Example 41 : Human Clinical Trial Adjunctive Therapy with Human Anti-109P1 D4 Antibody and Chemotherapeutic Agent
A phase I human clinical trial is initiated to assess the safety of six intravenous doses of a human anti-109P1 D4 antibody in connection with the treatment of a solid tumor, e.g., a cancer of a tissue listed in Table I. In the study, the safety of single doses of anti-109P1 D4 antibodies when utilized as an adjunctive therapy to an antineoplastic or chemotherapeutic agent as defined herein, such as, without limitation: cisplatin, topotecan, doxorubicin, adriamycin, taxol, or the like, is assessed. The trial design includes delivery of six single doses of an anti-109P1 D4 antibody with dosage of antibody escalating from approximately about 2δ mg/m 2to about 276 mg/m 2over the course of the treatment in accordance with the following schedule:
Day O Day 7 Day 14 Day 21 Day 28 Day 3δ
mAb Dose 26 75 125 175 225 275 mg/m 2 mg/m 2 mg/m 2 mg/m 2 mg/m 2 mg/m 2
Chemotherapy + + + + + + (standard dose)
Patients are closely followed for one-week following each administration of antibody and chemotherapy. In particular, patients are assessed for the safety concerns mentioned above: (i) cytokine release syndrome, i.e., hypotension, fever, shaking, chills; (ii) the development of an immunogenic response to the material (i.e., development of human antibodies by the patient to the human antibody therapeutic, or HAHA response); and, (iii) toxicity to normal cells that express 109P1D4. Standard tests and follow-up are utilized to monitor each of these safety concerns. Patients are also assessed for clinical outcome, and particularly reduction in tumor mass as evidenced by MRI or other imaging. The anti-109P1D4 antibodies are demonstrated to be safe and efficacious, Phase II trials confirm the efficacy and refine optimum dosing.
Example 42: Human Clinical Trial: Monotherapy with Human Anti-109P1D4 Antibody
Anti-109P1D4 antibodies are safe in connection with the above-discussed adjunctive trial, a Phase II human clinical trial confirms the efficacy and optimum dosing for monotherapy. Such trial is accomplished, and entails the same safety and outcome analyses, to the above-described adjunctive trial with the exception being that patients do not receive chemotherapy concurrently with the receipt of doses of anti-109P1 D4 antibodies.
Example 43: Human Clinical Trial: Diagnostic Imaging with Anti-109P1D4 Antibody
Once again, as the adjunctive therapy discussed above is safe within the safety criteria discussed above, a human clinical trial is conducted concerning the use of anti-109P1D4 antibodies as a diagnostic imaging agent. The protocol is designed in a substantially similar manner to those described in the art, such as in Divgi ef al. J. Natl. Cancer Inst. 83:97-104 (1991). The antibodies are found to be both safe and efficacious when used as a diagnostic modality.
Example 44: 109P1D4 Functional Assays
I. Phosphorylation of 109P1D4 on tyrosine residues
One hallmark of the cancer cell phenotype is the active signal transduction of surface bound receptor molecules, such as the EGF receptor, through tyrosine phosphorylation of their cytoplasmic domains and their subsequent interaction with cytosolic signaling molecules. To address the possibility that 109P1D4 is phosphorylated on its cytoplamsic tyrosine residues, 293T cells were transfected with the 109P1 D4 gene in an expression plasmid such that the 109P1 D4 gene was fused with a Myc/His tag, and were then stimulated with pervanadate (a 1:1 mixture of a3V04 and H2O2). After solubilization of the cells in Triton X-100, the 109P1D4 protein was immunoprecipitated with anti-His polyclonal antibody (pAb), subjected to SDS-PAGE and Western blotted with anti-phosphotyrosine. Equivalent immunoprecipitates were Western blotted with anti-His antibody. In Figure 22, 109P1D4 exhibits tyrosine phosphorylation only upon cell treatment with pervanadate and not without treatment. This suggests that pervanadate, which inhibits intracellular protein tyrosine phosphatases (PTPs), allows the accumulation of phosphotyrosine (tyrosine kinase activity) on 109P1D4. Further, a large amount of the 109P1D4 protein is sequestered into the insoluble fraction upon pervanadate activation, suggesting its association with cytoskeletal components. Similar effects of partial insolubility in Triton X-100 have been observed for cadherins, proteins that are related to protocadherins based on homology of their extracellular domains. Cadherins are known to interact with cytoskeletal proteins including actin, which are not readily soluble in the detergent conditions used in this study. Together, these data indicate that 109P1 D4 is a surface receptor with the capacity to be phosphorylated on tyrosine and to bind to signaling molecules that possess SH2 or PTB binding domains, including but not limited to, phospholipase-Cγ1, Grb2, She, Crk, Pl-3-kinase p85 subunit, rasGAP, Src-family kinases and abl-family kinases. Such interactions are important for downstream signaling through 109P1 D4, leading to changes in adhesion, proliferation, migration or elaboration of secreted factors. In addition, 109P1 D4 protein interacts with cytoskeletal components such as actin that facilitates its cell adhesion functions. These phenotypes are enhanced in 109P1D4 expressing tumor cells and contribute to their increased capacity to metastasize and grow in vivo.
Thus, when 109P1D4 plays a role in cell signaling and phosphorylation, it is used as a target for diagnostic, prognostic, preventative and/or therapeutic purposes.
Example 45: 109P1D4 RNA Interference (RNAi) RNA interference (RNAi) technology is implemented to a variety of cell assays relevant to oncology. RNAi is a post-transcriptional gene silencing mechanism activated by double-stranded RNA (dsRNA). RNAi induces specific mRNA degradation leading to changes in protein expression and subsequently in gene function. In mammalian cells, these dsRNAs called short interfering RNA (siRNA) have the correct composition to activate the RNAi pathway targeting for degradation, specifically some mRNAs. See, Elbashir S.M., ef. al, Duplexes of 21 -nucleotide RNAs Mediate RNA interference in Cultured Mammalian Cells, Nature 411(6836):494-8 (2001). Thus, RNAi technology is used successfully in mammalian cells to silence targeted genes.
Loss of cell proliferation control is a hallmark of cancerous cells; thus, assessing the role of 109P1D4 in cell survival/proliferation assays is relevant. Accordingly, RNAi was used to investigate the function of the 109P1D4 antigen. To generate siRNA for 109P1 D4, algorithms were used that predict oligonucleotides that exhibit the critical molecular parameters (G:C content, melting temperature, etc.) and have the ability to significantly reduce the expression levels of the 109P1D4 protein when introduced into cells. Accordingly, three targeted sequences for the 109P1D4 siRNA are: 6' AAGAGGATACTGGTGAGATCT 3' (SEQ ID NO: 57)(oligo 109P1D4.a), δ' AAGAGCAATGGTGCTGGTAAA 3' (SEQ ID NO: 58)(oligo 109P1D4.C), and 5' AACACCAGAAGGAGACAAGAT 3' (SEQ ID NO: 69)(oligo 109P1D4.d). In accordance with this Example, 109P1 D4 siRNA compositions are used that comprise siRNA (double stranded, short interfering RNA) that correspond to the nucleic acid ORF sequence of the 109P1 D4 protein or subsequences thereof. Thus, siRNA subsequences are used in this manner are generally 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35 or more than 35 contiguous RNA nucleotides in length. These siRNA sequences are complementary and non-complementary to at least a portion of the mRNA coding sequence. In a preferred embodiment, the subsequences are 19-25 nucleotides in length, most preferably 21-23 nucleotides in length. In preferred embodiments, these siRNA achieve knockdown of 109P1D4 antigen in cells expressing the protein and have functional effects as described below.
The selected siRNAs (109P1D4.a, 109P1D4.C, 109P1D4.d oligos) were tested in LNCaP cells in the 3H-thymidine incorporation assay (measures cellular proliferation). Moreover, the oligonucleotides achieved knockdown of 109P1D4 antigen in cells expressing the protein and had functional effects as described below using the following protocols.
Mammalian siRNA transfections: The day before siRNA transfection, the different cell lines were plated in media (RPM1 1640 with 10% FBS w/o antibiotics) at 2x103 cells/well in 80 μ (96 well plate format) for the proliferation assay. In parallel with the 109P1 D4 specific siRNA oligo, the following sequences were included in every experiment as controls: a) Mock transfected cells with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) and annealing buffer (no siRNA); b) Luciferase-4 specific siRNA (targeted sequence: δ'-AAGGGACGAAGACGAACACUUCTT-3') (SEQ ID NO: 60); and, c) Egδ specific siRNA (targeted sequence: δ'-AACTGAAGACCTGAAGACAATAA-S') (SEQ ID NO: 61). SiRNAs were used at 10nM and μg/ml Lipofectamine 2000 final concentration.
The procedure was as follows: The siRNAs were first diluted in OPTIMEM (serum-free transfection media, Invitrogen) at 0.1 μM (10-fold concentrated) and incubated 5-10 min RT. Lipofectamine 2000 was diluted at 10 μg/ml (10- fold concentrated) for the total number transfections and incubated 5-10 minutes at room temperature (RT). Appropriate amounts of diluted 10-fold concentrated Lipofectamine 2000 were mixed 1 :1 with diluted 10-fold concentrated siRNA and incubated at RT for 20-30" (5-fold concentrated transfection solution). 20 μls of the 5-fold concentrated transfection solutions were added to the respective samples and incubated at 37°C for 96 hours before analysis.
3H-Thymidine incorporation assay: The proliferation assay is a 3H-thymidine incorporation method for determining the proliferation of viable cells by uptake and incorporation of label into DNA.
The procedure was as follows: Cells growing in log phase are trypsinized, washed, counted and plated in 96-well plates at 1000-4000 cells/well in 10% FBS. After 4-8 hrs, the media is replaced. The cells are incubated for 24-72 hrs, pulsed with 3H-Thy at 1.5 μCi/ml for 14 hrs, harvested onto a filtermat and counted in scintillation cocktail on a Microbeta trilux or other counter.
In order to address the function of 109P1 D4 in cells, 109P1 D4 was silenced by transfecting the endogenously expressing 109P1D4 cell line (LNCaP) with the 109P1D4 specific siRNAs (109P1D4.a, 109P1D4.C, and 109P1D4.d) along with negative siRNA controls (Luc4, targeted sequence not represented in the human genome), a positive siRNA control (targeting Eg5) and no siRNA oligo (LF2K) (Figure 23). The results indicated that when these cells are treated with siRNA specifically targeting the 109P1 D4 mRNA, the resulting "109P1 D4 deficient cells" showed diminished cell proliferation as measured by this assay (e.g., see oligo 109P1D4.a treated cells).
These data indicate that 109P1 D4 plays an important role in the proliferation of cancer cells and that the lack of 109P1D4 clearly decreases the survival potential of these cells. It is to be noted that 109P1D4 is constitutively expressed in many tumor cell lines. 109P1 D4 serves a role in malignancy; its expression is a primary indicator of disease, where such disease is often characterized by high rates of uncontrolled cell proliferation and diminished apoptosis. Correlating cellular phenotype with gene knockdown following RNAi treatments is important, and allows one to draw valid conclusions and rule out toxicity or other non-specific effects of these reagents. To this end, assays to measure the levels of expression of both protein and mRNA for the target after RNAi treatments are important, including Western blotting, FACS staining with antibody, immunoprecipitation, Northern blotting or RT-PCR (Taqman or standard methods). Any phenotypic effect of the siRNAs in these assays should be correlated with the protein and/or mRNA knockdown levels in the same cell lines. 109P1D4 protein is reduced after treatment with siRNA oligos described above (e.g., 109P1D4.a, etc.)
A method to analyze 109P1 D4 related cell proliferation is the measurement of DNA synthesis as a marker for proliferation. Labeled DNA precursors (i.e. 3H-Thymidine) are used and their incorporation to DNA is quantified. Incorporation of the labeled precursor into DNA is directly proportional to the amount of cell division occurring in the culture. Another method used to measure cell proliferation is performing clonogenic assays. In these assays, a defined number of cells are plated onto the appropriate matrix and the number of colonies formed after a period of growth following siRNA treatment is counted.
In 109P1D4 cancer target validation, complementing the cell survival/proliferation analysis with apoptosis and cell cycle profiling studies are considered. The biochemical hallmark of the apoptotic process is genomic DNA fragmentation, an irreversible event that commits the cell to die. A method to observe fragmented DNA in cells is the immunological detection of histone-complexed DNA fragments by an immunoassay (i.e. cell death detection ELISA) which measures the enrichment of histone-complexed DNA fragments (mono- and oligo-nucleosomes) in the cytoplasm of apoptotic cells. This assay does not require pre-labeling of the cells and can detect DNA degradation in cells that do not proliferate in vitro (i.e. freshly isolated tumor cells).
The most important effector molecules for triggering apoptotic cell death are caspases. Caspases are proteases that when activated cleave numerous substrates at the carboxy-terminal site of an aspartate residue mediating very early stages of apoptosis upon activation. All caspases are synthesized as pro-enzymes and activation involves cleavage at aspartate residues. In particular, caspase 3 seems to play a central role in the initiation of cellular events of apoptosis. Assays for determination of caspase 3 activation detect early events of apoptosis. Following RNAi treatments, Western blot detection of active caspase 3 presence or proteolytic cleavage of products (i.e. PARP) found in apoptotic cells further support an active induction of apoptosis. Because the cellular mechanisms that result in apoptosis are complex, each has its advantages and limitations. Consideration of other criteria/endpoints such as cellular morphology, chromatin condensation, membrane blebbing, apoptotic bodies help to further support cell death as apoptotic. Since not all the gene targets that regulate cell growth are anti-apoptotic, the DNA content of permeabilized cells is measured to obtain the profile of DNA content or cell cycle profile. Nuclei of apoptotic cells contain less DNA due to the leaking out to the cytoplasm (sub-G1 population). In addition, the use of DNA stains (i.e., propidium iodide) also differentiate between the different phases of the ceil cycle in the cell population due to the presence of different quantities of DNA in G0/G1 , S and G27M. In these studies the subpopulations can be quantified.
For the 109P1 D4 gene, RNAi studies facilitate the understanding of the contribution of the gene product in cancer pathways. Such active RNAi molecules have use in identifying assays to screen for mAbs that are active anti-tumor therapeutics. Further, siRNA are administered as therapeutics to cancer patients for reducing the malignant growth of several cancer types, including those listed in Table I. When 109P1D4 plays a role in cell survival, cell proliferation, tumorigenesis, or apoptosis, it is used as a target for diagnostic, prognostic, preventative and/or therapeutic purposes.
Throughout this application, various website data content, publications, patent applications and patents are referenced. (Websites are referenced by their Uniform Resource Locator, or URL, addresses on the World Wide Web.)
The present invention is not to be limited in scope by the embodiments disclosed herein, which are intended as single illustrations of individual aspects of the invention, and any that are functionally equivalent are within the scope of the invention. Various modifications to the models and methods of the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and teachings, and are similarly intended to fall within the scope of the invention. Such modifications or other embodiments can be practiced without departing from the true scope and spirit of the invention.
TABLES:
TABLE I: Tissues that Express 109P1 D4 when malignant:
Prostate
Bladder
Kidney
Colon
Lymphoma
Lung
Pancreas
Ovary
Breast
Uterus
Stomach
Rectum
Cervix
Lymph Node
Bone
TABLE II: Amino Acid Abbreviations
Figure imgf000125_0001
TABLE III: Amino Acid Substitution Matrix
Adapted from the GCG Software 9.0 BLOSUM62 amino acid substitution matrix (block substitution matrix). The higher the value, the more likely a substitution is found in related, natural proteins. (See world wide web URL ikp.unibe.ch/manual/blosum62.html)
A C D E F G H I K M N P Q R S T V W Y .
4 0 -2 -1 -2 0 -2 -1 -1 -1 -1 -2 -1 -1 -1 1 0 0 -3 -2 A
9 -3 -4 -2 -3 -3 -1 -3 -1 -1 -3 -3 -3 -3 -1 -1 -1 -2 -2 C
6 2 -3 -1 -1 -3 -1 -4 -3 1 -1 0 -2 0 -1 -3 -4 -3 D
5 -3 -2 0 -3 1 -3 -2 0 -1 2 0 0 -1 -2 -3 -2 E
6 -3 -1 0 -3 0 0 -3 -4 -3 -3 -2 -2 -1 1 3 F
6 -2 -4 -2 -4 -3 0 -2 -2 -2 0 -2 -3 -2 -3 G
8 -3 -1 -3 -2 1 -2 0 0 -1 -2 -3 -2 2 H
4 -3 2 1 -3 -3 -3 -3 -2 -1 3 -3 -1 1
5 -2 -1 0 -1 1 2 0 -1 -2 -3 -2 K
4 2 -3 -3 -2 -2 -2 -1 1 -2 -1 L
5 -2 -2 0 -1 -1 -1 1 -1 -1 M
6 -2 0 0 1 0 -3 -4 -2 N
7 -1 -2 -1 -1 -2 -4 -3 P
5 1 0 -1 -2 -2 -1 Q
5 -1 -1 -3 -3 -2 R
4 1 -2 -3 -2 S
5 0 -2 -2 T
4 -3 -1 V
11 2
7 Y
TABLE IV:
HLA Class l/l! Motifs/Supermotifs
TABLE IV (A): HLA Class I Supermotifs/Motifs
Figure imgf000127_0001
Bolded residues are preferred, italicized residues are less preferred: A peptide is considered motif-bearing if it has primary anchors at each primary anchor position for a motif or supermotif as specified in the above table.
TABLE IV (B): HLA Class II Supermotif
Figure imgf000127_0002
TABLE IV (C): HLA Class II Motifs
MOTIFS 1° anchor 1 2 3 4 5 1° anchor 6 7 8 9
DR4 preferred FMYL/V M T I VSTCPAUM MH MH deleterious W R WDE
DR1 preferred wuvvπ PAMQ VMATSPL/C M AVM deleterious C CH FD CWD GDE D
DR7 preferred WVWY M W A IVMSACTPL M IV deleterious C G GRD N G
DR3 MOTIFS 1° anchor 1 2 3 1° anchor 4 5 1° anchor 6
Motif a preferred LIVMFY D
Motif b preferred LIVMFAY DNQEST KRH
DR Supermotif MFL/VWY VMSTACPL/
Italicized residues indicate less preferred or "tolerated" residues
TABLE IV (D): HLA Class I Supermotifs
POSITION: 1 C-terminus
SUPERMOTIFS
A1 1° Anchor 1° Anchor TILWWS - FWY
A2 1° Anchor 1° Anchor LIVMΛTQ LIVMAT .
A3 Preferred 1° Anchor YFW YFW YFW P 1° Anchor VSMATL/ (4/5) (3/5) (4/5) (4/5) RK deleterious DE (3/5); DE P (δ/5) (4/5)
A24 1° Anchor 1° Anchor YFWIVLMT FIYW M
B7 Preferred FWY (5/5) 1° Anchor FWY FWY 1°Anchor
LIVM (3/5) P (4/5) (3/5) VLFMWYA deleterious DE (3/5); DE G QN DE
P(5/5); (3/5) (4/5) (4/5) (4/5)
6(4/5);
A(3/5);
QN(3/δ)
B27 1° Anchor 1°Anchor RHK FYLWMIVA
B44 1° Anchor 1° Anchor ED FWYLIMVA
B58 1 " Anchor 1° Anchor ATS FWYL/VMA
B62 1° Anchor 1° Anchor QLIVMP FWMIVLA
Italicized residues indicate less preferred or "tolerated" residues TABLE IV (E): HLA Class I Motifs
POSITION 1 2 3 4 5 6 7 8 9 C- terminus or C-terminus
A1 preferred GFYW 1 "Anchor DEA YFW P DEQN YFW 1°Anchor 9-mer STM Y deleterious DE RHKLIVMP A G A
A1 preferred GRHK ASTCLIVM 1°Anchor GSTC ASTC LIVM DE 1°Anchor 9-mer DEAS Y deleterious A RHKDEPYFW DE PQN RHK PG GP
A1 preferred YFW 1 "Anchor DEAQN A YFWQN PASTC GDE P rAnchor 10- STM Y mer deleterious GP RHKGLIVM DE RHK QNA RHKYFW RHK A
A1 preferred YFW STCLIVM 1°Anchor A YFW PG G YFW 1°Anchor 10- DEAS Y mer deleterious RHK RHKDEPYFW P G PRHK QN
A2.1 preferred YFW 1 "Anchor YFW STC YFW A P 1°Anchor 9-mer LUIVQAT VLIMAT deleterious DEP DERKH RKH DERKH
POSITION: 1 2 3 4 δ 6 7 8 9 C- Terminus
A2.1 preferred AYFW 1 "Anchor LVIM G G FYWL rAnchor 10- LMIVQAT VIM VLIMAT mer deleterious DEP DE RKHA P RKH DERKHRKH
A3 preferred RHK 1 "Anchor YFW PRHKYF A YFW P 1°Anchor LMVISATFCGD W KYRHE4 deleterious DEP DE
A11 preferred A 1°Anchor YFW YFW A YFW YFW P 1°Anchor VTLMISAGNCD KRYH r deleterious DEP A G
A24 preferred YFWRHK 1 "Anchor STC YFW YFW 1°Anchor 9-mer YFW/W FLIW deleterious DEG DE G QNP DERHKG AQN
A24 Preferred 1°Anchor P YFWP P rAnchor 10- YFWJW FLIW mer
Deleterious GDE QN RHK DE A QN DEA
A3101 Preferred RHK 1°Anchor YFW P YFW YFW AP 1°Anchor MVTALIS RK Deleterious DEP DE ADE DE DE DE
A3301 Preferred 1"Anchor YFW AYFW 1°Anchor MVALF/SI RK
Deleterious GP DE
A6801 Preferred YFWSTC rAnchor YFWLIV YFW P rAnchor AVTMSLI M RK deleterious GP DEG RHK A
B0702Preferred RHKFWY 1°Anchor RHK RHK RHK RHK PA 1°Anchor P LMFWYAI
V deleterious DEQNP DEP DE DE GDE QN DE POSITION 1 C- terminus or C-terminus
A1 preferred GFYW 1°Anchor DEA YFW P DEQN YFW rAnchor 9-mer STM Y deleterious DE RHKLIVMP A G A
A1 preferred GRHK ASTCLIVM rAnchor GSTC ASTC LIVM DE 1°Anchor 9-mer DEAS Y deleterious A RHKDEPYFW DE PQN RHK PG GP
B3501 Preferred FWYLIVM 1°Anchor FWY FWY 1°Anchor P LMFWY/V
A deleterious AGP
B51 Preferred LIVMFWY rAnchor FWY STC FWY FWY rAnchor LNFWYA M deleterious AGPDER DE G DEQN GDE HKSTC
B5301 preferred LIVMFWY 1 "Anchor FWY STC FWY LIVMFWYFWY 1"Anchor P IMFWY/A V deleterious AGPQN G RHKQN DE
B5401 preferred FWY 1°Anchor FWYLIVM LIVM ALIVM FWYA 1 "Anchor P P ATIVLMF WY deleterious GPQNDE GDESTC RHKDE DE QNDGE DE
TABLE IV (F):
Figure imgf000131_0001
Figure imgf000131_0002
Figure imgf000131_0003
Figure imgf000132_0001
Table VI: Post-translational modifications of 109P1D4
0-qlycosylation sites 231 S 238 S 240 T 266 T
346 467 551 552 555 596 652 654
660 T
790 795 798 804 808 923 927 954 979 982 983 985 S
986 S 990 S
999 T
1000 T 1006 S 1017 S 1020 T
Serine phosphorylation sites
50 DLNLSLIPN (SEQ ID NO: 62)
147 VINISIPEN (SEQ ID NO: 63)
152 IPENSAINS (SEQ ID NO: 64)
238 ILQVSVTDT (SEQ ID NO: 65)
257 EIEVSIPEN (SEQ ID NO: 66)
428 LDYESTKEY (SEQ ID NO: 67)
480 PENNSPGIQ (SEQ ID NO: 68)
489 LTKVSAMDA (SEQ ID NO: 69)
495 MDADSGPNA (SEQ ID NO: 70)
559 TVFVSIIDQ (SEQ ID NO: 71)
567 QNDNSPVFT (SEQ ID NO: 72)
608 AVTLSILDE (SEQ ID NO: 73)
630 RPNISFDRE (SEQ ID NO: 74)
638 EKQESYTFY (SEQ ID NO: 75)
652 GGRVSRSSS (SEQ ID NO: 76)
654 RVSRSSSAK (SEQ ID NO: 77)
656 VSRSSSAKV (SEQ ID NO: 78)
656 SRSSSAKVT (SEQ ID NO: 79)
714 EVRYSIVGG (SEQ ID NO: 80)
789 LVRKSTEAP (SEQ ID NO: 81) 805 ADVSSPTSD (SEQ ID NO: 82) 808 SSPTSDYVK (SEQ ID NO: 83) 852 NKQNSEWAT (SEQ ID NO: 84) 877 KKKHSPKNL (SEQ ID NO: 85) 898 DDVDSDGNR (SEQ ID NO: 86) 932 FKPDSPDLA (SEQ ID NO: 87) 941 RHYKSASPQ (SEQ ID NO: 88) 943 YKSASPQPA (SEQ ID NO: 89)
982 ISKCSSSSS (SEQ ID NO: 90)
983 SKCSSSSSD (SEQ ID NO: 91)
984 KCSSSSSDP (SEQ ID NO: 92)
985 CSSSSSDPY (SEQ ID NO: 93) 990 SDPYSVSDC (SEQ ID NO: 94) 1006 EVPVSVHTR (SEQ ID NO: 95)
Threonine phosphorylation sites 29 EKNYTIREE (SEQ ID NO: 96) 81 IEEDTGEIF (SEQ ID NO: 97) 192 DVIETPEGD (SEQ ID NO: 98) 252 VFKETEIEV (SEQ ID NO: 99) 310 TGLITIKEP (SEQ ID NO: 100) 320 DREETPNHK (SEQ ID NO: 101) 551 VPPLTSNVT (SEQ ID NO: 102)
790 VRKSTEAPV (SEQ ID NO: 103) 856 SEWATPNPE (SEQ ID NO: 104) 924 NWVTTPTTF (SEQ ID NO: 105) 927 TTPTTFKPD (SEQ ID NO: 106) 999 GYPVTTFEV (SEQ ID NO: 107) 1000 YPVTTFEVP (SEQ ID NO: 108)
Tyrosine phosphorylation sites
67 FKLVYKTGD (SEQ ID NO: 109) 158 INSKYTLPA (SEQ ID NO: 110) 215 EKDTYVMKV (SEQ ID NO: 111) 359 IDIRYIVNP (SEQ ID NO: 112) 423 ETAAYLDYE (SEQ ID NO: 113) 426 AYLDYESTK (SEQ ID NO: 114) 432 STKEYAIKL (SEQ ID NO: 115) 536 KEDKYLFTI (SEQ ID NO: 116) 599 TDPDYGDNS (SEQ ID NO: 117) 642 SYTFYVKAE (SEQ ID NO: 118) 682 SNCSYELVL (SEQ ID NO: 119) 713 AEVRYSIVG (SEQ ID NO: 120) 810 PTSDYVKIL (SEQ ID NO: 121) 919 TMGKYNWVT (SEQ ID NO: 122) 989 SSDPYSVSD (SEQ ID NO: 123) 996 SDCGYPVTT (SEQ ID NO: 124)
Table VII: Search Peptides
109P1D4 v.1 - 9-mers, 10-mers and 15-mers (SEQ ID NO: 125)
MDLLSGTYIF AVL ACWFH SGAQEKNYTI REEMPENVLI GDLLKDLNLS LIPN SLTTA 60
MQFKLVYKTG DVPLIRIEED' TGEIFTTGAR IDREKLCAGI PRDEHCFYEV EVAILPDEIF 120
RLVKIRFLIE DINDNAPLFP ATVINISIPE NSAINSKYTL PAAVDPDVGI NGVQNYELIK 180
SQNIFGLDVI ETPEGDKMPQ LIVQKELDRE EKDTYVMKVK VEDGGFPQRS STAILQVSVT 240
DTNDNHPVFK ETEIEVSIPE NAPVGTSVTQ LHATDADIGE NAKIHFSFSN LVSNIARRLF 300
HLNATTGLIT IKEP DREET PNHK VLAS DGGLMPARAM VLVNVTDVND NVPSIDIRYI 360
VNPVNDTWL SENIPLNTKI ALITVTD DA DHNGRVTCFT DHEIPFRLRP VFSNQFL ET 420
AAY DYESTK EYAIKLLAAD AGKPPLNQSA MLFIKVKDEN DNAPVFTQSF VTVSIPENNS 480
PGIQLTKVSA MDADSGPNAK INYLLGPDAP PEFSLDCRTG MLTWKK DR EKEDKYLFTI 540
LAKDNGVPPL TSNVTVFVSI IDQNDNSPVF THNEYNFYVP EN PRHGTVG ITVTDPDYG 600
DNSAVTLSIL DENDDFTIDS QTGVIRPNIS FDREKQESYT FYVKAEDGGR VSRSSSAKVT 660
INWDVNDNK PVFIVPPSNC SYE VLPSTN PGTWFQVIA VDNDTGMNAE VRYSIVGGNT 720
RDLFAIDQET GNITLMEKCD VTDLGLHRV VKANDLGQPD SLFSWIVN FVNESVTNAT 780 INELVRKST EAPVTPNTEI ADVSSPTSDY VKI VAAVAG TITWWIFI TAVVRCRQAP 840
HLKAAQKNKQ NSEWATPNPE NRQMIMMKKK KKKKKHSPKN LLLNFVTIEE TKADDVDSDG 900
NRVTLD PID EEQT GKYN WVTTPTTFKP DSPDLARHYK SASPQPAFQI QPETP NSKH 960
HIIQE PLDN TFVACDSISK CSSSSSDPYS VSDCGYPVTT FEVPVSVHTR PVGIQVSNTT 1020
F 1021
109P1D4 v.2 (both ends diff from v.1) N' terminal 9-mers aa -30 to 8
MRTERQ VLIQIFQVLCGLIQQTVTSVPGMDLLSGTY ( SEQ ID NO : 126 )
10-mers aa -30 to 9
MRTERQWVLIQIFQVLCGLIQQTVTSVPGMDLLSGTYI ( SEQ ID NO : 127 )
15-mers aa -30 to 14
MRTERQ VLIQIFQVLCGLIQQTVTSVPGMDLLSGTYIFAVLL ( SEQ ID NO : 128 )
109P1D4 V.2
C Terminal
9 mers: aa 1004 to 1025
PVSVHTRPTDSRTSTIEICSEI (SEQ ID NO: 129)
10 mers: aa 1003 to 1025
VPVSVHTRPTDSRTSTIEICSEI (SEQ ID NO: 130)
15 mers: aa 997 to 1025
VTTFEVPVSVHTRPTDSRTSTIEICSEI ( SEQ ID NO : 131 )
109P1D4 V.3
9 mers: aa 1004 to 1347 (SEQ ID NO: 132)
PVSVHTRPPMKEVVRSCTPMKESTTMEI IHPQPQRKSEGKVAGKSQRRVTFHLPEGSQESSSDG GLGDHDAGSLTSTSHGLPLGYPQEEYFDRATPSNRTEGDGNSDPESTFIPGLKKAAEITVQPTVE EASDNCTQECLIYGHSDACWMPASLDHSSSSQAQASALCHSPPLSQASTQHHSPRVTQTIALCHS PPVTQTIALCHSPPPIQVSALHHSPPLVQATALHHSPPSAQASALCYSPPLAQAAAISHSSPLPQ VIALHRSQAQSSVSLQQG VQGADGLCSVDQGVQGSATSQFYTMSERLHPSDDSIKVIPLTTFTP RQQARPSRGDSPMEEHPL
10 mers: aa 1003 to 1347 (SEQ ID NO: 133)
VPVSVHTRPPMKEVVRSCTPMKESTTMEIWIHPQPQRKSEGKVAGKSQRRVTFHLPEGSQESSSD GGLGDHDAGSLTSTSHGLPLGYPQEEYFDRATPSNRTEGDGNSDPESTFIPG KKAAEITVQPTV EEASDNCTQECLIYGHSDAC MPASLDHSSSSQAQASALCHSPPLSQASTQHHSPRVTQTIALCH SPPVTQTIALCHSPPPIQVSALHHSPP VQATALHHSPPSAQASALCYSPPLAQAAAISHSSPLP QVIALHRSQAQSSVSLQQGWVQGADGLCSVDQGVQGSATSQFYTMSERLHPSDDSIKVIPLTTFT PRQQARPSRGDSPMEEHPL
15 mers: aa 998 to 1347 (SEQ ID NO: 134)
VTTFEVPVSV HTRPPMKEVV RSCTPMKEST TMEIWIHPQP QRKSEGKVAG KSQRRVTFHL PEGSQESSSD GGLGDHDAGS LTSTSHGLPL GYPQEEYFDR ATPSNRTEGD GNSDPESTFI PGLKKAAEIT VQPTVEEASD NCTQECLIYG HSDACWMPAS LDHSSSSQAQ ASALCHSPPL SQASTQHHSP RVTQTIALCH SPPVTQTIAL CHSPPPIQVS ALHHSPPLVQ ATALHHSPPS AQASALCYSP PLAQAAAISH SSPLPQVIAL HRSQAQSSVS LQQGWVQGAD GLCSVDQGVQ GSATSQFYTM SERLHPSDDS IKVIPLTTFT PRQQARPSRG DSPMEEHPL
109P1D4 v.4 (deleting 10 aa, 1039-1048, from v.1) 9-mers aa 1031-1056 (deleting 10 aa, 1039-1048, from v.1)
IWIHPQPQSQRRVTFH ( SEQ ID NO : 135 )
10-mers aa 1030- 1057 (deleting 10 aa, 1039-1048, from v.1)
EIWIHPQPQSQRRVTFHL ( SEQ ID NO : 136 )
15-mers aa 1025- 1062 (deleting 10 aa, 1039-1048, from v.1)
ESTTMEIWIHPQPQSQRRVTFHLPEGSQ ( SEQ ID NO : 137 )
109P1D4 v.5 (deleting 37 aa, 1012-1048, from v.1) 9-mers aa 1004-1056 (deleting 37 aa, 1012-1048, from v.1)
PVSVHTRPSQRRVTFH ( SEQ ID NO : 138 )
10-mers aa 1003-1057 (deleting 37 aa, 1012-1048, from v.1)
VPVSVHTRPSQRRVTFHL ( SEQ ID NO : 139 )
15-mers aa 998-1062 (deleting 37 aa, 1012-1048, from v.1)
VTTFEVPVSVHTRPSQRRVTFHLPEGSQ ( SEQ I D NO : 140 )
109P1D4 v.6 (both ends diff from v.1)
N' terminal
9-mers: aa -23 to 10 (excluding 1 and 2)
MTVGFNSDISSWRVNTTNCHKCLLSGTYIF ( SEQ ID NO : 141 )
10-mers: aa -23 to 11 (excluding 1 and 2)
MTVGFNSDISSVVRVNTTNCHKCLLSGTYI FA ( SEQ ID NO : 142 )
15-mers: aa -23 to 17 (excluding 1 and 2)
MTVGFNSDISSVVRVNTTNCHKCLLSGTYIFAVLLVC ( SEQ ID NO : 143 )
109P1D4 V.6
C terminal
9-mers: aa 1004-1016
PVSVHTRPTDSRT ( SEQ ID NO : 144 )
10-mers: aa 1003-1016
VPVSVHTRPTDSRT ( SEQ ID NO : 145 )
15-mers: aa 998-1016
VTTFEVPVSVHTRPTDSRT ( SEQ ID NO : 146 )
109P1D4 v.7 (N-terminal 21 aa diff from those in v.6) N' terminal
9-mers aa-21 to 10 (excluding 1 and 2)
MFRVGFLIISSSSSLSPLLLVSWRVNTT (SEQ ID NO: 147)
10-mers aa -21 to 11 (excluding 1 and 2)
MFRVGFLIISSSSSLSPLLLVSWRVNTTN (SEQ ID NO: 148)
15-mers aa -21 to 16 (excluding 1 and 2)
MFRVGFLIISSSSSLSPLLLVSWRVNTTNCHKCL (SEQ ID NO: 149)
109P1D4V.8
9-mers aa 1099-1126 (excluding 1117 and 1118)
TFIPGLKKEITVQPTV (SEQ ID NO: 150)
10-mers aa 1098-1127 (excluding 1117 and 1118)
STFIPGLKKEITVQPTVE (SEQ ID NO: 151)
15-mers aa 1093-1131 (excluding 1117 and 1118)
NSDPESTFIPGLKKEITVQPTVEEASDN (SEQ ID NO: 152)
109P1D4 v.1, v.2 and v.3 SNP variants
A15V
9-mers
TYIFAVLLVCVVFHSGA (SEQ ID NO: 153)
10-mers
GTYIFAVLLVCVVFHSGAQ (SEQ ID NO: 154)
15-mers
MDLLSGTYIFAVLLVCVVFHSGAQEKNYT (SEQ ID NO: 155)
109P1D4 v.1, v.2 and v.3 SNP variants
M34I
9-mers
KNYTIREEIPENVLIGD (SEQ ID NO: 156)
10-mers
EKNYTIREEIPENVLIGDL (SEQ ID NO: 157)
15-mers
HSGAQEKNYTIREEIPENVLIGDLLKDLN (SEQ ID NO: 158)
109P1D4 v.1, v.2 and v.3 SNP variants
M34I and D42N
9-mers
KNYTIREEIPENVLIGN (SEQ ID NO: 159)
10-mers
EKNYTIREEIPENVLIGNL (SEQ ID NO: 160)
15-mers
HSGAQEKNYTIREEIPENVLIGNLLKDLN (SEQ ID NO: 161)
109P1D4 v.1, v.2 and v.3 SNP variants
D42N
9-mers
MPENVLIGNLLKDLNLS (SEQ ID NO: 162)
10-mers
EMPENVLIGNLLKDLNLSL (SEQ ID NO: 163)
15-mers
YTIREEMPENVLIGNLLKDLNLSLIPNKS (SEQ ID NO: 164)
109P1D4 v.1, v.2 and v.3 SNP variants
D42N and M34I
9-mers
IPENVLIGNLLKDLNLS (SEQ ID NO: 165)
10-mers
EIPENVLIGNLLKDLNLSL (SEQ ID NO: 166) 15-mers
YTIREEIPENVLIGNLLKDLNLSLIPNKS ( SEQ ID NO : 167 )
109P1D4 v.1, v.2 and v.3 SNP variants
A60T
9-mers
IPNKSLTTTMQFKLVYK (SEQ ID NO: 168)
10-mers
LIPNKSLTTTMQFKLVYKT (SEQ ID NO: 169)
15-mers
DLNLSLIPNKSLTTTMQFKLVYKTGDVPLI (SEQ ID NO: 170)
109P1D4 v.1, v.2 and v.3 SNP variants
1154V
9-mers
ISIPENSAVNSKYTLPA (SEQ ID NO: 171)
10-mers
NISIPENSAVNSKYTLPAA (SEQ ID NO: 172)
15-mers
PATVINISIPENSAVNSKYTLPAAVDPDV (SEQ ID NO: 173)
109P1D4 v.1, v.2 and v.3 SNP variants
V292I
9-mers
IHFSFSNLISNIARRLF (SEQ ID NO: 174)
10-mers
KIHFSFSNLISNIARRLFH (SEQ ID NO: 175)
15-mers
IGENAKIHFSFSNLISNIARRLFHLNATT (SEQ ID NO : 176 )
109P1 D4 v.1 , v.2 and v.3 SNP variants
T420N
9-mers
FSNQFLLENAAYLDYES (SEQ ID NO: 177)
10-mers
VFSNQFLLENAAYLDYEST (SEQ ID NO: 178)
15-mers
FRLRPVFSNQFLLENAAYLDYESTKEYAI (SEQ ID NO: 179)
109P1D4 v.1, v.2 and v.3 SNP variants
T486M
9-mers
NNSPGIQLMKVSAMDAD (SEQ ID NO: 180)
10-mers
ENNSPGIQLMKVSAMDADS (SEQ ID NO: 181)
15-mers
TVSIPENNSPGIQLMKVSAMDADSGPNAK (SEQ ID NO: 182)
109P1D4 v.1, v.2 and v.3 SNP variants
T486M and M491T
9-mers
NNSPGIQLMKVSATDAD (SEQ ID NO: 183)
10-mers
ENNSPGIQLMKVSATDADS (SEQ ID NO: 184)
15-mers
TVSIPENNSPGIQLMKVSATDADSGPNAK (SEQ ID NO: 185)
109P1D4 v.1 , v.2 and v.3 SNP variants T486M and M491T and K500E 15-mers
TVSIPENNSPGIQLMKVSATDADSGPNAE (SEQ ID NO : 186 ) 109P1D4 v.1, v.2 and v.3 SNP variants
T486M and K500E
15-mers
TVSIPENNSPGIQLMKVSAMDADSGPNAE (SEQ ID NO: 187)
109P1D4 v.1, v.2 and v.3 SNP variants
M491T
9-mers
IQLTKVSATDADSGPNA (SEQ ID NO: 188)
10-mers
GIQLTKVSATDADSGPNAK (SEQ ID NO: 189)
15-mers
ENNSPGIQLTKVSATDADSGPNAKINYLL (SEQ ID NO: 190)
109P1D4 v.1, v.2 and v.3 SNP variants
M491TandT486M
9-mers
IQLNKVSATDADSGPNA (SEQ ID NO: 191)
10-mers
GIQLNKVSATDADSGPNAK (SEQ ID NO: 192)
15-mers
ENNSPGIQLNKVSATDADSGPNAKINYLL (SEQ ID NO: 193)
109P1D4 v.1, v.2 and v.3 SNP variants M491T and T486M and K500E 10-mers
GIQLNKVSATDADSGPNAE (SEQ ID NO: 194)
15-mers
ENNSPGIQLNKVSATDADSGPNAEINYLL (SEQ ID NO: 195)
109P1D4 v.1, v.2 and v.3 SNP variants
M491TandK500E
15-mers
ENNSPGIQLTKVSATDADSGPNAEINYLL (SEQ ID NO: 196)
109P1D4 v.1, v.2 and v.3 SNP variants
K500E
9-mers
DADSGPNAEINYLLGPD (SEQ ID NO: 197)
10-mers
MDADSGPNAEINYLLGPDA (SEQ ID NO: 198)
1δ-mers
TKVSAMDADSGPNAEINYLLGPDAPPEFS (SEQ ID NO: 199)
109P1D4 v.1, v.2 and v.3 SNP variants
K500E and M491T
10-mers
TDADSGPNAEINYLLGPDA (SEQ ID NO: 200)
15-mers
TKVSATDADSGPNAEINYLLGPDAPPEFS (SEQ ID NO: 201)
109P1D4 v.1, v.2 and v.3 SNP variants
Kδ00EandM491TandT486M
1δ-mers
MKVSATDADSGPNAEINYLLGPDAPPEFS (SEQ ID NO: 202)
109P1D4 v.1, v.2 and v.3 SNP variants
KδOOE and T486M
1δ-mers
MKVSAMDADSGPNAEINYLLGPDAPPEFS (SEQ ID NO: 203)
109P1D4 v.1, v.2 and v.3 SNP variants C517R 9-mers
APPEFSLDRRTGMLTW (SEQ ID NO: 204)
10-mers
DAPPEFSLDRRTGMLTVVK (SEQ ID NO: 205)
15-mers
INYLLGPDAPPEFSLDRRTGMLTVVKKLDRE (SEQ ID NO: 206)
109P1D4 v.1, v.2 and v.3 SNP variants
N576K
9-mers
PVFTHNEYKFYVPENLP (SEQ ID NO: 207)
10-mers
SPVFTHNEYKFYVPENLPR (SEQ ID NO: 208) 15-mers
DQNDNSPVFTHNEYKFYVPENLPRHGTVG (SEQ ID NO: 209)
109P1D4 v.1, v.2 and v.3 SNP variants
S678Y
9-mers
KPVFIVPPYNCSYELVLPS (SEQ ID NO: 210)
10-mers
NKPVFIVPPYNCSYELVLPST (SEQ ID NO: 211)
15-mers
VDVNDNKPVFIVPPYNCSYELVLPSTNPG (SEQ ID NO: 212)
109P1D4 v.1, v.2 and v.3 SNP variants
S678Y and C680Y
9-mers
KPVFIVPPYNYSYELVLPS (SEQ ID NO: 213)
10-mers
NKPVFIVPPYNYSYELVLPST (SEQ ID NO: 214)
15-mers
VDVNDNKPVFIVPPYNYSYELVLPSTNPG (SEQ ID NO: 215)
109P1D4 v.1, v.2 and v.3 SNP variants
C680Y
9-mers
VFIVPPSNYSYELVLPS (SEQ ID NO: 216)
10-mers
PVFIVPPSNYSYELVLPST (SEQ ID NO: 217)
15-mers
VNDNKPVFIVPPSNYSYELVLPSTNPGTV (SEQ ID NO: 218)
109P1D4 v.1, v.2 and v.3 SNP variants
C680YandS678Y
9-mers
VFIVPPYNYSYELVLPS (SEQ ID NO: 219)
10-mers
PVFIVPPYNYSYELVLPST (SEQ ID NO: 220)
15-mers
VNDNKPVFIVPPYNYSYELVLPSTNPGTV (SEQ ID NO: 221)
109P1D4 v.1, v.2 and v.3 SNP variants
T790I
9-mers
INELVRKSIEAPVTPNT (SEQ ID NO: 222)
10-mers
LINELVRKSIEAPVTPNTE (SEQ ID NO: 223)
15-mers
VTNATLINELVRKSIEAPVTPNTEIADVS (SEQ ID NO: 224) 109P1D4 v.1, v.2 and v.3 SNP variants
K846M
9-mers
HLKAAQKNMQNSEWATP (SEQ ID NO: 225) 10-mers
PHLKAAQKNMQNSEWATPN (SEQ ID NO: 226)
15-mers
RCRQAPHLKAAQKNMQNSEWATPNPENRQ (SEQ ID NO: 227)
109P1D4 v.1, v.2 and v.3 SNP variants
F855V
9-mers
SPKNLLLNVVTIEETKA (SEQ ID NO: 228)
10-mers
HSPKNLLLNVVTIEETKAD (SEQ ID NO: 229)
15-mers
KKKKKHSPKNLLLNVVTIEETKADDVDSD (SEQ ID NO: 230)
109P1D4 v.1, v.2 and v.3 SNP variants
S958L
9-mers
IQPETPLNLKHHIIQEL (SEQ ID NO: 231)
10-mers
QIQPETPLNLKHHIIQELP (SEQ ID NO: 232)
15-mers
PQPAFQIQPETPLNLKHHIIQELPLDNTF (SEQ ID NO: 233)
109P1D4 v.1, v.2 and v.3 SNP variants
K980N
9-mers
FVACDSISNCSSSSSDP (SEQ ID NO: 234)
10-mers
TFVACDSISNCSSSSSDPY (SEQ ID NO: 235)
15-mers
LPLDNTFVACDSISNCSSSSSDPYSVSDC (SEQ ID NO: 236)
Tables VIII -XXI:
Figure imgf000141_0004
Figure imgf000141_0001
Figure imgf000141_0002
Figure imgf000141_0003
Figure imgf000142_0004
Figure imgf000142_0002
Figure imgf000142_0003
Figure imgf000142_0001
Figure imgf000142_0005
Figure imgf000143_0001
Figure imgf000143_0002
Figure imgf000143_0003
Figure imgf000143_0004
Figure imgf000144_0002
Figure imgf000144_0005
Figure imgf000144_0001
Figure imgf000144_0003
Figure imgf000144_0004
Figure imgf000145_0004
Figure imgf000145_0001
Figure imgf000145_0002
Figure imgf000145_0003
Figure imgf000145_0005
Figure imgf000146_0003
Figure imgf000146_0001
Figure imgf000146_0002
Figure imgf000146_0004
Figure imgf000147_0004
Figure imgf000147_0001
Figure imgf000147_0002
Figure imgf000147_0003
Figure imgf000147_0005
Figure imgf000148_0003
Figure imgf000148_0001
Figure imgf000148_0002
Figure imgf000148_0004
Figure imgf000149_0004
Figure imgf000149_0001
Figure imgf000149_0002
Figure imgf000149_0003
Figure imgf000149_0005
Figure imgf000150_0003
Figure imgf000150_0001
Figure imgf000150_0002
Figure imgf000150_0004
Figure imgf000151_0004
Figure imgf000151_0001
Figure imgf000151_0002
Figure imgf000151_0003
Figure imgf000151_0005
Figure imgf000152_0002
Figure imgf000152_0004
Figure imgf000152_0005
Figure imgf000152_0001
Figure imgf000152_0003
Figure imgf000153_0002
Figure imgf000153_0001
Figure imgf000153_0004
Figure imgf000153_0005
Figure imgf000153_0003
Figure imgf000154_0002
Figure imgf000154_0004
Figure imgf000154_0005
Figure imgf000154_0001
Figure imgf000154_0003
Figure imgf000155_0002
Figure imgf000155_0004
Figure imgf000155_0001
Table VIII -109P1D4V.2 C Terminal-A1 -9-mers
Figure imgf000155_0003
Figure imgf000155_0005
Figure imgf000156_0004
Figure imgf000156_0005
Figure imgf000156_0002
Figure imgf000156_0003
Figure imgf000156_0001
Figure imgf000156_0006
Figure imgf000157_0005
Figure imgf000157_0002
Figure imgf000157_0008
Figure imgf000157_0006
Figure imgf000157_0003
Figure imgf000157_0009
Figure imgf000157_0007
Figure imgf000157_0004
Figure imgf000157_0001
Figure imgf000157_0010
Figure imgf000158_0001
Figure imgf000158_0007
Figure imgf000158_0004
Figure imgf000158_0002
Figure imgf000158_0008
Figure imgf000158_0005
Figure imgf000158_0006
Figure imgf000158_0009
Figure imgf000158_0003
Figure imgf000159_0005
Figure imgf000159_0002
Figure imgf000159_0008
Figure imgf000159_0006
Figure imgf000159_0003
Figure imgf000159_0009
Figure imgf000159_0007
Figure imgf000159_0004
Figure imgf000159_0001
Figure imgf000159_0010
Figure imgf000160_0005
Figure imgf000160_0008
Figure imgf000160_0002
Figure imgf000160_0009
Figure imgf000160_0006
Figure imgf000160_0003
Figure imgf000160_0010
Table XI-109P1D4V.6 C' terminal-A0201 -10-mers
Each peptide is a portion of SEQ
ID NO: 13; each start position is
Figure imgf000160_0007
specified, the length of peptide is
10 amino acids, and the end position for each peptide is the start position plus nine.
Figure imgf000160_0001
Figure imgf000160_0004
Figure imgf000161_0002
Figure imgf000161_0006
Figure imgf000161_0010
Figure imgf000161_0003
Figure imgf000161_0007
Figure imgf000161_0011
Figure imgf000161_0004
Figure imgf000161_0008
Figure imgf000161_0001
Figure imgf000161_0012
Table XX-109P1D4V.6 C terminal-B3501 -9-mers
Each peptide is a portion of SEQ
ID NO: 13; each start position is specified, the length of peptide is 9 amino acids, and the end position
Figure imgf000161_0009
for each peptide is the start
Figure imgf000161_0005
position plus eight.
Figure imgf000162_0002
Figure imgf000162_0005
Figure imgf000162_0003
Figure imgf000162_0007
Figure imgf000162_0004
Figure imgf000162_0008
Figure imgf000162_0001
Figure imgf000162_0006
Figure imgf000163_0004
Figure imgf000163_0002
Figure imgf000163_0003
Figure imgf000163_0001
Figure imgf000163_0005
Figure imgf000163_0006
Figure imgf000164_0007
Figure imgf000164_0005
Figure imgf000164_0003
Figure imgf000164_0006
Figure imgf000164_0001
Figure imgf000164_0008
Figure imgf000164_0002
Figure imgf000164_0004
Figure imgf000165_0004
Figure imgf000165_0002
Figure imgf000165_0006
Figure imgf000165_0005
Figure imgf000165_0003
Figure imgf000165_0001
Figure imgf000165_0007
Figure imgf000166_0004
Figure imgf000166_0002
Figure imgf000166_0006
Figure imgf000166_0005
Figure imgf000166_0001
Figure imgf000166_0003
Figure imgf000166_0007
Figure imgf000167_0004
Figure imgf000167_0002
Figure imgf000167_0006
Figure imgf000167_0005
Figure imgf000167_0003
Figure imgf000167_0001
Figure imgf000167_0007
Figure imgf000168_0004
Figure imgf000168_0002
Figure imgf000168_0006
Figure imgf000168_0005
Figure imgf000168_0003
Figure imgf000168_0001
Figure imgf000168_0007
Figure imgf000169_0004
Figure imgf000169_0007
Figure imgf000169_0002
Figure imgf000169_0005
Figure imgf000169_0008
Figure imgf000169_0006
Figure imgf000169_0001
Figure imgf000169_0003
Figure imgf000169_0009
Figure imgf000170_0001
Figure imgf000170_0007
Figure imgf000170_0004
Figure imgf000170_0002
Figure imgf000170_0008
Figure imgf000170_0005
Figure imgf000170_0006
Figure imgf000170_0003
Figure imgf000170_0009
Figure imgf000171_0002
Figure imgf000171_0003
Figure imgf000171_0004
Figure imgf000171_0001
Tables XXII -XLIX:
Figure imgf000172_0003
Figure imgf000172_0002
Figure imgf000172_0004
Figure imgf000172_0001
Figure imgf000173_0001
Figure imgf000173_0002
Figure imgf000173_0003
Figure imgf000174_0001
Figure imgf000174_0002
Figure imgf000174_0003
Figure imgf000175_0001
TableXXIV
109P1D4V.1
A0203-9- mers
No Results Found.
Table XXV- 109P1D4v.1-A3-9-mers
Each peptide is a portion of SEQ ID NO:
3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.
Figure imgf000175_0003
Figure imgf000175_0004
Figure imgf000175_0002
Figure imgf000176_0002
Table XXVI- 109P1D4V.1 A26-9-mers
Each peptide is a portion of SEQ ID NO:
3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start
Figure imgf000176_0001
position plus eight.
Figure imgf000176_0003
Figure imgf000177_0003
Table XXVII-109P1D4 v.1-B0702-9-mers
Each peptide is a portion of SEQ ID NO:
3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.
Figure imgf000177_0001
Figure imgf000177_0002
583 LPRHGTVGL 25
Figure imgf000178_0003
Figure imgf000178_0001
Figure imgf000178_0002
Figure imgf000178_0004
Figure imgf000179_0003
Figure imgf000179_0001
Figure imgf000179_0002
Figure imgf000179_0004
Figure imgf000180_0002
Table XXX-
109P1D4V.1-
B2705-9-mers
Each peptide is a portion of SEQ ID NO:
3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.
Figure imgf000180_0001
120 FRLVKIRFL 26
Figure imgf000180_0003
Figure imgf000181_0001
Figure imgf000181_0002
Figure imgf000181_0003
Figure imgf000182_0003
Table XXXI-109P1D4v,1 B2709-9-mers
Figure imgf000182_0001
Figure imgf000182_0002
Figure imgf000183_0001
Figure imgf000183_0002
Figure imgf000183_0003
Figure imgf000184_0002
Figure imgf000184_0001
Figure imgf000184_0004
Figure imgf000184_0003
Figure imgf000185_0001
Table XXXIIII-109P1D4 v.1-B5101-9-mers
Each peptide is a portion of SEQ ID NO:
3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight.
136 APLFPATVI 27
22 GAQEKNYTI 26
Figure imgf000185_0002
Figure imgf000185_0003
Figure imgf000186_0003
Figure imgf000186_0001
Figure imgf000186_0002
Figure imgf000186_0004
Figure imgf000187_0001
Table XXXV-109P1D4 V.1-A0201 -10-mers
Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine
3 LLSGTYIFAV 29
761 SLFSVVIVNL 29
Figure imgf000187_0002
Figure imgf000187_0003
Figure imgf000188_0003
Table XXXVI-109P1D4 v.1-A0203-10-mers
Figure imgf000188_0001
Figure imgf000188_0002
Figure imgf000189_0001
Figure imgf000189_0002
Figure imgf000189_0003
Figure imgf000190_0001
Figure imgf000190_0002
Figure imgf000190_0003
Figure imgf000190_0004
Figure imgf000191_0001
Figure imgf000191_0002
Figure imgf000191_0003
Table XXXVII-109P1D4 v.1-A3-10-mers
Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine
997 PVTTFEVPVS 14
Figure imgf000192_0001
Figure imgf000192_0002
Figure imgf000192_0003
Table XXXVIII-109P1D4 v.1-A26-10-mers
Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine
972 FVACDSISKC 16
1006 SVHTRPVGIQ 16,
Figure imgf000193_0004
Figure imgf000193_0001
Table
XLI-
109P1D4 v.1- B1510- 10-mers
No Results
Figure imgf000193_0002
Figure imgf000193_0003
Found.
Figure imgf000194_0001
Figure imgf000194_0002
Figure imgf000194_0003
Figure imgf000194_0004
Figure imgf000195_0004
Figure imgf000195_0002
Figure imgf000195_0003
Figure imgf000195_0001
Figure imgf000196_0001
Figure imgf000196_0003
Figure imgf000196_0004
Figure imgf000196_0002
Figure imgf000197_0003
Figure imgf000197_0001
Figure imgf000197_0002
Figure imgf000197_0004
Figure imgf000198_0002
Figure imgf000198_0001
Figure imgf000198_0003
Figure imgf000199_0001
Figure imgf000199_0002
Figure imgf000199_0003
Figure imgf000200_0001
Figure imgf000200_0002
Figure imgf000200_0003
Figure imgf000200_0004
661
89SCΪ0/l700ZSfl/X3cI SΪS860/1700Z OΛV
Figure imgf000201_0001
Figure imgf000201_0002
Figure imgf000201_0003
Figure imgf000201_0004
ooz
89£Cl0/l700ZSfl/13I SΪS860/1700Z OΛV
Figure imgf000202_0005
Figure imgf000202_0003
Figure imgf000202_0001
Figure imgf000202_0004
TableXXIII
109P1D4V.2
C Terminal-A0201
9-mers
Figure imgf000202_0006
TableXXVI-109P1D4 v.2 C Terminal-A26
Figure imgf000202_0002
9-mers Table XXVIII Each peptide is a
109P1D4v.2 portion of SEQ ID
C'Terminal-BOδ NO: 5; each start
9-mers position is specified, the length of peptide
Each peptide is a portion of SEQ ID is 9 amino acids, and
NO: 5; each start the end position for each peptide is the position is specified, start position plus the length of peptide is 9 amino acids, eight and the end position for each peptide is 11 SRTSTIEIC 131 the start position plus eight VHTRPTDSR 12
6]lTRPTDSRTS][Ϊ2
8 PTDSRTSTI 141 Ϊ4|| STIEICSEI 1|Ϊ2|
10 DSRTSTIEI 13 oll DSRTSTIEJ fs
Ϊ4.1 STIEICSEI l|ϊϊ TllRPTDSRTs irβ
Figure imgf000203_0001
ϊliSVHTRPTDSlfϊ Iό "jI 8 PTDSRTSTI 8
5~]|HTRPTDSRT|[7
TableXXVII
109P1D4V.2
CTerminal-B0702 Table XXIX
9-mers 109P1D4V.2
C' Terminal-B1510-
Each peptide is a 9-mers portion of SEQ ID
NO: 5; each start Each peptide is a position is specified, portion of SEQ iD the length ofpeptide NO: 5; each start is 9 amino acids, position is and the end position specified, the for each peptide is length of peptide is the start position 9 amino acids, and plus eight the end position for each peptide is the start position plus
7|)RPTDSRTST||Ϊ9 eight ϊl|PVSVHTRPT]fϊbl )|HTRPTDSRf][9 4J|VHTRPTDSR|[TT Toll DSRTSTIEl~l[9 lj|PVSVHTRPT.[4
5||HTRPTDSRT|[4
Table XXVIII 6JlTRPTDSRTS|[
109P1D4V.2
C'Terminal-B08
9-mers Table XXX
109P1D4V.2
Each peptide is a C* Terminal-B2705 portion of SEQ ID 9-mers
NO: 5; each start position is specified, the length of peptide
Figure imgf000203_0002
is 9 amino acids, and the end position Table XXXII for each peptide is 109P1D4V.2 the start position C'Terminal-B4402 plus eight 9-mers
Figure imgf000204_0003
Figure imgf000204_0006
Table XXXIX
109P1D4V.2
Figure imgf000204_0005
CTerminal-B0702
10-mers
Each peptide is a portion of SEQ ID NO:
5; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine
Figure imgf000204_0001
Figure imgf000204_0004
Table XXXVII T]|VPVSVHTRPT|[Ϊ8
109P1D4v.2 - C 8 || RPTDSRTSTTl[Ϊ8
Table XXXIV Terminal-A3-10-mers
10 TDSRTSTIEI 9 109P1D4V.2 Each peptide is a C Terminal-A1 -10- portion of SEQ ID mers NO: 5; each start
Each peptide is a position is specified, portion of SEQ ID the length of peptide NO: 5; each start is 10 amino acids, position is specified, and the end position the length of peptide for each peptide is is 10 amino acids, the start position plus and the end position nine for each peptide is the start position plus nine '4 SVHTRPTDSR 17ι
Figure imgf000204_0002
2 PVSVHTRPTD 15
9|| PIDSRTSTIE"[[Ϊ6 m RPIDSRISTΪ1|Ϊ2
6 HTRPTDSRTS 10! 6||HTRPTDSRTSl[Ϊ0
Figure imgf000205_0005
Figure imgf000205_0003
Figure imgf000205_0004
Figure imgf000205_0002
Figure imgf000205_0001
Figure imgf000205_0006
Figure imgf000206_0002
Figure imgf000206_0004
Figure imgf000206_0003
Figure imgf000206_0006
Figure imgf000206_0001
Figure imgf000206_0005
Figure imgf000207_0001
Figure imgf000207_0003
Figure imgf000207_0005
Figure imgf000207_0002
Table XXX-109P1D4 v.2 N' terminal-B2705
9-mers
Each peptide is a portion of SEQ ID
NO: 5; each start position is specified,
Figure imgf000207_0004
the length of peptide is 9 amino acids, and the end position for Table XXXII each peptide is the 109P1D4V.2 N' start position plus terminal-B4402-9- eight mers
Figure imgf000207_0006
Table XXXIII
109P1D4V.2 N' terminal-B5101-
9mers
Each peptide is a portion of SEQ ID
NO: 5; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight
24l|VTSVPGMDL|| 7 5|| TSVPG DLΓJΓ
Figure imgf000208_0004
Figure imgf000208_0006
Figure imgf000208_0001
Figure imgf000208_0002
Figure imgf000208_0007
Table XXXIX
109P1D4V.2 N' terminal-B0702-10mer
Figure imgf000208_0005
Figure imgf000208_0003
Table XLIII
109P1D4V.2
N' terminal-
B2709-
10mer
No Results Found.
Figure imgf000209_0005
Figure imgf000209_0003
Table XL 109P1D4V.2 N' terminal- B08-10mers
Figure imgf000209_0004
No Results Found.
Figure imgf000209_0001
• v
Figure imgf000209_0002
Table XLVI-109P1D4V.2
N' terminal -DRB1 0101
15-mers
Table XLII 109P1D4V.2 Each peptide is a portion of N' terminal- SEQ ID NO: 5; each start
B2705- position is specified, the length 10mer ofpeptide is 15 amino acids, and the end position for each peptide is the start position
No Results plus fourteen
Figure imgf000209_0006
Found.
Table XLVIII-109P1D4V.2 N'
27 VPGMDLLSGTYIFAV 3 terminal-DRB1 0401 -15-mers
21 QQTVTSVPGMDLLSG 31
Figure imgf000210_0001
Figure imgf000210_0003
Table XXIII-109P1D4 V.3-A0201 -9-mers
Each peptide is a portion of SEQ ID NO:
7; each start position is specified, the length of peptide is 9 amino acids, and the end position for each
Figure imgf000210_0002
peptide is the start position plus eight
Figure imgf000210_0004
Figure imgf000211_0004
Figure imgf000211_0002
Figure imgf000211_0001
Figure imgf000211_0003
Figure imgf000212_0002
Figure imgf000212_0004
Table XXVIII-109P1D4 v.3-B08-9-mers
Figure imgf000212_0001
Figure imgf000212_0003
Table XXVIII-109P1D4 v,3-B08-9-mers
Each peptide is a portion of SEQ ID NO:
7; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight
278 GWVQGADGL 11
Figure imgf000213_0003
Table XXX-109P1D4 v.3-B2705-9-mers
Each peptide is a portion of SEQ ID NO:
7; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start
Figure imgf000213_0001
Figure imgf000213_0002
position plus eight
Figure imgf000214_0003
Figure imgf000214_0004
Figure imgf000214_0001
Figure imgf000214_0002
Figure imgf000215_0003
Figure imgf000215_0001
Table XXXIV
109P1D4V.3-A1
Table XXXIII
10-mers
109P1D4v.3-B5101
9-mers
Figure imgf000215_0002
Figure imgf000216_0001
Figure imgf000216_0004
Table XXXVI
109P1D4V.3-A0203
10-mers
Each peptide is a portion of SEQ ID NO:
7; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine
Figure imgf000216_0002
Figure imgf000216_0003
Figure imgf000217_0001
Figure imgf000217_0003
Table XXXVIII
109P1D4V.3-A26
10-mers
Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine
Figure imgf000217_0002
Figure imgf000217_0004
Table XXXVIII Table XLII
109P1D4V.3-A26 109P1D4V.3-
10-mers B2705
10-mers
Each peptide is a portion of SEQ ID NO: 7; each start position is No Results specified, the length of Found. peptide is 10 amino acids, and the end position for each peptide Table XLIII is the start position plus 109P1D4V.3- nine B2709
10-mers
300 SQFYTMSERL 13
303 YTMSERLHPS 13 No Results Found.
Figure imgf000218_0003
Figure imgf000218_0001
Figure imgf000218_0002
Figure imgf000218_0004
Figure imgf000219_0001
Figure imgf000219_0002
Figure imgf000219_0003
Figure imgf000219_0004
Figure imgf000220_0002
Figure imgf000220_0004
Figure imgf000220_0001
Figure imgf000220_0003
Figure imgf000220_0005
Figure imgf000221_0001
Table XXIII
109P1D4V.4-A0201
9-mers
Each peptide is a portion of SEQ ID
NO: 9; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight
2J|WIHPQEQSQ||Ϊ2l
5]|PQPQSQRRV]| 7 l|| IWIHPQPQS][6
Figure imgf000221_0004
Figure imgf000221_0002
Table XXII
109P1D4v.4-A1 Table XXV
9-mers 109P1D4V.4 A3-9-mers
Figure imgf000221_0003
Table XXVIII Table XXXI
109P1D4V.4-B08 109P1D4V.4
9-mers B2709-9-mers
Each peptide is a Each peptide is a portion of SEQ ID portion of SEQ ID
NO: 9; each start NO: 9; each start position is specified, position is the length of peptide specified, the is 9 amino acids, length of peptide is and the end position 9 amino acids, and for each peptide is the end position for the start position each peptide is the plus eight start position plus eight
7|| PQSQRRVTF~l|Ϊ5
5[|PQPQSQRRVl[9l
8l|QSQRRVTFH~l[9
Figure imgf000222_0002
Til PQSQRRVTF] [9] 4"l|HPQPQSQRRl[7 lj| IWlHPQPQSlϋ
Table XXVI
109P1D4V.4-A26 Table XXIX
109P1D4V.4 Table XXXII
9-mers
B1510-9-mers 109P1D4V.4
Each peptide is a B4402-9-mers portion of SEQ ID Each peptide is a t portion of SEQ ID Each peptide is a
NO: 9; each star n is NO: 9; each start portion of SEQ ID positio position is specified, NO: 9; each start specified, the length of peptide is the length of position is specified, the length of
9 amino acids, and peptide is 9 amino the end position acids, and the end peptide is 9 amino end for each peptide is position for each acids, and the the start position peptide is the start position for each plus eight position plus eight peptide is the start position plus eight
7l|PQSQRRVTF|[9 3 IHPQPQSQR 14 21 7[|PQSQRRVTF][loi
7||PQSQRRVTF lWIHPQPQSQ 1 ϊ]| IWIHPQPQS][4 Tj| IWIHPQPQSlfδ]
Table XXXIII
109P1D4V.4-B5101
9-mers
Each peptide is a portion of SEQ ID
NO: 9; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight
l|QPQSQRRVtl|Ϊ4
5l|PQPQSQRRV|[Ϊ2"
4[|HPQPQSQRR1[V[
Figure imgf000222_0001
Figure imgf000222_0003
Figure imgf000223_0002
Figure imgf000223_0001
Table XLV Each peptide is a
109P1D4V.4- portion of SEQ ID
B5101 NO: 11; each start
10-mers position is specified, the length of peptide is 9 amino
No Results acids, and the end Found. position for each peptide is the start position plus eight
Figure imgf000224_0003
5 HTRPSQRRV H6'
3llSVHTRESQ |[6
Table XXIV
109P1D4V.5
A0203-9- mers
No Results Found
Table XXV
109P1D4V.5-A3
9-mers
Each peptide is a portion of SEQ ID
Figure imgf000224_0004
NO: 11; each start position is specified,
Figure imgf000224_0001
the length of
Table XXII peptide is 9 amino
109P1D4V.5-A1 acids, and the end
9-mers position for each
Each peptide is a peptide is the start portion of SEQ ID position plus eight
NO: 11; each start position is specified, the length of 3l|SVHTRPSQR][24 peptide is 9 amino l|RPSQRRVTF][Ϊ9 acids, and the end position for each peptide is the start Table XXVI position plus eight 109P1D4V.5-A26
9-mers
Each peptide is a
5l|HIRPSQRRVl| 0 portion of SEQ ID
^VSVHTRPSQ
Figure imgf000224_0002
NO: 11; each start
8[|PSQRRVIFHl 5 position is specified, the length of
Table XLVIII-109P1D4V.4 peptide is 9 amino DRB1 0401-15-mers Table XXIII acids, and the end
Each peptide is a portion of 109P1D4V.5 position for each
SEQ ID NO: 9; each start A0201 -9-mers peptide is the start position is specified, the position plus eight length of peptide is 15 amino acids, and the end position for each peptide is the start 3]|SVHTRPSQR[[Ϊ3 position plus fourteen 1 PVSVHTRPS 10, Table XXVI
109P1D4V.5-A26 Table XXXII
9-mers 109P1D4V.5
Each peptide is a B4402-9-mers portion of SEQ ID Each peptide is a
NO: 11; each start portion of SEQ ID position is specified, NO: 11; each start the length of position is specified, peptide is 9 amino the length of acids, and the end peptide is 9 amino position for each acids, and the end peptide is the start position for each position plus eight peptide is the start position plus eight
^HTRPSQRRV
TIIRPSQRRVTF"
Figure imgf000225_0001
7]|RPSQRRVTF 15
3 SVHTRPSQR
Table XXVII
109P1D4V.5 Table XXXIII
B0702-9-mers 109P1D4V.5
B5101 -9-mers
Each peptide is a portion of SEQ ID Each peptide is a
NO: 11; each start portion of SEQ ID position is specified, NO: 11; each start the length of position is specified, peptide is 9 amino the length of acids, and the end peptide is 9 amino position for each acids, and the end peptide is the start position for each position plus eight peptide is the start position plus eight
7||RPSQRRVTF" 22
7l|RPSQRRVTFl[Ϊ3
UllHTRPSQRRVp δllHTRPSQRRVlJϊl"
Table XXVIII 6l|TRPSQRRVT][6 109P1D4V.5
Figure imgf000225_0002
B08-9-mers Table XXXIV
Each peptide is a Table XXXI 109P1D4V.5-A1 portion of SEQ ID 109P1D4V.5 10-mers
NO: 11; each start B2709-9-mers Each peptide is a position is specified,
Each peptide is a portion of SEQ ID the length of portion of SEQ ID NO: 11; each start peptide is 9 amino
NO: 11; each start position is specified, acids, and the end position is specified, the length of peptide position for each the length of is 10 amino acids, peptide is the start peptide is 9 amino and the end position position plus eight acids, and the end for each peptide is position for each the start position plus
7 RPSQRRVTF 21 peptide is the start nine position plus eight
3||SVHTRPSQR|[Ϊ0
6l|HIRPSQRRVTl[Ϊ2
TIIRPSQRRVTF" 13 l|VSVHTRPSQRl[5
Table XXIX
109P1D4V.5 llTRPSQRRVf||ϊϊ
B1510-9-mers 5l|HTRPSQRRV|[Ϊ0 Table XLII
109P1D4V.5
B2705-10- mers
No Results Found.
Table XLIII
109P1D4V.5
B2709-10- mers
No Results Found.
Figure imgf000226_0005
Figure imgf000226_0003
Table XLIV
109P1D4V.5-B4402
Table 10-mers
XXXVI Each peptide is a
109P1D4V.5 portion of SEQ ID
A0203-10- NO: 11; each start mers position is specified, the length of peptide is 10 amino acids,
No Results and the end position Found. for each peptide is the start position plus nine
7 TRPSQRRVTF 14
9 PSQRRVTFHL 12
Figure imgf000226_0006
Figure imgf000226_0001
Table XLVI-109P1D4V.5 DRB1 0101 -15-mers
Each peptide is a portion of
Figure imgf000226_0002
SEQ ID NO: 11; each start position is specified, the
Figure imgf000226_0004
Table XLI length of peptide is 15 amino
109P1D4V.5 acids, and the end position for
Table XXXVIII B1510-10- each peptide is the start
109P1D4v.5-A26-10- mers position plus fourteen mers
No Results 4 FEVPVSVHTRPSQRR 22, Found. 3]|TFEVPVSVHTRPSQR||Ϊ7 Table XXIII
109P1D4V.6
C terminal-A0201
9-mers
Each peptide is a portion of SEQ ID
NO: 13; each start position is specified, the length of peptide is
9 amino acids, and the end position for each peptide is the start position plus eight
Figure imgf000227_0001
3]|SVHTRPTDS
Figure imgf000227_0004
[4||VHTRPIDSR1Γ5
Table XXII
109P1D4V.6 Table XXIV
C terminal-A1 109P1D4V.6
9-mers C terminal-
A0203
Each peptide is a
9-mers portion of SEQ ID
NO: 13; each start position is No Results specified, the Found. length of peptide is
9 amino acids, and the end position for Table XXV each peptide is the 109P1D4V.6 start position plus C terminal-A3 eight 9-mers
Each peptide is a:
5 HTRPTDSRT 10 portion of SEQ ID
NO: 13; each start
2l|VSVHTRPTD[[6 position is specified, the
Figure imgf000227_0002
length of peptide is
Table XXIII
9 amino acids, and
109P1D4V.6 the end position for
C terminal-A0201 each peptide is the
9-mers start position plus
Each peptide is a eight portion of SEQ ID
NO: 13; each start position is 3 SVHTRPTDS 15 specified, the T1|PVSVHIRPTJ[iθ length of peptide is
9 amino acids, and 4l|VHIRPIDSR the end position for 5l|HTRPTDSRT each peptide is the start position plus eight Table XXVI 109P1D4V.6 C terminal
5l|HTRPTDSRT|[iθ| A26-9-mers
||PVSVHIRPT||T
Figure imgf000227_0003
Each peptide is a Table XXXI portion of SEQ ID Table XXIX 109P1D4V.6
NO: 13; each start 109P1D4V.6 C terminal-B2709 position is C terminal 9-mers specified, the B1510-9-mers Each peptide is a length of peptide is portion of SEQ ID
9 amino acids, and Each peptide is a
NO: 13; each start the end position for portion of SEQ ID position is each peptide is the NO: 13; each start specified, the start position plus position is length of peptide eight specified, the length of peptide is is 9 amino acids,
9 amino acids, and and the end the end position for position for each
3||SVHTRPTDS1|ΪΪ each peptide is the peptide is the start
1 PVSVHTRPT 10 start position plus position plus eight
5l|HTRPTDSRτl|Tθ eight ξ]|VSVHTRPTD 2||VSVHTRPTDl[2|
[J]|VHTRPTDSR1[ΪΪ 1||HTRPTDSRT||2!
Table XXVII lj|PVSVHTRPT|["4~ 4.|VHTRPTDSR1[Ϊ
109P1D4V.6 ||HTRPTDSRT||T
C terminal-B0702
9-mers Table XXXII
109P1D4V.6
Each peptide is a Table XXX portion of SEQ ID 109P1D4V.6 C terminal-B4402
NO: 13; each start C terminal-B2705 9-mers position is 9-mers Each peptide is a specified, the Each peptide is a portion of SEQ ID length of peptide is portion of SEQ ID NO: 13; each start
9 amino acids, and NO: 13; each start position is the end position for position is specified, the each peptide is the specified, the length of peptide start position plus length of peptide is is 9 amino acids, eight 9 amino acids, and and the end the end position for position for each each peptide is the peptide is the start
T||PVSVHTRPT|[Ϊ0| start position plus position plus eight
^|HTRPTDSRT][9 eight
4 VHTRPTDSR 4 3l|SVHTRPTDSl|4]
4 VHTRPTDSR 12 llPVSVHTRPTlJfl
Table XXVIII 5J|HTRPTDSRT|[5 5]|HTRPTDSRT|[3l
109P1D4V.6
C terminal-B08 2]|VSVHTRPTD|[2]
9-mers Table XXXI 4.|VHTRPTDSR|U
109P1D4V.6
Each peptide is a C terminal-B2709 portion of SEQ ID 9-mers Table XXXIII
NO: 13; each start 109P1D4V.6 position is Each peptide is a
C' terminal-B5101 specified, the portion of SEQ ID
9-mers length of peptide is NO: 13; each start
9 amino acids, and position is the end position for specified, the each peptide is the length of peptide start position plus is 9 amino acids, eight and the end position for each peptide is the start
3l|SVHTRPTDS||ϊθ| position plus eight
5.|HTRPTDSRT|["7
Figure imgf000229_0001
Each peptide is a portion of Each peptide is a
SEQ ID NO: 13; each start portion of SEQ ID position is specified, the NO: 13; each start length of peptide is 15 amino position is specified, acids, and the end position the length of peptide for each peptide is the start is 9 amino acids, position plus fourteen and the end position for each peptide is the start position ξ[|EVPVSVHTRPTDSRT||Ϊ6 plus eight
3]|TFEVPVSVHTRPTDS|[Ϊ0 lllVTTFEVPVSVHTRPTlf ) 6 NSDISSVVR 15
121 HKCLLSGTY 15
Table XLVIII-109P1D4V.6 1 MTVGFNSDI 8
C terminal-DRB1 0401
15-mers 17 TTNCHKCLL 8
Each peptide is a portion of 18 TNCHKCLLS 8
SEQ ID NO: 13; each start position is specified, the length of peptide is 15 amino acids, and the end position for each peptide is the start
Figure imgf000230_0001
position plus fourteen
Table XLV
109P1D4V.6 T|| VTTFEVPVSVHTRPT |[22
C terminal-
4l|FEVPVSVHTRPTDSR||Ϊ8
B5101
10-mers 3l|TFEVPVSVHTRPTDS|[Ϊ4 l|EVPVSVHTRPTDSRT||Ϊ4
No Results Found. Table XLIX-109P1D4V.6
C terminal-DRB1 1101
15-mers
Table XLVI-109P1D4V.6
C' terminal-DRB1 0101 Each peptide is a portion of
15-mers SEQ ID NO: 13; each start position is specified, the
Each peptide is a portion of length of peptide is 15 amino
SEQ ID NO: 13; each start acids, and the end position position is specified, the for each peptide is the start length of peptide is 15 amino position plus fourteen acids, and the end position for each peptide is the start position plus fourteen 3l|TFEVPVSVHTRPTDS|[25
5l|EVPVSVHTRPTDSRT||l~5"
3 TFEVPVSVHTRPTDS 17 1 VTTFEVPVSVHTRPT 13
T.|VTTFEVPVSVHTRPT||Ϊ6
Figure imgf000230_0002
4||FEVPVSVHTRPTDSR|[Ϊ4 Table XXII
5[|EVPVSVHTRPTDSRT 109P1D4v,6 Table XXIV
N' terminal-A1 109P1D4V.6
9-mers N' terminal-
TableXLVII-109P1D4v,6 A0203
C terminal-DRB1 0301 9-mers
15-mers
No Results Found.
Figure imgf000231_0001
Figure imgf000231_0004
Figure imgf000231_0003
Table XXVIII
109P1D4V.6
N' terminal-B08
9-mers
Each peptide is a portion of SEQ ID
NO: 13; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight
10 SSWRVNTT 12!
23 CLLSGTYIF 12
16 NTTNCHKCL 11
17 TTNCHKCLL 10
18 TNCHKCLLS 10
Figure imgf000231_0005
20 CHKCLLSGT 10
12 WRVNTTNC, Table XXXI
109P1D4V.6 ϊl|MTVGFNSDΪ[[7
Figure imgf000231_0002
N' terminal-B2709 ^2|| KCLLSGTYll[τ 9-mers
Figure imgf000232_0005
Table XXXVI
109P1D4V.6
N' terminal-A0203
10-mers
Each peptide is a portion of SEQ ID
NO: 13; each start position is specified,
Figure imgf000232_0001
Figure imgf000232_0003
the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine
23 CLLSGTYIFA 10
Figure imgf000232_0004
Table XXXV
109P1D4V.6
Figure imgf000232_0002
N' terminal-A0201
10-mers
Table XXXIII
Each peptide is a
109P1D4V.6 portion of SEQ ID NO:
N' terminal-B5101
13; each start position
9-mers is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine
Figure imgf000232_0006
Figure imgf000233_0003
Figure imgf000233_0002
Figure imgf000233_0001
Table XXII-
109P1D4V.7
N' terminal-A1
9-mers
Each peptide is a portion of SEQ ID
NO: 15; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight
14 SLSPLLLVS 14
1 MFRVGFLII 11
9 ISSSSSLSP 10
Figure imgf000234_0003
Figure imgf000234_0001
11 SSSSLSPLL 8
Figure imgf000234_0004
Table XXII-
109P1D4V.7
N' terminal-A1
9-mers
Each peptide is a
Figure imgf000234_0002
portion of SEQ ID
NO: 15; each start
Table XLVIII-109P1D4V.6 position is specified,
N' terminal-DRB1 0401 the length of
Figure imgf000234_0005
15-mers peptide is 9 amino acids, and the end Table XXIV-
Each peptide is a portion of position for each 109P1D4V.7
SEQ ID NO: 13; each start peptide is the start position is specified, the N' terminal- position plus eight A0203 length of peptide is 15 amino
9-mers acids, and the end position for each peptide is the start 13 SSLSPLLLV 15 position plus fourteen
12 SSSLSPLLL 14 No Results Found. Table XXVIII
109P1D4V.7
N' terminal-B08
9-mers
Each peptide is a portion of SEQ ID
NO: 15; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight
19 LLVSWRVN 7
Table XXIX
109P1D4V.7
N' terminal-B1510
9-mers
Each peptide is a portion of SEQ ID
NO: 15; each start position is specified, the length of peptide
Figure imgf000235_0003
is 9 amino acids,
Figure imgf000235_0001
and the end position for each peptide is the start position
■ plus eight
11 SSSSLSPLL 12
12 SSSLSPLLL 12
10 SSSSSLSPL 11
7|| LIISSSSSL 1[ϊo
T8l|LLLVSVVRV|["6
Table XXX
109P1D4V.T
N' terminal-B2T05
9-mers
Each peptide is a portion of SEQ ID
NO: 15; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position
Figure imgf000235_0002
plus eight
ΪTJ|PLLLVSVVRl[ΪT
Figure imgf000235_0004
Y\ UISSSSSL][Ϊ6~
Figure imgf000236_0004
Figure imgf000236_0003
Table XXXIII
Figure imgf000236_0001
109P1D4V.7
N' terminal-B5101
9-mers
Each peptide is a portion of SEQ ID
NO: 15; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight
16 SPLLLVSW 25
Ϊ8l|LLLVSWRV][Ϊ7 ϊ]| MFRVGFUI |[Ϊ3
Ϊ5||LSPLLLVSV|[Ϊ3
Figure imgf000236_0005
Table XXXIV
109P1D4V.7 Table
N' terminal-A1 XXXVI
10-mers 109P1D4V.7
N' terminal
A0203-10- mers
Figure imgf000236_0002
No Results Found.
Figure imgf000237_0002
Figure imgf000237_0004
Figure imgf000237_0005
Figure imgf000237_0003
Figure imgf000237_0001
XLVIII-109P1D4V.7 Table XXII
Table XLVI-109P1D4V.T
109P1D4V.8-A1
N" terminal-DRBl 0101 N' terminal-DRB1 0401
9-mers
15-mers 15-mers eptide is a portion of Each peptide is a
Each peptide is a portion of Each p portion of SEQ ID
SEQ ID NO: 15; each start SEQ ID NO: 15; each start
NO: 17; each start position is specified, the position is specified, the position is length of peptide is 15 amino length of peptide is 15 amino specified, the acids, and the end position for acids, and the end position fori length of peptide each peptide is the start each peptide is the start position plus fourteen position plus fourteen is 9 amino acids, and the end position for each
T]| MFRVGFUISSSSSLJ25I peptide is the start position plus eight
Til VGFUISSSSSLSPL~||25
Ϊ2lfSSSLSPLLLVSWRV |l24 τl|KKEITVQPT|lϊϊ
Ϊ5|| LSPLLLVSWRVNTT] |23
1 TFIPGLKKE il GFUISSSSSLSPLL ||22 ll FLIISSSSSLSPLLL ||22
Table XXIII
9 II ISSSSSLSPLLLVSV ||22' 109P1D4V.8
20 LVSWRVNTTNCHKC1 22 A0201 -9-mers
1\\ FRVGFUISSSSSLS H2I Each peptide is a portion of SEQ ID
13 SSLSPLLLVSWRVN ||17| NO: 17; each start position is
Table XLVII-109P1D4V.7 specified, the
N' terminal-DRB1 0301 | length of peptide is
15-mers
Figure imgf000238_0001
9 amino acids, and the end position
Each peptide is a portion of for each peptide is
SEQ ID NO: 15; each start the start position position is specified, the plus eight length of peptide is 15 amino jacids, and the end position for) each peptide is the start 2l| FIPGLKKEI II2 position plus fourteen | IKEITVQPTV
5]|GLKKElTVQl|Ϊ4
4 1| VGFLIISSSSSLSPL]|20]
4l|PGLK ilTV||Ϊ2
Ϊ7|| PLLLVSVVRVNTTNC ||20
15|| LSPLLLVSWRVNTT ||1 Table XXIV
5 || GFLIISSSSSLSPLJJI14J 109P1D4V.8
A0203-9-
6 II FUISSSSSLSPLLLJ|13 mers
12ll SSSLSPLLLVSWRV ||13
9 ISSSSSLSPLLLVSV ||12|
No Results
16|| SPLLLVSWRVNTTN] [12] Found
20 LVSWRVNTTNCHKC]^
21HVSWRVNTTNCHKCL||12J Table XXV
31ΓRVGFUISSSSSLSP]|II 109P1D4V.8 A3-9-mers
HSSSSSLSPLLLVS 11
18 LLLVSWRVNTTNCH 11
||Tl| MFRVGFUISSSSSL ||ϊθ|
Figure imgf000238_0002
[Til UISSSSSLSPLLLV ||lθ| Table XXVII Table XXIX
109P1D4V.8 109P1D4V.8
B0702-9-mers B1510-9-mers
Each peptide is a Each peptide is a portion of SEQ ID portion of SEQ ID
NO: 17; each start NO: 17; each position is start position is specified, the specified, the length of peptide length of peptide is 9 amino acids, is 9 amino acids, and the end and the end position for each position for each peptide is the start peptide is the position plus eight start position plus eight
7l|KKEITVQPT||~9
Ϊ1|TF1PGLKKE|[4
Figure imgf000239_0001
2.1 FIPGLKKEI |jl
3]| IPGLKKEIT
Table XXVI βJlLKKElTVQPlJf 109P1D4V.8 A26-9-mers
Each peptide is a Table XXX portion of SEQ ID 109P1D4V.8
NO: 17; each start B2705-9-mers position is Each peptide is a specified, the portion of SEQ ID length of peptide NO: 17; each start is 9 amino acids, position is and the end specified, the position for each length of peptide is peptide is the start 9 amino acids, and position plus eight the end position for each peptide is the start position
1 TFIPGLKKE 11 plus eight
2 FIPGLKKEI 5
,6 LKKEITVOP 5 5l|GLKKEITVQ||Ϊ2,
8||KEITVQPTV|
Figure imgf000239_0002
2l| FIPGLKKEI |lϊϊ
Table XXIX 8|lKElTVQPTVl[T
Table XXVII 109P1D4V.8 ϊllTFIPGLKKElli
109P1D4V.8 B1510-9-mers
B0702-9-mers 4JlPGLKKEITV| Y
Each peptide is a
Each peptide is a portion of SEQ ID portion of SEQ ID NO: 17; each Table XXXI
NO: 17; each start start position is 109P1D4V.8 position is specified, the B2709-9-mers specified, the length of peptide length of peptide is 9 amino acids, is 9 amino acids, and the end and the end position for each position for each peptide is the peptide is the start start position plus position plus eight eight
jj|| IPGLKKEIT |[Ϊ8 ^GLKKEITVQ Each peptide is a Table XXXIV portion of SEQ ID 109P1D4V.8
NO: 17; each start A1 -10-mers position is
Each peptide is a specified, the portion of SEQ ID length of peptide NO: 17; each start is 9 amino acids, position is specified, and the end the length of position for each peptide is 10 amino peptide is the start acids, and the end position plus eight ition for each pepti de is the start
8l|KEITVQPTV|[Ϊ2 posi ition plus nine
4 PGLKKEITV 10
Figure imgf000240_0002
ξ|| FIPGLKKEI [ 8 I||SIFIPGLKKE|[ΪO
8l|KKEITVQPTV|[iθ
Table XXXVIIl
Table XXXII 109P1D4V.8
109P1D4V.8 A26-10-mers
B4402-9-mers Each peptide is a
Each peptide is a portion of SEQ ID portion of SEQ ID NO: 17; each start
NO: 17; each start position is position is specified, the length specified, the of peptide is 10 length of peptide amino acids, and is 9 amino acids, the end position for and the end each peptide is the position for each start position plus peptide is the start nine position plus eight ϊ]|STFlPGLKKE]fΪ8
8llKElTVQPTVl[Ϊ6
2ll FIPGLKKEI [|Ϊ2
Ϊ1|TFIPGLKKE||Ϊ0
Table XXXIII
109P1D4V.8
B5101 -9-mers
Each peptide is a
Figure imgf000240_0001
portion of SEQ ID NO: 17; each start Table position is XXXVI specified, the 109P1D4V.8 length of peptide A0203-10- is 9 amino acids, mers and the end position for each peptide is the start No Results position plus eight Found.
4 PGLKKEITV 21 Table XXXVII
Figure imgf000240_0003
109P1D4V.8 i2 FIPGLKKEI 14 A3-10-mers Table XL
3|[ IPGLKKEIT ||Ϊ3 109P1D4V.8
B08-10-
8 KEITVQPTV 131 mers No Results Table XLV Found. 109P1D4v.8 B5101 -10- mers
Table XLI
109P1D4V.8
B1510-10- No Results mers Found.
No Results Found.
Table XLII
109P1D4V.8
B2705-10- mers
No Results Found.
Figure imgf000241_0003
Table XLI 11 Table XLIX-109P1D4V.8
109P1D4V.8 DRB1 1101-15-mers
B2709-10- Each peptide is a portion mers of SEQ ID NO: 17; each start position is specified,
Figure imgf000241_0001
the length of peptide is 15
No Results amino acids, and the end Found. position for each peptide is the start position plus
Table XLIV fourteen
109P1D4V.8 βl|STFlPGLKKEITVQP|[2Ϊ
B4402-10-mers oj|ESTFIPGLKKEiTVQ|[Ϊ8
Each peptide is a portion of SEQ ID 9||lPGLKKElTVQPTVE 12
NO: 17; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine
9||KEITVQPTVEl[ΪT
Figure imgf000241_0002
2.| TFIPGLKKEil[Ϊ6 Table L: Protein Characteristics of 109P1D4
109PlD4 var.l Bioinformatic URL on World Wide Web Outcome Program
ORF ORF finder 846-3911 bp (includes stop codon) Protein length 1021aa
Transmembrane TM Pred xh.embnet.org/ 3 TM helices (aa3-aa23, aa756- region aa776, aa816-aa834), N terminus intracellular
HMMTop .enzim.hu/hmmtop/ no TM, N terminus extracellular
Sosui .genome.ad.jp/SOSui/ 3 TM helices (2-24aa, 756-778aa, 810-832aa), N terminus extracellular
TMHMM .cbs.dtu.dk/services/TMHMM 1TM helix (813-835aa), N terminus extracellular
Signal Peptide Signal P .cbs.dtu.dk/services/SignalP/ yes pi pI/MW tool .expasy.ch/tools/ pi 4.81
Molecular weight pI MW tool .expasy.ch/tools/ 112.7 kDa
Localization PSORT psort.nibb.ac.jp/ Plasma membrane
PSORT II psort.nibb.ac.jp/ 67% endoplasmic reticulum
Motifs Pfam .sanger.ac.uk/Pfam/ Cadherin domain
Prints .biochem.ucl . ac.uk/ Cadherin domain, DNA topoiso- Merase 4B, sonic hedgehog Blocks .blocks.fhcrc.org/ Cadherin domain, ribosomal protein LIOE, ribulose biphos- phate carboxylase (large chain), omithine decarboxylase antizyme protein phosphatase 2C subfamily
Table LI. Exon boundaries of transcript 109P1D4 v.1
Figure imgf000242_0001
Table Lll(a). Nucleotide sequence of transcript variant 109P1 D4 v.2 (SEQ ID NO: 237) cccctttctc cccctcggtt aagtccctcc ccctcgccat tcaaaagggc tggctcggca 60 ctggctcctt gcagtcggcg aactgtcggg gcgggaggag ccgtgagcag tagctgcact 120 cagctgcccg cgcggcaaag aggaaggcaa gccaaacaga gtgcgcagag tggcagtgcc 180 agcggcgaca caggcagcac aggcagcccg ggctgcctga atagcctcag aaacaacctc 240 agcgactccg gctgctctgc ggactgcgag ctgtggcggt agagcccgct acagcagtcg 300 cagtctccgt ggagcgggcg gaagcctttt ttctcccttt cgtttacctc ttcattctac 360 tctaaaggca tcgttattag gaaaatcctg ttgcgaataa gaaggattcc acagatcaca 420 taccggagag gttttgcctc agctgctctc aactttgtaa tcttgtgaag aagctgacaa 480 gcttggctga ttgcagagca ctatgaggac tgaacgacag tgggttttaa ttcagatatt 540 tcaagtgttg tgcgggttaa tacaacaaac tgtaacaagt gtacctggta tggacttgtt 600 gtccgggacg tacattttcg cggtcctgct agcatgcgtg gtgttccact ctggcgccca 660 ggagaaaaac tacaccatcc gagaagaaat gccagaaaac gtcctgatag gcgacttgtt 720 gaaagacctt aacttgtcgc tgattccaaa caagtccttg acaactgcta tgcagttcaa 780 gctagtgtac aagaccggag atgtgccact gattcgaatt gaagaggata ctggtgagat 840 cttcactact ggcgctcgca ttgatcgtga gaaattatgt gctggtatcc caagggatga 900 gcattgcttt tatgaagtgg aggttgccat tttgccggat gaaatattta gactggttaa 960 gatacgtttt ctgatagaag atataaatga taatgcacca ttgttcccag caacagttat 1020 caacatatca attccagaga actcggctat aaactctaaa tatactctcc cagcggctgt 1080 tgatcctgac gtaggaataa acggagttca aaactacgaa ctaattaaga gtcaaaacat 1140 ttttggcctc gatgtcattg aaacaccaga aggagacaag atgccacaac tgattgttca 1200 aaaggagtta gatagggaag agaaggatac ctacgtgatg aaagtaaagg ttgaagatgg 1260 tggctttcct caaagatcca gtactgctat tttgcaagtg agtgttactg atacaaatga 1320 caaccaccca gtctttaagg agacagagat tgaagtcagt ataccagaaa atgctcctgt 1380 aggcacttca gtgacacagc tccatgccac agatgctgac ataggtgaaa atgccaagat 1440 ccacttctct ttcagcaatc tagtctccaa cattgccagg agattatttc acctcaatgc 1500 caccactgga cttatcacaa tcaaagaacc actggatagg gaagaaacac caaaccacaa 1560 gttactggtt ttggcaagtg atggtggatt gatgccagca agagcaatgg tgctggtaaa 1620 tgttacagat gtcaatgata atgtcccatc cattgacata agatacatcg tcaatcctgt 1680 caatgacaca gttgttcttt cagaaaatat tccactcaac accaaaattg ctctcataac 1740 tgtgacggat aaggatgcgg accataatgg cagggtgaca tgcttcacag atcatgaaat 1800 ccctttcaga ttaaggccag tattcagtaa tcagttcctc ctggagactg cagcatatct 1860 tgactatgag tccacaaaag aatatgccat taaattactg gctgcagatg ctggcaaacc 1920 tcctttgaat cagtcagcaa tgctcttcat caaagtgaaa gatgaaaatg acaatgctcc 1980 agttttcacc cagtctttcg taactgtttc tattcctgag aataactctc ctggcatcca 2040 gttgacgaaa gtaagtgcaa tggatgcaga cagtgggcct aatgctaaga tcaattacct 2100 gctaggccct gatgctccac ctgaattcag cctggattgt cgtacaggca tgctgactgt 2160 agtgaagaaa ctagatagag aaaaagagga taaatattta ttcacaattc tggcaaaaga 2220 taacggggta ccacccttaa ccagcaatgt cacagtcttt gtaagcatta ttgatcagaa 2280 tgacaatagc ccagttttca ctcacaatga atacaacttc tatgtcccag aaaaccttcc 2340 aaggcatggt acagtaggac taatcactgt aactgatcct gattatggag acaattctgc 2400 agttacgctc tccattttag atgagaatga tgacttcacc attgattcac aaactggtgt 2460 catccgacca aatatttcat ttgatagaga aaaacaagaa tcttacactt tctatgtaaa 2520 ggctgaggat ggtggtagag tatcacgttc ttcaagtgcσ aaagtaacca taaatgtggt 2580 tgatgtcaat gacaacaaac cagttttcat tgtccctcct tccaactgtt cttatgaatt 2640 ggttctaccg tccactaatc caggcacagt ggtctttcag gtaattgctg ttgacaatga 2700 cactggcatg aatgcagagg ttcgttacag cattgtagga ggaaacacaa gagatctgtt 2760 tgcaatcgac caagaaacag gcaacataac attgatggag aaatgtgatg ttacagacct 2820 tggtttacac agagtgttgg tcaaagctaa tgacttagga cagcctgatt ctctcttcag 2880 tgttgtaatt gtcaatctgt tcgtgaatga gtcggtgacc aatgctacac tgattaatga 2940 actggtgcgc aaaagcactg aagcaccagt gaccccaaat actgagatag ctgatgtatc 3000 ctcaccaact agtgactatg tcaagatcct ggttgcagct gttgctggca ccataactgt 3060 cgttgtagtt attttcatca ctgctgtagt aagatgtcgc caggcaccac accttaaggc 3120 tgctcagaaa aacaagcaga attctgaatg ggctacccca aacccagaaa acaggcagat 3180 gataatgatg aagaaaaaga aaaagaagaa gaagcattcc cctaagaact tgctgcttaa 3240 ttttgtcact attgaagaaa ctaaggcaga tgatgttgac agtgatggaa acagagtcac 3300 actagacctt cctattgatc tagaagagca aacaatggga aagtacaatt gggtaactac 3360 acctactact ttcaagcccg acagccctga tttggcccga cactacaaat ctgcctctcc 3420 acagcctgcc ttccaaattc agcctgaaac tcccctgaat tcgaagcacc acatcatcca 3480 agaactgcct ctcgataaca cctttgtggc ctgtgactct atctccaagt gttcctcaag 3540 cagttcagat ccctacagcg tttctgactg tggctatcca gtgacgacct tcgaggtacc 3600 tgtgtccgta cacaccagac cgactgattc caggacatca actattgaaa tctgcagtga 3660 gatataactt tctaggaaca acaaaattcc attccccttc caaaaaattt caatgattgt 3720 gatttcaaaa ttaggctaag atcattaatt ttgtaatcta gatttcccat tataaaagca 3780 agcaaaaatc atcttaaaaa tgatgtccta gtgaaccttg tgctttcttt agctgtaatc 3840 tggcaatgga aatttaaaat ttatggaaga gacagtgcag cacaataaca gagtactctc 3900 atgctgtttc tctgtttgct ctgaatcaac agccatgatg taatataagg ctgtcttggt 3960 gtatacactt atggttaata tatcagtcat gaaacatgca attacttgcc ctgtctgatt 4020 gttgaataat taaaacatta tctccaggag tttggaagtg agctgaacta gccaaactac 4080 tctctgaaag gtatccaggg caagagacat ttttaagacc ccaaacaaac aaaaaacaaa 4140 accaaaacac tctggttcag tgttttgaaa atattcacta acataatatt gctgagaaaa 4200 tcatttttat tacccaccac tctgcttaaa agttgagtgg gccgggcgcg gtggctcacg 4260 cctgtaatcc cagcactttg ggaggccgag gcgggtggat cacgaggtca ggagattgag 4320 accatcctgg ctaacacggt gaaaccccat ctccactaaa aatacaaaaa attagcctgg 4380 cgtggtggcg ggcgcctgta gtcccagcta ctcgggaggc tgaggcagga gaatagcgtg 4440 aacccgggag gcggagcttg cagtgagccg agatggcgcc actgcactcc agcctgggtg 4500 acagagcaag actctgtctc aaaaagaaaa aaatgttcaa tgatagaaaa taattttact 4560 aggtttttat gttgattgta ctcatgctgt tccactcctt ttaattatta aaaagttatt 4620 tttggctggg tgtggtggct cacacctgta atcccagcac tttgggaggc cgaggtgggt 4680 ggatcacctg aggtcaggag ttcaagacca gtctggccaa cat 4723 Table Llll(a). Nucleotide sequence alignment of 109P1D4 v.1 (SEQ ID NO: 238) and 109P1D4 v.2 (SEQ ID NO: 239)
Score = 5920 bits (30T9), Expect = O.OIdentities = 3079/3079 (100%) Strand = Plus / Plus
V.l : 800 agtgttgtgcgggttaatacaacaaactgtaacaagtgtacctggtatggacttgttgtc 859
I III II II II II I I I I I I II I I I llll I II I III I II II II M I I M II M II II M II I V.2 : 544 agtgttgtgcgggttaatacaacaaactgtaacaagtgtacctggtatggacttgttgtc 603
V.l : 860 cgggacgtacattttcgcggtcctgctagcatgcgtggtgttccactctggcgcccagga 919
II II II II II I I I I I I II I I I I I llll I II II I I I II II II I I I I I I II II I I I I II II I V.2 : 604 cgggacgtacattttcgcggtcctgctagcatgcgtggtgttccactctggcgcccagga 663
V.l : 920 gaaaaactacaccatccgagaagaaatgccagaaaacgtcctgataggcgacttgttgaa 979
II II I I II II II I I I I II I I I I I I llll II llll I I I II III I I I I I I I II I I I I II II I V.2 : 664 gaaaaactacaccatccgagaagaaatgccagaaaacgtcctgataggcgacttgttgaa 723
V.l : 980 agaccttaacttgtcgctgattccaaacaagtccttgacaactgctatgcagttcaagct 1039
I I llll II II III I I I II I I I I I III II II II II I II II II I I I I I I II II I II I I II I I V.2 : 724 agaccttaacttgtcgctgattccaaacaagtccttgacaactgctatgcagttcaagct 783
V.l : 1040 agtgtacaagaccggagatgtgccactgattcgaattgaagaggatactggtgagatctt 1099
I I III I II II II I I I I II I I I I I III II II II II II I II I I I I I I II I I I I I I I I II I I I V.2 : 784 agtgtacaagaccggagatgtgccactgattcgaattgaagaggatactggtgagatctt 843
V.l : 1100 cactactggcgctcgcattgatcgtgagaaattatgtgctggtatcccaagggatgagca 1159
II III I II II II I I I I I I I I I I I I III I Mil I I II I I II I I I I I I I I II I I I I) I I II I V.2 : 844 cactactggcgctcgcattgatcgtgagaaattatgtgctggtatcccaagggatgagca 903
V.l : 1160 ttgcttttatgaagtggaggttgccattttgccggatgaaatatttagactggttaagat 1219 llll I I Ml I II I I I II I I I I I I llll I III II I II II III I I I II I I II I I I II I I III V.2 : 904 ttgcttttatgaagtggaggttgccattttgccggatgaaatatttagactggttaagat 963
V.l : 1220 acgttttctgatagaagatataaatgataatgcaccattgttcccagcaacagttatcaa 1279
I III I I III III I I II I II II I I I I I I I I III I I I I II III I II II I III I I III I M II
V.2 : 964 aogttttctgatagaagatataaatgataatgcaccattgttcccagcaacagttatcaa 1023
V.l : 1280 catatcaattccagagaaotcggctataaactctaaatatactctcccagcggctgttga 1339
II I II I II I II II I I I II I II I I III II II II II I II II I II II II II I I II II II I II I
V.2 : 1024 catatcaattccagagaactcggctataaactctaaatatactctcccagcggctgttga 1083
V.l : 1340 tcctgacgtaggaataaacggagttcaaaactacgaactaattaagagtcaaaacatttt 1399
I llll II I II II I I I M I I I I I I MM I III II I II II II I I I I I III II I I I II I I III V.2 : 1084 tcctgacgtaggaataaacggagttcaaaactacgaactaattaagagtcaaaacatttt 1143
V.l : 1400 tggcctcgatgtcattgaaacaccagaaggagacaagatgccacaactgattgttcaaaa 1459
1 I III I II I II I II II I I I II I llll II I I II II I I II II I I II II I I II II II I I II II V.2 : 1144 tggcctcgatgtcattgaaacaccagaaggagacaagatgccacaactgattgttcaaaa 1203
V.l : 1460 ggagttagatagggaagagaaggatacctacgtgatgaaagtaaaggttgaagatggtgg 1519
I II II I I I I I I II I I I I I I II I I III I I II III I II I I II II I I I I II II II I I II I II I
V.2 : 1204 ggagttagatagggaagagaaggatacctacgtgatgaaagtaaaggttgaagatggtgg 1263
V.l : 1520 ctttcctcaaagatccagtactgctattttgcaagtgagtgttactgatacaaatgacaa 1579
II II I I I I I II I I I II I I 1 I 1 I II I II II III I I II II II II I I II I 111 I I I I II II II
V.2 : 1264 ctttcctcaaagatccagtactgctattttgcaagtgagtgttactgatacaaatgacaa 1323 V.l : 1580 ccacccagtctttaaggagacagagattgaagtcagtataccagaaaatgctcctgtagg 1639
I II I II II MM I II II I I I I II II I II II II II llll II II II I II I II I I I I II llll V.2 : 1324 ccacccagtctttaaggagacagagattgaagtcagtataccagaaaatgctcctgtagg 1383
V.l : 1640 cacttcagtgacacagctccatgccacagatgctgacataggtgaaaatgccaagatcca 1699
1 I I I 1 I I 1 I I I I 1 I I 1 M 1 I I I I I I 1 I 1 I I 1 I I I I I I I I I II I I I ! 11 I I I I I I I I I I I I V.2 : 1384 cacttcagtgacacagctccatgccacagatgctgacataggtgaaaatgccaagatcca 1443
V.l : 1700 cttctctttcagcaatctagtctccaacattgccaggagattatttcacctcaatgccac 1759
III 11 II II II II I I I II I I I 111 II I II II II II III II I I I II I I I I 111 I I I II II 1 V.2 : 1444 cttctctttcagcaatctagtctccaacattgccaggagattatttcacctcaatgccac 1503
V.l : 1760 cactggacttatcacaatcaaagaaccactggatagggaagaaacaccaaaccacaagtt 1819
III II I I II II II II I I I I I I II I II I I III II II I II II I I I II I II I I II I I I II II I V.2 : 1504 cactggacttatcacaatcaaagaaccactggatagggaagaaacaccaaaccacaagtt 1563
V.l : 1820 actggttttggcaagtgatggtggattgatgccagcaagagcaatggtgctggtaaatgt 1879
I I I I I II I II III II II I I I I II I I II I II II MM III II I I I II I I II llll II I II I V.2 : 1564 actggttttggcaagtgatggtggattgatgccagcaagagcaatggtgctggtaaatgt 1623
V.l : 1880 tacagatgtcaatgataatgtcccatccattgacataagatacatcgtcaatcctgtcaa 1939
I I I I I II I I I I I I II III I I I I II II II II II II II III I I I II 111 I 1 I I I II II llll V.2 : 1624 tacagatgtcaatgataatgtcccatccattgacataagatacatcgtcaatcctgtcaa 1683
V.l : 1940 tgacacagttgttctttcagaaaatattccactcaacaccaaaattgctctcataactgt 1999
II II I I I I I I I I I II I I I I I I I II II I I II II II II III I I I II I I II II I I I I II II I I V.2 : 1684 tgacacagttgttctttcagaaaatattccactcaacaccaaaattgctctcataactgt 1743
V.l : 2000 gacggataaggatgcggaccataatggcagggtgacatgcttcacagatcatgaaatccc 2059
I I II II I I II I I I I II I I I I I I MM II llll II II MM I I II I I II II I I II II llll V.2 : 1744 gacggataaggatgcggaccataatggcagggtgacatgcttcacagatcatgaaatccc 1803
V.l : 2060 tttcagattaaggccagtattcagtaatcagttcctcctggagactgcagcatatcttga 2119
I I I I I II I I I I I I I I I I I I I I I III I II I I I III I I I I I I I I II I I I I I II I I I II I II I
V.2 : 1804 tttcagattaaggccagtattcagtaatcagttcctcctggagactgcagcatatcttga 1863
V.l : 2120 ctatgagtccacaaaagaatatgccattaaattactggctgcagatgctggcaaacctcc 2179
II II II I I I I I I I II I II I I I I II I I I III I I I II I I I I I I I II I I I II I I I II I I I II I
V.2 : 1864 ctatgagtccacaaaagaatatgccattaaattactggctgcagatgctggcaaacctcc 1923
V.l : 2180 tttgaatcagtcagcaatgctcttcatcaaagtgaaagatgaaaatgacaatgctccagt 2239
I I I II II II II I I II II I I I I II II II I II I II II I II I II I II II I II I II I I I II II 1 V.2 : 1924 tttgaatcagtcagcaatgctcttcatcaaagtgaaagatgaaaatgacaatgctccagt 1983
V.l : 2240 tttcacccagtctttcgtaactgtttctattcctgagaataactctcctggcatccagtt 2299
I II I II II I II II I I I I I I I I II I I II I III I I I I II I III I II I I I II I II I I I I I I II V.2 : 1984 tttcacccagtctttcgtaactgtttctattcctgagaataactctcctggcatccagtt 2043
V.l : 2300 gacgaaagtaagtgcaatggatgcagacagtgggcctaatgctaagatcaattacctgct 2359
I II I II I I I II II I II I I I I I II III I I I II III I I I III I I I II I II I I II I I II II II V.2 : 2044 gacgaaagtaagtgcaatggatgcagacagtgggcctaatgctaagatcaattacctgct 2103
V.l : 2360 aggccctgatgctccacctgaattcagcctggattgtcgtacaggcatgctgactgtagt 2419
I II I I II II I II I II I I I I I I I II I I I II III II II II 11 I I I I I I II I I I I II II Ml I V.2 : 2104 aggccctgatgctccacctgaattcagcctggattgtcgtacaggcatgctgactgtagt 2163 V.l : 2420 gaagaaactagatagagaaaaagaggataaatatttattcacaattctggcaaaagataa 2479
MIMMMIMMMMIMMIMMIMIMMMMMMMIMMMMIMM V.2 : 2164 gaagaaactagatagagaaaaagaggataaatatttattcacaattctggcaaaagataa 2223
V.l : 2480 cggggtaccacccttaaccagcaatgtcacagtctttgtaagcattattgatcagaatga 2539
II Mill II III Mill Ml I I II II I II II II II I I MM II II II M M I I II M II I V.2 : 2224 cggggtaccacccttaaccagcaatgtcacagtctttgtaagcattattgatcagaatga 2283
V.l : 2540 caatagcccagttttcactcacaatgaatacaacttctatgtcccagaaaaccttccaag 2599
II I I MM II 11 II II II I llll I I MM M II II I II I II III llll I II I I I I II I V.2 : 2284 caatagcccagttttcactcacaatgaatacaacttctatgtcccagaaaaccttccaag 2343
V.l : 2600 gcatggtacagtaggactaatcactgtaactgatcctgattatggagacaattctgcagt 2659
II Mlllll Ml II I II III I I II II I I II I II II I I Ml II I II II II II I I I I II IM V.2 : 2344 gcatggtacagtaggactaatcactgtaactgatcctgattatggagacaattctgcagt 2403
V.l : 2660 tacgctctccattttagatgagaatgatgacttcaccattgattcacaaactggtgtcat 2719
I I I MIMIIMM MM I I II I I I I III I II Mill III II II II II III II I I M I V.2 : 2404 tacgctctccattttagatgagaatgatgacttcaccattgattcacaaactggtgtcat 2463
V.l : 2720 ccgaccaaatatttcatttgatagagaaaaacaagaatcttacactttctatgtaaaggc 2779
I I I MUM MM llll IM I III II II II I II II I I III II I II II II II II I I I 111 I V.2 : 2464 ccgaccaaatatttcatttgatagagaaaaacaagaatcttacactttctatgtaaaggc 2523
V.l : 2780 tgaggatggtggtagagtatcacgttcttcaagtgccaaagtaaccataaatgtggttga 2839
I I I I III II II I Mill I I Ml I I I I II I III II II I II I I llll llll II I I I II II) I V.2 : 2524 tgaggatggtggtagagtatcacgttcttcaagtgccaaagtaaccataaatgtggttga 2583
V.l : 2840 tgtcaatgacaacaaaccagttttcattgtccctccttccaactgttcttatgaattggt 2899
I III llll Mill Mill I I I I II II I I I I III II II I I II I II II MM II I I III I II V.2 : 2584 tgtcaatgacaacaaaccagttttcattgtccctccttccaactgttcttatgaattggt 2643
V.l : 2900 tctaccgtccactaatccaggcaoagtggtctttcaggtaattgctgttgacaatgacac 2959
111 III II Mill II II II III I I III I II III II I II II II II II llll II I I I II II I V.2 : 2644 tctaccgtccactaatccaggcacagtggtctttcaggtaattgctgttgacaatgacac 2703
V.l : 2960 tggcatgaatgcagaggttcgttacagcattgtaggaggaaacacaagagatctgtttgc 3019
I I 1 II I II II III llll I 1 II I I I I III I llll II I I III II II I II I II I I I I I II I I I V.2 : 2704 tggcatgaatgcagaggttcgttacagcattgtaggaggaaacacaagagatctgtttgc 2763
V.l : 3020 aatcgaccaagaaacaggcaacataacattgatggagaaatgtgatgttacagaccttgg 3079
I I I II III I III III II I I II I I I I I II I II I II II I III I III llll II I I I I I MM I V.2 : 2764 aatcgaccaagaaacaggcaacataacattgatggagaaatgtgatgttacagaccttgg 2823
V.l : 3080 tttacacagagtgttggtcaaagctaatgacttaggacagcctgattctctcttcagtgt 3139
I I II III II I I llll II II III I II I II I III II II I III I I II llll II I I I I I II I I I V.2 : 2824 tttacacagagtgttggtcaaagctaatgacttaggacagcctgattctctcttcagtgt 2883
V.l : 3140 tgtaattgtcaatctgttcgtgaatgagtcggtgaccaatgctacactgattaatgaact 3199
I I I llll II III III II I I I II I II I I II I II II 11 I llll III III III I 1 I I I llll I V.2 : 2884 tgtaattgtcaatctgttcgtgaatgagtcggtgaccaatgctacactgattaatgaact 2943
V.l : 3200 ggtgcgcaaaagcactgaagcaccagtgaccccaaatactgagatagctgatgtatcctc 3259
MIMMMMIIMMM llll 1 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I Mlllll V.2 : 2944 ggtgcgcaaaagcactgaagcaccagtgaccccaaatactgagatagctgatgtatcctc 3003 V.l : 3260 accaactagtgactatgtcaagatcctggttgcagctgttgctggcaccataactgtcgt 3319
I 1 I I I I II I I 11 MM llll II III II II II II I I I II I II II I II II I II II I I II III V.2 : 3004 accaactagtgactatgtcaagatcctggttgcagctgttgctggcaccataactgtcgt 3063
V.l : 3320 tgtagttattttcatcactgctgtagtaagatgtcgccaggcaccacaccttaaggctgc 3379
1) I I I I II II II llll II I I II I II II I I II II I I I II Ml I II II II II I II I I II II I V.2 : 3064 tgtagttattttcatcactgctgtagtaagatgtcgccaggcaccacaccttaaggctgc 3123
V.l : 3380 tcagaaaaacaagcagaattctgaatgggctaccccaaacccagaaaacaggcagatgat 3439
I I I II II I MM II Mill II II III I I II III I I II III II I Ml II II II I I II II II V.2 : 3124 tcagaaaaacaagcagaattctgaatgggctaccccaaacccagaaaacaggcagatgat 3183
V.l : 3440 aatgatgaagaaaaagaaaaagaagaagaagcattcccctaagaacttgctgcttaattt 3499
I I I II II I II Mill II II II llll I II II II II III II I II I II II III II I III I II I V.2 : 3184 aatgatgaagaaaaagaaaaagaagaagaagcattcccctaagaacttgctgcttaattt 3243
V.l : 3500 tgtcactattgaagaaactaaggcagatgatgttgacagtgatggaaacagagtcacact 3559
I I I I II III II Hill I II III III II I I II II II llll I I II I I llll I II II llll II V.2 : 3244 tgtcactattgaagaaactaaggcagatgatgttgacagtgatggaaacagagtcacact 3303
V.l : 3560 agaccttcctattgatctagaagagcaaacaatgggaaagtacaattgggtaactacacc 3619
I I II II I I I I II II II MM I I I II II I I I I I I I I I I I I II I II II II I I I II I I Mill
V.2 : 3304 agaccttcctattgatctagaagagcaaacaatgggaaagtacaattgggtaactacacc 3363
V.l : 3620 tactactttcaagcccgacagccctgatttggcccgacactacaaatctgcctctccaca 3679
II I I I I II I II I II I II II II llll II I II II I I I III II II I II I I I I I II I 1 II III I
V.2 : 3364 tactactttcaagcccgacagccctgatttggcccgacactacaaatctgcctctccaca 3423
V.l : 3680 gcctgccttccaaattcagcctgaaactcccctgaattcgaagcaccacatcatccaaga 3739
I I II II III I llll I II I II II III II I I II II I 1 I I II III Mill II II I II I I MM V.2 : 3424 gcctgccttccaaattcagcctgaaactcccctgaattcgaagcaccacatcatccaaga 3483
V.l : 3740 actgcctctcgataacacctttgtggcctgtgactctatctccaagtgttcctcaagcag 3799
I I II I MM II III I I III II II I II I I II II I I I II I III II I I II I II II II Ml II 1 V.2 • : 3484 actgcctctcgataacacctttgtggcctgtgactctatctccaagtgttcctcaagcag 3543
V.l : 3800 ttcagatccctacagcgtttctgactgtggctatccagtgacgaccttcgaggtacctgt 3859
I I II II II I I II llll II I I II II I II II II II II II II II I I III II I llll I I llll I V.2 : 3544 ttcagatccctacagcgtttctgactgtggctatccagtgacgaccttcgaggtacctgt 3603
V.l : 3860 gtccgtacacaccagaccg 3878
I I I I II I II II III I II II V.2 : 3604 gtccgtacacaccagaccg 3622
Table LΙV(a). Peptidesequences of protein coded by 109P1D4v.2 (SEQ ID NO: 240)
MRTERQWVLI QIFQVLCGLI QQTVTSVPGM DLLSGTYIFA VLLACVVFHS GAQEKNYTIR 60
EEMPENVLIG DLLKDLNLSL IPNKSLTTAM QFKLVYKTGD VPLIRIEEDT GEIFTTGARI 120
DREKLCAGIP RDEHCFYEVE VAILPDEIFR LVKIRFLIED INDNAPLFPA TVINISIPEN 180
SAINSKYTLP AAVDPDVGIN GVQNYELIKS QNIFGLDVIE TPEGDKMPQL IVQKELDREE 240
KDTYVMKVKV EDGGFPQRSS TAILQVSVTD TNDNHPVFKE TEIEVSIPEN APVGTSVTQL 300
HATDADIGEN AKIHFSFSNL VSNIARRLFH LNATTGLITI KEPLDREETP NHKLLVLASD 360
GGLMPARAMV LVNVTDVNDN VPSIDIRYIV NPVNDTVVLS ENIPLNTKIA LITVTDKDAD 420
HNGRVTCFTD HEIPFRLRPV FSNQFLLETA AYLDYESTKE YAIKLLAADA GKPPLNQSAM 480
LFIKVKDEND NAPVFTQSFV TVSIPENNSP GIQLTKVSAM DADSGPNAKI NYLLGPDAPP 540
EFSLDCRTGM LTVVKKLDRE KEDKYLFTIL AKDNGVPPLT SNVTVFVSII DQNDNSPVFT 600 HNEYNFYVPE NLPRHGTVGL ITVTDPDYGD NSAVTLSILD ENDDFTIDSQ TGVIRPNISF 660
DREKQESYTF YVKAEDGGRV SRSSSAKVTI NWDVNDNKP VFIVPPSNCS YELVLPSTNP 720
GTVVFQVIAV DNDTGMNAEV RYSIVGGNTR DLFAIDQETG NITLMEKCDV TDLGLHRVLV 780
KANDLGQPDS LFSWIVNLF VNESVTNATL INELVRKSTE APVTPNTEIA DVSSPTSDYV 840
KILVAAVAGT ITWWIFIT AWRCRQAPH LKAAQKNKQN SEWATPNPEN RQMIMMKKKK 900
KKKKHSPKNL LLNFVTIEET KADDVDSDGN RVTLDLPIDL EEQTMGKYNW VTTPTTFKPD 960
SPDLARHYKS ASPQPAFQIQ PETPLNSKHH IIQELPLDNT FVACDSISKC SSSSSDPYSV 1020
SDCGYPVTTF EVPVSVHTRP TDSRTSTIEI CSEI 1054
Table LV(a). Amino acid sequence alignment of 109P1D4 v.1 (SEQ ID NO: 241) and 109P1D4 v.2 (SEQ ID NO: 242)
Score = 2006 bits (5197), Expect = O.OIdentities = 1012/1017 (99%), Positives = 1013/1017 (99%)
V.l 1 MDLLSGTYIFAVLLACWFHSGAQEKNYTIREEMPENVLIGDLLKDLNLSLIPNKSLTTA 60
MDLLSGTYIFAVLLACWFHSGAQEKNYTIREEMPENVLIGDLLKDLNLSLIPNKSLTTA V.2 30 DLLSGTYIFAVLLACWFHSGAQEK YTIREEMPENV IGDLLKDLNLSLIPNKSLTTA 89 V.l 61 MQFKLVYKTGDVP 1RIEEDTGEIFTTGARIDREKLCAGIPRDEHCFYEVEVAILPDEIF 120
MQFKLVYKTGDVPLIRIEEDTGEIFTTGARIDREK CAGIPRDEHCFYEVEVAILPDEIF V.2 90 MQFKLVYKTGDVPLIRIEEDTGEIFTTGARIDREKLCAGIPRDEHCFYEVEVAI PDEIF 149 V.l 121 RLVKIRF IEDINDNAP FPATVINISIPENSAINSKYTLPAAVDPDVGINGVQNYELIK 180
RLVKIRFLIEDINDNAPLFPATVINISIPENSAINSKYTLPAAVDPDVGINGVQNYELIK V.2 150 RLVKIRF IEDINDNAPLFPATVINISIPENSAINSKYTLPAAVDPDVGINGVQNYELIK 209 V.l 181 SQNIFGLDVIETPEGDK PQLIVQKELDREEKDTYVMKVKVEDGGFPQRSSTAILQVSVT 240
SQNIFGLDVIETPEGDKMPQLIVQKELDREEKDTYVMPVKVEDGGFPQRSSTAILQVSVT V.2 210 SQNIFGLDVIETPEGDKMPQLIVQKE DREEKDTYVMKVKVEDGGFPQRSSTAILQVSVT 269 V.l 241 DTNDNHPVFKETEIEVSIPENAPVGTSVTQLHATDADIGENAKIHFSFSNLVSNIARRLF 300
DTNDNHPVFKETEIEVSIPENAPVGTSVTQLHATDADIGENAKIHFSFSNLVSNIARR F V.2 270 DTNDNHPVFKETEIEVSIPENAPVGTSVTQ HATDADIGENAKIHFSFSN VSNIARR F 329 V.l 301 HLNATTGLITIKEPLDREETPNHKLLVLASDGGLMPARAMVLVNVTDVNDNVPSIDIRYI 360
HLNATTGLITIKEPLDREETPNHKLLVLASDGGLMPARAMV VNVTDVNDNVPSIDIRYI V.2 330 HLNATTGLITIKEPLDREETPNHK VLASDGGL PARAMVLVNVTDVNDNVPSIDIRYI 389 V.l 361 VNPV DTVVLSENIP NTKIALITVTDKDADHNGRVTCFTDHEIPFRLRPVFSNQFLLET 420
VNPV DTW SENIPLNTKIALITVTDKDADHNGRVTCFTDHEIPFRLRPVFSNQFLLET V.2 390 V PVNDTWLSENIPLNTKIA ITVTDKDADHNGRVTCFTDHEIPFRLRPVFSNQFLLET 449 V.l 421 AAYLDYESTKEYAIKLLAADAGKPPLNQSAMLFIKVKDENDNAPVFTQSFVTVSIPENNS 480
AAYLDYESTKEYAIKLLAADAGKPP NQSA LFIKVKDENDNAPVFTQSFVTVSIPENNS V.2 450 AAYLDYESTKEYAIKLLAADAGKPPLNQSAMLFIKVKDENDNAPVFTQSFVTVSIPENNΞ 509 V.l 481 PGIQLTKVSAMDADSGPNAKINYLLGPDAPPEFSLDCRTGM TWKKLDREKEDKY FTI 540
PGIQLTKVSAMDADSGPNAKINYLLGPDAPPEFSLDCRTGM TWKK DREKEDKYLFTI V.2 510 PGIQLTKVSAMDADSGPNAKINYLLGPDAPPEFSLDCRTGMLTWKKLDREKEDKYLFTI 569 V.l 541 LAKDNGVPPLTSNVTVFVSIIDQNDNSPVFTHNEYNFYVPENLPRHGTVGLITVTDPDYG 600
LAKDNGVPPLTSNVTVFVSIIDQNDNSPVFTHNEYNFYVPENLPRHGTVGLITVTDPDYG V.2 570 LAKDNGVPP TSNVTVFVSIIDQNDNSPVFTHNEYNFYVPEN PRHGTVG ITVTDPDYG 629 .l 601 DNSAVT SILDENDDFTIDSQTGVIRPNISFDREKQESYTFYVKAEDGGRVSRSSSAKVT 660
DNSAVT SILDENDDFTIDSQTGVIRPNISFDREKQESYTFYVKAEDGGRVSRSSSAKVT V.2 630 DNSAVTLSILDENDDFTIDSQTGVIRPNISFDREKQESYTFYVKAEDGGRVSRSSSAKVT 689 V.l 661 INWDVNDNKPVFIVPPSNCSYELVLPSTNPGTVVFQVIAVDNDTG NAEVRYSIVGGNT 720
INVVDVNDNKPVFIVPPSNCSYELVLPSTNPGTWFQVIAVDNDTGMNAEVRYSIVGGNT V.2 690 INWDVNDNKPVFIVPPSNCSYE VLPSTNPGTWFQVIAVDNDTGMNAEVRYSIVGGNT 749 V.l 721 RDLFAIDQETGNITLMEKCDVTDLG HRVLVKAND GQPDS FSWIVNLFVNESVTNAT 780
RDLFAIDQETGNITL EKCDVTDLG HRVLVKANDLGQPDSLFSWIVNLFVNESVTNAT V.2 750 RDLFAIDQETGNITLMEKCDVTDLGLHRVLVKANDLGQPDSLFSWIVNLFVNESVTNAT 809 V.l 781 LINE VRKSTEAPVTPNTEIADVSSPTSDYVKI VAAVAGTITWWIFITAWRCRQAP 840
LINE VRKSTEAPVTPNTEIADVSSPTSDYVKILVAAVAGTITWWIFITAWRCRQAP V.2 810 LINELVRKSTEAPVTPNTEIADVSSPTSDYVKILVAAVAGTITWWIFITAWRCRQAP 869 V.l 841 HLKAAQKNKQNSE ATPNPENRQMIMMKKKKKKKKHSPKNLLLNFVTIEETKADDVDSDG 900
HLKAAQKNKQNSEWATPNPENRQMI MKKKKKKKKHSPKNLLLNFVTIEETKADDVDSDG V.2 870 HLK7AAQKNKQNSEWATPNPENRQMIMMKKKKKKKKHSPK LLLNFVTIEETKADDVDSDG 929 V.l 901 NRVTLDLP1DLEEQTMGKYN VTTPTTFKPDSPDLARHYKSASPQPAFQIQPETP NSKH 960
NRVTLDLPIDLEEQT GKYNWVTTPTTFKPDSPD ARHYKSASPQPAFQ1QPETPLNSKH V.2 930 NRVTLDLPIDLEEQTMGKYN VTTPTTFKPDSPDLARHYKSASPQPAFQIQPETP NSKH 989 V.l 961 HIIQELPLDNTFVACDSISKCSSSSSDPYSVSDCGYPVTTFEVPVSVHTRPVGIQVS 1017
HIIQELPLDNTFVACDSISKCSSSSSDPYSVSDCGYPVTTFEVPVSVHTRP + S V.2 990 HIIQELP DNTFVACDSISKCSSSSSDPYSVSDCGYPVTTFEVPVSVHTRPTDSRTS 1046
Table Lll(b). Nucleotide sequence of transcript variant 109P1 D4 v.3 (SEQ ID NO: 243) ctggtggtcc agtacctcca aagatatgga atacactcct gaaatatcct gaaaactttt 60 ttttttcaga atcctttaat aagcagttat gtcaatctga aagttgctta cttgtacttt 120 atattaatag ctattcttgt ttttcttatc caaagaaaaa tcctctaatc cccttttcac 180 atgatagttg ttaccatgtt taggcattag tcacatcaac ccctctcctc tcccaaactt 240 ctcttcttca aatcaaactt tattagtccc tcctttataa tgattccttg cctcgtttta 300 tccagatcaa ttttttttca ctttgatgcc cagagctgaa gaaatggact actgtataaa 360 ttattcattg ccaagagaat aattgcattt taaacccata ttataacaaa gaataatgat 420 tatattttgt gatttgtaac aaataccctt tattttccct taactattga attaaatatt 480 ttaattattt gtattctctt taactatctt ggtatattaa agtattatct tttatatatt 540 tatcaatggt ggacactttt ataggtactc tgtgtcattt ttgatactgt aggtatctta 600 tttcatttat ctttattctt aatgtacgaa ttcataatat ttgattcaga acaaatttat 660 cactaattaa cagagtgtca attatgctaa catctcattt actgatttta atttaaaaca 720 gtttttgtta acatgcatgt ttagggttgg cttcttaata atttcttctt cctcttctct 780 ctctcctctt cttttggtca gtgttgtgcg ggttaataca acaaactgta acaagtgtac 840 ctggtatgga cttgttgtcc gggacgtaca ttttcgcggt cctgctagca tgcgtggtgt 900 tccactctgg cgcccaggag aaaaactaca ccatccgaga agaaatgcca gaaaacgtcc 960 tgataggcga cttgttgaaa gaccttaact tgtcgctgat tccaaacaag tccttgacaa 1020 ctgctatgca gttcaagcta gtgtacaaga ccggagatgt gccactgatt cgaattgaag 1080 aggatactgg tgagatcttc actactggcg ctcgcattga tcgtgagaaa ttatgtgctg 1140 gtatcccaag ggatgagcat tgcttttatg aagtggaggt tgccattttg ccggatgaaa 1200 tatttagact ggttaagata cgttttctga tagaagatat aaatgataat gcaccattgt 1260 tcccagcaac agttatcaac atatcaattc cagagaactc ggctataaac tctaaatata 1320 ctctcccagc ggctgttgat cctgacgtag gaataaacgg agttcaaaac tacgaactaa 1380 ttaagagtca aaacattttt ggcctcgatg tcattgaaac accagaagga gacaagatgc 1440 cacaactgat tgttcaaaag gagttagata gggaagagaa ggatacctac gtgatgaaag 1500 taaaggttga agatggtggc tttcctcaaa gatccagtac tgctattttg caagtgagtg 1560 ttactgatac aaatgacaac cacccagtct ttaaggagac agagattgaa gtcagtatac 1620 cagaaaatgc tcctgtaggc acttcagtga cacagctcca tgccacagat gctgacatag 1680 gtgaaaatgc caagatccac ttctctttca gcaatctagt ctccaacatt gccaggagat 1740 tatttcacct caatgccacc actggactta tcacaatcaa agaaccactg gatagggaag 1800 aaacaccaaa ccacaagtta ctggttttgg caagtgatgg tggattgatg ccagcaagag 1860 caatggtgct ggtaaatgtt acagatgtca atgataatgt cccatccatt gacataagat 1920 acatcgtcaa tcctgtcaat gacacagttg ttctttcaga aaatattcca ctcaacacca 1980 aaattgctct cataactgtg acggataagg atgcggacca taatggcagg gtgacatgct 2040 tcacagatca tgaaatccct ttcagattaa ggccagtatt cagtaatcag ttcctcctgg 2100 agactgcagc atatcttgac tatgagtcca caaaagaata tgccattaaa ttactggctg 2160 cagatgctgg caaacctcct ttgaatcagt cagcaatgct cttcatcaaa gtgaaagatg 2220 aaaatgacaa tgctccagtt ttcacccagt ctttcgtaac tgtttctatt cctgagaata 2280 actctcctgg catccagttg acgaaagtaa gtgcaatgga tgcagacagt gggcctaatg 2340 ctaagatcaa ttacctgcta ggccctgatg ctccacctga attcagcctg gattgtcgta 2400 caggcatgct gactgtagtg aagaaactag atagagaaaa agaggataaa tatttattca 2460 caattctggc aaaagataac ggggtaccac ccttaaccag caatgtcaca gtctttgtaa 2520 gcattattga tcagaatgac aatagcccag ttttcactca caatgaatac aacttctatg 2580 tcccagaaaa ccttccaagg catggtacag taggactaat cactgtaact gatcctgatt 2640 atggagacaa ttctgcagtt acgctctcca ttttagatga gaatgatgac ttcaccattg 2700 attcacaaac tggtgtcatc cgaccaaata tttcatttga tagagaaaaa caagaatctt 2760 acactttcta tgtaaaggct gaggatggtg gtagagtatc acgttcttca agtgccaaag 2820 taaccataaa tgtggttgat gtcaatgaca acaaaccagt tttcattgtc cctccttcca 2880 actgttctta tgaattggtt ctaccgtcca ctaatccagg cacagtggtc tttcaggtaa 2940 ttgctgttga caatgacact ggcatgaatg cagaggttcg ttacagcatt gtaggaggaa 3000 acacaagaga tctgtttgca atcgaccaag aaacaggcaa cataacattg atggagaaat 3060 gtgatgttac agaccttggt ttacacagag tgttggtcaa agctaatgac ttaggacagc 3120 ctgattctct cttcagtgtt gtaattgtca atctgttcgt gaatgagtcg gtgaccaatg 3180 ctacactgat taatgaactg gtgcgcaaaa gcactgaagc accagtgacc ccaaatactg 3240 agatagctga tgtatcctca ccaactagtg actatgtcaa gatcctggtt gcagctgttg 3300 ctggcaccat aactgtcgtt gtagttattt tcatcactgc tgtagtaaga tgtcgccagg 3360 caccacacct taaggctgct cagaaaaaca agcagaattc tgaatgggct accccaaacc 3420 cagaaaacag gcagatgata atgatgaaga aaaagaaaaa gaagaagaag cattccccta 3480 agaacttgct gcttaatttt gtcactattg aagaaactaa ggcagatgat gttgacagtg 3540 atggaaacag agtcacacta gaccttccta ttgatctaga agagcaaaca atgggaaagt 3600 acaattgggt aactacacct actactttca agcccgacag ccctgatttg gcccgacact 3660 acaaatctgc ctctccacag cctgccttcc aaattcagcc tgaaactccc ctgaattcga 3720 agcaccacat catccaagaa ctgcctctcg ataacacctt tgtggcctgt gactctatct 3780 ccaagtgttc ctcaagcagt tcagatccct acagcgtttc tgactgtggc tatccagtga 3840 cgaccttcga ggtacctgtg tccgtacaca ccagaccgcc aatgaaggag gttgtgcgat 3900 cttgcacccc catgaaagag tctacaacta tggagatctg gattcatccc caaccacagc 3960 ggaaatctga agggaaagtg gcaggaaagt cccagcggcg tgtcacattt cacctgccag 4020 aaggctctca ggaaagcagc agtgatggtg gactgggaga ccatgatgca ggcagcctta 4080 ccagcacatc tcatggcctg ccccttggct atcctcagga ggagtacttt gatcgtgcta 4140 cacccagcaa tcgcactgaa ggggatggca actccgatcc tgaatctact ttcatacctg 4200 gactaaagaa agctgcagaa ataactgttc aaccaactgt ggaagaggcc tctgacaact 4260 gcactcaaga atgtctcatc tatggccatt ctgatgcctg ctggatgccg gcatctctgg 4320 atcattccag ctcttcgcaa gcacaggcct ctgctctatg ccacagccca ccactgtcac 4380 aggcctctac tcagcaccac agcccacgag tgacacagac cattgctctc tgccacagcc 4440 ctccagtgac acagaccatc gcattgtgcc acagcccacc accgatacag gtgtctgctc 4500 tccaccacag tcctcctcta gtgcaggcta ctgcacttca ccacagccca ccatcagcac 4560 aggcctcagc cctctgctac agccctcctt tagcacaggc tgctgcaatc agccacagct 4620 ctcctctgcc acaggttatt gccctccatc gtagtcaggc ccaatcatca gtcagtttgc 4680 agcaaggttg ggtgcaaggt gctgatgggc tatgctctgt tgatcaggga gtgcaaggta 4740 gtgcaacatc tcagttttac accatgtctg aaagacttca tcccagtgat gattcaatta 4800 aagtcattcc tttgacaacc ttcactccac gccaacaggc cagaccgtcc agaggtgatt 4860 cccccattat ggaagaacat cccttgtaaa gctaaaatag ttacttcaaa ttttcagaaa 4920 agatgtatat agtcaaaatt taagatacaa ttccaatgag tattctgatt atcagatttg 4980 taaataacta tgtaaataga aacagatacc agaataaatc tacagctaga cccttagtca 5040 atagttaacc aaaaaattgc aatttgttta attcagaatg tgtatttaaa aagaaaagga 5100 atttaacaat ttgcatcccc ttgtacagta aggcttatca tgacagagcg cactatttct 5160 gatgtacagt attttttgtt gtttttatca tcatgtgcaa tattactgat ttgtttccat 5220 gctgattgtg tggaaccagt atgtagcaaa tggaaagcct agaaatatct tattttctaa 5280 gtttaccttt agtttaccta aacttttgtt cagataacgt taaaaggtat acgtactcta 5340 gccttttttt gggctttctt tttgattttt gtttgttgtt ttcagttttt ttgttgttgt 5400 tagtgagtct cccttcaaaa tacgcagtag gtagtgtaaa tactgcttgt ttgtgtctct 5460 ctgctgtcat gttttctacc ttattccaat actatattgt tgataaaatt tgtatataca 5520 ttttcaataa agaatatgta taaactgtac agatctagat ctacaaccta tttctctact 5580 ctttagtaga gttcgagaca cagaagtgca ataactgccc taattaagca actatttgtt 5640 aaaaagggcc tctttttact ttaatagttt agtgtaaagt acatcagaaa taaagctgta 5700 tctgccattt taagcctgta gtccattatt acttgggtct ttacttctgg gaatttgtat 5760 gtaacagcct agaaaattaa aaggaggtgg atgcatccaa agcacgagtc acttaaaata 5820 tcgacggtaa actactattt tgtagagaaa ctcaggaaga tttaaatgtt gatttgacag 5880 ctcaataggc tgttaccaaa gggtgttcag taaaaataac aaatacatgt aactgtagat 5940 aaaaccatat actaaatcta taagactaag ggatttttgt tattctagct caacttactg 6000 aagaaaacca ctaataacaa caagaatatc aggaaggaac ttttcaagaa atgtaattat 6060 aaatctacat caaacagaat tttaaggaaa aatgcagagg gagaaataag gcacatgact 6120 gcttcttgca gtcaacaaga aataccaata acacacacag aacaaaaacc atcaaaatct 6180 catatatgaa ataaaatata ttcttctaag caaagaaaca gtactattca tagaaaacat 6240 tagttttctt ctgttgtctg ttatttcctt cttgtatcct cttaactggc cattatcttg 6300 tatgtgcaca ttttataaat gtacagaaac atcaccaact taattttctt ccatagcaaa 6360 actgagaaaa taccttgttt cagtataaca ctaaaccaag agacaattga tgtttaatgg 6420 gggcggttgg ggtggggggg ggagtcaata tctcctattg attaacttag acatagattt 6480 tgtaatgtat aacttgatat ttaatttatg attaaactgt gtgtaaattt tgtaacataa 6540 actgtggtaa ttgcataatt tcattggtga ggatttccac tgaatattga gaaagtttct 6600 tttcatgtgc ccagcaggtt aagtagcgtt ttcagaatat acattattcc catccattgt 6660 aaagttcctt aagtcatatt tgactgggcg tgcagaataa cttcttaact tttaactatc 6720 agagtttgat taataaaatt aattaatgtt ttttctcctt cgtgttgtta atgttccaag 6780 ggatttggag catactggtt ttccaggtgc atgtgaatcc cgaaggactg atgatatttg 6840 aatgtttatt aaattattat catacaaatg tgttgatatt gtggctattg ttgatgttga 6900 aaattttaaa cttggggaag attaagaaaa gaaccaatag tgacaaaaat cagtgcttcc 6960 agtagatttt agaacattct ttgcctcaaa aaacctgcaa agatgatgtg agattttttc 7020 ttgtgtttta attattttca cattttctct ctgcaaaact ttagttttct gatgatctac 7080 acacacacac acacacacac gtgcacacac acacacattt aaatgatata aaaagaagag 7140 gttgaaagat tattaaataa cttatcaggc atctcaatgg ttactatcta tgttagtgaa 7200 aatcaaatag gactcaaagt tggatatttg ggatttttct tctgacagta taatttattg 7260 agttactagg gaggttctta aatcctcata tctggaaact tgtgacgttt tgacaccttt 7320 cctatagatg atataggaat gaaccaatac gcttttatta ccctttctaa ctctgatttt 7380 ataatcagac ttagattgtg tttagaatat taaatgactg ggcaccctct tcttggtttt 7440 taccagagag gctttgaatg gaagcaggct gagagtagcc aaagaggcaa ggggtattag 7500 cccagttatt ctcccctatg ccttccttct ctttctaagc gtccactagg tctggccttg 7560 gaaacctgtt acttctaggg cttcagatct gatgatatct ttttcatcac attacaagtt 7620 atttctctga ctgaatagac agtggtatag gttgacacag cacacaagtg gctattgtga 7680 tgtatgatgt atgtagtcct acaactgcaa aacgtcttac tgaaccaaca atcaaaaaat 7740 ggttctgttt taaaaaggat tttgtttgat ttgaaattaa aacttcaagc tgaatgactt 7800 atatgagaat aatacgttca atcaaagtag ttattctatt ttgtgtccat attccattag 7860 attgtgatta ttaattttct agctatggta ttactatatc acacttgtga gtatgtattc 7920 aaatactaag tatcttatat gctacgtgca tacacattct tttcttaaac tttacctgtg 7980 ttttaactaa tattgtgtca gtgtattaaa aattagcttt tacatatgat atctacaatg 8040 taataaattt agagagtaat tttgtgtatt cttatttact taacatttta cttttaatta 8100 tgtaaatttg gttagaaaat aataataaat ggttagtgct attgtgtaat ggtagcagtt 8160 acaaagagcc tctgccttcc caaactaata tttatcacac atggtcatta aatgggaaaa 8220 aaatagacta aacaaatcac aaattgttca gttcttaaaa tgtaattatg tcacacacac 8280 aaaaaatcct tttcaatcct gagaaaatta aaggcgtttt actcacatgg ctatttcaac 8340 attagttttt tttgtttgtt tctttttcat ggtattactg aaggtgtgta tactccctaa 8400 tacacattta tgaaaatcta cttgtttagg cttttattta tactcttctg atttatattt 8460 tttattataa ttattatttc ttatctttct tcttttatat tttttggaaa ccaaatttat 8520 agttagttta ggtaaacttt ttattatgac cattagaaac tattttgaat gcttccaact 8580 ggctcaattg gccgggaaaa catgggagca agagaagctg aaatatattt ctgcaagaac 8640 ctttctatat tatgtgccaa ttaccacacc agatcaattt tatgcagagg ccttaaaata 8700 ttctttcaca gtagctttct tacactaacc gtcatgtgct tttagtaaat atgattttta 8760 aaagcagttc aagttgacaa cagcagaaac agtaacaaaa aaatctgctc agaaaaatgt 8820 atgtgcacaa ataaaaaaaa ttaatggcaa ttgtttagtg attgtaagtg atacttttta 8880 aagagtaaac tgtgtgaaat ttatactatc cctgcttaaa atattaagat ttttatgaaa 8940 tatgtattta tgtttgtatt gtgggaagat tcctcctctg tgatatcata cagcatctga 9000 aagtgaacag tatcccaaag cagttccaac catgctttgg aagtaagaag gttgactatt 9060 gtatggccaa ggatggcagt atgtaatcca gaagcaaact tgtattaatt gttctatttc 9120 aggttctgta ttgcatgttt tcttattaat atatattaat aaaagttatg agaaat 9176
Table Llll(b). Nucleotide sequence alignment of 109P1D4 v.1 (SEQ ID NO: 244) and 109P1D4 v.3 (SEQ ID NO: 245)
Score = 7456 bits (3878), Expect = O.OIdentities = 3878/3878 (100%) Strand = Plus / Plus
V . l ctggtggtccagtacctccaaagatatggaatacactcctgaaatatcctgaaaactttt 60 II II I I II I I lllll II II II III II I I I I II I I I I I I I II I II II II I II I II II I I I I
V . 3 ctggtggtccagtacctccaaagatatggaatacactcctgaaatatcctgaaaactttt 60
V.l : 61 ttttttcagaatcctttaataagcagttatgtcaatctgaaagttgcttacttgtacttt 120
II I I I I II I I II III II llll I II II I I I I I I I I I II I I II I llll II I II III II I 111
V.3 : 61 ttttttcagaatcctttaataagcagttatgtcaatctgaaagttgcttacttgtacttt 120
V.l 121 atattaatagctattcttgtttttcttatccaaagaaaaatcctctaatccccttttcac 180 II I I I I II I I I Ml I II II II I II II I I II I I I I I II I I I II I I II II I II III II I I II V.3 121 atattaatagctattcttgtttttcttatccaaagaaaaatcctctaatccccttttcac 180
V.l : 181 atgatagttgttaccatgtttaggcattagtcacatcaacccctctcctctcccaaactt 240 II I I I I II I I II I II II II I II II II I II II I I I I II I I I II I III I I I II I I I II II II V.3 : 181 atgatagttgttaccatgtttaggcattagtcacatcaacccctctcctctcccaaactt 240
V.l : 241 ctcttcttcaaatcaaactttattagtccctcctttataatgattccttgcctcgtttta 300
III 111 I I 111 I I 111) I II I II MM III I II I I MM I I II I I I I III I II III II II
V.3 : 241 ctcttcttcaaatcaaactttattagtccctcctttataatgattccttgcctcgtttta 300
V.l : 301 tccagatcaattttttttcactttgatgcccagagctgaagaaatggactactgtataaa 360
II I II I I II I I I I I I II I I I llll I III II II I I I llll I III I I I I I II III II II II I
V.3 : 301 tccagatcaattttttttcactttgatgcccagagctgaagaaatggactactgtataaa 360
V.l : 361 ttattcattgccaagagaataattgcattttaaacccatattataacaaagaataatgat 420
II III I 11 I I I I I I I II I II III III 11 II II II II II I II I I II 1 I II II I II II III I
V.3 : 361 ttattcattgccaagagaataattgcattttaaacccatattataacaaagaataatgat 420
V.l : 421 tatattttgtgatttgtaacaaataccctttattttcccttaactattgaattaaatatt 480
II I II I II I II I I I I II I II I II Mlllll II I I I II I II I I II I I I III I lllll MM
V.3 : 421 tatattttgtgatttgtaacaaataccctttattttcccttaactattgaattaaatatt 480
V.l : 481 ttaattatttgtattctctttaactatcttggtatattaaagtattatcttttatatatt 540
I III I I I I I I II 1 llll I I I Mill III II II II II I II II II I I I I II I 1 II II III I I
V.3 : 481 ttaattatttgtattctctttaactatcttggtatattaaagtattatcttttatatatt 540
V.l : 541 tatcaatggtggacacttttataggtactctgtgtcatttttgatactgtaggtatctta 600
I I I I I I I 11 II I I II II II I 1 I I II II II llll I I III I 11 II II I III I I I I 1 I I I II I
V.3 : 541 tatcaatggtggacacttttataggtactctgtgtcatttttgatactgtaggtatctta 600
V.l : 601 tttcatttatctttattcttaatgtacgaattcataatatttgattcagaacaaatttat 660
I II I II I I I I I I I I Ml II I lllll III II II II II II II I I I I I I I I II II I II II I I I
V.3 : 601 tttcatttatctttattcttaatgtacgaattcataatatttgattcagaacaaatttat 660
V.l : 661 cactaattaacagagtgtcaattatgctaacatctcatttactgattttaatttaaaaca 720
I II I I I II I I I I I I I I I II I I I I II II III II I I II II I I I I I I I I II II I I I Mlllll
V.3 : 661 cactaattaacagagtgtcaattatgctaacatctcatttactgattttaatttaaaaca 720
V.l : 721 gtttttgttaacatgcatgtttagggttggcttcttaataatttcttcttcctcttctct 780
II I II I I I II II II Ml I I I II llll II I I II II I I 1 I I II II I I I I II II I I I II I II I
V.3 : 721 gtttttgttaacatgcatgtttagggttggcttcttaataatttcttcttcctcttctct 780
V.l : 781 ctctcctcttcttttggtcagtgttgtgcgggttaatacaacaaactgtaacaagtgtac 840
III I II II I I I I I I II I II I lllll II Mill II II II I I I I II I I III I III II II II I
V.3 : 781 ctctcctcttcttttggtcagtgttgtgcgggttaatacaacaaactgtaacaagtgtac 840
V.l : 841 ctggtatggacttgttgtccgggacgtacattttcgcggtcctgctagcatgcgtggtgt 900
II II I I II II I I I I 111 I I I II I I III III II I I II II II II I II II I II I II II II III
V.3 : 841 ctggtatggacttgttgtccgggacgtacattttcgcggtcctgctagcatgcgtggtgt 900
V.l : 901 tccactctggcgcccaggagaaaaactacaccatccgagaagaaatgccagaaaacgtcc 960
II II I I I 111 II 1 I III I I I II I II II II II II I I I II I I II II I I I II II I II III II I
V.3 : 901 tccactctggcgcccaggagaaaaactacaccatccgagaagaaatgccagaaaacgtcc 960
V.l : 961 tgataggcgacttgttgaaagaccttaacttgtcgctgattccaaacaagtccttgacaa 1020
II I II I I I I I II I I III I I I II III II I I I II I I II II I I I I II I I III II I I II I I II I
V.3 : 961 tgataggcgacttgttgaaagaccttaacttgtcgctgattccaaacaagtccttgacaa 1020
V.l : 1021 ctgctatgcagttcaagctagtgtacaagaccggagatgtgccactgattcgaattgaag 1080 II I II I II M II I II II I I I I I I I I II II I II II I II I I II I 1 II I I I II II II I I I II I V.3 : 1021 ctgctatgcagttcaagctagtgtacaagaccggagatgtgccactgattcgaattgaag 1080
V.l : 1081 aggatactggtgagatcttcactactggcgctcgcattgatcgtgagaaattatgtgctg 1140
I II III I II II M II I I I I I I I I III II II II I I II llll Ml I I II I I II I I I I I III I V.3 : 1081 aggatactggtgagatcttcactactggcgctcgcattgatcgtgagaaattatgtgctg 1140
V.l : 1141 gtatcccaagggatgagcattgcttttatgaagtggaggttgccattttgccggatgaaa 1200
I II I II II I II III I I I II II I II III I llll I llll II II I I I I I I I II I I 111 I III I
V.3 : 1141 gtatcccaagggatgagcattgcttttatgaagtggaggttgccattttgccggatgaaa 1200
V.l : 1201 tatttagactggttaagatacgttttctgatagaagatataaatgataatgcaccattgt 1260
I llll 1 II II II I II I I I I I I II III I II III II II I I II I II II I I I I I I I I I I I I III V.3 : 1201 tatttagactggttaagatacgttttctgatagaagatataaatgataatgcaccattgt 1260
V.l : 1261 tcccagcaacagttatcaacatatcaattccagagaactcggctataaactctaaatata 1320
II I III I I II I II I I II I 1 I I I I III I III II II II II I II II I I II I I II I I I I I III I
V.3 : 1261 tcccagcaacagttatcaacatatcaattccagagaactcggctataaactctaaatata 1320
V.l : 1321 ctctcccagcggctgttgatcctgacgtaggaataaacggagttcaaaactacgaactaa 1380
I II I I I II I llll II I I I I II I I I I II I llll lllll llll I I II I I I II I II II II III
V.3 : 1321 ctctcccagcggctgttgatcctgacgtaggaataaacggagttcaaaactacgaactaa 1380
V.l : 1381 ttaagagtcaaaacatttttggcctcgatgtcattgaaacaccagaaggagacaagatgc 1440
II I III II II I I II I I II I II I I III II llll II II I II I II I I I I III II I I I I I III I
V.3 : 1381 ttaagagtcaaaacatttttggcctcgatgtcattgaaacaccagaaggagacaagatgc 1440
V.l : 1441 cacaactgattgttcaaaaggagttagatagggaagagaaggatacctacgtgatgaaag 1500
I I II I I III I III I I II II II I I I I II I MM I II II II II I MM I I II I II II II Ml V.3 : 1441 cacaactgattgttcaaaaggagttagatagggaagagaaggatacctacgtgatgaaag 1500
V.l : 1501 taaaggttgaagatggtggctttcctcaaagatccagtactgctattttgcaagtgagtg 1560
I I I I II I II 111111 II II I I I 1 III II llll I II II II II I I I I II II I I I I II I III I V.3 : 1501 taaaggttgaagatggtggctttcctcaaagatccagtactgctattttgcaagtgagtg 1560
V.l : 1561 ttactgatacaaatgacaaccacccagtctttaaggagacagagattgaagtcagtatac 1620 llll I I II I I I II I I I I II I I I I II II II I II II II II II II I I I I I I I II II I I II II I V.3 : 1561 ttactgatacaaatgacaaccacccagtctttaaggagacagagattgaagtcagtatac 1620
V.l : 1621 cagaaaatgctcctgtaggcacttcagtgacacagctccatgccacagatgctgacatag 1680
I II I I I II I II II I I I I I I II I I II II I llll II II I III I I III I I I I I I II I I II III V.3 : 1621 cagaaaatgctcctgtaggcacttcagtgacacagctccatgccacagatgctgacatag 1680
V.l : 1681 gtgaaaatgccaagatccacttctctttcagcaatctagtctccaacattgccaggagat 1740
I II I II I II I II II 111 I I I I I I II I II llll I II II II II II I III llll I II I I I II I V.3 : 1681 gtgaaaatgccaagatccacttctctttcagcaatctagtctccaacattgccaggagat 1740
V.l : 1741 tatttcacctcaatgccaccactggacttatcacaatcaaagaaccactggatagggaag 1800
I I I II I II I I II I I I I II I I I I I MM II I I I II III II I I II I I II II II I I II I III I V.3 : 1741 tatttcacctcaatgccaccactggacttatcacaatcaaagaaccactggatagggaag 1800
V.l : 1801 aaacaccaaaccacaagttactggttttggcaagtgatggtggattgatgccagcaagag 1860
III III) Mill IIIIIM II IMIIMMMMMMI
V.3 : 1801 aaacaccaaaccacaagttactggttttggcaagtgatggtggattgatgccagcaagag 1860 V.l : 1861 caatggtgctggtaaatgttacagatgtcaatgataatgtcccatccattgacataagat 1920
II I I lllll II I II Ml I I II II I I I II II II I I III I I II II II II llll I I I II I I II
V.3 : 1861 caatggtgctggtaaatgttacagatgtcaatgataatgtcccatccattgacataagat 1920
V.l : 1921 acatcgtcaatcctgtcaatgacacagttgttctttcagaaaatattccactcaacacca 1980
II II llll II MM llll I I I II I I I II II I I II I III II I II II I II II II I I II II II
V.3 : 1921 acatcgtcaatcctgtcaatgacacagttgttctttcagaaaatattccactcaacacca 1980
V.l : 1981 aaattgctctcataactgtgacggataaggatgcggaccataatggcagggtgacatgct 2040
I I I II II III Ml II II II I I I I I II I II II I III II II II II I II I Ml I III I II II I
V.3 : 1981 aaattgctctcataactgtgacggataaggatgcggaccataatggcagggtgacatgct 2040
V.l : 2041 tcacagatcatgaaatccctttcagattaaggccagtattcagtaatcagttcctcctgg 2100
I I I II I III llll II II II I I I II I II I II I I I II I I II I I I II I II MM I I MM I II
V.3 : 2041 tcacagatcatgaaatccctttcagattaaggccagtattcagtaatcagttcctcctgg 2100
V.l : 2101 agactgcagcatatcttgactatgagtccacaaaagaatatgccattaaattactggctg 2160
II I I I Mil lllll III I I 11 II I I 1 II II II II III II III I II llll II I I I II II II
V.3 : 2101 agactgcagcatatcttgactatgagtccacaaaagaatatgccattaaattactggctg 2160
V.l : 2161 cagatgctggcaaacctcctttgaatcagtcagcaatgctcttcatcaaagtgaaagatg 2220
I I II MM MUM II II I I I II I II II II I I II I II I II II I II I II I II II I II II II
V.3 : 2161 cagatgctggcaaacctcctttgaatcagtcagcaatgctcttcatcaaagtgaaagatg 2220
V.l : 2221 aaaatgacaatgctccagttttcacccagtctttcgtaactgtttctattcctgagaata 2280
I I II llll llll I II II II I I II II II III I I II I I I I III II II I II III I II II I II I
V.3 : 2221 aaaatgacaatgctccagttttcacccagtctttcgtaactgtttctattcctgagaata 2280
V.l : 2281 actctcctggcatccagttgacgaaagtaagtgcaatggatgcagacagtgggcctaatg 2340
I II Mill I IM I II II II I I I I II I I II II I III I II II I II II II III I I II I II II I
V.3 : 2281 actctcctggcatccagttgacgaaagtaagtgcaatggatgcagacagtgggcctaatg 2340
V.l : 2341 ctaagatcaattacctgctaggccctgatgctccacctgaattcagcctggattgtcgta 2400
II I I llll II II ill I II I I I II I I II II I II I 111 II I II II II II II II II II II I
V.3 : 2341 ctaagatcaattacctgctaggccctgatgctccacctgaattcagcctggattgtcgta 2400
V.l : 2401 caggcatgctgactgtagtgaagaaactagatagagaaaaagaggataaatatttattca 2460
II I I I III II III II II M II II II I I II II I II I I II II I II II I III II II I II II II
V.3 : 2401 caggcatgctgactgtagtgaagaaactagatagagaaaaagaggataaatatttattca 2460
V.l : 2461 caattctggcaaaagataacggggtaccacccttaaccagcaatgtcacagtctttgtaa 2520
II I I I I III II III II II I II II II II III II II I 11 I II II I II III I II I II II II II
V.3 : 2461 caattctggcaaaagataacggggtaccacccttaaccagcaatgtcacagtctttgtaa 2520
V.l : 2521 gcattattgatcagaatgacaatagcccagttttcactcacaatgaatacaacttctatg 2580
I I I II II II II I II II II I I I II II I II I II III I I) I II II II I I III II I I I II II I I
V.3 : 2521 gcattattgatcagaatgacaatagcccagttttcactcacaatgaatacaacttctatg 2580
V.l : 2581 tcccagaaaaccttccaaggcatggtacagtaggactaatcactgtaactgatcctgatt 2640
I I I llll II III II II II I I I II I I I II II I I II II I III II II I I MM I I II II III I V.3 : 2581 tcccagaaaaccttccaaggcatggtacagtaggactaatcactgtaactgatcctgatt 2640
V.l 2641 atggagacaattctgcagttacgctctccattttagatgagaatgatgacttcaccattg 2700 I 1 MMM II II I I II II I I I II I I II I II I II II I I II I II II I II III I I I II II II I V.3 2641 atggagacaattctgcagttacgctctccattttagatgagaatgatgacttcaccattg 2700 V.l : 2701 attcacaaactggtgtcatccgaccaaatatttcatttgatagagaaaaacaagaatctt 2760
I II I I I II III II I II I I II I II I II II I I II I I II I Ml III MM I III I II II II I I
V.3 : 2701 attcacaaactggtgtcatccgaccaaatatttcatttgatagagaaaaacaagaatctt 2760
V.l : 2761 acactttctatgtaaaggctgaggatggtggtagagtatcacgttcttcaagtgccaaag 2820
I I I I I I II II llll II II Ml II II I II I I II II II I I II II I I II I llll I II II III I
V.3 : 2761 acactttctatgtaaaggctgaggatggtggtagagtatcacgttcttcaagtgccaaag 2820
V.l : 2821 taaccataaatgtggttgatgtcaatgacaacaaaccagttttcattgtccctccttcca 2880
I I I II II I II I III II II III II MM I II I Ml II II Ml I I MM II I I III Ml II I
V.3 : 2821 taaccataaatgtggttgatgtcaatgacaacaaaccagttttcattgtccctccttcca 2880
V.l : 2881 actgttcttatgaattggttctaccgtccactaatccaggcacagtggtctttcaggtaa 2940
I I I I I III I II III II II Ml I III II I II I I II I II I III I I III Ml II I I II II III
V.3 : 2881 actgttcttatgaattggttctaccgtccactaatccaggcacagtggtctttcaggtaa 2940
V.l : 2941 ttgctgttgacaatgacactggcatgaatgcagaggttcgttacagcattgtaggaggaa 3000
I II I I I II I II II I II III III II I I I I II II I I II I I I II I II II I II II II II II III
V.3 : 2941 ttgctgttgacaatgacactggcatgaatgcagaggttcgttacagcattgtaggaggaa 3000
V.l : 3001 acacaagagatctgtttgcaatcgaccaagaaacaggcaacataacattgatggagaaat 3060
I I II I I II I I III II II Ml II I I II I II II I I I II I I I I II I II II II I I III I Ml II
V.3 : 3001 acacaagagatctgtttgcaatcgaccaagaaacaggcaacataacattgatggagaaat 3060
V.l : 3061 gtgatgttacagaccttggtttacacagagtgttggtcaaagctaatgacttaggacagc 3120
I II I I Ml II I III II II II Ml III Ml II II I II I I II II III I I I I I II II Mill I
V.3 : 3061 gtgatgttacagaccttggtttacacagagtgttggtcaaagctaatgacttaggacagc 3120
V.l : 3121 ctgattctctcttcagtgttgtaattgtcaatctgttcgtgaatgagtcggtgaccaatg 3180
I I I I I III I I I II 111111 III I II II I I III II I II I II II I III II I II II II I I II I
V.3 : 3121 ctgattctctcttcagtgttgtaattgtcaatctgttcgtgaatgagtcggtgaccaatg 3180
V.l : 3181 ctacactgattaatgaactggtgcgcaaaagcactgaagcaccagtgaccccaaatactg 3240
I I I I I III I II II I I I I Ml II I I II I II I II I III I I II I II II II I I I II II II III I
V.3 : 3181 ctacactgattaatgaactggtgcgcaaaagcactgaagcaccagtgaccccaaatactg 3240
V.l : 3241 agatagctgatgtatcctcaccaactagtgactatgtcaagatcctggttgcagctgttg 3300
I I I I I Ml II I I II II II III II II I II II I I II I II I II II MM I II II II II II III
V.3 : 3241 agatagctgatgtatcctcaccaactagtgactatgtcaagatcctggttgcagctgttg 3300
V.l : 3301 ctggcaccataactgtcgttgtagttattttcatcactgctgtagtaagatgtcgccagg 3360
I I II I I I I I llll II I I Ml III I I I I II I II I I II I I I I I MM I llll II II II Ml I
V.3 : 3301 ctggcaccataactgtcgttgtagttattttcatcactgctgtagtaagatgtcgccagg 3360
V.l : 3361 caccacaccttaaggctgctcagaaaaacaagcagaattctgaatgggctaccccaaacc 3420
I II I I III I I III II I III llll M II I II II II I II I I I I I II II II I II II II I II II
V.3 : 3361 caccacaccttaaggctgctcagaaaaacaagcagaattctgaatgggctaccccaaacc 3420
V.l : 3421 cagaaaacaggcagatgataatgatgaagaaaaagaaaaagaagaagaagcattccccta 3480
I I I II I I I I II II I II II II I II I I II II I II I I II I I I II I III II I III I II I II II I
V.3 : 3421 cagaaaacaggcagatgataatgatgaagaaaaagaaaaagaagaagaagcattccccta 3480
V.l : 3481 agaacttgctgcttaattttgtcactattgaagaaactaaggcagatgatgttgacagtg 3540
I II I I I II I I I II II I II II II I II II I II II II I I I 1 II MM II II Ml II II III I I
V.3 : 3481 agaacttgctgcttaattttgtcactattgaagaaactaaggcagatgatgttgacagtg 3540 V.l : 3541 atggaaacagagtcacactagaccttcctattgatctagaagagcaaacaatgggaaagt 3600
I II I II II II II 1 II I III Mill II II I II I I I I II II MM II II III I II II III I I V.3 : 3541 atggaaacagagtcacactagaccttcctattgatctagaagagcaaacaatgggaaagt 3600
V.l : 3601 acaattgggtaactacacctactactttcaagcccgacagccctgatttggcccgacact 3660
I MM I II II II I I II II I Mlllll I II II I II I II III I II II I M I II II llll I V.3 : 3601 acaattgggtaactacacctactactttcaagcccgacagccctgatttggcccgacact 3660
V.l : 3661 acaaatctgcctctccacagcctgccttccaaattcagcctgaaactcccctgaattcga 3720
I II II II II I II I I II II I MM I I I I II I II I I I II I II 1 II I I II llll I II I llll I V.3 : 3661 acaaatctgcctctccacagcctgccttccaaattcagcctgaaactcccctgaattcga 3720
V.l : 3721 agcaccacatcatccaagaactgcctctcgataacacctttgtggcctgtgactctatct 3780
I II II I I II 1 I I I I II I II MM II I II I II I II I II I III II I I I I llll I III III II
V.3 : 3721 agcaccacatcatccaagaactgcctctcgataacacctttgtggcctgtgactctatct 3780
V.l : 3781 ccaagtgttcctcaagcagttcagatccctacagcgtttctgactgtggctatccagtga 3840
II II I II I I I I II II II II llll II II llll I II II II I II I III I I II I I I III llll I
V.3 : 3781 ccaagtgttcctcaagcagttcagatccctacagcgtttctgactgtggctatccagtga 3840
V.l : 3841 cgaccttcgaggtacctgtgtccgtacacaccagaccg 3878
I II II I I II I I I I I II I II llll MM I I Ml II I II I V.3 : 3841 cgaccttcgaggtacctgtgtccgtacacaccagaccg 3878
Table LΙV(b). Peptide sequences of protein coded by 109P1D4v.3 (SEQ ID NO: 246)
MDLLSGTYIF AVLLACWFH SGAQEKNYTI REEMPENVLI GDLLKDLNLS LIPNKSLTTA 60
MQFKLVYKTG DVPLIRIEED TGEIFTTGAR IDREKLCAGI PRDEHCFYEV EVAILPDEIF 120
RLVKIRFLIE DINDNAPLFP ATVINISIPE NSAINSKYTL PAAVDPDVGI NGVQNYELIK 180
SQNIFGLDVI ETPEGDKMPQ LIVQKELDRE EKDTYVMKVK VEDGGFPQRS STAILQVSVT 240
DTNDNHPVFK ETEIEVSIPE NAPVGTSVTQ LHATDADIGE NAKIHFSFSN LVSNIARRLF 300
HLNATTGLIT IKEPLDREET PNHKLLVLAS DGGLMPARAM VLVNVTDVND NVPSIDIRYI 360
VNPVNDTWL SENIPLNTKI ALITVTDKDA DHNGRVTCFT DHEIPFRLRP VFSNQFLLET 420
AAYLDYESTK EYAIKLLAAD AGKPPLNQSA MLFIKVKDEN DNAPVFTQSF VTVSIPENNS 480
PGIQLTKVSA MDADSGPNAK INYLLGPDAP PEFSLDCRTG MLTWKKLDR EKEDKYLFTI 540
LAKDNGVPPL TSNVTVFVSI IDQNDNSPVF THNEYNFYVP ENLPRHGTVG LITVTDPDYG 600
DNSAVTLSIL DENDDFTIDS QTGVIRPNIS FDREKQESYT FYVKAEDGGR VSRSSSAKVT 660
INWDVNDNK PVFIVPPSNC SYELVLPSTN PGTWFQVIA VDNDTGMNAE VRYSIVGGNT 720
RDLFAIDQET GNITLMEKCD VTDLGLHRVL VKANDLGQPD SLFSWIVNL FVNESVTNAT 780
LINELVRKST EAPVTPNTEI ADVSSPTSDY VKILVAAVAG TITWWIFI TAWRCRQAP 840
HLKAAQKNKQ NSEWATPNPE NRQMIMMKKK KKKKKHSPKN LLLNFVTIEE TKADDVDSDG 900
NRVTLDLPID LEEQTMGKYN WVTTPTTFKP DSPDLARHYK SASPQPAFQI QPETPLNSKH 960
HIIQELPLDN TFVACDSISK CSSSSSDPYS VSDCGYPVTT FEVPVSVHTR PPMKEVVRSC 1020
TPMKESTTME IWIHPQPQRK SEGKVAGKSQ RRVTFHLPEG SQESSSDGGL GDHDAGSLTS 1080
TSHGLPLGYP QEEYFDRATP SNRTEGDGNS DPESTFIPGL KKAAEITVQP TVEEASDNCT 1140
QECLIYGHSD ACWMPASLDH SSSSQAQASA LCHSPPLSQA STQHHSPRVT QTIALCHSPP 1200
VTQTIALCHS PPPIQVSALH HSPPLVQATA LHHSPPSAQA SALCYSPPLA QAAAISHSSP 1260
LPQVIALHRS QAQSSVSLQQ GWVQGADGLC SVDQGVQGSA TSQFYTMSER LHPSDDSIKV 1320
IPLTTFTPRQ QARPSRGDSP IMEEHPL 1347
Table LV(b). Amino acid sequence alignment of 109P1 D4 v.1 (SEQ ID NO: 247) and 109P1 D4 v.3 (SEQ ID NO: 248)
Score = 2005 bits (5195), Expect = O.OIdentities = 1011/1011 (100%), Positives = 1011/1011 (100%)
V.l : 1 MDLLSGTYIFAVLLACVVFHSGAQEKNYTIREEMPENVLIGDL KDLN SLIPNKSLTTA 60
MDL SGTYIFAVL ACVVFHSGAQEK YTIREEMPENVLIGD LKD NLSLIPNKS TTA V.3 : 1 MDLLSGTYIFAVL ACWFHSGAQEK YTIREEMPENVLIGD LKD NLS IPNKS TTA 60
V.l : 61 QFKLVYKTGDVPLIRIEEDTGEIFTTGARIDREKLCAGIPRDEHCFYEVEVAILPDEIF 120 MQFKLVYKTGDVPLIRIEEDTGEIFTTGARIDREKLCAGIPRDEHCFYEVEVAILPDEIF 61 MQFKLVYKTGDVPLIRIEEDTGEIFTTGARIDREKLCAGIPRDEHCFYEVEVAILPDEIF 120
121 RLVKIRFLIEDINDNAPLFPATVINISIPENSAINSKYTLPAAVDPDVGINGVQNYELIK 180
RLVKIRFLIEDINDNAPLFPATVINISIPENSAINSKYTLPAAVDPDVGINGVQNYELIK 121 RLVKIRFLIEDINDNAPLFPATVINISIPENSAINSKYTLPAAVDPDVGINGVQNYELIK 180
181 SQNIFGLDVIETPEGDKMPQ IVQKELDREEKDTYVMKVKVEDGGFPQRSSTAILQVSVT 240
SQNIFGLDVIETPEGDKMPQLIVQKELDREEKDTYVMKVKVEDGGFPQRSSTAILQVSVT 181 SQNIFGLDVIETPEGDKMPQ IVQKELDREEKDTYV KV VEDGGFPQRSSTAILQVSVT 240
241 DTNDNHPVFKETEIEVSIPENAPVGTSVTQLHATDADIGENAKIHFSFSNLVSNIARRLF 300
DTNDNHPVFKETEIEVSIPENAPVGTSVTQ HATDADIGENAKIHFSFSNLVSNIARRLF 241 DTNDNHPVFKETEIEVSIPENAPVGTSVTQLHATDADIGENAKIHFSFSN VSNIARRLF 300
301 HLNATTGLITI EPLDREETPNHKL VLASDGGLMPARAMVLVNVTDVNDNVPSIDIRYI 360
HLNATTGLITIKEPLDREETPNHKL VLASDGGLMPARAMVLVVTDVNDNVPSIDIRYI 301 H NATTGLITIKEPLDREETPNHKLLVLASDGGLMPARAVLVNVTDVNDNVPSIDIRYI 360
361 V PVNDTWLSENIP NTKIALITVTDKDADHNGRVTCFTDHEIPFRLRPVFSNQFLLET 420
VNPV DTW SENIPLNTKIALITVTDKDADHNGRVTCFTDHEIPFRLRPVFSNQFLLET 361 VNPVNDTW SENIPLNTKIALITVTDKDADHNGRVTCFTDHEIPFRLRPVFSNQFLLET 420
421 AAYLDYESTKEYAIKLLAADAGKPP NQSAM FIKVKDENDNAPVFTQSFVTVSIPENNS 480
AAY DYESTKEYAIKLLAADAGKPPLNQSAMLFIKVKDENDNAPVFTQSFVTVSIPENNS 421 AAYLDYESTKEYAIKLLAADAGKPPLNQSAMLFIKVKDENDNAPVFTQSFVTVSIPENNS 480
481 PGIQ TKVSAMDADSGPNAKINYLLGPDAPPEFSLDCRTGMLTVVKKLDREKEDKYLFTI 540
PGIQLTKVSAMDADSGPNAKINY LGPDAPPEFSLDCRTGM TWKKLDREKEDKY FTI 481 PGIQLTKVSAMDADSGPNAKINYLLGPDAPPEFSLDCRTGMLTVVKKLDREKEDKYLFTI 540
541 LAKDNGVPPLTSNVTVFVSIIDQNDNSPVFTHNEYNFYVPENLPRHGTVG ITVTDPDYG 600
LAKDNGVPPLTSNVTVFVSIIDQNDNSPVFTHNEYNFYVPENLPRHGTVGLITVTDPDYG 541 LAKDNGVPPLTSNVTVFVSIIDQNDNSPVFTHNEYNFYVPENLPRHGTVGLITVTDPDYG 600
601 DNSAVTLSILDENDDFTIDSQTGVIRPNISFDREKQESYTFYVKAEDGGRVSRSSSAKVT 660
DNSAVT SILDENDDFTIDSQTGVIRPNISFDREKQESYTFYVKAEDGGRVSRSSSAKVT 601 DNSAVTLSILDENDDFTIDSQTGVIRPNISFDREKQESYTFYVKAEDGGRVSRΞSSAKVT 660
661 INWDVNDNKPVFIVPPSNCSYE V PSTNPGTWFQVIAVDNDTG NAEVRYSIVGGNT 720
INVVDV DNKPVFIVPPSNCSYELVLPSTNPGTWFQVIAVDNDTGMNAEVRYSIVGGNT 661 INVVDV DNKPVFIVPPSNCSYELVLPSTNPGTWFQVIAVDNDTGMNAEVRYSIVGGNT 720
721 RDLFAIDQETGNITLMEKCDVTDLGLHRVLVKANDLGQPDS FSWIV LFVNESVTNAT 780
RD FAIDQETGNITLMEKCDVTDLG HRVLVKAND GQPDSLFSWIVN FV ESVTNAT 721 RDLFAIDQETGNITLMEKCDVTDLG HRVLVKANDLGQPDSLFSWIVNLFVNESVTNAT 780
781 INELVRKSTEAPVTPNTEIADVSSPTSDYVKILVAAVAGTITVVVVIFITAWRCRQAP 840
LINELVRKSTEAPVTPNTEIADVSSPTSDYVKILVAAVAGTITVVWIFITAWRCRQAP 781 INELVRKSTEAPVTPNTEIADVSSPTSDYVKI VAAVAGTITWWIFITAVVRCRQAP 840
841 HLKAAQK KQNSEWATPNPENRQMI MKKKKKKKKHSPKN LLNFVTIEETKADDVDSDG 900
Table Lll(c). Nucleotide sequence of transcript variant 109P1 D4 v.4 (SEQ ID NO: 249) ctggtggtcc agtacctcca aagatatgga atacactcct gaaatatcct gaaaactttt 60 ttttttcaga atcctttaat aagcagttat gtcaatctga aagttgctta cttgtacttt 120 atattaatag ctattcttgt ttttcttatc caaagaaaaa tcctctaatc cccttttcac 180 atgatagttg ttaccatgtt taggcattag tcacatcaac ccctctcctc tcccaaactt 240 ctcttcttca aatcaaactt tattagtccc tcctttataa tgattccttg cctcgtttta 300 tccagatcaa ttttttttca ctttgatgcc cagagctgaa gaaatggact actgtataaa 360 ttattcattg ccaagagaat aattgcattt taaacccata ttataacaaa gaataatgat 420 tatattttgt gatttgtaac aaataccctt tattttccct taactattga attaaatatt 480 ttaattattt gtattctctt taactatctt ggtatattaa agtattatct tttatatatt 540 tatcaatggt ggacactttt ataggtactc tgtgtcattt ttgatactgt aggtatctta 600 tttcatttat ctttattctt aatgtacgaa ttcataatat ttgattcaga acaaatttat 660 cactaattaa cagagtgtca attatgctaa catctcattt actgatttta atttaaaaca 720 gtttttgtta acatgcatgt ttagggttgg cttcttaata atttcttctt cctcttctct 780 ctctcctctt cttttggtca gtgttgtgcg ggttaataca acaaactgta acaagtgtac 840 ctggtatgga cttgttgtcc gggacgtaca ttttcgcggt cctgctagca tgcgtggtgt 900 tccactctgg cgcccaggag aaaaactaca ccatccgaga agaaatgcca gaaaacgtcc 960 tgataggcga cttgttgaaa gaccttaact tgtcgctgat tccaaacaag tccttgacaa 1020 ctgctatgca gttcaagcta gtgtacaaga ccggagatgt gccactgatt cgaattgaag 1080 aggatactgg tgagatcttc actactggcg ctcgcattga tcgtgagaaa ttatgtgctg 1140 gtatcccaag ggatgagcat tgcttttatg aagtggaggt tgccattttg ccggatgaaa 1200 tatttagact ggttaagata cgttttctga tagaagatat aaatgataat gcaccattgt 1260 tcccagcaac agttatcaac atatcaattc cagagaactc ggctataaac tctaaatata 1320 ctctcccagc ggctgttgat cctgacgtag gaataaacgg agttcaaaac tacgaactaa 1380 ttaagagtca aaacattttt ggcctcgatg tcattgaaac accagaagga gacaagatgc 1440 cacaactgat tgttcaaaag gagttagata gggaagagaa ggatacctac gtgatgaaag 1500 taaaggttga agatggtggc tttcctcaaa gatccagtac tgctattttg caagtgagtg 1560 ttactgatac aaatgacaac cacccagtct ttaaggagac agagattgaa gtcagtatac 1620 cagaaaatgc tcctgtaggc acttcagtga cacagctcca tgccacagat gctgacatag 1680 gtgaaaatgc caagatccac ttctctttca gcaatctagt ctccaacatt gccaggagat 1740 tatttcacct caatgccacc actggactta tcacaatcaa agaaccactg gatagggaag 1800 aaacaccaaa ccacaagtta ctggttttgg caagtgatgg tggattgatg ccagcaagag 1860 caatggtgct ggtaaatgtt acagatgtca atgataatgt cccatccatt gacataagat 1920 acatcgtcaa tcctgtcaat gacacagttg ttctttcaga aaatattcca ctcaacacca 1980 aaattgctct cataactgtg acggataagg atgcggacca taatggcagg gtgacatgct 2040 tcacagatca tgaaatccct ttcagattaa ggccagtatt cagtaatcag ttcctcctgg 2100 agactgcagc atatcttgac tatgagtcca caaaagaata tgccattaaa ttactggctg 2160 cagatgctgg caaacctcct ttgaatcagt cagcaatgct cttcatcaaa gtgaaagatg 2220 aaaatgacaa tgctccagtt ttcacccagt ctttcgtaac tgtttctatt cctgagaata 2280 actctcctgg catccagttg acgaaagtaa gtgcaatgga tgcagacagt gggcctaatg 2340 ctaagatcaa ttacctgcta ggccctgatg ctccacctga attcagcctg gattgtcgta 2400 caggcatgct gactgtagtg aagaaactag atagagaaaa agaggataaa tatttattca 2460 caattctggc aaaagataac ggggtaccac ccttaaccag caatgtcaca gtctttgtaa 2520 gcattattga tcagaatgac aatagcccag ttttcactca caatgaatac aacttctatg 2580 tcccagaaaa ccttccaagg catggtacag taggactaat cactgtaact gatcctgatt 2640 atggagacaa ttctgcagtt acgctctcca ttttagatga gaatgatgac ttcaccattg 2700 attcacaaac tggtgtcatc cgaccaaata tttcatttga tagagaaaaa caagaatctt 2760 acactttcta tgtaaaggct gaggatggtg gtagagtatc acgttcttca agtgccaaag 2820 taaccataaa tgtggttgat gtcaatgaca acaaaccagt tttcattgtc cctccttcca 2880 actgttctta tgaattggtt ctaccgtcca ctaatccagg cacagtggtc tttcaggtaa 2940 ttgctgttga caatgacact ggcatgaatg cagaggttcg ttacagcatt gtaggaggaa 3000 acacaagaga tctgtttgca atcgaccaag aaacaggcaa cataacattg atggagaaat 3060 gtgatgttac agaccttggt ttacacagag tgttggtcaa agctaatgac ttaggacagc 3120 ctgattctct cttcagtgtt gtaattgtca atctgttcgt gaatgagtcg gtgaccaatg 3180 ctacactgat taatgaactg gtgcgcaaaa gcactgaagc accagtgacc ccaaatactg 3240 agatagctga tgtatcctca ccaactagtg actatgtcaa gatcctggtt gcagctgttg 3300 ctggcaccat aactgtcgtt gtagttattt tcatcactgc tgtagtaaga tgtcgccagg 3360 caccacacct taaggctgct cagaaaaaca agcagaattc tgaatgggct accccaaacc 3420 cagaaaacag gcagatgata atgatgaaga aaaagaaaaa gaagaagaag cattccccta 3480 agaacttgct gcttaatttt gtcactattg aagaaactaa ggcagatgat gttgacagtg 3540 atggaaacag agtcacacta gaccttccta ttgatctaga agagcaaaca atgggaaagt 3600 acaattgggt aactacacct actactttca agcccgacag ccctgatttg gcccgacact 3660 acaaatctgc ctctccacag cctgccttcc aaattcagcc tgaaactccc ctgaattcga 3720 agcaccacat catccaagaa ctgcctctcg ataacacctt tgtggcctgt gactctatct 3780 ccaagtgttc ctcaagcagt tcagatccct acagcgtttc tgactgtggc tatccagtga 3840 cgaccttcga ggtacctgtg tccgtacaca ccagaccgcc aatgaaggag gttgtgcgat 3900 cttgcacccc catgaaagag tctacaacta tggagatctg gattcatccc caaccacagt 3960 cccagcggcg tgtcacattt cacctgccag aaggctctca ggaaagcagc agtgatggtg 4020 gactgggaga ccatgatgca ggcagcctta ccagcacatc tcatggcctg ccccttggct 4080 atcctcagga ggagtacttt gatcgtgcta cacccagcaa tcgcactgaa ggggatggca 4140 actccgatcc tgaatctact ttcatacctg gactaaagaa agctgcagaa ataactgttc 4200 aaccaactgt ggaagaggcc tctgacaact gcactcaaga atgtctcatc tatggccatt 4260 ctgatgcctg ctggatgccg gcatctctgg atcattccag ctcttcgcaa gcacaggcct 4320 ctgctctatg ccacagccca ccactgtcac aggcctctac tcagcaccac agcccacgag 4380 tgacacagac cattgctctc tgccacagcc ctccagtgac acagaccatc gcattgtgcc 4440 acagcccacc accgatacag gtgtctgctc tccaccacag tcctcctcta gtgcaggcta 4500 ctgcacttca ccacagccca ccatcagcac aggcctcagc cctctgctac agccctcctt 4560 tagcacaggc tgctgcaatc agccacagct ctcctctgcc acaggttatt gccctccatc 4620 gtagtcaggc ccaatcatca gtcagtttgc agcaaggttg ggtgcaaggt gctgatgggc 4680 tatgctctgt tgatcaggga gtgcaaggta gtgcaacatc tcagttttac accatgtctg 4740 aaagacttca tcccagtgat gattcaatta aagtcattcc tttgacaacc ttcactccac 4800 gccaacaggc cagaccgtcc agaggtgatt cccccattat ggaagaacat cccttgtaaa 4860 gctaaaatag ttacttcaaa ttttcagaaa agatgtatat agtcaaaatt taagatacaa 4920 ttccaatgag tattctgatt atcagatttg taaataacta tgtaaataga aacagatacc 4980 agaataaatc tacagctaga cccttagtca atagttaacc aaaaaattgc aatttgttta 5040 attcagaatg tgtatttaaa aagaaaagga atttaacaat ttgcatcccc ttgtacagta 5100 aggcttatca tgacagagcg cactatttct gatgtacagt attttttgtt gtttttatca 5160 tcatgtgcaa tattactgat ttgtttccat gctgattgtg tggaaccagt atgtagcaaa 5220 tggaaagcct agaaatatct tattttctaa gtttaccttt agtttaccta aacttttgtt 5280 cagataacgt taaaaggtat acgtactcta gccttttttt gggctttctt tttgattttt 5340 gtttgttgtt ttcagttttt ttgttgttgt tagtgagtct cccttcaaaa tacgcagtag 5400 gtagtgtaaa tactgcttgt ttgtgtctct ctgctgtcat gttttctacc ttattccaat 5460 actatattgt tgataaaatt tgtatataca ttttcaataa agaatatgta taaactgtac 5520 agatctagat ctacaaccta tttctctact ctttagtaga gttcgagaca cagaagtgca 5580 ataactgccc taattaagca actatttgtt aaaaagggcc tctttttact ttaatagttt 5640 agtgtaaagt acatcagaaa taaagctgta tctgccattt taagcctgta gtccattatt 5700 acttgggtct ttacttctgg gaatttgtat gtaacagcct agaaaattaa aaggaggtgg 5760 atgcatccaa agcacgagtc acttaaaata tcgacggtaa actactattt tgtagagaaa 5820 ctcaggaaga tttaaatgtt gatttgacag ctcaataggc tgttaccaaa gggtgttcag 5880 taaaaataac aaatacatgt aactgtagat aaaaccatat actaaatcta taagactaag 5940 ggatttttgt tattctagct caacttactg aagaaaacca ctaataacaa caagaatatc 6000 aggaaggaac ttttcaagaa atgtaattat aaatctacat caaacagaat tttaaggaaa 6060 aatgcagagg gagaaataag gcacatgact gcttcttgca gtcaacaaga aataccaata 6120 acacacacag aacaaaaacc atcaaaatct catatatgaa ataaaatata ttcttctaag 6180 caaagaaaca gtactattca tagaaaacat tagttttctt ctgttgtctg ttatttcctt 6240 cttgtatcct cttaactggc cattatcttg tatgtgcaca ttttataaat gtacagaaac 6300 atcaccaact taattttctt ccatagcaaa actgagaaaa taccttgttt cagtataaca 6360 ctaaaccaag agacaattga tgtttaatgg gggcggttgg ggtggggggg ggagtcaata 6420 tctcctattg attaacttag acatagattt tgtaatgtat aacttgatat ttaatttatg 6480 attaaactgt gtgtaaattt tgtaacataa actgtggtaa ttgcataatt tcattggtga 6540 ggatttccac tgaatattga gaaagtttct tttcatgtgc ccagcaggtt aagtagcgtt 6600 ttcagaatat acattattcc catccattgt aaagttcctt aagtcatatt tgactgggcg 6660 tgcagaataa cttcttaact tttaactatc agagtttgat taataaaatt aattaatgtt 6720 ttttctcctt cgtgttgtta atgttccaag ggatttggag catactggtt ttccaggtgc 6780 atgtgaatcc cgaaggactg atgatatttg aatgtttatt aaattattat catacaaatg 6840 tgttgatatt gtggctattg ttgatgttga aaattttaaa cttggggaag attaagaaaa 6900 gaaccaatag tgacaaaaat cagtgcttcc agtagatttt agaacattct ttgcctcaaa 6960 aaacctgcaa agatgatgtg agattttttc ttgtgtttta attattttca cattttctct 7020 ctgcaaaact ttagttttct gatgatctac acacacacac acacacacac gtgcacacac 7080 acacacattt aaatgatata aaaagaagag gttgaaagat tattaaataa cttatcaggc 7140 atctcaatgg ttactatcta tgttagtgaa aatcaaatag gactcaaagt tggatatttg 7200 ggatttttct tctgacagta taatttattg agttactagg gaggttctta aatcctcata 7260 tctggaaact tgtgacgttt tgacaccttt cctatagatg atataggaat gaaccaatac 7320 gcttttatta ccctttctaa ctctgatttt ataatcagac ttagattgtg tttagaatat 7380 taaatgactg ggcaccctct tcttggtttt taccagagag gctttgaatg gaagcaggct 7440 gagagtagcc aaagaggcaa ggggtattag cccagttatt ctcccctatg ccttccttct 7500 ctttctaagc gtccactagg tctggccttg gaaacctgtt acttctaggg cttcagatct 7560 gatgatatct ttttcatcac attacaagtt atttctctga ctgaatagac agtggtatag 7620 gttgacacag cacacaagtg gctattgtga tgtatgatgt atgtagtcct acaactgcaa 7680 aacgtcttac tgaaccaaca atcaaaaaat ggttctgttt taaaaaggat tttgtttgat 7740 ttgaaattaa aacttcaagc tgaatgactt atatgagaat aatacgttca atcaaagtag 7800 ttattctatt ttgtgtccat attccattag attgtgatta ttaattttct agctatggta 7860 ttactatatc acacttgtga gtatgtattc aaatactaag tatcttatat gctacgtgca 7920 tacacattct tttcttaaac tttacctgtg ttttaactaa tattgtgtca gtgtattaaa 7980 aattagcttt tacatatgat atctacaatg taataaattt agagagtaat tttgtgtatt 8040 cttatttact taacatttta cttttaatta tgtaaatttg gttagaaaat aataataaat 8100 ggttagtgct attgtgtaat ggtagcagtt acaaagagcc tctgccttcc caaactaata 8160 tttatcacac atggtcatta aatgggaaaa aaatagacta aacaaatcac aaattgttca 8220 gttcttaaaa tgtaattatg tcacacacac aaaaaatcct tttcaatcct gagaaaatta 8280 aaggcgtttt actcacatgg ctatttcaac attagttttt tttgtttgtt tctttttcat 8340 ggtattactg aaggtgtgta tactccctaa tacacattta tgaaaatcta cttgtttagg 8400 cttttattta tactcttctg atttatattt tttattataa ttattatttc ttatctttct 8460 tcttttatat tttttggaaa ccaaatttat agttagttta ggtaaacttt ttattatgac 8520 cattagaaac tattttgaat gcttccaact ggctcaattg gccgggaaaa catgggagca 8580 agagaagctg aaatatattt ctgcaagaac ctttctatat tatgtgccaa ttaccacacc 8640 agatcaattt tatgcagagg ccttaaaata ttctttcaca gtagctttct tacactaacc 8700 gtcatgtgct tttagtaaat atgattttta aaagcagttc aagttgacaa cagcagaaac 8760 agtaacaaaa aaatctgctc agaaaaatgt atgtgcacaa ataaaaaaaa ttaatggcaa 8820 ttgtttagtg attgtaagtg atacttttta aagagtaaac tgtgtgaaat ttatactatc 8880 cctgcttaaa atattaagat ttttatgaaa tatgtattta tgtttgtatt gtgggaagat 8940 tcctcctctg tgatatcata cagcatctga aagtgaacag tatcccaaag cagttccaac 9000 catgctttgg aagtaagaag gttgactatt gtatggccaa ggatggcagt atgtaatcca 9060 gaagcaaact tgtattaatt gttctatttc aggttctgta ttgcatgttt tcttattaat 9120 atatattaat aaaagttatg agaaat 9146
Table LI II (c). Nucleotide sequence alignment of 109P1D4 v.1 (SEQ ID NO: 250) and 109P1D4 v.4 (SEQ ID NO: 251)
Score = 7456 bits (3878), Expect = O.OIdentities = 3878/3878 (100%) Strand = Plus / Plus
V . l ctggtggtccagtacctccaaagatatggaatacactcctgaaatatcctgaaaactttt 60 I III II II Mill lllll lllll I I I llll II I II II III llll I II II I III I I II I II
V . 4 ctggtggtccagtacctccaaagatatggaatacactcctgaaatatcctgaaaactttt 60
V.l : 61 ttttttcagaatcctttaataagcagttatgtcaatctgaaagttgcttacttgtacttt 120
I I I 111 I II Mill II III Mill I II I III I 1 II Ml I I II MM I II MM I I I MM V.4 : 61 ttttttcagaatcctttaataagcagttatgtcaatctgaaagttgcttacttgtacttt 120
V.l : 121 atattaatagctattcttgtttttcttatccaaagaaaaatcctctaatccccttttcac 180
I lllll II II MM llll II III II I I II MM II II II II MM II II II II I I II I II
V.4 : 121 atattaatagctattcttgtttttcttatccaaagaaaaatcctctaatccccttttcac 180
V.l : 181 atgatagttgttaccatgtttaggcattagtcacatcaacccctctcctctcccaaactt 240
I II II I I I I MM I MM I III II II II II II I II Mill II III II II II I II II I I 11
V.4 : 181 atgatagttgttaccatgtttaggcattagtcacatcaacccctctcctctcccaaactt 240
V.l : 241 ctcttcttcaaatcaaactttattagtccctcctttataatgattccttgcctcgtttta 300
I I I II I II I III II II Mill II I I I II II II I II II II II III II II llll III II II I
V.4 : 241 ctcttcttcaaatcaaactttattagtccctcctttataatgattccttgcctcgtttta 300
V.l : 301 tccagatcaattttttttcactttgatgcccagagctgaagaaatggactactgtataaa 360
I II II I I II llll lllll lllll II I I MM II II I I II II III ill II II I I II II I
V.4 : 301 tccagatcaattttttttcactttgatgcccagagctgaagaaatggactactgtataaa 360
V.l : 361 ttattcattgccaagagaataattgcattttaaacccatattataacaaagaataatgat 420 lllll I I I III II II III II II lllll I II II I II I I II I III II llll II II I I II II I V.4 : 361 ttattcattgccaagagaataattgcattttaaacccatattataacaaagaataatgat 420
V.l : 421 tatattttgtgatttgtaacaaataccctttattttcccttaactattgaattaaatatt 480
I I II I II I Mill I Mlllll II I I III II II I I I III III III II II II II II I I I II I
V.4 : 421 tatattttgtgatttgtaacaaataccctttattttcccttaactattgaattaaatatt 480
V.l : 481 ttaattatttgtattctctttaactatcttggtatattaaagtattatcttttatatatt 540
I I 111 I II I lllll 111 llll II I I I I II II I III II II II III II II II II I III I II I
V.4 : 481 ttaattatttgtattctctttaactatcttggtatattaaagtattatcttttatatatt 540
V.l : 541 tatcaatggtggacacttttataggtactctgtgtcatttttgatactgtaggtatctta 600 I II II I I II III II II II III II I II I II II II I II II II I II II III I III I I 111 II I V.4 : 541 tatcaatggtggacacttttataggtactctgtgtcatttttgatactgtaggtatctta 600
V.l : 601 tttcatttatctttattcttaatgtacgaattcataatatttgattcagaacaaatttat 660
II I I 11 II I I I III I III MM II II I lllll 11 III II I II I I lllll II II I
V.4 : 601 tttcatttatctttattcttaatgtacgaattcataatatttgattcagaacaaatttat 660
V.l : 661 cactaattaacagagtgtcaattatgctaacatctcatttactgattttaatttaaaaca 720
II I I I I II I I I Ml II II MM I llll I II I II II I II II I II II I Ml I II II I II II I
V.4 : 661 cactaattaacagagtgtcaattatgctaacatctcatttactgattttaatttaaaaca 720
V.l : 721 gtttttgttaacatgcatgtttagggttggcttcttaataatttcttcttcctcttctct 780
II I I I Ml I I I II II I I I I III I llll I II II I II I II I II II III III I II II I II II I
V.4 : 721 gtttttgttaacatgcatgtttagggttggcttcttaataatttcttcttcctcttctct 780
V.l : 781 ctctcctcttcttttggtcagtgttgtgcgggttaatacaacaaactgtaacaagtgtac 840
II I I I I II I I I III II III II I I llll I 1 III I II I II II II II I I II I I I I I I I II III
V.4 : 781 ctctcctcttcttttggtcagtgttgtgcgggttaatacaacaaactgtaacaagtgtac 840
V.l : 841 ctggtatggacttgttgtccgggacgtacattttcgcggtcctgctagcatgcgtggtgt 900
I I I I I II II I I I II II I II Ml I I llll I II II II I II I II I II I I I II I I I II III III
V.4 : 841 ctggtatggacttgttgtccgggacgtacattttcgcggtcctgctagcatgcgtggtgt 900
V.l : 901 tccactctggcgcccaggagaaaaactacaccatccgagaagaaatgccagaaaacgtcc 960
II I 11 II I I I I I I II II II) I I I I I I lllll I I II I) II 11 I II 11 I I II I II II II I II
V.4 : 901 tccactctggcgcccaggagaaaaactacaccatccgagaagaaatgccagaaaacgtcc 960
V.l : 961 tgataggcgacttgttgaaagaccttaacttgtcgctgattccaaacaagtccttgacaa 1020
11 I I I I I II I I III I I I II II II I I II I I II I 111 I I II II I I II II I I I II I I
V.4 : 961 tgataggcgacttgttgaaagaccttaacttgtcgctgattccaaacaagtccttgacaa 1020
V.l : 1021 ctgctatgcagttcaagctagtgtacaagaccggagatgtgccactgattcgaattgaag 1080
I I I I I II II I I I I III II I II lllll I I I I I I I II I I I 11 II I I I I I I II I I I III I II I
V.4 : 1021 ctgctatgcagttcaagctagtgtacaagaccggagatgtgccactgattcgaattgaag 1080
V.l : 1081 aggatactggtgagatcttcactactggcgctcgcattgatcgtgagaaattatgtgctg 1140
I II I I I I II I I III I I III Ml MM II III I 1 II II I I I II I I II I II I II I II II I II
V.4 : 1081 aggatactggtgagatcttcactactggcgctcgcattgatcgtgagaaattatgtgctg 1140
V.l : 1141 gtatcccaagggatgagcattgcttttatgaagtggaggttgccattttgccggatgaaa 1200
I I I I I I I II I II II I I I II II II II llll III I I III I I II I Ml I II I II II I II II I I
V.4 : 1141 gtatcccaagggatgagcattgcttttatgaagtggaggttgccattttgccggatgaaa 1200
V.l : 1201 tatttagactggttaagatacgttttctgatagaagatataaatgataatgcaccattgt 1260
I I I I I II II I I I llll II Ml I llll MM II I I II I llll II I I II I II I II II I I II I
V.4 : 1201 tatttagactggttaagatacgttttctgatagaagatataaatgataatgcaccattgt 1260
V.l : 1261 tcccagcaacagttatcaacatatcaattccagagaactcggctataaactctaaatata 1320
II I II I I II I II II I lllll I II lllll III I III I II I I II II II I II I I I Ml III II
V.4 : 1261 tcccagcaacagttatcaacatatcaattccagagaactcggctataaactctaaatata 1320
V.l : 1321 ctctcccagcggctgttgatcctgacgtaggaataaacggagttcaaaactacgaactaa 1380
I I I I I I I II I I I I I II I I III III I III III II I I II I I I II II I I II I II II I II I II I
V.4 : 1321 ctctcccagcggctgttgatcctgacgtaggaataaacggagttcaaaactacgaactaa 1380
V.l : 1381 ttaagagtcaaaacatttttggcctcgatgtcattgaaacaccagaaggagacaagatgc 1440 III II I I Ml I II I I I I I I II I II II II III II I I III I 1 Ml II II I I I I II III I III V.4 : 1381 ttaagagtcaaaacatttttggcctcgatgtcattgaaacaccagaaggagacaagatgc 1440
V.l : 1441 cacaactgattgttcaaaaggagttagatagggaagagaaggatacctacgtgatgaaag 1500
I Ml I II I II I II II II I I II II III II I llll I I I II I II II II I I I II I I II II II I I V.4 : 1441 cacaactgattgttcaaaaggagttagatagggaagagaaggatacctacgtgatgaaag 1500
V.l : 1501 taaaggttgaagatggtggctttcctcaaagatccagtactgctattttgcaagtgagtg 1560
I III I II I II I II I II II 1 II II I III Ml II I II I I I II I I II I I I II I I I II II III 1 V.4 : 1501 taaaggttgaagatggtggctttcctcaaagatccagtactgctattttgcaagtgagtg 1560
V.l 1561 ttactgatacaaatgacaaccacccagtctttaaggagacagagattgaagtcagtatac 1620 III II I I II II I I II I I I III I II III I II II III II I I I II II I I I II I I I II I II III V.4 1561 ttactgatacaaatgacaaccacccagtctttaaggagacagagattgaagtcagtatac 1620
V.l : 1621 cagaaaatgctcctgtaggcacttcagtgacacagctccatgccacagatgctgacatag 1680
I II I I I I II II I I II I I I I II I Ml III II II I I II II I II II I II I I II I III V.4 : 1621 cagaaaatgctcctgtaggcacttcagtgacacagctccatgccacagatgctgacatag 1680
V.l : 1681 gtgaaaatgccaagatccacttctctttcagcaatctagtctccaacattgccaggagat 1740
I lllll I I II II I II II I I II I I III III I II I I I I I I I I II II I II I I II I II I I III I V.4 : 1681 gtgaaaatgccaagatccacttctctttcagcaatctagtctccaacattgccaggagat 1740
V.l : 1741 tatttcacctcaatgccaccactggacttatcacaatcaaagaaccactggatagggaag 1800
I II II I I I I I I I II II I I I I I I II III III I I I II I I I II I II I I I II I I I I II I I I III V.4 : 1741 tatttcacctcaatgccaccactggacttatcacaatcaaagaaccactggatagggaag 1800
V.l : 1801 aaacaccaaaccacaagttactggttttggcaagtgatggtggattgatgccagcaagag 1860
I II II II II II I II II II I II I MM II III II I I III I I II II I II II II I I II I 11 II V.4 : 1801 aaacaccaaaccacaagttactggttttggcaagtgatggtggattgatgccagcaagag 1860
V.l : 1861 caatggtgctggtaaatgttacagatgtcaatgataatgtcccatccattgacataagat 1920
III II I I I I II II I I I I I I I I 1 I I II I III II I I I I II I I I II II I II I I I III V.4 : 1861 caatggtgctggtaaatgttacagatgtcaatgataatgtcccatccattgacataagat 1920
V.l : 1921 acatcgtcaatcctgtcaatgacacagttgttctttcagaaaatattccactcaacacca 1980
I III I I I II II I I II I I I I II I II llll llll I II II II I I II II II I II I I II I I III I
V.4 : 1921 acatcgtcaatcctgtcaatgacacagttgttctttcagaaaatattccactcaacacca 1980
V.l : 1981 aaattgctctcataactgtgacggataaggatgcggaccataatggcagggtgacatgct 2040
II I II II I II I I I II II I II II II III III I II I I I II I I II I I I I I II II I I II II II I
V.4 : 1981 aaattgctctcataactgtgacggataaggatgcggaccataatggcagggtgacatgct 2040
V.l : 2041 tcacagatcatgaaatccctttcagattaaggccagtattcagtaatcagttcctcctgg 2100
I III I II III I I II I I I I I II II III II I III I II II I II I II II II I II I II II II III V.4 : 2041 tcacagatcatgaaatccctttcagattaaggccagtattcagtaatcagttcctcctgg 2100
V.l : 2101 agactgcagcatatcttgactatgagtccacaaaagaatatgccattaaattactggctg 2160
I II II I I II II I I I I II I I I I I II III II II II I II II I I II II I I I II I I I I II I II I I V.4 : 2101 agactgcagcatatcttgactatgagtccacaaaagaatatgccattaaattactggctg 2160
V.l : 2161 cagatgctggcaaacctcctttgaatcagtcagcaatgctcttcatcaaagtgaaagatg 2220
III II II I II I I I I I II I I II I I I II III I II I II I I II I I II II I I I II I II II I I III V.4 : 2161 cagatgctggcaaacctcctttgaatcagtcagcaatgctcttcatcaaagtgaaagatg 2220 V.l : 2221 aaaatgacaatgctccagttttcacccagtctttcgtaactgtttctattcctgagaata 2280
MMMMMMMMMMMMIIMMMMMMMMMMM lllll Mlllll V.4 : 2221 aaaatgacaatgctccagttttcacccagtctttcgtaactgtttctattcctgagaata 2280
V.l : 2281 actctcctggcatccagttgacgaaagtaagtgcaatggatgcagacagtgggcctaatg 2340
I I I MM II II III II II I II II II II II III II I II II II II I 11 II II II I M M Ml V.4 : 2281 actctcctggcatccagttgacgaaagtaagtgcaatggatgcagacagtgggcctaatg 2340
V.l : 2341 ctaagatcaattacctgctaggccctgatgctccacctgaattcagcctggattgtcgta 2400
I Ml II I II II llll II I II II II I II I II II II II lllll I I II II II II I I II II II I V.4 : 2341 ctaagatcaattacctgctaggccctgatgctccacctgaattcagcctggattgtcgta 2400
V.l : 2401 caggcatgctgactgtagtgaagaaactagatagagaaaaagaggataaatatttattca 2460
I I Ml III I III I III I I II II II I I I II III I I I II II I I I II II II I I I II I II II II V.4 : 2401 caggcatgctgactgtagtgaagaaactagatagagaaaaagaggataaatatttattca 2460
V.l : 2461 caattctggcaaaagataacggggtaccacccttaaccagcaatgtcacagtctttgtaa 2520
I I llll Ml II III II II I II I I I I I II I III II I II II II II 11 Ml I I 111 MM III V.4 : 2461 caattctggcaaaagataacggggtaccacccttaaccagcaatgtcacagtctttgtaa 2520
V.l : 2521 gcattattgatcagaatgacaatagcccagttttcactcacaatgaatacaacttctatg 2580
I II I II I II I I llll II I II II II I 11 III 11 II II I I II II II II I II II I I I I I II II V.4 : 2521 gcattattgatcagaatgacaatagcccagttttcactcacaatgaatacaacttctatg 2580
V.l : 2581 tcccagaaaaccttccaaggcatggtacagtaggactaatcactgtaactgatcctgatt 2640
I I I III II I II II Mill I II II I II II I III I I I I I II II II I I I II I I II I II I I II I V.4 : 2581 tcccagaaaaccttccaaggcatggtacagtaggactaatcactgtaactgatcctgatt 2640
V.l : 2641 atggagacaattctgcagttacgctctccattttagatgagaatgatgacttcaccattg 2700
I II I I II I I I II I II I II II I II I II II I 1 II II II I I I I I I II I II I II I 11 II II I I I V.4 : 2641 atggagacaattctgcagttacgctctccattttagatgagaatgatgacttcaccattg 2700
V.l : 2701 attcacaaactggtgtcatccgaccaaatatttcatttgatagagaaaaacaagaatctt 2760
I II I II III II III I III I I I II I I I Mill I I II II II II I I II I I I I I I II 1 V.4 : 2701 attcacaaactggtgtcatccgaccaaatatttcatttgatagagaaaaacaagaatctt 2760
V.l : 2761 acactttctatgtaaaggctgaggatggtggtagagtatcacgttcttcaagtgccaaag 2820
I I llll II I I Ml II II I II II 111 II I II I II I Ml II II I I I II II I I I I I I II I II I V.4 : 2761 acactttctatgtaaaggctgaggatggtggtagagtatcacgttcttcaagtgccaaag 2820
V.l : 2821 taaccataaatgtggttgatgtcaatgacaacaaaccagttttcattgtccctccttcca 2880
I I III I I llll llll I I I I II III I II II II III II II II I I I III I III I I I I I II I I I V.4 : 2821 taaccataaatgtggttgatgtcaatgacaacaaaccagttttcattgtccctccttcca 2880
V.l : 2881 actgttcttatgaattggttctaccgtccactaatccaggcacagtggtctttcaggtaa 2940 llll I I I II II III I I I I II II I I I I I II Ml I II II I lllll II I II I I II I I III I II V.4 : 2881 actgttcttatgaattggttctaccgtccactaatccaggcacagtggtctttcaggtaa 2940
V.l : 2941 ttgctgttgacaatgacactggcatgaatgcagaggttcgttacagcattgtaggaggaa 3000
I II III III II II II II I II II II I I I III I I II II I II II I II II II I I II I I I I I II I V.4 : 2941 ttgctgttgacaatgacactggcatgaatgcagaggttcgttacagcattgtaggaggaa 3000
V.l : 3001 acacaagagatctgtttgcaatcgaccaagaaacaggcaacataacattgatggagaaat 3060
I II Ml II I I III II II I I I I I I I I II I II II II II II II II II II I I I I I I I I II I II I V.4 : 3001 acacaagagatctgtttgcaatcgaccaagaaacaggcaacataacattgatggagaaat 3060 V. l : 3061 gtgatgttacagaccttggtttacacagagtgttggtcaaagctaatgacttaggacagc 3120
I I I 1 1 1 I I I I I I 1 I 1 1 1 1 I I 1 I I 1 1 I I I I I 1 I 1 I I 1 I 1 I 1 I I I I I I I 1 I 1 I I 1 I M I I 1 1
V.4 : 3061 gtgatgttacagaccttggtttacacagagtgttggtcaaagctaatgacttaggacagc 3120
V.l : 3121 ctgattctctcttcagtgttgtaattgtcaatctgttcgtgaatgagtcggtgaccaatg 3180
II I II II I II II lllll I II III III I II II II II I I II I Ml II II I II II I II I I II I
V.4 : 3121 ctgattctctcttcagtgttgtaattgtcaatctgttcgtgaatgagtcggtgaccaatg 3180
V.l : 3181 ctacactgattaatgaactggtgcgcaaaagcactgaagcaccagtgaccccaaatactg 3240
I I I I II II I lllll lllll III II II II II I I I I 11 II II I I II II I II II I II I II I II
V.4 : 3181 ctacactgattaatgaactggtgcgcaaaagcactgaagcaccagtgaccccaaatactg 3240
V.l : 3241 agatagctgatgtatcctcaccaactagtgactatgtcaagatcctggttgcagctgttg 3300
II I II II I I lllll III II I I II I II I II I I II I II I I I 111 II II I I II Ml I
V.4 : 3241 agatagctgatgtatcctcaccaactagtgactatgtcaagatcctggttgcagctgttg 3300
V.l : 3301 ctggcaccataactgtcgttgtagttattttcatcactgctgtagtaagatgtcgccagg 3360
II I I Ml llll Ml I I Ml Ml II I I II III I I II I II II I I I 111 I II I II I I II I II I V.4 : 3301 ctggcaccataactgtcgttgtagttattttcatcactgctgtagtaagatgtcgccagg 3360
V.l : 3361 caccacaccttaaggctgctcagaaaaacaagcagaattctgaatgggctaccccaaacc 3420
I I I I II III Mill llll I II III II llll 11 I II I I I I II II II II II II I II I III II V.4 : 3361 caccacaccttaaggctgctcagaaaaacaagcagaattctgaatgggctaccccaaacc 3420
V.l : 3421 cagaaaacaggcagatgataatgatgaagaaaaagaaaaagaagaagaagcattccccta 3480
I I I I II I II llll llll I I I 111 II II II II II II I II I I I I I II I II II I I I I II II I I V.4 : 3421 cagaaaacaggcagatgataatgatgaagaaaaagaaaaagaagaagaagcattccccta 3480
V.l : 3481 agaacttgctgcttaattttgtcactattgaagaaactaaggcagatgatgttgacagtg 3540
I I I III I I III II III III III II II II II I I I I I III II II I I I I II II I II III I I II
V.4 : 3481 agaacttgctgcttaattttgtcactattgaagaaactaaggcagatgatgttgacagtg 3540
V.l : 3541 atggaaacagagtcacactagaccttcctattgatctagaagagcaaacaatgggaaagt 3600
II II III II II III I MM II Ml II I III III I I MM I II II II I I I II I I I II I II I
V.4 : 3541 atggaaacagagtcacactagaccttcctattgatctagaagagcaaacaatgggaaagt 3600
V.l : 3601 acaattgggtaactacacctactactttcaagcccgacagccctgatttggcccgacact 3660
II I I II I II Mill I I II Ml II II llll II llll I I II I II II II I II II I II II I II I V.4 : 3601 acaattgggtaactacacctactactttcaagcccgacagccctgatttggcccgacact 3660
V.l : 3661 acaaatctgcctctccacagcctgccttccaaattcagcctgaaactcccctgaattcga 3720
I I I I II I I I II III llll III II II II II I I I I II I III I II III II II II I II 111 I II
V.4 : 3661 acaaatctgcctctccacagcctgccttccaaattcagcctgaaactcccctgaattcga 3720
V.l : 3721 agcaccacatcatccaagaactgcctctcgataacacctttgtggcctgtgactctatct 3780
II I I II I II II III II II III II II I) II I II I II II 111 I I I I I II II I I I II I I II II
V.4 : 3721 agcaccacatcatccaagaactgcctctcgataacacctttgtggcctgtgactctatct 3780
V.l : 3781 ccaagtgttcctcaagcagttcagatccctacagcgtttctgactgtggctatccagtga 3840
I I I MM II lllll llll II II II I I II II II I I II I I II I II II I II II I I I II II I II
V.4 : 3781 ccaagtgttcctcaagcagttcagatccctacagcgtttctgactgtggctatccagtga 3840
V.l : 3841 cgaccttcgaggtacctgtgtccgtacacaccagaccg 3878
II I I II I II Mill III II I II I I I I II II II II I I II
V.4 : 3841 cgaccttcgaggtacctgtgtccgtacacaccagaccg 3878 Table LIV(c). Peptide sequences of protein coded by 109P1D4 v.4 (SEQ ID NO: 252)
MDLLSGTYIF AVLLACWFH SGAQEKNYTI REEMPENVLI GDLLKDLNLS LIPNKSLTTA 60 MQFKLVYKTG DVPLIRIEED TGEIFTTGAR IDREKLCAGI PRDEHCFYEV EVAILPDEIF 120 RLVKIRFLIE DINDNAPLFP ATVINISIPE NSAINSKYTL PAAVDPDVGI NGVQNYELIK 180 SQNIFGLDVI ETPEGDKMPQ LIVQKELDRE EKDTYVMKVK VEDGGFPQRS STAILQVSVT 240 DTNDNHPVFK ETEIEVSIPE NAPVGTSVTQ LHATDADIGE NAKIHFSFSN LVSNIARRLF 300 HLNATTGLIT IKEPLDREET PNHKLLVLAS DGGLMPARAM VLVNVTDVND NVPSIDIRYI 360 VNPVNDTWL SENIPLNTKI ALITVTDKDA DHNGRVTCFT DHEIPFRLRP VFSNQFLLET 420 AAYLDYESTK EYAIKLLAAD AGKPPLNQSA MLFIKVKDEN DNAPVFTQSF VTVSIPENNS 480 PGIQLTKVSA MDADSGPNAK INYLLGPDAP PEFSLDCRTG MLTWKKLDR EKEDKYLFTI 540 LAKDNGVPPL TSNVTVFVSI IDQNDNSPVF THNEYNFYVP ENLPRHGTVG LITVTDPDYG 600 DNSAVTLSIL DENDDFTIDS QTGVIRPNIS FDREKQESYT FYVKAEDGGR VSRSSSAKVT 660 INWDVNDNK PVFIVPPSNC SYELVLPSTN PGTVVFQVIA VDNDTGMNAE VRYSIVGGNT 720 RDLFAIDQET GNITLMEKCD VTDLGLHRVL VKANDLGQPD SLFSVVIVNL FVNESVTNAT 780 LINELVRKST EAPVTPNTEI ADVSSPTSDY VKILVAAVAG TITWWIFI TAVVRCRQAP 840 HLKAAQKNKQ NSEWATPNPE NRQMIMMKKK KKKKKHSPKN LLLNFVTIEE TKADDVDSDG 900 NRVTLDLPID LEEQTMGKYN WVTTPTTFKP DSPDLARHYK SASPQPAFQI QPETPLNSKH 960 HIIQELPLDN TFVACDSISK CSSSSSDPYS VSDCGYPVTT FEVPVSVHTR PPMKEWRSC 1020 TPMKESTTME IWIHPQPQSQ RRVTFHLPEG SQESSSDGGL GDHDAGSLTS TSHGLPLGYP 1080 QEEYFDRATP SNRTEGDGNS DPESTFIPGL KKAAEITVQP TVEEASDNCT QECLIYGHSD 1140 ACWMPASLDH SSSSQAQASA LCHSPPLSQA STQHHSPRVT QTIALCHSPP VTQTIALCHS 1200 PPPIQVSALH HSPPLVQATA LHHSPPSAQA SALCYSPPLA QAAAISHSSP LPQVIALHRS 1260 QAQSSVSLQQ GWVQGADGLC SVDQGVQGSA TSQFYTMSER LHPSDDSIKV IPLTTFTPRQ 1320 QARPSRGDSP IMEEHPL 1337
Table LV(c). Amino acid sequence alignment of 109P1 D4 v.1 (SEQ ID NO: 253) and 109P1 D4 v.4 (SEQ ID NO: 254)
Score = 2005 bits (5195), Expect = O.OIdentities = 1011/1011 (100%), Positives = 1011/1011 (100%)
1 MDLLSGTYIFAVL ACWFHSGAQEK YTIREEMPENVLIGDLLKD NLSLIPNKSLTTA 60
MDLLSGTYIFAVLLACWFHSGAQEK YTIREEMPENVL1GDLLKDLNLSLIPNKS TTA 1 MDLLSGTYIFAV LACVVFHSGAQEKNYTIREEMPENVLIGDLLKDLNLSLIPNKSLTTA 60
61 MQFKLVYKTGDVPLIRIEEDTGEIFTTGARIDREK CAGIPRDEHCFYEVEVAILPDEIF 120
MQFKLVYKTGDVPLIRIEEDTGEIFTTGARIDREKLCAGIPRDEHCFYEVEVAILPDEIF 61 MQFKLVYKTGDVPLIRIEEDTGEIFTTGARIDREKLCAGIPRDEHCFYEVEVAILPDEIF 120
121 RLVKIRFLIEDINDNAPLFPATVINISIPENSAINSKYTLPAAVDPDVGINGVQNYE IK 180
RLVKIRFLIEDINDNAPLFPATVINISIPENSAINSKYTLPAAVDPDVGINGVQNYELIK 121 RLVKIRF IEDINDNAP FPATVINISIPENSAINSKYT PAAVDPDVGINGVQNYE IK 180
181 SQNIFGLDVIETPEGDKMPQLIVQ ELDREEKDTYVMKVKVEDGGFPQRSSTAI QVSVT 240
SQNIFGLDVIETPEGDKMPQ IVQKELDREEKDTYVMKVIVEDGGFPQRSSTAI QVSVT 181 SQNIFGLDVIETPEGDKMPQLIVQKELDREEKDTYVMKVKVEDGGFPQRSSTAILQVSVT 240
241 DTNDNHPVFKETEIEVSIPENAPVGTSVTQLHATDADIGENAKIHFSFSN VSNIARRLF 300
DTNDNHPVFKETEIEVSIPENAPVGTSVTQLHATDADIGENAKIHFSFSNLVSNIARRLF 241 DTNDNHPVFKETEIEVSIPENAPVGTSVTQLHATDADIGENAKIHFSFSN VSNIARRLF 300
301 HLNATTGLITIKEPLDREETPNHKLLVLASDGGLMPARAMVLV VTDVNDNVPSIDIRYI 360
HLNATTGLITIKEPLDREETPNHK LVLASDGGLMPARAMVLVNVTDVNDNVPSIDIRYI 301 HLNATTG ITIKEP DREETPNHKLLV ASDGG PARAMVLVNVTDVNDNVPSIDIRYI 360
361 VNPVNDTVVLSENIPLNTKIALITVTDKDADHNGRVTCFTDHEIPFR RPVFSNQFLLET 420
VNPVNDTWLSENIPLNTKIALITVTDKDADHNGRVTCFTDHEIPFRLRPVFSNQFLLET 361 VNPVNDTWLSENIPLNTKIALITVTDKDADHNGRVTCFTDHEIPFRLRPVFSNQFLLET 420
421 AAYLDYESTKEYAIKLLAADAGKPPLNQSAM FIKVKDENDNAPVFTQSFVTVSIPENNS 480
AAYLDYESTKEYAIKLLAADAGKPP NQSAMLFIKVKDENDNAPVFTQSFVTVSIPENNS 421 AAYLDYESTKEYAIKLLAADAGKPPLNQSAMLFIKVKDENDNAPVFTQSFVTVSIPENNS 480
481 PGIQLTKVSAMDADSGPNAKINYL GPDAPPEFSLDCRTG LTWKK DREKEDKY FTI 540 PGIQLTKVSAMDADSGPNAKINYL GPDAPPEFS DCRTG LTWKKLDREKEDKYLFTI V.4 481 PGIQLTKVSAMDADSGPNAKINYLLGPDAPPEFSLDCRTGM TWKKLDREKEDKY FTI 540 V.l 541 LAKDNGVPP TSNVTVFVS1IDQNDNSPVFTHNEYNFYVPENLPRHGTVGLITVTDPDYG 600
LAKDNGVPPLTSNVTVFVSIIDQNDNSPVFTHNEYNFYVPENLPRHGTVG ITVTDPDYG V.4 541 LAKDNGVPPLTSNVTVFVSIIDQNDNSPVFTHNEYNFYVPEN PRHGTVGLITVTDPDYG 600 V.l 601 DNSAVTLSILDENDDFTIDSQTGVIRPNISFDREKQESYTFYVKAEDGGRVSRSSSAKVT 660
DNSAVTLSILDENDDFTIDSQTGVIRPNISFDREKQESYTFYVKAEDGGRVSRSSSAKVT V.4 601 DNSAVTLSILDENDDFTIDSQTGVIRPNISFDREKQESYTFYVKAEDGGRVSRSSSAKVT 660 V.l 661 INWDVNDNKPVFIVPPSNCSYELVLPSTNPGTWFQVIAVDNDTGMNAEVRYSIVGGNT 720
INWDVNDNKPVFIVPPSNCSYELV PSTNPGTWFQVIAVDNDTGMNAEVRYSIVGGNT V.4 661 INWDVNDNKPVFIVPPSNCSYELVLPSTNPGTWFQVIAVDNDTGM AEVRYSIVGGNT 720 V.l 721 RDLFAIDQETGNITLMEKCDVTD GLHRVLVKANDLGQPDSLFSWIVNLFVNESVTNAT 780
RDLFAIDQETGNITLMEKCDVTDLGLHRVLVKANDLGQPDSLFSWIVNLFVNESVTNAT V.4 721 RDLFAIDQETGNITLMEKCDVTDLGLHRVLVKANDLGQPDSLFSWIVNLFVNESVTNAT 780 V.l 781 LINELVRKSTEAPVTPNTEIADVSSPTSDYVKILVAAVAGTITWWIFITAWRCRQAP 840
LINELVRKSTEAPVTPNTEIADVSSPTSDYVKILVAAVAGTITWWIFITAWRCRQAP V.4 781 INELVRKSTEAPVTPNTEIADVSSPTSDYVKILVAAVAGTITWWIFITAWRCRQAP 840 V.l 841 HLKAAQK KQNSEWATPNPENRQMIMMKKKKK KKHSPKNLLLNFVTIEET ADDVDSDG 900
HLKAAQKNKQNSEWATPNPENRQMI MKKKKKKKKHSPKNLLLNFVTIEETKADDVDSDG V.4 841 HLKAAQKNKQNSEWATPNPENRQMI KKKKKKKKHSPKNLLLNFVTIEETKADDVDSDG 900 V.l 901 NRVTLDLPIDLEEQTMGKYNWVTTPTTFKPDSPDLARHYKSASPQPAFQIQPETPLNSKH 960
NRVTLDLPIDLEEQTMGKYNWVTTPTTFKPDSPDLARHYKSASPQPAFQIQPETPLNSKH V.4 901 NRVTLDLPIDLEEQTMGKYN VTTPTTFKPDSPDLARHYKSASPQPAFQIQPETPLNSKH 960 V.l 961 HIIQE PLDNTFVACDSISKCSSSSSDPYSVSDCGYPVTTFEVPVSVHTRP 1011
HIIQE PLDNTFVACDSISKCSSSSSDPYSVSDCGYPVTTFEVPVSVHTRP V.4 961 HIIQELPLDNTFVACDSISKCSSSSSDPYSVSDCGYPVTTFEVPVSVHTRP 1011
Table Lll(d). Nucleotide sequence of transcript variant 109P1 D4 v.5 (SEQ ID NO: 255) ctggtggtcc agtacctcca aagatatgga atacactcct gaaatatcct gaaaactttt 60 ttttttcaga atcctttaat aagcagttat gtcaatctga aagttgctta cttgtacttt 120 atattaatag ctattcttgt ttttcttatc caaagaaaaa tcctctaatc cccttttcac 180 atgatagttg ttaccatgtt taggcattag tcacatcaac ccctctcctc tcccaaactt 240 ctcttcttca aatcaaactt tattagtccc tcctttataa tgattccttg cctcgtttta 300 tccagatcaa ttttttttca ctttgatgcc cagagctgaa gaaatggact actgtataaa 360 ttattcattg ccaagagaat aattgcattt taaacccata ttataacaaa gaataatgat 420 tatattttgt gatttgtaac aaataccctt tattttccct taactattga attaaatatt 480 ttaattattt gtattctctt taactatctt ggtatattaa agtattatct tttatatatt 540 tatcaatggt ggacactttt ataggtactc tgtgtcattt ttgatactgt aggtatctta 600 tttcatttat ctttattctt aatgtacgaa ttcataatat ttgattcaga acaaatttat 660 cactaattaa cagagtgtca attatgctaa catctcattt actgatttta atttaaaaca 720 gtttttgtta acatgcatgt ttagggttgg cttcttaata atttcttctt cctcttctct 780 ctctcctctt cttttggtca gtgttgtgcg ggttaataca acaaactgta acaagtgtac 840 ctggtatgga cttgttgtcc gggacgtaca ttttcgcggt cctgctagca tgcgtggtgt 900 tccactctgg cgcccaggag aaaaactaca ccatccgaga agaaatgcca gaaaacgtcc 960 tgataggcga cttgttgaaa gaccttaact tgtcgctgat tccaaacaag tccttgacaa 1020 ctgctatgca gttcaagcta gtgtacaaga ccggagatgt gccactgatt cgaattgaag 1080 aggatactgg tgagatcttc actactggcg ctcgcattga tcgtgagaaa ttatgtgctg 1140 gtatcccaag ggatgagcat tgcttttatg aagtggaggt tgccattttg ccggatgaaa 1200 tatttagact ggttaagata cgttttctga tagaagatat aaatgataat gcaccattgt 1260 tcccagcaac agttatcaac atatcaattc cagagaactc ggctataaac tctaaatata 1320 ctctcccagc ggctgttgat cctgacgtag gaataaacgg agttcaaaac tacgaactaa 1380 ttaagagtca aaacattttt ggcctcgatg tcattgaaac accagaagga gacaagatgc 1440 cacaactgat tgttcaaaag gagttagata gggaagagaa ggatacctac gtgatgaaag 1500 taaaggttga agatggtggc tttcctcaaa gatccagtac tgctattttg caagtgagtg 1560 ttactgatac aaatgacaac cacccagtct ttaaggagac agagattgaa gtcagtatac 1620 cagaaaatgc tcctgtaggc acttcagtga cacagctcca tgccacagat gctgacatag 1680 gtgaaaatgc caagatccac ttctctttca gcaatctagt ctccaacatt gccaggagat 1740 tatttcacct caatgccacc actggactta tcacaatcaa agaaccactg gatagggaag 1800 aaacaccaaa ccacaagtta ctggttttgg caagtgatgg tggattgatg ccagcaagag 1860 caatggtgct ggtaaatgtt acagatgtca atgataatgt cccatccatt gacataagat 1920 acatcgtcaa tcctgtcaat gacacagttg ttctttcaga aaatattcca ctcaacacca 1980 aaattgctct cataactgtg acggataagg atgcggacca taatggcagg gtgacatgct 2040 tcacagatca tgaaatccct ttcagattaa ggccagtatt cagtaatcag ttcctcctgg 2100 agactgcagc atatcttgac tatgagtcca caaaagaata tgccattaaa ttactggctg 2160 cagatgctgg caaacctcct ttgaatcagt cagcaatgct cttcatcaaa gtgaaagatg 2220 aaaatgacaa tgctccagtt ttcacccagt ctttcgtaac tgtttctatt cctgagaata 2280 actctcctgg catccagttg acgaaagtaa gtgcaatgga tgcagacagt gggcctaatg 2340 ctaagatcaa ttacctgcta ggccctgatg ctccacctga attcagcctg gattgtcgta 2400 caggcatgct gactgtagtg aagaaactag atagagaaaa agaggataaa tatttattca 2460 caattctggc aaaagataac ggggtaccac ccttaaccag caatgtcaca gtctttgtaa 2520 gcattattga tcagaatgac aatagcccag ttttcactca caatgaatac aacttctatg 2580 tcccagaaaa ccttccaagg catggtacag taggactaat cactgtaact gatcctgatt 2640 atggagacaa ttctgcagtt acgctctcca ttttagatga gaatgatgac ttcaccattg 2700 attcacaaac tggtgtcatc cgaccaaata tttcatttga tagagaaaaa caagaatctt 2760 acactttcta tgtaaaggct gaggatggtg gtagagtatc acgttcttca agtgccaaag 2820 taaccataaa tgtggttgat gtcaatgaca acaaaccagt tttcattgtc cctccttcca 2880 actgttctta tgaattggtt ctaccgtcca ctaatccagg cacagtggtc tttcaggtaa 2940 ttgctgttga caatgacact ggcatgaatg cagaggttcg ttacagcatt gtaggaggaa 3000 acacaagaga tctgtttgca atcgaccaag aaacaggcaa cataacattg atggagaaat 3060 gtgatgttac agaccttggt ttacacagag tgttggtcaa agctaatgac ttaggacagc 3120 ctgattctct cttcagtgtt gtaattgtca atctgttcgt gaatgagtcg gtgaccaatg 3180 ctacactgat taatgaactg gtgcgcaaaa gcactgaagc accagtgacc ccaaatactg 3240 agatagctga tgtatcctca ccaactagtg actatgtcaa gatcctggtt gcagctgttg 3300 ctggcaccat aactgtcgtt gtagttattt tcatcactgc tgtagtaaga tgtcgccagg 3360 caccacacct taaggctgct cagaaaaaca agcagaattc tgaatgggct accccaaacc 3420 cagaaaacag gcagatgata atgatgaaga aaaagaaaaa gaagaagaag cattccccta 3480 agaacttgct gcttaatttt gtcactattg aagaaactaa ggcagatgat gttgacagtg 3540 atggaaacag agtcacacta gaccttccta ttgatctaga agagcaaaca atgggaaagt 3600 acaattgggt aactacacct actactttca agcccgacag ccctgatttg gcccgacact 3660 acaaatctgc ctctccacag cctgccttcc aaattcagcc tgaaactccc ctgaattcga 3720 agcaccacat catccaagaa ctgcctctcg ataacacctt tgtggcctgt gactctatct 3780 ccaagtgttc ctcaagcagt tcagatccct acagcgtttc tgactgtggc tatccagtga 3840 cgaccttcga ggtacctgtg tccgtacaca ccagaccgtc ccagcggcgt gtcacatttc 3900 acctgccaga aggctctcag gaaagcagca gtgatggtgg actgggagac catgatgcag 3960 gcagccttac cagcacatct catggcctgc cccttggcta tcctcaggag gagtactttg 4020 atcgtgctac acccagcaat cgcactgaag gggatggcaa ctccgatcct gaatctactt 4080 tcatacctgg actaaagaaa gctgcagaaa taactgttca accaactgtg gaagaggcct 4140 ctgacaactg cactcaagaa tgtctcatct atggccattc tgatgcctgc tggatgccgg 4200 catctctgga tcattccagc tcttcgcaag cacaggcctc tgctctatgc cacagcccac 4260 cactgtcaca ggcctctact cagcaccaca gcccacgagt gacacagacc attgctctct 4320 gccacagccc tccagtgaca cagaccatcg cattgtgcca cagcccacca ccgatacagg 4380 tgtctgctct ccaccacagt cctcctctag tgcaggctac tgcacttcac cacagcccac 4440 catcagcaca ggcctcagcc ctctgctaca gccctccttt agcacaggct gctgcaatca 4500 gccacagctc tcctctgcca caggttattg ccctccatcg tagtcaggcc caatcatcag 4560 tcagtttgca gcaaggttgg gtgcaaggtg ctgatgggct atgctctgtt gatcagggag 4620 tgcaaggtag tgcaacatct cagttttaca ccatgtctga aagacttcat cccagtgatg 4680 attcaattaa agtcattcct ttgacaacct tcactccacg ccaacaggcc agaccgtcca 4740 gaggtgattc ccccattatg gaagaacatc ccttgtaaag ctaaaatagt taσttcaaat 4800 tttcagaaaa gatgtatata gtcaaaattt aagatacaat tccaatgagt attctgatta 4860 tcagatttgt aaataactat gtaaatagaa acagatacca gaataaatct acagctagac 4920 ccttagtcaa tagttaacca aaaaattgca atttgtttaa ttcagaatgt gtatttaaaa 4980 agaaaaggaa tttaacaatt tgcatcccct tgtacagtaa ggcttatcat gacagagcgc 5040 actatttctg atgtacagta ttttttgttg tttttatcat catgtgcaat attactgatt 5100 tgtttccatg ctgattgtgt ggaaccagta tgtagcaaat ggaaagccta gaaatatctt 5160 attttctaag tttaccttta gtttacctaa acttttgttc agataacgtt aaaaggtata 5220 cgtactctag cctttttttg ggctttcttt ttgatttttg tttgttgttt tcagtttttt 5280 tgttgttgtt agtgagtctc ccttcaaaat acgcagtagg tagtgtaaat actgcttgtt 5340 tgtgtctctc tgctgtcatg ttttctacct tattccaata ctatattgtt gataaaattt 5400 gtatatacat tttcaataaa gaatatgtat aaactgtaca gatctagatc tacaacctat 5460 ttctctactc tttagtagag ttcgagacac agaagtgcaa taactgccct aattaagcaa 5520 ctatttgtta aaaagggcct ctttttactt taatagttta gtgtaaagta catcagaaat 5580 aaagctgtat ctgccatttt aagcctgtag tccattatta cttgggtctt tacttctggg 5640 aatttgtatg taacagccta gaaaattaaa aggaggtgga tgcatccaaa gcacgagtca 5700 cttaaaatat cgacggtaaa ctactatttt gtagagaaac tcaggaagat ttaaatgttg 5760 atttgacagc tcaataggct gttaccaaag ggtgttcagt aaaaataaca aatacatgta 5820 actgtagata aaaccatata ctaaatctat aagactaagg gatttttgtt attctagctc 5880 aacttactga agaaaaccac taataacaac aagaatatca ggaaggaact tttcaagaaa 5940 tgtaattata aatctacatc aaacagaatt ttaaggaaaa atgcagaggg agaaataagg 6000 cacatgactg cttcttgcag tcaacaagaa ataccaataa cacacacaga acaaaaacca 6060 tcaaaatctc atatatgaaa taaaatatat tcttctaagc aaagaaacag tactattcat 6120 agaaaacatt agttttcttc tgttgtctgt tatttccttc ttgtatcctc ttaactggcc 6180 attatcttgt atgtgcacat tttataaatg tacagaaaca tcaccaactt aattttcttc 6240 catagcaaaa ctgagaaaat accttgtttc agtataacac taaaccaaga gacaattgat 6300 gtttaatggg ggcggttggg gtgggggggg gagtcaatat ctcctattga ttaacttaga 6360 catagatttt gtaatgtata acttgatatt taatttatga ttaaactgtg tgtaaatttt 6420 gtaacataaa ctgtggtaat tgcataattt cattggtgag gatttccact gaatattgag 6480 aaagtttctt ttcatgtgcc cagcaggtta agtagcgttt tcagaatata cattattccc 6540 atccattgta aagttcctta agtcatattt gactgggcgt gcagaataac ttcttaactt 6600 ttaactatca gagtttgatt aataaaatta attaatgttt tttctccttc gtgttgttaa 6660 tgttccaagg gatttggagc atactggttt tccaggtgca tgtgaatccc gaaggactga 6720 tgatatttga atgtttatta aattattatc atacaaatgt gttgatattg tggctattgt 6780 tgatgttgaa aattttaaac ttggggaaga ttaagaaaag aaccaatagt gacaaaaatc 6840 agtgcttcca gtagatttta gaacattctt tgcctcaaaa aacctgcaaa gatgatgtga 6900 gattttttct tgtgttttaa ttattttcac attttctctc tgcaaaactt tagttttctg 6960 atgatctaca cacacacaca cacacacacg tgcacacaca cacacattta aatgatataa 7020 aaagaagagg ttgaaagatt attaaataac ttatcaggca tctcaatggt tactatctat 7080 gttagtgaaa atcaaatagg actcaaagtt ggatatttgg gatttttctt ctgacagtat 7140 aatttattga gttactaggg aggttcttaa atcctcatat ctggaaactt gtgacgtttt 7200 gacacctttc ctatagatga tataggaatg aaccaatacg cttttattac cctttctaac 7260 tctgatttta taatcagact tagattgtgt ttagaatatt aaatgactgg gcaccctctt 7320 cttggttttt accagagagg ctttgaatgg aagcaggctg agagtagcca aagaggcaag 7380 gggtattagc ccagttattc tcccctatgc cttccttctc tttctaagcg tccactaggt 7440 ctggccttgg aaacctgtta cttctagggc ttcagatctg atgatatctt tttcatcaca 7500 ttacaagtta tttctctgac tgaatagaca gtggtatagg ttgacacagc acacaagtgg 7560 ctattgtgat gtatgatgta tgtagtccta caactgcaaa acgtcttact gaaccaacaa 7620 tcaaaaaatg gttctgtttt aaaaaggatt ttgtttgatt tgaaattaaa acttcaagct 7680 gaatgactta tatgagaata atacgttcaa tcaaagtagt tattctattt tgtgtccata 7740 ttccattaga ttgtgattat taattttcta gctatggtat tactatatca cacttgtgag 7800 tatgtattca aatactaagt atcttatatg ctacgtgcat acacattctt ttcttaaact 7860 ttacctgtgt tttaactaat attgtgtcag tgtattaaaa attagctttt acatatgata 7920 tctacaatgt aataaattta gagagtaatt ttgtgtattc ttatttactt aacattttac 7980 ttttaattat gtaaatttgg ttagaaaata ataataaatg gttagtgcta ttgtgtaatg 8040 gtagcagtta caaagagcct ctgccttccc aaactaatat ttatcacaca tggtcattaa 8100 atgggaaaaa aatagactaa acaaatcaca aattgttcag ttcttaaaat gtaattatgt 8160 cacacacaca aaaaatcctt ttcaatcctg agaaaattaa aggcgtttta ctcacatggc 8220 tatttcaaca ttagtttttt ttgtttgttt ctttttcatg gtattactga aggtgtgtat 8280 actccctaat acacatttat gaaaatctac ttgtttaggc ttttatttat actcttctga 8340 tttatatttt ttattataat tattatttct tatctttctt cttttatatt ttttggaaac 8400 caaatttata gttagtttag gtaaactttt tattatgacc attagaaact attttgaatg 8460 cttccaactg gctcaattgg ccgggaaaac atgggagcaa gagaagctga aatatatttc 8520 tgcaagaacc tttctatatt atgtgccaat taccacacca gatcaatttt atgcagaggc 8580 cttaaaatat tctttcacag tagctttctt acactaaccg tcatgtgctt ttagtaaata 8640 tgatttttaa aagcagttca agttgacaac agcagaaaca gtaacaaaaa aatctgctca 8700 gaaaaatgta tgtgcacaaa taaaaaaaat taatggcaat tgtttagtga ttgtaagtga 8760 tactttttaa agagtaaact gtgtgaaatt tatactatcc ctgcttaaaa tattaagatt 8820 tttatgaaat atgtatttat gtttgtattg tgggaagatt cctcctctgt gatatcatac 8880 agcatctgaa agtgaacagt atcccaaagc agttccaacc atgctttgga agtaagaagg 8940 ttgactattg tatggccaag gatggcagta tgtaatccag aagcaaactt gtattaattg 9000 ttctatttca ggttctgtat tgcatgtttt cttattaata tatattaata aaagttatga 9060 gaaat 9065 Table Llll(d). Nucleotide sequence alignment of 109P1 D4 v.1 (SEQ ID NO: 256) and 109P1 D4 v.5 (SEQ ID NO: 257)
Score = 7456 bits (3878), Expect = O.OIdentities = 3878/3878 (100%) Strand = Plus / Plus
V.l : 1 ctggtggtccagtacctccaaagatatggaatacactcctgaaatatcctgaaaactttt 60
I I II I II II I II I I I II II llll I I II I II III I II I II llll I I I I II II llll II III V.5 : 1 ctggtggtccagtacctccaaagatatggaatacactcctgaaatatcctgaaaactttt 60
V.l : 61 ttttttcagaatcctttaataagcagttatgtcaatctgaaagttgcttacttgtacttt 120
I I II II I I I II I I I I III I II II II III II I I I III I llll I I I I I I I II I II II II II I
V.5 : 61 ttttttcagaatcctttaataagcagttatgtcaatctgaaagttgcttacttgtacttt 120
V.l : 121 atattaatagctattcttgtttttcttatccaaagaaaaatcctctaatccccttttcac 180
II II II II I II I I I II I 11 I I II III III II I II I I I II II I I 1 I I II II Ml I II I II I
V.5 : 121 atattaatagctattcttgtttttcttatccaaagaaaaatcctctaatccccttttcac 180
V.l : 181 atgatagttgttaccatgtttaggcattagtcacatcaacccctctcctctcccaaactt 240
II II 1 I I I I I I II I I II I I I I II II I I II I I II I I I II I I I I I I II 11 I- 1 I II I II I II I V.5 : 181 atgatagttgttaccatgtttaggcattagtcacatcaacccctctcctctcccaaactt 240
V.l : 241 ctcttcttcaaatcaaactttattagtccctcctttataatgattccttgcctcgtttta 300
I II I I I I I II I I I I I III I I II I I II I III II I I II II I I II I I I I I I I II I I I III I I I V.5 : 241 ctcttcttcaaatcaaactttattagtccctcctttataatgattccttgcctcgtttta 300
V.l : 301 tccagatcaattttttttcactttgatgcccagagctgaagaaatggactactgtataaa 360
I I II I I I I I II II I I I II II I lllll II III I I II I II 11 II II II II II I II II I I II I V.5 : 301 tccagatcaattttttttcactttgatgcccagagctgaagaaatggactactgtataaa 360
V.l : 361 ttattcattgccaagagaataattgcattttaaacccatattataacaaagaataatgat 420
I II II I I II I I I I I I II I II I Mill llll II I I I I II I I III I II I I II II II I I I Ml
V.5 : 361 ttattcattgccaagagaataattgcattttaaacccatattataacaaagaataatgat 420
V.l : 421 tatattttgtgatttgtaacaaataccctttattttcccttaactattgaattaaatatt 480
II II II I I II I I I I II II I I I llll I I I I II I II I II I I I llll II I I II I II II llll I
V.5 : 421 tatattttgtgatttgtaacaaataccctttattttcccttaactattgaattaaatatt 480
V.l : 481 ttaattatttgtattctctttaactatcttggtatattaaagtattatcttttatatatt 540
II I I II I II I I I I I I II I II I llll I II II II II II I I I Ml I I II II II II II I II III V.5 : 481 ttaattatttgtattctctttaactatcttggtatattaaagtattatcttttatatatt 540
V.l : 541 tatcaatggtggacacttttataggtactctgtgtcatttttgatactgtaggtatctta 600
I III I II I I II I I I II II I I Mill I I I II I I I I I 111 II II II I II I II I I I II II II I V.5 : 541 tatcaatggtggacacttttataggtactctgtgtcatttttgatactgtaggtatctta 600
V.l : 601 tttcatttatctttattcttaatgtacgaattcataatatttgattcagaacaaatttat 660
I I I I II I I I II II I I III I I II I III I III II II II I I I I I II I I II II I I I I II II II 1
V.5 : 601 tttcatttatctttattcttaatgtacgaattcataatatttgattcagaacaaatttat 660
V.l : 661 cactaattaacagagtgtcaattatgctaacatctcatttactgattttaatttaaaaca 720
II I I II 1 II I I I II I II II II II Ml II I II II I I III I I I II I I I I I II II I llll III
V.5 : 661 cactaattaacagagtgtcaattatgctaacatctcatttactgattttaatttaaaaca 720
V.l : 721 gtttttgttaacatgcatgtttagggttggcttcttaataatttcttcttcctcttctct 780
I I II I I I I I II I II II I I I I I llll II I II II I II I I II I II II II II II I II II I I I II V.5 : 721 gtttttgttaacatgcatgtttagggttggcttcttaataatttcttcttcctcttctct 780
V.l : 781 ctctcctcttcttttggtcagtgttgtgcgggttaatacaacaaactgtaacaagtgtac 840 V.5 : 781 ctctcctcttcttttggtcagtgttgtgcgggttaatacaacaaactgtaacaagtgtac 840
V.l : 841 ctggtatggacttgttgtccgggacgtacattttcgcggtcctgctagcatgcgtggtgt 900
II II I I II II II II I II I I I I I II III II Ml I I II I 111 II I II 111 II I I II I II III V.5 : 841 ctggtatggacttgttgtccgggacgtacattttcgcggtcctgctagcatgcgtggtgt 900
V.l : 901 tccactctggcgcccaggagaaaaactacaccatccgagaagaaatgccagaaaacgtcc 960
I II II II I II II I I II I I I I II II II I II III II I II II M I I I M I I II I II II II III V.5 : 901 tccactctggcgcccaggagaaaaactacaccatccgagaagaaatgccagaaaacgtcc 960
V.l : 961 tgataggcgacttgttgaaagaccttaacttgtcgctgattccaaacaagtccttgacaa 1020
I II II II II I II I I II II I I II I I II I II I II II I llll II I I II I II II II II I I I I II
V.5 : 961 tgataggcgacttgttgaaagaccttaacttgtcgctgattccaaacaagtccttgacaa 1020
V.l : 1021 ctgctatgcagttcaagctagtgtacaagaccggagatgtgccactgattcgaattgaag 1080
II II I II II II I I I I II II I I I llll II I I I II II III II I II II I II III I III II II I
V.5 : 1021 ctgctatgcagttcaagctagtgtacaagaccggagatgtgccactgattcgaattgaag 1080
V.l : 1081 aggatactggtgagatcttcactactggcgctcgcattgatcgtgagaaattatgtgctg 1140
II II M I II II I II II II I I I I I III I I II III I II II II II II I II II I I 11 I I I llll V.5 : 1081 aggatactggtgagatcttcactactggcgctcgcattgatcgtgagaaattatgtgctg 1140
V.l : 1141 gtatcccaagggatgagcattgcttttatgaagtggaggttgccattttgccggatgaaa 1200 llll II II II I I II I llll II I I I II I I I III I II II II II I I I I I I I I II I II I II II I V.5 : 1141 gtatcccaagggatgagcattgcttttatgaagtggaggttgccattttgccggatgaaa 1200
V.l : 1201 tatttagactggttaagatacgttttctgatagaagatataaatgataatgcaccattgt 1260
I llll II II I I I II I I I I I I I I llll I I I III I II II I I I I I I I II 11 I II I I II II II I V.5 : 1201 tatttagactggttaagatacgttttctgatagaagatataaatgataatgcaccattgt 1260
V.l : 1261 tcccagcaacagttatcaacatatcaattccagagaactcggctataaactctaaatata 1320
II II II I II II I II II I II I I I llll II II II MM II I I I I II II I I II I II I I II III
V.5 : 1261 tcccagcaacagttatcaacatatcaattccagagaactcggctataaactctaaatata 1320
V.l : 1321 ctctcccagcggctgttgatcctgacgtaggaataaacggagttcaaaactacgaactaa 1380
III I II llll I I II I II II II I I I I I I II II I II I I I II II II II II II II I I I I II II I
V.5 : 1321 ctctcccagcggctgttgatcctgacgtaggaataaacggagttcaaaactacgaactaa 1380
V.l : 1381 ttaagagtcaaaacatttttggcctcgatgtcattgaaacaccagaaggagacaagatgc 1440
II II II I II I II I I II I I 1 I I I I III II llll I I II II II I II II I I I III II I I I MM V.5 : 1381 ttaagagtcaaaacatttttggcctcgatgtcattgaaacaccagaaggagacaagatgc 1440
V.l : 1441 cacaactgattgttcaaaaggagttagatagggaagagaaggatacctacgtgatgaaag 1500
I llll I I I II I I II I 11 II I I I II II I II Ml I II I I II II I I I II I I II II I II III I I V.5 : 1441 cacaactgattgttcaaaaggagttagatagggaagagaaggatacctacgtgatgaaag 1500
V.l : 1501 taaaggttgaagatggtggctttcctcaaagatccagtactgctattttgcaagtgagtg 1560
MMMIMMMIMMIIIMMMMIMMIMMMIIM II IMIMIMMM V.5 : 1501 taaaggttgaagatggtggctttcctcaaagatccagtactgctattttgcaagtgagtg 1560
V.l : 1561 ttactgatacaaatgacaaccacccagtctttaaggagacagagattgaagtcagtatac 1620
I III I II I II II I I I I II I I I I I I I II I Ml I I II II I I II I I I I I II II I I I II I II I I V.5 : 1561 ttactgatacaaatgacaaccacccagtctttaaggagacagagattgaagtcagtatac 1620 V.l : 1621 cagaaaatgctcctgtaggcacttcagtgacacagctccatgccacagatgctgacatag 1680
II I I I Mlllll II I I I II I II I I I MM I I II II II II llll II I I II III V.5 : 1621 cagaaaatgctcctgtaggcacttcagtgacacagctccatgccacagatgctgacatag 1680
V.l : 1681 gtgaaaatgccaagatccacttctctttcagcaatctagtctccaacattgccaggagat 1740
II II III III MM I II II II II I II M I III II II I II I II II Ml II MM 11 I Ml I V.5 : 1681 gtgaaaatgccaagatccacttctctttcagcaatctagtctccaacattgccaggagat 1740
V.l : 1741 tatttcacctcaatgccaccactggacttatcacaatcaaagaaccactggatagggaag 1800
II I I I MM III II II II II II II I II I II I I I II Ml I II II II I I llll II I I II III V.5 : 1741 tatttcacctcaatgccaccactggacttatcacaatcaaagaaccactggatagggaag 1800
V.l : 1801 aaacaccaaaccacaagttactggttttggcaagtgatggtggattgatgccagcaagag 1860
I I II I II I I I 111 II II I I II II I II I I I II I II II I II I II M I II llllll I I I llll
V.5 : 1801 aaacaccaaaccacaagttactggttttggcaagtgatggtggattgatgccagcaagag 1860
V.l : 1861 caatggtgctggtaaatgttacagatgtcaatgataatgtcccatccattgacataagat 1920
II I I I MM Ml II MM II II I I I III II I I I III II I II II II I Mill I I I Ml Ml
V.5 : 1861 caatggtgctggtaaatgttacagatgtcaatgataatgtcccatccattgacataagat 1920
V.l : 1921 acatcgtcaatcctgtcaatgacacagttgttctttcagaaaatattccactcaacacca 1980
I I II I II II II II II I I I I II III II Mill II I II II II I III II I I I 11 II I
V.5 : 1921 acatcgtcaatcctgtcaatgacacagttgttctttcagaaaatattccactcaacacca 1980
V.l : 1981 aaattgctctcataactgtgacggataaggatgcggaccataatggcagggtgacatgct 2040
II I I 11 III I II I I II II III I II I I III II I II I MM II llll I I I II III I
V.5 : 1981 aaattgctctcataactgtgacggataaggatgcggaccataatggcagggtgacatgct 2040
V.l : 2041 tcacagatcatgaaatccctttcagattaaggccagtattcagtaatcagttcctcctgg 2100
II II I MM III II II II I I I I M II II I 11 I II II II I II 111 II I llll I I I II 111 I V.5 : 2041 tcacagatcatgaaatccctttcagattaaggccagtattcagtaatcagttcctcctgg 2100
V.l : 2101 agactgcagcatatcttgactatgagtccacaaaagaatatgccattaaattactggctg 2160
I III I II I) I I I II I II II I II I 1 I I I II I I I II III II I I I I I I I I II I I I I I IM I I I V.5 : 2101 agactgcagcatatcttgactatgagtccacaaaagaatatgccattaaattactggctg 2160
V.l : 2161 cagatgctggcaaacctcctttgaatcagtcagcaatgctcttcatcaaagtgaaagatg 2220
I I II III II I Mill I III I II I I I II I II I I I II II I I I II II II llll II I II I I II I
V.5 : 2161 cagatgctggcaaacctcctttgaatcagtcagcaatgctcttcatcaaagtgaaagatg 2220
V.l : 2221 aaaatgacaatgctccagttttcacccagtctttcgtaactgtttctattcctgagaata 2280
II II II I II MM Ml II II I II I II II II I I I llll I II II II II I I I II III
V.5 : 2221 aaaatgacaatgctccagttttcacccagtctttcgtaactgtttctattcctgagaata 2280
V.l : 2281 actctcctggcatccagttgacgaaagtaagtgcaatggatgcagacagtgggcctaatg 2340
I II II II I II Ml I II II I I I I I I II II I II I II llll II I III I 1 III I I I I I I II II I V.5 : 2281 actctcctggcatccagttgacgaaagtaagtgcaatggatgcagacagtgggcctaatg 2340
V.l : 2341 ctaagatcaattacctgctaggccctgatgctccacctgaattcagcctggattgtcgta 2400
I I I I Ml II llll I II I II II II I II I II II I II II II I II II II I II Ml I I I II III I
V.5 : 2341 ctaagatcaattacctgctaggccctgatgctccacctgaattcagcctggattgtcgta 2400
V.l : 2401 caggcatgctgactgtagtgaagaaactagatagagaaaaagaggataaatatttattca 2460
II lllll IMIMIIIMMIMMMMIMMMM
V.5 : 2401 caggcatgctgactgtagtgaagaaactagatagagaaaaagaggataaatatttattca 2460 V.l : 2461 caattctggcaaaagataacggggtaccacccttaaccagcaatgtcacagtctttgtaa 2520
I II II II I II I I Ml llll III II I II I I I II I I I I MM I II I III II I I II II II II I
V.5 : 2461 caattctggcaaaagataacggggtaccacccttaaccagcaatgtcacagtctttgtaa 2520
V.l : 2521 gcattattgatcagaatgacaatagcccagttttcactcacaatgaatacaacttctatg 2580
I II II II I II I MM lllll II III I II I I II I I I II III I I II III II I II I II II II I
V.5 : 2521 gcattattgatcagaatgacaatagcccagttttcactcacaatgaatacaacttctatg 2580
V.l : 2581 tcccagaaaaccttccaaggcatggtacagtaggactaatcactgtaactgatcctgatt 2640
II I II II 1 II I MM MM I II Ml I I I I II II II II I II II I II II II I I I I I I II I II
V.5 : 2581 tcccagaaaaccttccaaggcatggtacagtaggactaatcactgtaactgatcctgatt 2640
V.l : 2641 atggagacaattctgcagttacgctctccattttagatgagaatgatgacttcaccattg 2700
I II II II I II II III II II I II III I I I I III II II I I II II I II II II II I I) I II I II
V.5 : 2641 atggagacaattctgcagttacgctctccattttagatgagaatgatgacttcaccattg 2700
V.l : 2701 attcacaaactggtgtcatccgaccaaatatttcatttgatagagaaaaacaagaatctt 2760
I I I I I III I I II III llll I I I I II I I I llll I II I I I II I I I II III I II I II Ml II I
V.5 : 2701 attcacaaactggtgtcatccgaccaaatatttcatttgatagagaaaaacaagaatctt 2760
V.l : 2761 acactttctatgtaaaggctgaggatggtggtagagtatcacgttcttcaagtgccaaag 2820
II I II I II I I III I I Mill I I I II I I) II II I II I I I II II I I II I I I I I I II I I II I I
V.5 : 2761 acactttctatgtaaaggctgaggatggtggtagagtatcacgttcttcaagtgccaaag 2820
V.l : 2821 taaccataaatgtggttgatgtcaatgacaacaaaccagttttcattgtccctccttcca 2880
I I I I I II I II I Mlllll II I III II II II I I I I I I I II II II I I II II I II I II II I I I
V.5 : 2821 taaccataaatgtggttgatgtcaatgacaacaaaccagttttcattgtccctccttcca 2880
V.l : 2881 actgttcttatgaattggttctaccgtccactaatccaggcacagtggtctttcaggtaa 2940
I I I II III II II I II llll I II III I I I I I I II II II I II II I II I 111 II I II II I I I I
V.5 : 2881 actgttcttatgaattggttctaccgtccactaatccaggcacagtggtctttcaggtaa 2940
V.l : 2941 ttgctgttgacaatgacactggcatgaatgcagaggttcgttacagcattgtaggaggaa 3000
I II II II I I I I II I II I I II I I II I I II I I II I I I I I II I I I I III II II I II II II I I I
V.5 : 2941 ttgctgttgacaatgacactggcatgaatgcagaggttcgttacagcattgtaggaggaa 3000
V.l : 3001 acacaagagatctgtttgcaatcgaccaagaaacaggcaacataacattgatggagaaat 3060
I I I I llll I I I II II III II I I II I II I I I II I I I II Ml II I II II I I II I II I II II I
V.5 : 3001 acacaagagatctgtttgcaatcgaccaagaaacaggcaacataacattgatggagaaat 3060
V.l : 3061 gtgatgttacagaccttggtttacacagagtgttggtcaaagctaatgacttaggacagc 3120
I II I I I II I I III III I I II II II II I II I III II I I I 111 I I llll I II Ml II II I II
V.5 : 3061 gtgatgttacagaccttggtttacacagagtgttggtcaaagctaatgacttaggacagc 3120
V.l : 3121 ctgattctctcttcagtgttgtaattgtcaatctgttcgtgaatgagtcggtgaccaatg 3180
I I I I I III I I II III llll I I III II II I I II I I I I I III II I II I II I I I II I II II I I
V.5 : 3121 ctgattctctcttcagtgttgtaattgtcaatctgttcgtgaatgagtcggtgaccaatg 3180
V.l : 3181 ctacactgattaatgaactggtgcgcaaaagcactgaagcaccagtgaccccaaatactg 3240
I I I I I I I I II I III II I III II II I I I I II I I I I I I II II II I llll I I I II I II II II I
V.5 : 3181 ctacactgattaatgaactggtgcgcaaaagcactgaagcaccagtgaccccaaatactg 3240
V.l : 3241 agatagctgatgtatcctcaccaactagtgactatgtcaagatcctggttgcagctgttg 3300
I I I I I III I I II I II llll I II II II II I I II I I I I III I I II II I II II I II I III I II
V.5 : 3241 agatagctgatgtatcctcaccaactagtgactatgtcaagatcctggttgcagctgttg 3300 V.l : 3301 ctggcaccataactgtcgttgtagttattttcatcactgctgtagtaagatgtcgccagg 3360
II 1 I II I I Mill II II II I II II II I I llll I II III II II II I II II II I II
V.5 : 3301 ctggcaccataactgtcgttgtagttattttcatcactgctgtagtaagatgtcgccagg 3360
V.l : 3361 caccacaccttaaggctgctcagaaaaacaagcagaattctgaatgggctaccccaaacc 3420
II I II I I I I III II I III llll II I I I II I I I I II I I I I I II III I I II II I II I I I II I
V.5 : 3361 caccacaccttaaggctgctcagaaaaacaagcagaattctgaatgggctaccccaaacc 3420
V.l : 3421 cagaaaacaggcagatgataatgatgaagaaaaagaaaaagaagaagaagcattccccta 3480
I I I III I I Mill I II II II I II I I II I II I I I II I III II II II I I I II I III I II I II
V.5 : 3421 cagaaaacaggcagatgataatgatgaagaaaaagaaaaagaagaagaagcattccccta 3480
V.l : 3481 agaacttgctgcttaattttgtcactattgaagaaactaaggcagatgatgttgacagtg 3540
I I I I I I I I I llll II III I I II II I I II II I I I II II II I II III I II I II II I I II I II
V.5 : 3481 agaacttgctgcttaattttgtcactattgaagaaactaaggcagatgatgttgacagtg 3540
V.l : 3541 atggaaacagagtcacactagaccttcctattgatctagaagagcaaacaatgggaaagt 3600
I I llll 1 I Mill I II II II III I I II I II I I I II I II Ml Ml II I II I III II I I II I
V.5 : 3541 atggaaacagagtcacactagaccttcctattgatctagaagagcaaacaatgggaaagt 3600
V.l : 3601 acaattgggtaactacacctactactttcaagcccgacagccctgatttggcccgacact 3660
II I II I I I I III I I I MM I II 11 I II II III III II II I II I II II I II I II I I I I II I
V.5 : 3601 acaattgggtaactacacctactactttcaagcccgacagccctgatttggcccgacact 3660
V.l : 3661 acaaatctgcctctccacagcctgccttccaaattcagcctgaaactcccctgaattcga 3720
II I I I II I I MM lllll II I I I I I I I II II I I I II I I I I I I II II II I I I I I 11 I I I I I
V.5 : 3661 acaaatctgcctctccacagcctgccttccaaattcagcctgaaactcccctgaattcga 3720
V.l : 3721 agcaccacatcatccaagaactgcctctcgataacacctttgtggcctgtgactctatct 3780
I 1 II I I I I I Ml I I III I II I II I I I I I I I I I I I II II II I III I I I II I II II I II I I I
V.5 : 3721 agcaccacatcatccaagaactgcctctcgataacacctttgtggcctgtgactctatct 3780
V.l : 3781 ccaagtgttcctcaagcagttcagatccctacagcgtttctgactgtggctatccagtga 3840
II I II I I II llll I I lllll II II II I III II II I I I I I II II llll I I I llll I I I II I
V.5 : 3781 ccaagtgttcctcaagcagttcagatccctacagcgtttctgactgtggctatccagtga 3840
V.l : 3841 cgaccttcgaggtacctgtgtccgtacacaccagaccg 3878
I I I II I I I lllll II II I II II I I I I 11 I I I 1 II I II I V.5 : 3841 cgaccttcgaggtacctgtgtccgtacacaccagaccg 3878
Table LΙV(d). Peptide sequences of protein coded by 109P1D4v.5 (SEQ ID NO: 258)
MDLLSGTYIF AVLLACWFH SGAQEKNYTI REEMPENVLI GDLLKDLNLS LIPNKSLTTA 60
MQFKLVYKTG DVPLIRIEED TGEIFTTGAR IDREKLCAGI PRDEHCFYEV EVAILPDEIF 120
RLVKIRFLIE DINDNAPLFP ATVINISIPE NSAINSKYTL PAAVDPDVGI NGVQNYELIK 180
SQNIFGLDVI ETPEGDKMPQ LIVQKELDRE EKDTYVMKVK VEDGGFPQRS STAILQVSVT 240
DTNDNHPVFK ETEIEVSIPE NAPVGTSVTQ LHATDADIGE NAKIHFSFSN LVSNIARRLF 300
HLNATTGLIT IKEPLDREET PNHKLLVLAS DGGLMPARAM VLVNVTDVND NVPSIDIRYI 360
VNPVNDT VL SENIPLNTKI ALITVTDKDA DHNGRVTCFT DHEIPFRLRP VFSNQFLLET 420
AAYLDYESTK EYAIKLLAAD AGKPPLNQSA MLFIKVKDEN DNAPVFTQSF VTVSIPENNS 480
PGIQLTKVSA MDADSGPNAK INYLLGPDAP PEFSLDCRTG MLTVVKKLDR EKEDKYLFTI 540
LAKDNGVPPL TSNVTVFVSI IDQNDNSPVF THNEYNFYVP ENLPRHGTVG LITVTDPDYG 600
DNSAVTLSIL DENDDFTIDS QTGVIRPNIS FDREKQESYT FYVKAEDGGR VSRSSSAKVT 660
INWDVNDNK PVFIVPPSNC SYELVLPSTN PGTWFQVIA VDNDTGMNAE VRYSIVGGNT 720
RDLFAIDQET GNITLMEKCD VTDLGLHRVL VKANDLGQPD SLFSVVIVNL FVNESVTNAT 780
LINELVRKST EAPVTPNTEI ADVSSPTSDY VKILVAAVAG TITVVVVIFI TAWRCRQAP 840 HLKAAQKNKQ NSEWATPNPE NRQMIMMKKK KKKKKHSPKN LLLNFVTIEE TKADDVDSDG 900
NRVTLDLPID LEEQTMGKYN WVTTPTTFKP DSPDLARHYK SASPQPAFQI QPETPLNSKH 960
HIIQELPLDN TFVACDSISK CSSSSSDPYS VSDCGYPVTT FEVPVSVHTR PSQRRVTFHL 1020
PEGSQESSSD GGLGDHDAGS LTSTSHGLPL GYPQEEYFDR ATPSNRTEGD GNSDPESTFI 1080
PGLKKAAEIT VQPTVEEASD NCTQECLIYG HSDACWMPAS LDHSSSSQAQ ASALCHSPPL 1140
SQASTQHHSP RVTQTIALCH SPPVTQTIAL CHSPPPIQVS ALHHSPPLVQ ATALHHSPPS 1200
AQASALCYSP PLAQAAAISH SSPLPQVIAL HRSQAQSSVS LQQGWVQGAD GLCSVDQGVQ 1260
GSATSQFYTM SERLHPSDDS IKVIPLTTFT PRQQARPSRG DSPIMEEHPL 1310
Table LV(d). Amino acid sequence alignment of 109P1D4 v.1 (SEQ ID NO: 259) and 109P1D4 v.5 (SEQ ID NO: 26
Score = 2005 bits (5195), Expect = O.OIdentities = 1011/1011 (100%), Positives = 1011/1011 (100%) .l 1 DLLSGTYIFAVLLACWFHSGAQEKNYTIREEMPENV IGDLLKDLNLSLIPNKSLTTA 60
MDLLSGTYIFAVLLACWFHSGAQEKNYTIREEMPENVLIGDLLKDLNLSLIPNKS TTA V.5 1 MDLLSGTYIFAVLLACVVFHSGAQEK YTIREE PENVLIGDLLKDLNLSLIPNKSLTTA 60 V.l 61 MQFKLVYKTGDVPLIRIEEDTGEIFTTGARIDREK CAGIPRDEHCFYEVEVAILPDEIF 120
MQFKLVYKTGDVPLIRIEEDTGEIFTTGARIDREKLCAGIPRDEHCFYEVEVAILPDEIF V.5 61 MQFKLVYKTGDVPLIRIEEDTGEIFTTGARIDREKLCAGIPRDEHCFYEVEVAILPDEIF 120 V.l 121 RLVKIRF IEDINDNAPLFPATVINISIPENSAINSKYTLPAAVDPDVGINGVQNYE IK 180
RLVKIRFLIEDINDNAPLFPATVINISIPENSAINSKYTLPAAVDPDVGINGVQNYELIK V.5 121 RLVKIRFLIEDINDNAPLFPATVINISIPENSAINSKYTLPAAVDPDVGINGVQNYELIK 180 V.l 181 SQNIFGLDVIETPEGDKMPQLIVQKELDREEKDTYVM VKVEDGGFPQRSSTAILQVSVT 240
SQNIFGLDVIETPEGDKMPQLIVQKELDREEKDTYVMKVKVEDGGFPQRSSTAILQVSVT V.5 181 SQNIFGLDVIETPEGDKMPQLIVQKE DREEKDTYV KVKVEDGGFPQRSSTAILQVSVT 240 V.l 241 DTNDNHPVFKETEIEVSIPENAPVGTSVTQLHATDADIGENAKIHFSFSNLVSNIARRLF 300
DTNDNHPVFKETEIEVSIPENAPVGTSVTQLHATDADIGENAKIHFSFSNLVSNIARRLF V.5 241 DTNDNHPVFKETEIEVSIPENAPVGTSVTQ HATDADIGENAKIHFSFSNLVSNIARRLF 300 V.l 301 H NATTGLITIKEPLDREETPNHKLLVLASDGGLMPARAMVLV VTDV DNVPSIDIRYI 360
HLNATTGLITIKEP DREETPNHKLLVLASDGGL PARAMVLVNVTDV DNVPSIDIRYI V.5 301 HLNATTG ITIKEPLDREETPNHKLLVLASDGG MPARAMV VNVTDVNDNVPSIDIRYI 360 V.l 361 V PV DTWLSENIP NTKIALITVTDKDADHNGRVTCFTDHEIPFR RPVFSNQFLLET 420
V PVNDTVV SENIPLNTKIALITVTDKDADHNGRVTCFTDHEIPFRLRPVFSNQFLLET V.5 361 VNPVNDTVVLSENIPLNTKIALITVTDKDADHNGRVTCFTDHEIPFRLRPVFSNQF LET 420 V.l 421 AAYLDYESTKEYAIKLLAADAGKPPLNQSAMLFIKVKDENDNAPVFTQSFVTVSIPENNS 480
AAYLDYESTKEYAIK LAADAGKPPLNQSAMLFIKVKDENDNAPVFTQSFVTVSIPENNS V.5 421 AAYLDYESTKEYAIKLLAADAGKPPLNQSAMLFIKVKDENDNAPVFTQSFVTVSIPENNS 480 V.l 481 PGIQLTKVSAMDADSGPNAKINYLLGPDAPPEFSLDCRTGMLTWKKLDREKEDKYLFTI 540
PGIQLTKVSAMDADSGPNAKINYL GPDAPPEFSLDCRTGMLTWKKLDREKEDKYLFTI V.5 481 PGIQLTKVSAMDADSGPNAKINYLLGPDAPPEFSLDCRTGMLTWKKLDREKEDKYLFTI 540 .l 541 LAKDNGVPP TSNVTVFVSIIDQNDNSPVFTHNEYNFYVPENLPRHGTVGLITVTDPDYG 600
LAKDNGVPPLTSNVTVFVS11DQNDNSPVFTHNEYNFYVPENLPRHGTVGLITVTDPDYG V.5 541 AKDNGVPP TSNVTVFVSIIDQNDNSPVFTHNEYNFYVPEN PRHGTVG ITVTDPDYG 600 V.l 601 DNSAVTLSILDENDDFTIDSQTGVIRPNISFDREKQESYTFYVKAEDGGRVSRSSSAKVT 660
DNSAVT SILDENDDFTIDSQTGVIRPNISFDREKQESYTFYVKAEDGGRVSRSSSAKVT V.5 601 DNSAVTLSILDENDDFTIDSQTGVIRPNISFDREKQESYTFYVKAEDGGRVSRSSSAKVT 660 V.l 661 INWDVNDNKPVFIVPPSNCSYELVLPSTNPGTWFQVIAVDNDTGMNAEVRYSIVGGNT 720
INWDVNDNKPVFIVPPSNCSYELVLPSTNPGTWFQVIAVDNDTGMNAEVRYSIVGGNT V.5 661 INWDVNDNKPVFIVPPSNCSYELVLPSTNPGTVVFQVIAVDNDTGMNAEVRYSIVGGNT 720 V.l 721 RDLFAIDQETGNIT MEKCDVTDLG HRVLVKA DLGQPDSLFSWIVNLFV ESVTNAT 780
RDLFAIDQETGNITL EKCDVTDLGLHRVLVKAND GQPDSLFSWIVNLFV ESVTNAT V.5 721 RDLFAIDQETGNITLMEKCDVTD GLHRVLVKANDLGQPDSLFSVVIVNLFVNESVTNAT 780 V.l 781 INELVRKSTEAPVTPNTEIADVSSPTSDYVKI VAAVAGTITWWIFITAWRCRQAP 840
LINELVRKSTEAPVTPNTEIADVSSPTSDYVKILVAAVAGTITVWVIFITAWRCRQAP V.5 781 LINELVRKSTEAPVTPNTEIADVSSPTSDYVKILVAAVAGTITWWIFITAWRCRQAP 840 V.l 841 HLKAAQKNKQNSEWATPNPENRQMI MKKKKKKKKHSPKNLLLNFVTIEETKADDVDSDG 900
HLKAAQKNKQNSEWATPNPENRQMIMMKKKKKKKKHSPKN LLNFVTIEETKADDVDSDG V.5 841 HLKAAQK KQNSE ATPNPENRQMIMMKKKKKKKKHSPKNLLLNFVTIEETKADDVDSDG 900 V.l 901 NRVTLDLPIDLEEQTMGKYNWVTTPTTFKPDSPDLARHYKSASPQPAFQIQPETPLNSKH 960
NRVTLD PIDLEEQTMG YN VTTPTTFKPDSPDLARHYKSASPQPAFQIQPETP NSKH V.5 901 NRVT DLPIDLEEQTMGKYN VTTPTTFKPDSPDLARHYKSASPQPAFQIQPETPLNSKH 960 V.l 961 HIIQE PLDNTFVACDSISKCSSSSSDPYSVSDCGYPVTTFEVPVSVHTRP 1011
HIIQELPLDNTFVACDSISKCSSSSSDPYSVSDCGYPVTTFEVPVSVHTRP V.5 961 HIIQE PLDNTFVACDSISKCSSSSSDPYSVSDCGYPVTTFEVPVSVHTRP 1011
Table Lll(e). Nucleotide sequence of transcript variant 109P1 D4 v.6 (SEQ ID NO: 261) ggcagtcggc gaactgtctg ggcgggagga gccgtgagca gtagctgcac tcagctgccc 60 gcgcggcaaa gaggaaggca agccaaacag agtgcgcaga gtggcagtgc cagcggcgac 120 acaggcagca caggcagccc gggctgcctg aatagcctca gaaacaacct cagcgactcc 180 ggctgctctg cggactgcga gctgtggcgg tagagcccgc tacagcagtc gcagtctccg 240 tggagcgggc ggaagccttt tttctccctt tcgtttacct cttcattcta ctctaaaggc 300 atcgttatta gagggtgctt aaaaagtaca gatcaactgg atggatgaat ggatggaaga 360 ggatggaata tcttaacaaa acacattttc cttaagtaaa ttcatgcata ctccaaataa 420 aatacagaat gtgaagtatc tctgaactgt gctgttgaat atggtagcta ctagctacat 480 gaaaatcctg ttgtgaataa gaaggattcc acagatcaca taccagagcg gttttgcctc 540 agctgctctc aactttgtaa tcttgtgaag aagctgacaa gcttggctga ttgcagtgca 600 ctatgaggac tgaatgacag tgggttttaa ttcagatatt tcaagtgttg tgcgggttaa 660 tacaacaaac tgtcacaagt gtttgttgtc cgggacgtac attttcgcgg tcctgctagt 720 atgcgtggtg ttccactctg gcgcccagga gaaaaactac accatccgag aagaaattcc 780 agaaaacgtc ctgataggca acttgttgaa agaccttaac ttgtcgctga ttccaaacaa 840 gtccttgaca actactatgc agttcaagct agtgtacaag accggagatg tgccactgat 900 tcgaattgaa gaggatactg gtgagatctt cactaccggc gctcgcattg atcgtgagaa 960 attatgtgct ggtatcccaa gggatgagca ttgcttttat gaagtggagg ttgccatttt 1020 gccggatgaa atatttagac tggttaagat acgttttctg atagaagata taaatgataa 1080 tgcaccattg ttcccagcaa cagttatcaa catatcaatt ccagagaact cggctataaa 1140 ctctaaatat actctcccag cggctgttga tcctgacgta ggcataaacg gagttcaaaa 1200 ctacgaacta attaagagtc aaaacatttt tggcctcgat gtcattgaaa caccagaagg 1260 agacaagatg ccacaactga ttgttcaaaa ggagttagat agggaagaga aggataccta 1320 tgtgatgaaa gtaaaggttg aagatggtgg ctttcctcaa agatccagta ctgctatttt 1380 gcaagtaagt gttactgata caaatgacaa ccacccagtc tttaaggaga cagagattga 1440 agtcagtata ccagaaaatg ctcctgtagg cacttcagtg acacagctcc atgccacaga 1500 tgctgacata ggtgaaaatg ccaagatcca cttctctttc agcaatctag tctccaacat 1560 tgccaggaga ttatttcacc tcaatgccac cactggactt atcacaatca aagaaccact 1620 ggatagggaa gaaacaccaa accacaagtt actggttttg gcaagtgatg gtggattgat 1680 gccagcaaga gcaatggtgc tggtaaatgt tacagatgtc aatgataatg tcccatccat 1740 tgacataaga tacatcgtca atcctgtcaa tgacacagtt gttctttcag aaaatattcc 1800 actcaacacc aaaattgctc tcataactgt gacggataag gatgcggacc ataatggcag 1860 ggtgacatgc ttcacagatc atgaaattcc tttcagatta aggccagtat tcagtaatca 1920 gttcctcctg gagaatgcag catatcttga ctatgagtcc acaaaagaat atgccattaa 1980 attactggct gcagatgctg gcaaacctcc tttgaatcag tcagcaatgc tcttcatcaa 2040 agtgaaagat gaaaatgaca atgctccagt tttcacccag tctttcgtaa ctgtttctat 2100 tcctgagaat aactctcctg gcatccagtt gatgaaagta agtgcaacgg atgcagacag 2160 tgggcctaat gctgagatca attacctgct aggccctgat gctccacctg aattcagcct 2220 ggatcgtcgt acaggcatgc tgactgtagt gaagaaacta gatagagaaa aagaggataa 2280 atatttattc acaattctgg caaaagataa tggggtacca cccttaacca gcaatgtcac 2340 agtctttgta agcattattg atcagaatga caatagccca gttttcactc acaatgaata 2400 caaattctat gtcccagaaa accttccaag gcatggtaca gtaggactaa tcactgtaac 2460 tgatcctgat tatggagaca attctgcagt tacgctctcc attttagatg agaatgatga 2520 cttcaccatt gattcacaaa ctggtgtcat ccgaccaaat atttcatttg atagagaaaa 2580 acaagaatct tacactttct atgtaaaggc tgaggatggt ggtagagtat cacgttcttc 2640 aagtgccaaa gtaaccataa atgtggttga tgtcaatgac aacaaaccag ttttcattgt 2700 ccctccttac aactattctt atgaattggt tctaccgtcc actaatccag gcacagtggt 2760 ctttcaggta attgctgttg acaatgacac tggcatgaat gcagaggttc gttacagcat 2820 tgtaggagga aacacaagag atctgtttgc aatcgaccaa gaaacaggca acataacatt 2880 gatggagaaa tgtgatgtta cagaccttgg tttacacaga gtgttggtca aagctaatga 2940 cttaggacag cctgattctc tcttcagtgt tgtaattgtc aatctgttcg tgaatgagtc 3000 agtgaccaat gctacactga ttaatgaact ggtgcgcaaa agcattgaag caccagtgac 3060 cccaaatact gagatagctg atgtatcctc accaactagt gactatgtca agatcctggt 3120 tgcagctgtt gctggcacca taactgtcgt tgtagttatt ttcatcactg ctgtagtaag 3180 atgtcgccag gcaccacacc ttaaggctgc tcagaaaaac atgcagaatt ctgaatgggc 3240 taccccaaac ccagaaaaca ggcagatgat aatgatgaag aaaaagaaaa agaagaagaa 3300 gcattcccct aagaacctgc tgcttaattt tgtcactatt gaagaaacta aggcagatga 3360 tgttgacagt gatggaaaca gagtcacact agaccttcct attgatctag aagagcaaac 3420 aatgggaaag tacaattggg taactacacc tactactttc aagcctgaca gccctgattt 3480 ggcccgacac tacaaatctg cctctccaca gcctgccttc caaattcagc ctgaaactcc 3540 cctgaatttg aagcaccaca tcatccaaga actgcctctc gataacacct ttgtggcctg 3600 tgactctatc tccaagtgtt cctcaagcag ttcagatccc tacagcgttt ctgactgtgg 3660 ctatccagtg acaaccttcg aggtacctgt gtccgtacac accagaccga ctgattccag 3720 gacatgaact attgaaatct gcagtgagat gtaactttct aggaacaaca aaattccatt 3780 ccccttccaa aaaatttcaa tggattgtga tttcaaaatt aggctaagat cattaatttt 3840 gtaatctaga tttcccatta taaaagcaag caaaaatcat cttaaaaatg atgtcctagt 3900 gaaccttgtg ctttctttag ctgtaatctg gcaatggaaa tttaaaattt atggaagaga 3960 cagtgcagca caataacaga gtactctcat gctgtttctc tgtttgctct gaatcaacag 4020 ccatgatgta atataaggct gtcttggtgt atacacttat ggttaatata tcagtcatga 4080 aacatgcaat tacttgccct gtctgattgt tgaataatta aaacattatc ttccaggagt 4140 ttggaagtga gctgaactag ccaaactact ctctgaaagg tatccagggc aagagacatt 4200 tttaagaccc caaacaaaca aaaaacaaaa ccaaaacact ctggttcagt gttttgaaaa 4260 tattcactaa cataatattg ctgagaaaat catttttatt acccaccact ctgcttaaaa 4320 gttgagtggg ccgggcgcgg tggctcacgc ctgtaatccc agcactttgg gaggccgagg 4380 cgggtggatc acgaggtcag gagattgaga ccatcctggc taacacggtg aaaccccatc 4440 tccactaaaa atacaaaaaa ttagcctggc gtggtggcgg gcgcctgtag tcccagctac 4500 tcgggaggct gaggcaggag aatagcgtga acccgggagg cggagcttgc agtgagccga 4560 gatggcgcca ctctgcactc cagcctgggt gacagagcaa gactctgtct caaaaagaaa 4620 aaaatgttca atgatagaaa ataattttac taggttttta tgttgattgt actcatggtg 4680 ttccactcct tttaattatt aaaaagttat ttttggggtg ggtgtggtgg ctcacaccgt 4740 aatcccagca ctttgggagg ccgaggtggg tggatcacct gaggtcagga gttcaagacc 4800 agtntggcca acatggcgaa accccgtttt 4830
Table Llll(e). Nucleotide sequence alignment of 109P1D4 v.1 (SEQ ID NO: 262) and 109P1D4 v.6 (SEQ ID NO: 263)
Score = 5676 bits (2952), Expect = O.OIdentities = 3002/3027 (99%) Strand = Plus / Plus
V. l 852 ttgttgtccgggacgtacattttcgcggtcctgctagcatgcgtggtgttccactctggc 911 llll II I I 111 I III I I I II I I I I II I I I II II I II I I I II I III I I I I II II I I I Ml
V . 6 683 ttgttgtccgggacgtacattttcgcggtcctgctagtatgcgtggtgttccactctggc 742
V.l : 912 gcccaggagaaaaactacaccatccgagaagaaatgccagaaaacgtcctgataggcgac 971
I I III I II I II II I II II III II I I II I Ml I I II I I II II III II I I I I I II II I II
V.6 : 743 gcccaggagaaaaactacaccatccgagaagaaattccagaaaacgtcctgataggcaac 802
V.l : 972 ttgttgaaagaccttaacttgtcgctgattccaaacaagtccttgacaactgctatgcag 1031
I III II II I I I llll I lllll I I I I II I II I II III II II III I III I I I I II I I I II I
V.6 : 803 ttgttgaaagaccttaacttgtcgctgattccaaacaagtccttgacaactactatgcag 862
V.l : 1032 ttcaagctagtgtacaagaccggagatgtgccactgattcgaattgaagaggatactggt 1091
I lllll I I I II I I I I II I I II II I I II I III I II II I I I I I I I II II I II II II I I I I II
V.6 : 863 ttcaagctagtgtacaagaccggagatgtgccactgattcgaattgaagaggatactggt 922
V.l : 1092 gagatcttcactactggcgctcgcattgatcgtgagaaattatgtgctggtatcccaagg 1151
I Ml II I I I I I I I I II II I II I I I II I III I 1 II I II II I II llll I I I I I II I I I I II
V.6 : 923 gagatcttcactaccggcgctcgcattgatcgtgagaaattatgtgctggtatcccaagg 982
V.l : 1152 gatgagcattgcttttatgaagtggaggttgccattttgccggatgaaatatttagactg 1211 V.6 : 983 gatgagcattgcttttatgaagtggaggttgccattttgccggatgaaatatttagactg 1042 V.l 1212 gttaagatacgttttctgatagaagatataaatgataatgcaccattgttcccagcaaca 1271 II 1 II I 111 III lllll II llll II I I I llll III II 111 I llll I I I II I II I I II Ml V.6 1043 gttaagatacgttttctgatagaagatataaatgataatgcaccattgttcccagcaaca 1102
.l 1272 gttatcaacatatcaattccagagaactcggctataaactctaaatatactctcccagcg 1331 M I I II I I II I I II I llll I II II I II I II II I II I II I I II II I I I III I I 11 I II I II V.6 1103 gttatcaacatatcaattccagagaactcggctataaactctaaatatactctcccagcg 1162
V.l : 1332 gctgttgatcctgacgtaggaataaacggagttcaaaactacgaactaattaagagtcaa 1391
I I I II II II III I II III II I II I I II II I 111 II I II I II I II I I II Ml I I I I I I II
V.6 : 1163 gctgttgatcctgacgtaggcataaacggagttcaaaactacgaactaattaagagtcaa 1222
V.l : 1392 aacatttttggcctcgatgtcattgaaacaccagaaggagacaagatgccacaactgatt 1451
I I I I II I II I II II III II II II I I II II II MM 111 I I I I III I II II I II MM II I
V.6 : 1223 aacatttttggcctcgatgtcattgaaacaccagaaggagacaagatgccacaactgatt 1282
V.l : 1452 gttcaaaaggagttagatagggaagagaaggatacctacgtgatgaaagtaaaggttgaa 1511
I I II II I I llll I I II II I III II II I II III I I I II I I Ml II I II I II II I
V.6 : 1283 gttcaaaaggagttagatagggaagagaaggatacctatgtgatgaaagtaaaggttgaa 1342
V.l : 1512 gatggtggctttcctcaaagatccagtactgctattttgcaagtgagtgttactgataca 1571
I I I I I I II I II II I I III II II I I I I llll I II I III I I I II I I II II II II II II II I
V.6 : 1343 gatggtggctttcctcaaagatccagtactgctattttgcaagtaagtgttactgataca 1402
V.l : 1572 aatgacaaccacccagtctttaaggagacagagattgaagtcagtataccagaaaatgct 1631
II I 11 II II Mill I II I I II I II I II II I II II II I II I I II II II I II II I II II I II
V.6 : 1403 aatgacaaccacccagtctttaaggagacagagattgaagtcagtataccagaaaatgct 1462
V.l : 1632 cctgtaggcacttcagtgacacagctccatgccacagatgctgacataggtgaaaatgcc 1691
II I I II I II III II III II II Ml 1 II I I I II II I III I I I I II I I I I II II I II II I II
V.6 : 1463 cctgtaggcacttcagtgacacagctccatgccacagatgctgacataggtgaaaatgcc 1522
V.l : 1692 aagatccacttctctttcagcaatctagtctccaacattgccaggagattatttcacctc 1751
I I I II I I II III II III II I I I II I II I I II I I I I I llll I I II III 111 II I II II I I I
V.6 : 1523 aagatccacttctctttcagcaatctagtctccaacattgccaggagattatttcacctc 1582
V.l : 1752 aatgccaccactggacttatcacaatcaaagaaccactggatagggaagaaacaccaaac 1811
1 I I I II I II III II III II III II I II I llll I I I I II) I I II I I I I II I II I II II II I
V.6 : 1583 aatgccaccactggacttatcacaatcaaagaaccactggatagggaagaaacaccaaac 1642
V.l : 1812 cacaagttactggttttggcaagtgatggtggattgatgccagcaagagcaatggtgctg 1871
I I I III I II III II III II I II II I II I II I I I II I II II I MM II II I II I II II II I
V.6 : 1643 cacaagttactggttttggcaagtgatggtggattgatgccagcaagagcaatggtgctg 1702
V.l : 1872 gtaaatgttacagatgtcaatgataatgtcccatccattgacataagatacatcgtcaat 1931
II I II I I II Ml I I I II II II II I I II III II I II I II II I II II I I I I I II I II II II I
V.6 : 1703 gtaaatgttacagatgtcaatgataatgtcccatccattgacataagatacatcgtcaat 1762
V.l : 1932 cctgtcaatgacacagttgttctttcagaaaatattccactcaacaccaaaattgctctc 1991
I I I II I I I I III I I III II I II II I I I I II II I I I I II II I II II I I II II I I II II II I
V.6 : 1763 cctgtcaatgacacagttgttctttcagaaaatattccactcaacaccaaaattgctctc 1822
V.l : 1992 ataactgtgacggataaggatgcggaccataatggcagggtgacatgcttcacagatcat 2051 I I I II I I II 111 II II I II II II I I II II II I III III II II II I I I llll I II II III I V.6 : 1823 ataactgtgacggataaggatgcggaccataatggcagggtgacatgcttcacagatcat 1882
V.l : 2052 gaaatccctttcagattaaggccagtattcagtaatcagttcctcctggagactgcagca 2111 lllll 1 I 111 I llll I II III II I I I II II I II III II I I 111 II II III V.6 : 1883 gaaattcctttcagattaaggccagtattcagtaatcagttcctcctggagaatgcagca 1942
V.l : 2112 tatcttgactatgagtccacaaaagaatatgccattaaattactggctgcagatgctggc 2171
I II II I I I I II I III II I Ml III II llll II I I I II II I I I I I I I II I I Ml I I II II 1 V.6 : 1943 tatcttgactatgagtccacaaaagaatatgccattaaattactggctgcagatgctggc 2002
V.l : 2172 aaacctcctttgaatcagtcagcaatgctcttcatcaaagtgaaagatgaaaatgacaat 2231
I I 11 I 111 I I 1 MMMIMMMMMMMM II I I I II II I II I I 1 II II 1 V.6 : 2003 aaacctcctttgaatcagtcagcaatgctcttcatcaaagtgaaagatgaaaatgacaat 2062
V.l : 2232 gctccagttttcacccagtctttcgtaactgtttctattcctgagaataactctcctggc 2291
I I I I II I II I I Ml II II III III Mill II 111 II II 11 II II I II II III Ml II II I V.6 : 2063 gctccagttttcacccagtctttcgtaactgtttctattcctgagaataactctcctggc 2122
V.l : 2292 atccagttgacgaaagtaagtgcaatggatgcagacagtgggcctaatgctaagatcaat 2351
I I I I II I Ml III II II II III II I II II Ml II II I I I Ml I II I II I V.6 : 2123 atccagttgatgaaagtaagtgcaacggatgcagacagtgggcctaatgctgagatcaat 2182
V.l : 2352 tacctgctaggccctgatgctccacctgaattcagcctggattgtcgtacaggcatgctg 2411
I II I 11 llll I Ml II II II I II I III I I I I I II II II I I II I I II II 111 I I I II I II V.6 : 2183 tacctgctaggccctgatgctccacctgaattcagcctggatcgtcgtacaggcatgctg 2242
V.l : 2412 actgtagtgaagaaactagatagagaaaaagaggataaatatttattcacaattctggca 2471
I II II II II II II I II II I I I III lllll I II M II M I I I II I II I II I llll I II III V.6 : 2243 actgtagtgaagaaactagatagagaaaaagaggataaatatttattcacaattctggca 2302
V.l : 2472 aaagataacggggtaccacccttaaccagcaatgtcacagtctttgtaagcattattgat 2531 l l II Ml II II III I II I llll I I I I I 111 II I I II I II I I II llll I II II I V.6 : 2303 aaagataatggggtaccacccttaaccagcaatgtcacagtctttgtaagcattattgat 2362
V.l : 2532 cagaatgacaatagcccagttttcactcacaatgaatacaacttctatgtcccagaaaac 2591
I I I I II I II I I II I II II II I llll II llll I I I II I II I I I I II I I I I Ml I II MM V.6 : 2363 cagaatgacaatagcccagttttcactcacaatgaatacaaattctatgtcccagaaaac 2422
V.l : 2592 cttccaaggcatggtacagtaggactaatcactgtaactgatcctgattatggagacaat 2651
I I II II I II I I I II I I I I llll I II I I I II III llll I I I II I llll III III I
V.6 : 2423 cttccaaggcatggtacagtaggactaatcactgtaactgatcctgattatggagacaat 2482
V.l : 2652 tctgcagttacgctctccattttagatgagaatgatgacttcaccattgattcacaaact 2711
II III I I II I I II II I I II I Ml III I III II I II I I II MM II I II I II II I
V.6 : 2483 tctgcagttacgctctccattttagatgagaatgatgacttcaccattgattcacaaact 2542
V.l : 2712 ggtgtcatccgaccaaatatttcatttgatagagaaaaacaagaatcttacactttctat 2771
I llll I I I I I I III I I I III I lllll lllll II III II I I I II I I II I II II II I III I 1 V.6 : 2543 ggtgtcatccgaccaaatatttcatttgatagagaaaaacaagaatcttacactttctat 2602
V.l : 2772 gtaaaggctgaggatggtggtagagtatcacgttcttcaagtgccaaagtaaccataaat 2831
I II I I 11 II I I III I III III III lllll II II II I II I I I I II III II I III I II llll V.6 : 2603 gtaaaggctgaggatggtggtagagtatcacgttcttcaagtgccaaagtaaccataaat 2662
V.l : 2832 gtggttgatgtcaatgacaacaaaccagttttcattgtccctccttccaactgttcttat 2891 MIMMIMMMMM II I I II I II 11 II II I II I I 1 M I I I 1 I lllll Mlllll
V.6 : 2663 gtggttgatgtcaatgacaacaaaccagttttcattgtccctccttacaactattcttat 2722
V.l : 2892 gaattggttctaccgtccactaatccaggcacagtggtctttcaggtaattgctgttgac 2951 I I I I 11 I I I I 11 I I I 1 I I I 1 I I 1 M 1 I I I 1 I I I I I I I I 111111 I I I I I 1 I I I I I I I I I I
V.6 : 2723 gaattggttctaccgtccactaatccaggcacagtggtctttcaggtaattgctgttgac 2782
V.l : 2952 aatgacactggcatgaatgcagaggttcgttacagcattgtaggaggaaacacaagagat 3011
I II I II MM 11 II I I I I I II I Mlllll I) II Ml I I II II II I I I I II I I I I I MM I
V.6 : 2783 aatgacactggcatgaatgcagaggttcgttacagcattgtaggaggaaacacaagagat 2842
V.l : 3012 ctgtttgcaatcgaccaagaaacaggcaacataacattgatggagaaatgtgatgttaca 3071
III II llll II I I I II I I I I I I Mill II llll I I II II II I I I I II III I II I I II II I
V.6 : 2843 ctgtttgcaatcgaccaagaaacaggcaacataacattgatggagaaatgtgatgttaca 2902
V.l : 3072 gaccttggtttacacagagtgttggtcaaagctaatgacttaggacagcctgattctctc 3131
II II III I llll I I I II I I II I II II I I II II II I I II llll I I I I I II I I I I I I llll I
V.6 : 2903 gaccttggtttacacagagtgttggtcaaagctaatgacttaggacagcctgattctctc 2962
V.l : 3132 ttcagtgttgtaattgtcaatctgttcgtgaatgagtcggtgaccaatgctacactgatt 3191
I lllll MM I I I II I I I I I I I MM II II I II Ml I I I II II II I II I I II I I II I I I
V.6 : 2963 ttcagtgttgtaattgtcaatctgttcgtgaatgagtcagtgaccaatgctacactgatt 3022
V.l : 3192 aatgaactggtgcgcaaaagcactgaagcaccagtgaccccaaatactgagatagctgat 3251
I II II MM I I I I I I I I II I I I I II I II II I 11 II I II II I II I II I II II I I II I I II
V.6 : 3023 aatgaactggtgcgcaaaagcattgaagcaccagtgaccccaaatactgagatagctgat 3082
V.l : 3252 gtatcctcaccaactagtgactatgtcaagatcctggttgcagctgttgctggcaccata 3311
II I II I I I 111 I II I I llll I I llll II I I III I II I I II II I I I II I III III
V.6 : 3083 gtatcctcaccaactagtgactatgtcaagatcctggttgcagctgttgctggcaccata 3142
V.l : 3312 actgtcgttgtagttattttcatcactgctgtagtaagatgtcgccaggcaccacacctt 3371
I I I I I III II I II II II I I I I I I III II llll I I II I II I I II 1 II I I II II II I III II ' V.6 : 3143 actgtcgttgtagttattttcatcactgctgtagtaagatgtcgccaggcaccacacctt 3202
V.l : 3372 aaggctgctcagaaaaacaagcagaattctgaatgggctaccccaaacccagaaaacagg 3431
II II llll II II I II I II I I II I II II llll I I I I II II I I I I II I II I I I II I llll I
V.6 : 3203 aaggctgctcagaaaaacatgcagaattctgaatgggctaccccaaacccagaaaacagg 3262
V.l : 3432 cagatgataatgatgaagaaaaagaaaaagaagaagaagcattcccctaagaacttgctg 3491
II I I llll II I I I II II I I II II II I II II II II I I II 11 I I I II I I II II II I lllll
V.6 : 3263 cagatgataatgatgaagaaaaagaaaaagaagaagaagcattcccctaagaacctgctg 3322
V.l : 3492 cttaattttgtcactattgaagaaactaaggcagatgatgttgacagtgatggaaacaga 3551
II II I I I llll II II I I I I I I II II I II II II II I I II II I I I I I I II I I I III I llll I
V.6 : 3323 cttaattttgtcactattgaagaaactaaggcagatgatgttgacagtgatggaaacaga 3382
V.l : 3552 gtcacactagaccttcctattgatctagaagagcaaacaatgggaaagtacaattgggta 3611
MMIMMMM II I I I II II I II I I I II II II I I I
V.6 : 3383 gtcacactagaccttcctattgatctagaagagcaaacaatgggaaagtacaattgggta 3442
V.l : 3612 actacacctactactttcaagcccgacagccctgatttggcccgacactacaaatctgcc 3671
II II llll II I II II I I I I I I II I I II III III I I II llll I II I I I I I I II I II III I
V.6 : 3443 actacacctactactttcaagcctgacagccctgatttggcccgacactacaaatctgcc 3502 V. l 3672 tctccacagcctgccttccaaattcagcctgaaactcccctgaattcgaagcaccacatc 3731 l l l l l l l l l l l l l I M M M M M M M M I M M M M M I M M '
V. 6 3503 tctccacagcctgccttccaaattcagcctgaaactcccctgaatttgaagcaccacatc 3562
V.l : 3732 atccaagaactgcctctcgataacacctttgtggcctgtgactctatctccaagtgttcc 3791
I I I I lllll II II llll II I III I I I I II I MM I Ml II II I 111 I II Ml I MM III V.6 : 3563 atccaagaactgcctctcgataacacctttgtggcctgtgactctatctccaagtgttcc 3622
V.l : 3792 tcaagcagttcagatccctacagcgtttctgactgtggctatccagtgacgaccttcgag 3851
I I I Ml III II II Ml II I I II II I I III I II II I llll I II III I Ml I V.6 : 3623 tcaagcagttcagatccctacagcgtttctgactgtggctatccagtgacaaccttcgag 3682
V.l : 3852 gtacctgtgtccgtacacaccagaccg 3878
I I II llll I Ml I III II I II II I II I V.6 : 3683 gtacctgtgtccgtacacaccagaccg 3709
Table LΙV(e). Peptidesequences of protein coded by 109P1D4 v.6 (SEQ ID NO: 264)
MTVGFNSDIS SVVRVNTTNC HKCLLSGTYI FAVLLVCWF HSGAQEKNYT IREEIPENVL 60 IGNLLKDLNL SLIPNKSLTT TMQFKLVYKT GDVPLIRIEE DTGEIFTTGA RIDREKLCAG 120 IPRDEHCFYE VEVAILPDEI FRLVKIRFLI EDINDNAPLF PATVINISIP ENSAINSKYT 180 LPAAVDPDVG INGVQNYELI KSQNIFGLDV IETPEGDKMP QLIVQKELDR EEKDTYVMKV 240 KVEDGGFPQR SSTAILQVSV TDTNDNHPVF KETEIEVSIP ENAPVGTSVT QLHATDADIG 300 ENAKIHFSFS NLVSNIARRL FHLNATTGLI TIKEPLDREE TPNHKLLVLA SDGGLMPARA 360 MVLVNVTDVN DNVPSIDIRY IVNPVNDTW LSENIPLNTK lALITVTDKD ADHNGRVTCF 420 TDHEIPFRLR PVFSNQFLLE NAAYLDYEST KEYAIKLLAA DAGKPPLNQS AMLFIKVKDE 480 NDNAPVFTQS FVTVSIPENN SPGIQLMKVS ATDADSGPNA EINYLLGPDA PPEFSLDRRT 540 GMLTWKKLD REKEDKYLFT ILAKDNGVPP LTSNVTVFVS IIDQNDNSPV FTHNEYKFYV 600 PENLPRHGTV GLITVTDPDY GDNSAVTLSI LDENDDFTID SQTGVIRPNI SFDREKQESY 660 TFYVKAEDGG RVSRSSSAKV TINWDVNDN KPVFIVPPYN YSYELVLPST NPGTWFQVI 720 AVDNDTGMNA EVRYSIVGGN TRDLFAIDQE TGNITLMEKC DVTDLGLHRV LVKANDLGQP 780 DSLFSWIVN LFVNESVTNA TLINELVRKS IEAPVTPNTE IADVSSPTSD YVKILVAAVA 840 GTITVWVIF ITAVVRCRQA PHLKAAQKNM QNSEWATPNP ENRQMIMMKK KKKKKKHSPK 900 NLLLNFVTIE ETKADDVDSD GNRVTLDLPI DLEEQTMGKY NWVTTPTTFK PDSPDLARHY 960 KSASPQPAFQ IQPETPLNLK HHIIQELPLD NTFVACDSIS KCSSSSSDPY SVSDCGYPVT 1020 TFEVPVSVHT RPTDSRT 1037
Table LV(e). Amino acid sequence alignment of 109P1D4 v.1 (SEQ ID NO: 265) and 109P1D4 v.6 (SEQ ID NO: 266)
Score = 1966 bits (5093), Expect = O.OIdentities = 994/1009 (98%), Positives = 997/1009 (98%) .l 3 L SGTYIFAVLLACWFHSGAQEKNYTIREE PENVLIGDLLKDLNLS IPNKS TTAMQ 62
LLSGTYIFAVLL CWFHSGAQEKNYTIREE+PENVLIG+LLKD NLSLIPNKSLTT MQ V.6 24 L SGTYIFAV LVCWFHSGAQEKNYTIREEIPENVLIGNLLKDLNLSLIPNKS TTTMQ 83 V.l 63 FK VYKTGDVPLIRIEEDTGEIFTTGARIDREK CAGIPRDEHCFYEVEVAILPDEIFRL 122
FKLVYKTGDVPLIRIEEDTGEIFTTGARIDREKLCAGIPRDEHCFYEVEVAI PDEIFRL V.6 84 FKLVYKTGDVPLIRIEEDTGEIFTTGARIDREKLCAGIPRDEHCFYEVEVAI PDEIFRL 143 V.l 123 VKIRFLIEDINDNAPLFPATVINISIPENSAINSKYTLPAAVDPDVGINGVQNYELIKSQ 182
VKIRFLIEDINDNAPLFPATVINISIPENSAINSKYTLPAAVDPDVGINGVQNYELIKSQ V.6 144 VKIRF IEDINDNAPLFPATVINISIPENSAINSKYT PAAVDPDVGINGVQNYELIKSQ 203 V.l 183 NIFG DVIETPEGDKMPQ IVQKELDREEKDTYVMKVKVEDGGFPQRSSTAI QVSVTDT 242
NIFGLDVIETPEGDKMPQLIVQKELDREEKDTYVMKVKVEDGGFPQRSSTAILQVSVTDT V.6 204 NIFGLDVIETPEGDK PQ IVQKELDREEKDTYVMKVKVEDGGFPQRSSTAI QVSVTDT 263 V.l 243 NDNHPVFKETEIEVSIPENAPVGTSVTQLHATDADIGENAKIHFSFSN VSNIARRLFHL 302
NDNHPVFKETEIEVSIPENAPVGTSVTQLHATDADIGENAKIHFSFSNLVSNIARRLFH V.6 264 NDNHPVFKETEIEVSIPENAPVGTSVTQLHATDADIGENAKIHFSFSNLVSNI RRLFH 323 V.l 303 NATTGLITIKEPLDREETPNHKLLVASDGGL PARAMVLVNVTDVNDNVPSIDIRYIVN 362
NATTGLITIKEP DREETPNHK LVLASDGGLMPARAMVLVNVTDVNDNVPSIDIRYIVN V.6 324 NATTGLITIKEP DREETPNHK LV ASDGG PARAMV VNVTDVNDNVPSIDIRYIVN 383 363 PVNDTWLSENIP NTKIALITVTDKDADHNGRVTCFTDHEIPFRLRPVFSNQFLLETAA 422
PVNDTWLSENXPLNTKIALITVTDKDADHNGRVTCFTDHEIPFRLRPVFSNQFL E AA 384 PVNDTW SENIPLNTKIALITVTDKDADHNGRVTCFTDHEIPFRLRPVFSNQF ENAA 443
423 Y DYESTKEYAIKLLAADAGKPPLNQSAMLFIKVKDENDNAPVFTQSFVTVSIPENNSPG 482
YLDYESTKEYAIKLLAADAGKPPLNQSAMLFIKVKDENDNAPVFTQSFVTVSIPENNSPG 444 YLDYESTKEYAIK AADAGKPP NQSAMLFIKVKDENDNAPVFTQSFVTVSIPENNSPG 503
483 IQLTKVSAMDADSGPNAKINY LGPDAPPEFSLDCRTGMLTWKKLDREKEDKY FTILA 542
IQL KVSA DADSGPNA+INYLLGPDAPPEFSLD RTGMLTWKKLDREKEDKY FTILA 504 IQLMKVSATDADSGPNAEINYL GPDAPPEFSLDRRTGMLTWKK DREKEDKYLFTILA 563
543 KDNGVPPLTSNVTVFVSIIDQNDNSPVFTHNEYNFYVPEN PRHGTVGLITVTDPDYGDN 602
KDNGVPPLTSNVTVFVSIIDQNDNSPVFTHNEY FYVPEN PRHGTVGLITVTDPDYGDN 564 KDNGVPPLTSNVTVFVSIIDQNDNSPVFTHNEYKFYVPEN PRHGTVGLITVTDPDYGDN 623
603 SAVTLSILDENDDFTIDSQTGVIRPN1SFDREKQESYTFYVKAEDGGRVSRSSSAKVTIN 662
SAVTLSILDENDDFTIDSQTGVIRPNISFDREKQESYTFYVKAEDGGRVSRSSSAKVTIN 624 SAVTLSILDENDDFTIDSQTGVIRPNISFDREKQESYTFYVKAEDGGRVSRSSSAKVTIN 683
663 VVDVNDNKPVFIVPPSNCSYE VLPSTNPGTWFQVIAVDNDTGMNAEVRYSIVGGNTRD 722
WDVNDNKPVFIVPP N SYELVLPSTNPGTWFQVIAVDNDTGMNAEVRYSIVGGNTRD 684 WDVNDNKPVFIVPPYNYSYE VLPSTNPGTWFQVIAVDNDTG NAEVRYSIVGGNTRD 743
723 LFAIDQETGNITLMEKCDVTDLGLHRVLVKAND GQPDSLFSWIVNLFVNESVTNAT I 782
LFAIDQETGNITLMEKCDVTDLGLHRVLVKANDLGQPDSLFSWIVN FVNESVTNATLI 744 LFAIDQETGNITLMEKCDVTDLGLHRVLVK7Λ DLGQPDS FSWIVNLFVNESVTNATLI 803
783 NELVRKSTEAPVTPNTEIADVSSPTSDYVKI VAAVAGTITVWVIFITAVVRCRQAPHL 842
NE VRKS EAPVTPNTEIADVSSPTSDYVKILVAAVAGTITVVWIFITAVVRCRQAPH 804 NELVRKSIEAPVTPNTEIADVSSPTSDYVKILVAAVAGTITWWIFITAWRCRQAPHL 863
843 KAAQKNKQNSE ATPNPENRQMIMMKKKKKKKKHSPKN LLNFVTIEETKADDVDSDGNR 902
KAAQKN QNSE ATPNPENRQMIMMKKKKKKKKHSPKNL NFVTIEETKADDVDSDGNR 864 KAAQKN QNSE ATPNPENRQMIMMKKKKKKKKHSPKNLL NFVTIEETKADDVDSDGNR 923
903 VTLDLPIDLEEQTMGKYN VTTPTTFKPDSPDLARHYKSASPQPAFQIQPETPLNSKHHI 962
VTLDLPIDLEEQTMGKYNWVTTPTTFKPDSPD ARHYKSASPQPAFQIQPETP N KHHI 924 VT DLPIDLEEQTMGKYNWVTTPTTFKPDSPDLARHYKSASPQPAFQIQPETPLNLKHHI 983
963 1QELP DNTFVACDSISKCSSSSSDPYSVSDCGYPVTTFEVPVSVHTRP 1011
IQELP DNTFVACDSISKCSSSSSDPYSVSDCGYPVTTFEVPVSVHTRP 984 IQELPLDNTFVACDSISKCSSSSSDPYSVSDCGYPVTTFEVPVSVHTRP 1032
Table Lll(f). Nucleotide sequence of transcript variant 109P1 D4 v.7 (SEQ ID NO: 267] I ggtggtccag tacctccaaa gatatggaat acactcctga aatatcctga aacctttttt 60 ttttcagaat cctttaataa gcagttatgt caatctgaaa gttgcttact tgtactttat 120 attaatagct attcttgttt ttcttatcca aagaaaaatc ctctaatccc cttttcacat 180 gatagttgtt accatgttta ggcgttagtc acatcaaccc ctctcctctc ccaaacttct 240 cttcttcaaa tcaaacttta ttagtccctc ctttataatg attccttgcc tccttttatc 300 cagatcaatt ttttttcact ttgatgccca gagctgaaga aatggactat tgtataaatt 360 attcattgcc aagagaataa ttgcatttta aacccatgtt ataacaaaga ataatgatta 420 tattttgtga tttgtaacaa atacccttta ttttccctta actattgaat taaatatttt 480 aattatttgt attctcttta actatcttgg tatattaaag tattatcttt tatatattta 540 tcaatggtgg acacttttat aggtactctg tgtcattttt gatactgtag gtatcttatt 600 tcatttatct ttattcttaa tgtacgaatt cataatattt gattcagaac agatttatca 660 ctaattaaca gagtgtcaat tatgctaaca tctcatttac tgattttaat ttaaaacagt 720 ttttgttaac atgcatgttt agggttggct tcttaataat ttcttcttcc tcttctctct 780 ctcctcttct tttggtcagt gttgtgcggg ttaatacaac aaactgtcac aagtgtttgt 840 tgtccgggac gtacattttc gcggtcctgc tagtatgcgt ggtgttccac tctggcgccc 900 aggagaaaaa ctacaccatc cgagaagaaa ttccagaaaa cgtcctgata ggcaacttgt 960 tgaaagacct taacttgtcg ctgattccaa acaagtcctt gacaactact atgcagttca 1020 agctagtgta caagaccgga gatgtgccac tgattcgaat tgaagaggat actggtgaga 1080 tcttcactac cggcgctcgc attgatcgtg agaaattatg tgctggtatc ccaagggatg 1140 agcattgctt ttatgaagtg gaggttgcca ttttgccgga tgaaatattt agactggtta 1200 agatacgttt tctgatagaa gatataaatg ataatgcacc attgttccca gcaacagtta 1260 tcaacatatc aattccagag aactcggcta taaactctaa atatactctc ccagcggctg 1320 ttgatcctga cgtaggcata aacggagttc aaaactacga actaattaag agtcaaaaca 1380 tttttggcct cgatgtcatt gaaacaccag aaggagacaa gatgccacaa ctgattgttc 1440 aaaaggagtt agatagggaa gagaaggata cctatgtgat gaaagtaaag gttgaagatg 1500 gtggctttcc tcaaagatcc agtactgcta ttttgcaagt aagtgttact gatacaaatg 1560 acaaccaccc agtctttaag gagacagaga ttgaagtcag tataccagaa aatgctcctg 1620 taggcacttc agtgacacag ctccatgcca cagatgctga cataggtgaa aatgccaaga 1680 tccacttctc tttcagcaat ctagtctcca acattgccag gagattattt cacctcaatg 1740 ccaccactgg acttatcaca atcaaagaac cactggatag ggaagaaaca ccaaaccaca 1800 agttactggt tttggcaagt gatggtggat tgatgccagc aagagcaatg gtgctggtaa 1860 atgttacaga tgtcaatgat aatgtcccat ccattgacat aagatacatc gtcaatcctg 1920 tcaatgacac agttgttctt tcagaaaata ttccactcaa caccaaaatt gctctcataa 1980 ctgtgacgga taaggatgcg gaccataatg gcagggtgac atgcttcaca gatcatgaaa 2040 ttcctttcag attaaggcca gtattcagta atcagttcct cctggagaat gcagcatatc 2100 ttgactatga gtccacaaaa gaatatgcca ttaaattact ggctgcagat gctggcaaac 2160 ctcctttgaa tcagtcagca atgctcttca tcaaagtgaa agatgaaaat gacaatgctc 2220 cagttttcac ccagtctttc gtaactgttt ctattcctga gaataactct cctggcatcc 2280 agttgatgaa agtaagtgca acggatgcag acagtgggcc taatgctgag atcaattacc 2340 tgctaggccc tgatgctcca cctgaattca gcctggatcg tcgtacaggc atgctgactg 2400 tagtgaagaa actagataga gaaaaagagg ataaatattt attcacaatt ctggcaaaag 2460 ataatggggt accaccctta accagcaatg tcacagtctt tgtaagcatt attgatcaga 2520 atgacaatag cccagttttc actcacaatg aatacaaatt ctatgtccca gaaaaccttc 2580 caaggcatgg tacagtagga ctaatcactg taactgatcc tgattatgga gacaattctg 2640 cagttacgct ctccatttta gatgagaatg atgacttcac cattgattca caaactggtg 2700 tcatccgacc aaatatttca tttgatagag aaaaacaaga atcttacact ttctatgtaa 2760 aggctgagga tggtggtaga gtatcacgtt cttcaagtgc caaagtaacc ataaatgtgg 2820 ttgatgtcaa tgacaacaaa ccagttttca ttgtccctcc ttacaactat tcttatgaat 2880 tggttctacc gtccactaat ccaggcacag tggtctttca ggtaattgct gttgacaatg 2940 acactggcat gaatgcagag gttcgttaca gcattgtagg aggaaacaca agagatctgt 3000 ttgcaatcga ccaagaaaca ggcaacataa cattgatgga gaaatgtgat gttacagacc 3060 ttggtttaca cagagtgttg gtcaaagcta atgacttagg acagcctgat tctctcttca 3120 gtgttgtaat tgtcaatctg ttcgtgaatg agtcagtgac caatgctaca ctgattaatg 3180 aactggtgcg caaaagcatt gaagcaccag tgaccccaaa tactgagata gctgatgtat 3240 cctcaccaac tagtgactat gtcaagatcc tggttgcagc tgttgctggc accataactg 3300 tcgttgtagt tattttcatc actgctgtag taagatgtcg ccaggcacca caccttaagg 3360 ctgctcagaa aaacatgcag aattctgaat gggctacccc aaacccagaa aacaggcaga ' 3420 tgataatgat gaagaaaaag aaaaagaaga agaagcattc ccctaagaac ctgctgctta 3480 atgttgtcac tattgaagaa actaaggcag atgatgttga cagtgatgga aacagagtca 3540 cactagacct tcctattgat ctagaagagc aaacaatggg aaagtacaat tgggtaacta 3600 cacctactac tttcaagcct gacagccctg atttggcccg acactacaaa tctgcctctc 3660 cacagcctgc cttccaaatt cagcctgaaa ctcccctgaa tttgaagcac cacatcatcc 3720 aagaactgcc tctcgataac acctttgtgg cctgtgactc tatctccaat tgttcctcaa 3780 gcagttcaga tccctacagc gtttctgact gtggctatcc agtgacaacc ttcgaggtac 3840 ctgtgtccgt acacaccaga ccgactgatt ccaggacatg aactattgaa atctgcagtg 3900 agatgtaact ttctaggaac aacaaaattc cattcccctt ccaaaaaatt tcaatgattg 3960 tgatttcaaa attaggctaa gatcattaat tttgtaatct agatttccca ttataaaagc 4020 aagcaaaaat catcttaaaa atgatgtcct agtgaacctt gtgctttctt tagctgtaat 4080 ctggcaatgg aaatttaaaa tttatggaag agacagtgca gcgcaataac agagtactct 4140 catgctgttt ctctgtttgc tctgaatcaa cagccatgat gtaatataag gctgtcttgg 4200 tgtatacact tatggttaat atatcagtca tgaaacatgc aattacttgc cctgtctgat 4260 tgttgaataa ttaaaacatt atctccagga gtttggaagt gagctgaact agccaaacta 4320 ctctctgaaa ggtatccagg gcaagagaca tttttaagac cccaaacaaa caaaaaacaa 4380 aaccaaaaca ctctggttca gtgttttgaa aatattgact aacataatat tgctgagaaa 4440 atcattttta ttacccacca ctctgcttaa aagttgagtg ggccgggcgc ggtggctcac 4500 gcctgtaatt ccagcacttt gggaggccga ggcgggtgga tcacgaggtc aggatattga 4560 gaccatcctg gctaacatgg tgaaacccca tctccactaa aaatacaaaa aattagctgg 4620 gcgtggtggc gggcgcctgt agtcccagct actcgggagg ctgaggcagg agaatggcgt 4680 gaacccggga ggcggagctt gcagtgagcc gagatggcgc cactgcactc cagcctgggt 4740 gacagagcaa gactctgtct caaaaagaaa aaaatgttca gtgatagaaa ataattttac 4800 taggttttta tgttgattgt actcatgctg ttccactcct tttaattatt aaaaagttat 4860 ttttggctgg gtgtggtggc tcatacctgt aatcccagca ctttgggagg ccgaggcggg 4920 tggatcacct gaggtcagga gttcaagacc agtctggcca acat 4964 Table Llll(f). Nucleotide sequence alignment of 109P1 D4 v.1 (SEQ ID NO: 268) and 109P1 D4 v.7 (SEQ ID NO: 269)
Score = 5664 bits (2946), Expect = O.OIdentities = 3000/3027 (99%) Strand = Plus / Plus
V.l : 852 ttgttgtccgggacgtacattttcgcggtcctgctagcatgcgtggtgttccactctggc 911
II II I I I I I I II Mill lllll Ml I I I III I I I I I 1 II III I llll II I II II III I I V.7 : 837 ttgttgtccgggacgtacattttcgcggtcctgctagtatgcgtggtgttccactctggc 896
V.l : 912 gcccaggagaaaaactacaccatccgagaagaaatgccagaaaacgtcctgataggcgac 971
I I II I I II II I II llll I I II III II 1 I I I II I I I II I I II II I II II II II llll II
V.7 : 897 gcccaggagaaaaactacaccatccgagaagaaattccagaaaacgtcctgataggcaac 956
V.l : 972 ttgttgaaagaccttaacttgtcgctgattccaaacaagtccttgacaactgctatgcag 1031
II II II I II II II I I I II II I I II I I I I I I II I I II II I I II II I II II I I
V.7 : 957 ttgttgaaagaccttaacttgtcgctgattccaaacaagtccttgacaactactatgcag 1016
V.l : 1032 ttcaagctagtgtacaagaccggagatgtgccactgattcgaattgaagaggatactggt 1091
I I I 1 I I I II I II I II III II II I III I I I I I II I I II II I I II I II II II II II I Mill
V.7 : 1017 ttcaagctagtgtacaagaccggagatgtgccactgattcgaattgaagaggatactggt 1076
V.l : 1092 gagatcttcactactggcgctcgcattgatcgtgagaaattatgtgctggtatcccaagg 1151
II I I I 1 I I I I II II I I III I I III II I II I I I I I II I III III II II II I II II MM I
V.7 : 1077 gagatcttcactaccggcgctcgcattgatcgtgagaaattatgtgctggtatcccaagg 1136
V.l : 1152 gatgagcattgcttttatgaagtggaggttgccattttgccggatgaaatatttagactg 1211
I I II I II I I I II I llll III I llll II I II I II I I II I II I I II II II I I I III I MM I V.7 : 1137 gatgagcattgcttttatgaagtggaggttgccattttgccggatgaaatatttagactg 1196
V.l : 1212 gttaagatacgttttctgatagaagatataaatgataatgcaccattgttcccagcaaca 1271
I I II I I I I II II llll I II II II II I II II I I II I II I II I I I I II II I I I III I llll I V.7 : 1197 gttaagatacgttttctgatagaagatataaatgataatgcaccattgttcccagcaaca 1256
V.l : 1272 gttatcaacatatcaattccagagaactcggctataaactctaaatatactctcccagcg 1331
I I I I I I I I II II llll I II II llll I I I II II I I I II II II I I I II II I II III I lllll V.7 : 1257 gttatcaacatatcaattccagagaactcggctataaactctaaatatactctcccagcg 1316
V.l : 1332 gctgttgatcctgacgtaggaataaacggagttcaaaactacgaactaattaagagtcaa 1391
I I I II I I I II II I III I II I llll II I II I II II II II I I II I II II II MM I Mill V.7 : 1317 gctgttgatcctgacgtaggcataaacggagttcaaaactacgaactaattaagagtcaa 1376
V.l : 1392 aacatttttggcctcgatgtcattgaaacaccagaaggagacaagatgccacaactgatt 1451
I I I II II II II I I III II I I II III II I I I I II II II II II II I II II II II II II Ml I
V.7 : 1377 aacatttttggcctcgatgtcattgaaacaccagaaggagacaagatgccacaactgatt 1436
V.l : 1452 gttcaaaaggagttagatagggaagagaaggatacctacgtgatgaaagtaaaggttgaa 1511
II I I I II II II I I lllll II II llll I I I I II I II II I I I II II I II II II II I I III I
V.7 : 1437 gttcaaaaggagttagatagggaagagaaggatacctatgtgatgaaagtaaaggttgaa 1496
V.l : 1512 gatggtggctttcctcaaagatccagtactgctattttgcaagtgagtgttactgataca 1571
II I I I I I I I I I I II llll II II llll I I I I II I I I I I I I I I II I II II II I I II I I II I V.7 : 1497 gatggtggctttcctcaaagatccagtactgctattttgcaagtaagtgttactgataca 1556
V.l : 1572 aatgacaaccacccagtctttaaggagacagagattgaagtcagtataccagaaaatgct 1631
I I I I I I I I II II I I I III I II II III II I II I I II I II I I I II II II II II II II llll I V.7 : 1557 aatgacaaccacccagtctttaaggagacagagattgaagtcagtataccagaaaatgct 1616 V.l : 1632 cctgtaggcacttcagtgacacagctccatgccacagatgctgacataggtgaaaatgcc 1691
III II I I II I II I I I I I llll I I III II II I II II II I II III I I I III II II I llll II V.7 : 1617 cctgtaggcacttcagtgacacagctccatgccacagatgctgacataggtgaaaatgcc 1676
V.l : 1692 aagatccacttctctttcagcaatctagtctccaacattgccaggagattatttcacctc 1751
MM I I llll II I II I I I II I I MM II II II I 111 III II Ml I I II III II II I llll V.7 : 1677 aagatccacttctctttcagcaatctagtctccaacattgccaggagattatttcacctc 1736
V.l : 1752 aatgccaccactggacttatcacaatcaaagaaccactggatagggaagaaacaccaaac 1811
II II II II II I I I II I I II 11 II llll I llll Ml II I II II II I II Ml III I I llll I
V.7 : 1737 aatgccaccactggacttatcacaatcaaagaaccactggatagggaagaaacaccaaac 1796
V.l : 1812 cacaagttactggttttggcaagtgatggtggattgatgccagcaagagcaatggtgctg 1871
MM I I I II I I I II I I I III I lllll llll I II II I Mill II I I II II II II II I llll V.7 : 1797 cacaagttactggttttggcaagtgatggtggattgatgccagcaagagcaatggtgctg 1856
V.l : 1872 gtaaatgttacagatgtcaatgataatgtcccatccattgacataagatacatcgtcaat 1931
I III I 1 II II II I I I I I I II I I Mill I II II III II I III II I I III I II llll lllll V.7 : 1857 gtaaatgttacagatgtcaatgataatgtcccatccattgacataagatacatcgtcaat 1916
V.l : 1932 cctgtcaatgacacagttgttctttcagaaaatattccactcaacaccaaaattgctctc 1991
MM II III I I I II I I II II I III I II III I I II II II I I II II III I II III I V.7 : 1917 cctgtcaatgacacagttgttctttcagaaaatattccactcaacaccaaaattgctctc 1976
V.l : 1992 ataactgtgacggataaggatgcggaccataatggcagggtgacatgcttcacagatcat 2051
III I I I III II I I I I I I II I I I II III II II I I I I II II I I II I 11 I lllll
V.7 : 1977 ataactgtgacggataaggatgcggaccataatggcagggtgacatgcttcacagatcat 2036
V.l : 2052 gaaatccctttcagattaaggccagtattcagtaatcagttcctcctggagactgcagca 2111
Mill I II I I I I II I 11 II I I llll I Ml I I III I I Ml II I I I I I II I II I II MM V.7 : 2037 gaaattcctttcagattaaggccagtattcagtaatcagttcctcctggagaatgcagca 2096
V.l : 2112 tatcttgactatgagtccacaaaagaatatgccattaaattactggctgcagatgctggc 2171
III 11 I I I I II I II II I II I I II llll I II) I I llll I III II II I II II llll I II I I I V.7 : 2097 tatcttgactatgagtccacaaaagaatatgccattaaattactggctgcagatgctggc 2156
V.l : 2172 aaacctcctttgaatcagtcagcaatgctcttcatcaaagtgaaagatgaaaatgacaat 2231
I I) I I I I I II I I I I I I III I I I III I II II II III I I II II II I I I I II llll II III I I V.7 : 2157 aaacctcctttgaatcagtcagcaatgctcttcatcaaagtgaaagatgaaaatgacaat 2216
V.l : 2232 gctccagttttcacccagtctttcgtaactgtttctattcctgagaataactctcctggc 2291 llll I II II I I I II I I II I I I I llll I III I III II I I II II I I I II II II II I II II II V.7 : 2217 gctccagttttcacccagtctttcgtaactgtttctattcctgagaataactctcctggc 2276
V.l : 2292 atccagttgacgaaagtaagtgcaatggatgcagacagtgggcctaatgctaagatcaat 2351
I II II MM I I II I I II II II III I II II II I II I I llll I I I I II I I I
V.7 : 2277 atccagttgatgaaagtaagtgcaacggatgcagacagtgggcctaatgctgagatcaat 2336
V.l : 2352 tacctgctaggccctgatgctccacctgaattcagcctggattgtcgtacaggcatgctg 2411
II II I I II II I I I II 1 I II I I II II II II 111 Ml II I I II I I I I I I II II II I Mill
V.7 : 2337 tacctgctaggccctgatgctccacctgaattcagcctggatcgtcgtacaggcatgctg 2396
V.l : 2412 actgtagtgaagaaactagatagagaaaaagaggataaatatttattcacaattctggca 2471
III I I I II 1 I 1 I I I I II II I I II II II I II II MM 111 llll I 1 II II III I llll III
V.7 : 2397 actgtagtgaagaaactagatagagaaaaagaggataaatatttattcacaattctggca 2456 V.l : 2472 aaagataacggggtaccacccttaaccagcaatgtcacagtctttgtaagcattattgat 2531 lllll I I II I I II I I II I III II I II II II III MM I I I I II II
V.7 : 2457 aaagataatggggtaccacccttaaccagcaatgtcacagtctttgtaagcattattgat 2516
V.l : 2532 cagaatgacaatagcccagttttcactcacaatgaatacaacttctatgtcccagaaaac 2591 lllll II II I I I III ll I I II I I
V.7 : 2517 cagaatgacaatagcccagttttcactcacaatgaatacaaattctatgtcccagaaaac 2576
V.l : 2592 cttccaaggcatggtacagtaggactaatcactgtaactgatcctgattatggagacaat 2651
I I II III Ml llll I I II 111 I Ml I I I III III II II II II III II I I II II I I llll I
V.7 : 2577 cttccaaggcatggtacagtaggactaatcactgtaactgatcctgattatggagacaat 2636
V.l : 2652 tctgcagttacgctctccattttagatgagaatgatgacttcaccattgattcacaaact 2711
II II III II I I I I I I II II I I) Ml I II II II II I I III II II II II I I II II I
V.7 : 2637 tctgcagttacgctctccattttagatgagaatgatgacttcaccattgattcacaaact 2696
V.l : 2712 ggtgtcatccgaccaaatatttcatttgatagagaaaaacaagaatcttacactttctat 2771 llll II II llll II I I II II I II I II I Ml II 111 II II II Ml II Mlllll I I II II I
V.7 : 2697 ggtgtcatccgaccaaatatttcatttgatagagaaaaacaagaatcttacactttctat 2756
V.l : 2772 gtaaaggctgaggatggtggtagagtatcacgttcttcaagtgccaaagtaaccataaat 2831
II II II Ml lllll I I II II I II I I II Ml 11 I II llll II I I III I I II III I Mill I
V.7 : 2757 gtaaaggctgaggatggtggtagagtatcacgttcttcaagtgccaaagtaaccataaat 2816
V.l : 2832 gtggttgatgtcaatgacaacaaaccagttttcattgtccctccttccaactgttcttat 2891 lllll I II I III I I I I II II I II II I I MM II II II llll I II II lllll I I llll I
V.7 : 2817 gtggttgatgtcaatgacaacaaaccagttttcattgtccctccttacaactattcttat 2876
V.l : 2892 gaattggttctaccgtccactaatccaggcacagtggtctttcaggtaattgctgttgac 2951
II I I III II Mill I 111 II I II I I I I 11 MM II IM II I I II II II I II II I
V.7 : 2877 gaattggttctaccgtccactaatccaggcacagtggtctttcaggtaattgctgttgac 2936
V.l : 2952 aatgacactggcatgaatgcagaggttcgttacagcattgtaggaggaaacacaagagat 3011
III II II I I lllll I I I I II 1 II II II III II I II llll II II I II II II II I I I II II I
V.7 : 2937 aatgacactggcatgaatgcagaggttcgttacagcattgtaggaggaaacacaagagat 2996
V.l : 3012 ctgtttgcaatcgaccaagaaacaggcaacataacattgatggagaaatgtgatgttaca 3071
111 II I II I III II I I I I II I III I I I III II I I I I II II llllll I III III I I I I II I
V.7 : 2997 ctgtttgcaatcgaccaagaaacaggcaacataacattgatggagaaatgtgatgttaca 3056
V.l : 3072 gaccttggtttacacagagtgttggtcaaagctaatgacttaggacagcctgattctctc 3131
II II II II Mill I II I I II I II I I I Mill I II II II Ml III I I I llll II I II I II I
V.7 : 3057 gaccttggtttacacagagtgttggtcaaagctaatgacttaggacagcctgattctctc 3116
V.l : 3132 ttcagtgttgtaattgtcaatctgttcgtgaatgagtcggtgaccaatgctacactgatt 3191
II II I II I II III I I I II I I I I I I I I II III I II llll I I MM I II I Ml I I II II 1 I
V.7 : 3117 ttcagtgttgtaattgtcaatctgttcgtgaatgagtcagtgaccaatgctacactgatt 3176
V.l : 3192 aatgaactggtgcgcaaaagcactgaagcaccagtgaccccaaatactgagatagctgat 3251
I II II II II II II I I III I I I I I 1 I Ml II I II III II I I II II II I I III I I II II II V.7 : 3177 aatgaactggtgcgcaaaagcattgaagcaccagtgaccccaaatactgagatagctgat 3236
V.l : 3252 gtatcctcaccaactagtgactatgtcaagatcctggttgcagctgttgctggcaccata 3311
II I II II II III I II II I I I I I I 11 I III II III III II I I I II II I I I II I I I V.7 : 3237 gtatcctcaccaactagtgactatgtcaagatcctggttgcagctgttgctggcaccata 3296 V.l : 3312 actgtcgttgtagttattttcatcactgctgtagtaagatgtcgccaggcaccacacctt 3371
I I I I III II llll II II II 111 II I I I III II I II I I II II MM II Ml I II II I II I I
V.7 : 3297 actgtcgttgtagttattttcatcactgctgtagtaagatgtcgccaggcaccacacctt 3356
V.l : 3372 aaggctgctcagaaaaacaagcagaattctgaatgggctaccccaaacccagaaaacagg 3431
I 1 I III I II II II I I II I I III II I II I I II I I I Ml II Ml 111 II Ml I II I II II I
V.7 : 3357 aaggctgctcagaaaaacatgcagaattctgaatgggctaccccaaacccagaaaacagg 3416
V.l : 3432 cagatgataatgatgaagaaaaagaaaaagaagaagaagcattcccctaagaacttgctg 3491
II I I II I I II I II II I I I III II III II III I MM II III II I I II I I I
V.7 : 3417 cagatgataatgatgaagaaaaagaaaaagaagaagaagcattcccctaagaacctgctg 3476
V.l : 3492 cttaattttgtcactattgaagaaactaaggcagatgatgttgacagtgatggaaacaga 3551 llllll II lllll II II II Mill I II I II II II II I 11 Ml II III II II II III II I
V.7 : 3477 cttaatgttgtcactattgaagaaactaaggcagatgatgttgacagtgatggaaacaga 3536
V.l : 3552 gtcacactagaccttcctattgatctagaagagcaaacaatgggaaagtacaattgggta 3611
I II II I I I I llll I III I I III II I I II llll I) I I I I II I I II II I Ml I llll I I I I I
V.7 : 3537 gtcacactagaccttcctattgatctagaagagcaaacaatgggaaagtacaattgggta 3596
V.l : 3612 actacacctactactttcaagcccgacagccctgatttggcccgacactacaaatctgcc 3671
I I I I II I II Ml II III I II I I I I I II III II I I II I II Mill I II I II II III II II
V.7 : 3597 actacacctactactttcaagcctgacagccctgatttggcccgacactacaaatctgcc 3656
V.l : 3672 tctccacagcctgccttccaaattcagcctgaaactcccctgaattcgaagcaccacatc 3731
I I I I II I I II) III II I II III I I II I Ml II II I I II II II I II I I II II II II I II I
V.7 : 3657 tctccacagcctgccttccaaattcagcctgaaactcccctgaatttgaagcaccacatc 3716
V.l : 3732 atccaagaactgcctctcgataacacctttgtggcctgtgactctatctccaagtgttcc 3791
I 11 I II I II I I II I I I 11 I III II I III I I II I II I I I II lllll II I I llll
V.7 : 3717 atccaagaactgcctctcgataacacctttgtggcctgtgactctatctccaattgttcc 3776
V.l : 3792 tcaagcagttcagatccctacagcgtttctgactgtggctatccagtgacgaccttcgag 3851
I II III I I II I I II I II II lllll I I II I I I I I I I I I llll llll I I Ml lllllllll
V.7 : 3777 tcaagcagttcagatccctacagcgtttctgactgtggctatccagtgacaaccttcgag 3836
V.l : 3852 gtacctgtgtccgtacacaccagaccg 3878
I 11 II III MM II III II I I III I I I V.7 : 3837 gtacctgtgtccgtacacaccagaccg 3863
Score = 1567 bits (815), Expect = O.OIdentities = 829/836 (99%) Strand = Plus / Plus
V.l : 3 ggtggtccagtacctccaaagatatggaatacactcctgaaatatcctgaaaactttttt 62
I I I I III I II II II II II II I I III I Ml I I I I I I I I I II Ml I I II I I I I V.7 : 1 ggtggtccagtacctccaaagatatggaatacactcctgaaatatcctgaaacctttttt 60
V.l : 63 ttttcagaatcctttaataagcagttatgtcaatctgaaagttgcttacttgtactttat 122
I I II Ml I I III II II II II II III I I II II I I I II I I II II llll I III I I II II II II V.7 : 61 ttttcagaatcctttaataagcagttatgtcaa^ctgaaagttgcttacttgtactttat 120
V.l : 123 attaatagctattcttgtttttcttatccaaagaaaaatcctctaatccccttttcacat 182
I I II Ml I I II I II II II II lllll I I II II I I I II II II I III Mlllll I II II II I I V.7 : 121 attaatagctattcttgtttttcttatccaaagaaaaatcctctaatccccttttcacat 180 V.l : 183 gatagttgttaccatgtttaggcattagtcacatcaacccctctcctctcccaaacttct 242
M MMIIIMI 11 II II II II I I II I II II II II II I I II 11 I II II V.7 : 181 gatagttgttaccatgtttaggcgttagtcacatcaacccctctcctctcccaaacttct 240
V.l : 243 cttcttcaaatcaaactttattagtccctcctttataatgattccttgcctcgttttatc 302
11 I II II II I I 1 I III I I II I III II I 11 II II I llll I I I II I II II I II I I III II I V.7 : 241 cttcttcaaatcaaactttattagtccctcctttataatgattccttgcctccttttatc 300
V.l : 303 cagatcaattttttttcactttgatgcccagagctgaagaaatggactactgtataaatt 362
I I III I I I I I I I II II I I I MM 11 II I II III I I II II II I II I III I II II I II II I V.7 : 301 cagatcaattttttttcactttgatgcccagagctgaagaaatggactattgtataaatt 360
V.l : 363 attcattgccaagagaataattgcattttaaacccatattataacaaagaataatgatta 422
I I II II I II II I II II I II II I II MM II Ml 11 I I I II 11 II II I Ml II II I II II
V.7 : 361 attcattgccaagagaataattgcattttaaacccatgttataacaaagaataatgatta 420
V.l : 423 tattttgtgatttgtaacaaataccctttattttcccttaactattgaattaaatatttt 482
II M I M II I I I I II I II III II I III II III I I llllll I II I II II I I II I I II I II I
V.7 : 421 tattttgtgatttgtaacaaataccctttattttcccttaactattgaattaaatatttt 480
V.l : 483 aattatttgtattctctttaactatcttggtatattaaagtattatcttttatatattta 542
I I I I I I I I I I I I II II I III III I II II III I I II I II II II M I I II I I II II I II II I V.7 : 481 aattatttgtattctctttaactatcttggtatattaaagtattatcttttatatattta 540
V.l : 543 tcaatggtggacacttttataggtactctgtgtcatttttgatactgtaggtatcttatt 602
I I I I II I I I II I II II I I II I II II I II III II I I II I I III I II II I I I II II I II II I V.7 : 541 tcaatggtggacacttttataggtactctgtgtcatttttgatactgtaggtatcttatt 600
V.l : 603 tcatttatctttattcttaatgtacgaattcataatatttgattcagaacaaatttatca 662
I I 11 I II I I III 11 II I I I I I I I llll I II I 11 II II I I II I I I I II I I II V.7 : 601 tcatttatctttattcttaatgtacgaattcataatatttgattcagaacagatttatca 660
V.l : 663 ctaattaacagagtgtcaattatgctaacatctcatttactgattttaatttaaaacagt 722
II 1111 I 11 II I II II I I I I I II II I I I III II I I I II I 111 I I III 11 II Ml I II II I V.7 : 661 ctaattaacagagtgtcaattatgctaacatctcatttactgattttaatttaaaacagt 720
V.l : 723 ttttgttaacatgcatgtttagggttggcttcttaataatttcttcttcctcttctctct 782
I I II II I II II I I I II lllll II llll I lllll Mill I I III II II 11 I I I III II II I V.7 : 721 ttttgttaacatgcatgtttagggttggcttcttaataatttcttcttcctcttctctct 780
V.l : 783 ctcctcttcttttggtcagtgttgtgcgggttaatacaacaaactgtaacaagtgt 838
I I I I I I I II II I II II I III I II lllll llll I I I II I I I I II II II II II II I I V.7 : 781 ctcctcttcttttggtcagtgttgtgcgggttaatacaacaaactgtcacaagtgt 836
Table LΙV(f). Peptide sequences of protein coded by 109P1D4v.7 (SEQ ID NO: 270)
MFRVGFLIIS SSSSLSPLLL VSWRVNTTN CHKCLLSGTY IFAVLLVCW FHSGAQEKNY 60
TIREEIPENV LIGNLLKDLN LSLIPNKSLT TTMQFKLVYK TGDVPLIRIE EDTGEIFTTG 120
ARIDREKLCA GIPRDEHCFY EVEVAILPDE IFRLVKIRFL IEDINDNAPL FPATVINISI 180
PENSAINSKY TLPAAVDPDV GINGVQNYEL IKSQNIFGLD VIETPEGDKM PQLIVQKELD 240
REEKDTYVMK VKVEDGGFPQ RSSTAILQVS VTDTNDNHPV FKETEIEVSI PENAPVGTSV 300
TQLHATDADI GENAKIHFSF SNLVSNIARR LFHLNATTGL ITIKEPLDRE ETPNHKLLVL 360
ASDGGLMPAR AMVLVNVTDV NDNVPSIDIR YIVNPVNDTV VLSENIPLNT KIALITVTDK 420
DADHNGRVTC FTDHEIPFRL RPVFSNQFLL ENAAYLDYES TKEYAIKLLA ADAGKPPLNQ 480
SAMLFIKVKD ENDNAPVFTQ SFVTVSIPEN NSPGIQLMKV SATDADSGPN AEINYLLGPD 540
APPEFSLDRR TGMLTWKKL DREKEDKYLF TILAKDNGVP PLTSNVTVFV SIIDQNDNSP 600
VFTHNEYKFY VPENLPRHGT VGLITVTDPD YGDNSAVTLS ILDENDDFTI DSQTGVIRPN 660 ISFDREKQES YTFYVKAEDG GRVSRSSSAK VTINWDVND NKPVFIVPPY NYSYELVLPS 720
TNPGTWFQV lAVDNDTGMN AEVRYSIVGG NTRDLFAIDQ ETGNITLMEK CDVTDLGLHR 780
VLVKANDLGQ PDSLFSWIV NLFVNESVTN ATLINELVRK SIEAPVTPNT EIADVSSPTS 840
DYVKILVAAV AGTIT WI FITAλΛ/RCRQ APHLKAAQKN MQNSEWATPN PENRQMIMMK 900
KKKKKKKHSP KNLLLNWTI EETKADDVDS DGNRVTLDLP IDLEEQTMGK YNWVTTPTTF 960
KPDSPDLARH YKSASPQPAF QIQPETPLNL KHHIIQELPL DNTFVACDSI SNCSSSSSDP 1020
YSVSDCGYPV TTFEVPVSVH TRPTDSRT 1048
Table LV(f). Amino acid sequence alignment of 109P1D4 v.1 (SEQ ID NO: 271) and 109P D4 v.7 (SEQ ID NO: 272)
Score = 1961 bits (5081), Expect = O.OIdentities = 992/1009 (98%), Positives = 995/1009 (98%)
V.l 3 LSGTYIFAVLLACWFHSGAQEKNYTIREEMPENVLIGDLLKD NLS IPNKSLTTAMQ 62
L SGTYIFAVLL CWFHSGAQEKNYTIREE+PENV IG+LLKDLNLSLIPNKSLTT MQ V.7 35 LLSGTYIFAVLLVCWFHSGAQEKNYTIREEIPENVLIGNLLKDLNLSLIPNKSLTTT Q 94 V.l 63 FKLVYKTGDVPLIRIEEDTGEIFTTGARIDREKLCAGIPRDEHCFYEVEVAILPDEIFRL 122
FKLVYKTGDVPLIRIEEDTGEIFTTGARIDREKLCAGIPRDEHCFYEVEVAILPDEIFR V.7 95 FKLVYKTGDVPLIRIEEDTGEIFTTGARIDREKLCAGIPRDEHCFYEVEVAILPDEIFRL 154 V.l 123 VKIRFLIEDINDNAP FPATVINISIPENSAINSKYTLPAAVDPDVGINGVQNYELIKSQ 182
VKIRFLIEDINDNAPLFPATVINISIPENSAINSKYTLPAAVDPDVGINGVQNYELIKSQ V.7 155 VKIRFLIEDINDNAPLFPATVINISIPENSAINSKYTLPAAVDPDVGINGVQNYELIKSQ 214 V.l 183 NIFGLDVIETPEGDKMPQLIVQKELDREEKDTYVMKVKVEDGGFPQRSSTAILQVSVTDT 242
NIFGLDVIETPEGDKMPQ IVQKELDREE DTYVMKV VEDGGFPQRSSTAILQVSVTDT V.7 215 NIFGLDVIETPEGDK PQLIVQKE DREEKDTYVMKVKVEDGGFPQRSSTAILQVSVTDT 274 V.l 243 NDNHPVFKETEIEVSIPENAPVGTSVTQLHATDADIGENAKIHFSFSNLVSNIARR FHL 302
NDNHPVFKETEIEVSIPENAPVGTSVTQ HATDADIGENAKIHFSFSNLVSNIARRLFHL V.7 275 NDNHPVFKETEIEVSIPENAPVGTSVTQLHATDADIGENAKIHFSFSNLVSNIARRLFHL 334 V.l 303 NATTGLITIKEP DREETPNHKLLVLASDGGLMPARAMVLVNVTDVNDNVPSIDIRYIVN 362
NATTGLITIKEPLDREETPNHKLLVLASDGGLMPARAMVLVNVTDVNDNVPSIDIRYIVN V.7 335 NATTGLITIKEPLDREETPNHKL VLASDGG MPARAMVLVNVTDVNDNVPSIDIRYIVN 394 V.l 363 PVNDTVVLSENIP NT IALITVTDKDADHNGRVTCFTDHEIPFRLRPVFSNQFL ETAA 422
PVNDTVVLSENIPLNTKIALITVTDKDADHNGRVTCFTDHEIPFRLRPVFSNQF LE AA V.7 395 PVNDTWLSENIPLNTKIALITVTDKDADHNGRVTCFTDHEIPFRLRPVFSNQFL ENAA 454 .l 23 Y DYESTKEYAIKL AADAGKPP NQSAMLFIKVKDENDNAPVFTQSFVTVSIPENNSPG 482
YLDYESTKEYAIKLLAADAGKPPLNQSAMLFIKVKDENDNAPVFTQSFVTVSIPENNSPG V.7 455 YLDYESTKEYAIKLLAADAGKPP NQSAMLFIKVKDENDNAPVFTQSFVTVSIPENNSPG 514 V.l 483 IQLTKVSAMDADSGPNAKINYLLGPDAPPEFSLDCRTGMLTWKKLDREKEDKY FTILA 542
IQL KVSA DADSGPNA+INYLLGPDAPPEFSLD RTGMLTWKKLDREKEDKYLFTILA V.7 515 IQ MKVSATDADSGPNAEINYL GPDAPPEFSLDRRTGMLTWKKLDREKEDKYLFTILA 574 V.l 543 KDNGVPPLTSNVTVFVSIIDQNDNSPVFTHNEYNFYVPENLPRHGTVGLITVTDPDYGDN 602
KDNGVPPLTSNVTVFVSIIDQNDNSPVFTHNEY FYVPEN PRHGTVG ITVTDPDYGDN V.7 575 KDNGVPPLTSNVTVFVSIIDQNDNSPVFTHNEYKFYVPENLPRHGTVGLITVTDPDYGDN 634 V.l 603 SAVTLSILDENDDFTIDSQTGVIRPNISFDREKQESYTFYVKAEDGGRVSRSSSAKVTIN 662
SAVT SILDENDDFTIDSQTGVIRPNISFDREKQESYTFYVKAEDGGRVSRSSSAKVTIN V.7 635 SAVTLSILDENDDFTIDSQTGVIRPNISFDREKQESYTFYVKAEDGGRVSRSSSAKVTIN 694 .l 663 WDVNDNKPVFIVPPSNCSYELVLPSTNPGTVVFQVIAVDNDTG NAEVRYSIVGGNTRD 722
WDV DNKPVFIVPP N SYELVLPSTNPGTVVFQVIAVDNDTGMNAEVRYSIVGGNTRD V.7 695 WDVNDNKPVFIVPPYNYSYELVLPΞTNPGTWFQVXAVDNDTG NAEVRYSIVGGNTRD 754 V.l 723 LFAIDQETGNITL EKCDVTDLG HRVLVKANDLGQPDSLFSWIVNLFVNESVTNATLI 782
LFAIDQETGNITLMEKCDVTDLGLHRV VKANDLGQPDSLFSWIVNLFVNESVTNATLI V.7 755 LFAIDQETGNIT MEKCDVTDLGLHRVLVKANDLGQPDSLFSWIVNLFVNESVTNATLI 814 V.l 783 NE VRKSTEAPVTPNTEIADVSSPTSDYVKILVAAVAGTITWWIFITAWRCRQAPH 842
NELVRKS EAPVTPNTEIADVSSPTSDYVKILVAAVAGTITVWVIFITAWRCRQAPHL V.7 815 NE VRKSIEAPVTPNTEIADVSSPTSDYVKILVAAVAGTITWWIFITAVVRCRQAPHL 874 V.l 843 KAAQK KQNSE ATPNPENRQMI MKKKKKKKKHSPKNLLLNFVTIEETKADDVDSDGNR 902 KAAQKN QNSEWATPNPENRQMIMMKKKKKKKKHSPKNLLLN VTIEETKADDVDSDGNR 875 KAAQKMQNSEWATPNPENRQMIM KKKKKKKKHSPKNLLLNWTIEETKADDVDSDGNR 934
903 VT DLPIDLEEQTMGKY VTTPTTFKPDSPD ARHYKSASPQPAFQIQPETPLNSKHHI 962
VTLDLPIDLEEQTMGKYNWVTTPTTFKPDSPDLARHYKSASPQPAFQIQPETPLN KHHI 935 VTLDLPIDLEEQTMGKYNWVTTPTTFKPDSPDLARHYKSASPQPAFQIQPETPLN KHHI 994
963 IQE PLDNTFVACDSISKCSSSSSDPYSVSDCGYPVTTFEVPVSVHTRP 1011
IQE PLDNTFVACDSIS CSSSSSDPYSVSDCGYPVTTFEVPVSVHTRP 995 IQELPLDNTFVACDSISNCSSSSSDPYSVSDCGYPVTTFEVPVSVHTRP 1043
Table Lll(g). Nucleotide sequence of transcript variant 109P1 D4 v.8 (SEQ ID NO: 273) ggtggtccag tacctccaaa gatatggaat acactcctga aatatcctga aacctttttt 60 ttttcagaat cctttaataa gcagttatgt caatctgaaa gttgcttact tgtactttat 120 attaatagct attcttgttt ttcttatcca aagaaaaatc ctctaatccc cttttcacat 180 gatagttgtt accatgttta ggcgttagtc acatcaaccc ctctcctctc ccaaacttct 240 cttcttcaaa tcaaacttta ttagtccctc ctttataatg attccttgcc tccttttatc 300 cagatcaatt ttttttcact ttgatgccca gagctgaaga aatggactat tgtataaatt 360 attcattgcc aagagaataa ttgcatttta aacccatgtt ataacaaaga ataatgatta 420 tattttgtga tttgtaacaa atacccttta ttttccctta actattgaat taaatatttt 480 aattatttgt attctcttta actatcttgg tatattaaag tattatcttt tatatattta 540 tcaatggtgg acacttttat aggtactctg tgtcattttt gatactgtag gtatcttatt 600 tcatttatct ttattcttaa tgtacgaatt cataatattt gattcagaac agatttatca 660 ctaattaaca gagtgtcaat tatgctaaca tctcatttac tgattttaat ttaaaacagt 720 ttttgttaac atgcatgttt agggttggct tcttaataat ttcttcttcc tcttctctct 780 ctcctcttct tttggtcagt gttgtgcggg ttaatacaac aaactgtcac aagtgtttgt 840 tgtccgggac gtacattttc gcggtcctgc tagtatgcgt ggtgttccac tctggcgccc 900 aggagaaaaa ctacaccatc cgagaagaaa ttccagaaaa cgtcctgata ggcaacttgt 960 tgaaagacct taacttgtcg ctgattccaa acaagtcctt gacaactact atgcagttca 1020 agctagtgta caagaccgga gatgtgccac tgattcgaat tgaagaggat actggtgaga 1080 tcttcactac cggcgctcgc attgatcgtg agaaattatg tgctggtatc ccaagggatg 1140 agcattgctt ttatgaagtg gaggttgcca ttttgccgga tgaaatattt agactggtta 1200 agatacgttt tctgatagaa gatataaatg ataatgcacc attgttccca gcaacagtta 1260 tcaacatatc aattccagag aactcggcta taaactctaa atatactctc ccagcggctg 1320 ttgatcctga cgtaggcata aacggagttc aaaactacga actaattaag agtcaaaaca 1380 tttttggcct cgatgtcatt gaaacaccag aaggagacaa gatgccacaa ctgattgttc 1440 aaaaggagtt agatagggaa gagaaggata cctatgtgat gaaagtaaag gttgaagatg 1500 gtggctttcc tcaaagatcc agtactgcta ttttgcaagt aagtgttact gatacaaatg 1560 acaaccaccc agtctttaag gagacagaga ttgaagtcag tataccagaa aatgctcctg 1620 taggcacttc agtgacacag ctccatgcca cagatgctga cataggtgaa aatgccaaga 1680 tccacttctc tttcagcaat ctagtctcca acattgccag gagattattt cacctcaatg 1740 ccaccactgg acttatcaca atcaaagaac cactggatag ggaagaaaca ccaaaccaca 1800 agttactggt tttggcaagt gatggtggat tgatgccagc aagagcaatg gtgctggtaa 1860 atgttacaga tgtcaatgat aatgtcccat ccattgacat aagatacatc gtcaatcctg 1920 tcaatgacac agttgttctt tcagaaaata ttccactcaa caccaaaatt gctctcataa 1980 ctgtgacgga taaggatgcg gaccataatg gcagggtgac atgcttcaca gatcatgaaa 2040 ttcctttcag attaaggcca gtattcagta atcagttcct cctggagaat gcagcatatc 2100 ttgactatga gtccacaaaa gaatatgcca ttaaattact ggctgcagat gctggcaaac 2160 ctcctttgaa tcagtcagca atgctcttca tcaaagtgaa agatgaaaat gacaatgctc 2220 cagttttcac ccagtctttc gtaactgttt ctattcctga gaataactct cctggcatcc 2280 agttgatgaa agtaagtgca acggatgcag acagtgggcc taatgctgag atcaattacc 2340 tgctaggccc tgatgctcca cctgaattca gcctggatcg tcgtacaggc atgctgactg 2400 tagtgaagaa actagataga gaaaaagagg ataaatattt attcacaatt ctggcaaaag 2460 ataatggggt accaccctta accagcaatg tcacagtctt tgtaagcatt attgatcaga 2520 atgacaatag cccagttttc actcacaatg aatacaaatt ctatgtccca gaaaaccttc 2580 caaggcatgg tacagtagga ctaatcactg taactgatcc tgattatgga gacaattctg 2640 cagttacgct ctccatttta gatgagaatg atgacttcac cattgattca caaactggtg 2700 tcatccgacc aaatatttca tttgatagag aaaaacaaga atcttacact ttctatgtaa 2760 aggctgagga tggtggtaga gtatcacgtt cttcaagtgc caaagtaacc ataaatgtgg 2820 ttgatgtcaa tgacaacaaa ccagttttca ttgtccctcc ttacaactat tcttatgaat 2880 tggttctacc gtccactaat ccaggcacag tggtctttca ggtaattgct gttgacaatg 2940 acactggcat gaatgcagag gttcgttaca gcattgtagg aggaaacaca agagatctgt 3000 ttgcaatcga ccaagaaaca ggcaacataa cattgatgga gaaatgtgat gttacagacc 3060 ttggtttaca cagagtgttg gtcaaagcta atgacttagg acagcctgat tctctcttca 3120 gtgttgtaat tgtcaatctg ttcgtgaatg agtcagtgac caatgctaca ctgattaatg 3180 aactggtgcg caaaagcatt gaagcaccag tgaccccaaa tactgagata gctgatgtat 3240 cctcaccaac tagtgactat gtcaagatcc tggttgcagc tgttgctggc accataactg 3300 tcgttgtagt tattttcatc actgctgtag taagatgtcg ccaggcacca caccttaagg 3360 ctgctcagaa aaacatgcag aattctgaat gggctacccc aaacccagaa aacaggcaga 3420 tgataatgat gaagaaaaag aaaaagaaga agaagcattc ccctaagaac ctgctgctta 3480 atgttgtcac tattgaagaa actaaggcag atgatgttga cagtgatgga aacagagtca 3540 cactagacct tcctattgat ctagaagagc aaacaatggg aaagtacaat tgggtaacta 3600 cacctactac tttcaagcct gacagccctg atttggcccg acactacaaa tctgcctctc 3660 cacagcctgc cttccaaatt cagcctgaaa ctcccctgaa tttgaagcac cacatcatcc 3720 aagaactgcc tctcgataac acctttgtgg cctgtgactc tatctccaat tgttcctcaa 3780 gcagttcaga tccctacagc gtttctgact gtggctatcc agtgacaacc ttcgaggtac 3840 ctgtgtccgt acacaccaga ccgtcccagc ggcgtgtcac atttcacctg ccagaaggct 3900 ctcaggaaag cagcagtgat ggtggactgg gagaccatga tgcaggcagc cttaccagca 3960 catcccatgg cctgcccctt ggctatcctc aggaggagta ctttgatcgt gctacaccca 4020 gcaatcgcac tgaaggggat ggcaactccg atcctgaatc tactttcata cctggactaa 4080 agaaagaaat aactgttcaa ccaactgtgg aagaggcctc tgacaactgc actcaagaat 4140 gtctcatcta tggccattct gatgcctgct ggatgccggc atctctggat cattccagct 42Q0 cttcacaagc acaggcctct gctctatgcc acagcccacc actgtcacag gcctctactc 4260 agcaccacag cccaccagtg acacagacca ttgttctctg ccacagccct ccagtgacac 4320 agaccatcgc attgtgccac agcccaccac cgatacaggt gtctgctctc caccacagtc 4380 ctcctctagt gcagggtact gcacttcacc acagcccacc atcagcacag gcctcagccc 4440 tctgctacag ccctccttta gcacaggctg ctgcaatcag ccacagctct tctctgccac 4500 aggttattgc cctccatcgt agtcaggccc aatcatcagt cagtttgcag caaggttggg 4560 tgcaaggtgc taatggacta tgctctgttg atcagggagt gcaaggtagt gcaacatctc 4620 agttttacac catgtctgaa agacttcatc ccagtgatga ttcaattaaa gtcattcctt 4680 tgacaacctt cgctccacgc caacaggcca gaccgtccag aggtgattcc cccattatgg 4740 aaacacatcc cttgtaaagc taaaatagtt acttcaaatt ttcagaaaag atgtatatag 4800 tcaaaattta agatacaatt ccaatgagta ttctgattat cagatttgta aataactatg 4860 taaatagaaa cagataccag aataaatcta cagctagacc cttagtcaat agttaaccaa 4920 aaaattgcaa tttgtttaat tcagaatgtg tatttaaaaa gaaaaggaat ttaacaattt 4980 gcatcccctt gtacagtaag gcttatcatg acagagcgta ctatttctga tgtacagtat 5040 tttttgttgt ttttatcatc atgtgcaata ttactgattt gtttccatgc tgattgtgtg 5100 gaaccagtat gtagcaaatg gaaagcctag aaatatctta ttttctaagt ttacctttag 5160 tttacctaaa cttttgttca gataatgtta aaaggtatac gtactctagc cttttttggg 5220 gctttctttt tgatttttgt ttgtggtttt cagttttttt gttgttgtta gtgagtctcc 5280 cttcaaaata cacagtaggt agtgtaaata ctgcttgttt gtgtctctct gctgtcatgt 5340 tttctacctt attccaatac tatattgttg ataaaatttg tatatacatt ttcaataaag 5400 aatatgtata aactgtacag atctagatct acaacctatt tctctactct ttagtagagt 5460 tcgagacaca gaagtgcaat aactgcccta attaagcaac tatttgttaa aaagggcccc 5520 tttttacttt aatagtttag tgtaaagtac atcagaaata aaactgtatc tgacatttta 5580 agcctgtagt ccattattac ttgggtcttt acttctggga atttgtatgt aacagcctag 5640 aaaattaaaa ggaggtggat gcatccaaag cacgagtcac ttaaaatatc gacggtaaac 5700 tactattttg tagagaaact caggaagatt taaatgttga tttgacagct caataggctg 5760 ttaccaaagg gtgttcagta aaaataacaa atacatgtaa ctgtagataa aaccacatac 5820 taaatctata agactaaggg atttttgtta ttctagctca acttactgaa gaaaaccact 5880 aataacaaca agaatatcag gaaggaactt ttcaagaaat gtaattataa atctacatca 5940 aacagaattt taaggaaaaa tgcagaggga gaaataaggc acatgactgc ttcttgcagt 6000 caagaagaaa taccaataac acacacagaa caaaaaccat caaaatctca tatatgaaat 6060 aaaatatatt cttctaagca aagaaacagt actattcata gaaaacatta gttttctcct 6120 gttgtctgtt atttccttct tttatcctct taactggcca ttatcttgta tgtgcacatt 6180 ttataaatgt acagaaacat caccaacttg attttcttcc atagcaaaac tgagaaaata 6240 ccttgtttca gtataacact aaaccaagag acaattgatg tttaatgggg gcggttgggg 6300 ttggggggga gtcaatatct cctattgatt aacttagaca tagattttgt aatgtataac 6360 ttgatattta atttatgatt aaactgtaat tttgtaacat aaactgtggt aattgcataa 6420 tttcattggt gaggatttcc tttgaatatt gagaaagttt cttttcatgt gcccagcagg 6480 ttaagtagcg ttttcagaat atacattatt cccatccatt gtaaagttcc ttaagtcata 6540 tttgactggg cgtgcagaat aacttcttaa ctattaacta tcagagtttg attaataaaa 6600 ttaattaatt ttttttctcc ttcgtgttgt taatgttcca agggatttgg agcatactgg 6660 ttttccaggt gcatgtgaat cccgaaggac tgatgatatt tgaatgttta ttaaattatt 6720 atcacacaaa tgtgttgata ttgtggctat tgttgatgtt gaaaattgta aacttgggga 6780 agattaagaa aagaaccaat agtgacaaaa atcagtgctt ccagtagatt ttagaacatt 6840 ctttgcctca aaaaacctgc aaagatgatg tgagattttt tcttgtgttt taattatttt 6900 cacattttct ctctgcaaac ctttagtttt ctgatgatct acacacacac atacacacac 6960 acacacacac acgtgcacac acacacattt aaaggatata aaaagaagag gttgaaagat 7020 tattaaataa cttatcaggc atctcaatgg ttactatcta tgttagtgaa aatcaaatag 7080 gactcaaagt tggatatttg ggatttttct tctgacagta taatttattg agttactagg 7140 gaggttctta aatcctcata tctggaaact tgtgaagttt tgacaccttt cctatagata 7200 taggaatgaa ccaatacgct tttattaccc tttctaactc tgattttata atcagactta 7260 gattgtgttt agaatattaa atgactgggc accctcttct tggtttttac cagagaggct 7320 ttgaatggaa gcaggctgag agtagccaaa gaggcaaggg gtattagccc agttattctc 7380 ccctatgcct tctcttccta agcgtccact aggtctggcc ttggaaatct gttacttcta 7440 cggcttcaga tctgatgata tctttttcat cacattacaa gttatttctt tgactgaata 7500 gacagtggta taggttgaca cagcacacaa gtggctattg tgatgtatga tgtatgtagt 7560 cccacaactg caaaacgtct tactgaagca acaatcgaaa aatggttctg ttttaaaaag 7620 gattttgttt gatttgaaat taaaacttca aactgaatga cttatatgag aataatatgt 7680 tcaatcaaag tagttattct attttgtgtc catattccat tagattgtga ttattaattt 7740 tctagctatg gtattactat atcacacttg tgagtatgta ttcaaatact aagtatctta 7800 tatgctacgt gcatacacat tcttttctta aactttacct gtgttttaac taatattgtg 7860 tcagtgtatt aaaaattagc ttttacatat gatatctaca atgtaataaa tttagagagt 7920 aattttgtgt attcttattt acttaacatt ttacttttaa ttatgtaaat ttggttagaa 7980 aataataata aatggttagt gctattgtgt aatggtagca gttacaaaga gcctctgcct 8040 tcccaaacta atatttatca cacatggtca ttaaatggga aaaaaataga ctaaacaaat 8100 cacaaattgt tcagttctta aaatgtaatt atgtcacaca cacaaaaaaa tccttttcaa 8160 tcctgagaaa attaaaggtg ttttactcac atggatattt caacattagt tttttttgtt 8220 tgtttctttt tcatggtatt actgaaggtg tgtatactcc ctaatacaca tttatgaaaa 8280 tctacttgtt tagactttta tttatactct tctgatttat attttttatt ataattatta 8340 tttcttatct tcttttatat tttttggaaa ccaaatttat agttagttta ggtaaacttt 8400 ttattatgac cattagaaac tattttgaat gtttccaact ggctcaattg gctgggaaaa 8460 catgggaaca agagaagctg aaatatattt ctgcaagaac ctttctatat tatgtgccaa 8520 ttaccacacc agatcaattt tatgcagagg ccttaaaata ttctttcaca gtagctttct 8580 tacactaacc gtcatgtgct tttagtaaat atgattttta aaagcagttc aagttgacaa 8640 cagcagaaac agtaacaaaa aaatctgctc agaaaaatgt atgtgcacaa ataaaaaaaa 8700 ttaatggcaa ttgtttagtg actgtaagtg atacttttta aagagtaaac tgtgtgaaat 8760 ttatactatc cctgcttaaa atattaagat ttttatgaaa tatgtattta tgtttgtatt 8820 gtgggaagat tcctcctctg tgatatcata cagcatctga aagtgaacag tatcccaaag 8880 cagttccaag catgctttgg aagtaagaag gttgactatt gtatggccaa ggatggcagt 8940 atgtaatcca gaagcaaact tgtattaatt gttctatttc aggttctgta ttgcatgttt 9000 tcttattaat atatattaat aaaagttatg agaaat 9036
Table LI I l(g). Nucleotide sequence alignment of 109P1D4 v.1 (SEQ ID NO: 274)and 109P1D4v.8 (SEQ ID NO: 275)
Score = 5664 bits (2946), Expect = O.OIdentities = 3000/3027 (99%) Strand = Plus / Plus
V.l 852 ttgttgtccgggacgtacattttcgcggtcctgctagcatgcgtggtgttccactctggc 911
I I I II I I I I I I I I II II II I II II II I llll II I I II I I I II II I I II I I I II II I II I
V.8 837 ttgttgtccgggacgtacattttcgcggtcctgctagtatgcgtggtgttccactctggc 896
V.l : 912 gcccaggagaaaaactacaccatccgagaagaaatgccagaaaacgtcctgataggcgac 971
I I II I I II I I I II II II I III I Ml I I llll II II II I II I II I I I II II I II II I II
V.8 : 897 gcccaggagaaaaactacaccatccgagaagaaattccagaaaacgtcctgataggcaac 956
V.l : 972 ttgttgaaagaccttaacttgtcgctgattccaaacaagtccttgacaactgctatgcag 1031
II II I II I II I I I II II I II II lllll III I II II II I I I I II I I I I II II
V.8 : 957 ttgttgaaagaccttaacttgtcgctgattccaaacaagtccttgacaactactatgcag 1016
V.l : 1032 ttcaagctagtgtacaagaccggagatgtgccactgattcgaattgaagaggatactggt 1091
II I I I II II I I I I I I II I MM lllll I II I II II I I I I II II I I I I II II I I I I I llll
V.8 : 1017 ttcaagctagtgtacaagaccggagatgtgccactgattcgaattgaagaggatactggt 1076
V.l : 1092 gagatcttcactactggcgctcgcattgatcgtgagaaattatgtgctggtatcccaagg 1151 II Mill Mlllll I I II II I II II I I II I II II I II II I II I I I I II II I I II II I I I V. 1077 gagatcttcactaccggcgctcgcattgatcgtgagaaattatgtgctggtatcccaagg 1136
V.l : 1152 gatgagcattgcttttatgaagtggaggttgccattttgccggatgaaatatttagactg 1211
II llll III III I I I I II I I I Ml lllll II II I II llll I I I M I II Ml I II I M I II V.8 : 1137 gatgagcattgcttttatgaagtggaggttgccattttgccggatgaaatatttagactg 1196
V.l : 1212 gttaagatacgttttctgatagaagatataaatgataatgcaccattgttcccagcaaca 1271
II Ml II Ml II I I I I II I I Ml lllll III Ml I III 11 I III III I II II II I II III V.8 : 1197 gttaagatacgttttctgatagaagatataaatgataatgcaccattgttcccagcaaca 1256
V.l : 1272 gttatcaacatatcaattccagagaactcggctataaactctaaatatactctcccagcg 1331
II Ml II I II II II I I Ml I Ml III Mill I II I II I II III I I I II I I MM V.8 : 1257 gttatcaacatatcaattccagagaactcggctataaactctaaatatactctcccagcg 1316
V.l 1332 gctgttgatcctgacgtaggaataaacggagttcaaaactacgaactaattaagagtcaa 1391
II III II I I 111111 II I I I II II lllll II I II II II II I Ml I I II I II I II I I II I V.8 1317 gctgttgatcctgacgtaggcataaacggagttcaaaactacgaactaattaagagtcaa 1376
V.l : 1392 aacatttttggcctcgatgtcattgaaacaccagaaggagacaagatgccacaactgatt 1451
I llll I III II I I I I II I I I II III II II 1 II I I I 11 II I I II I I I II I II I V.8 : 1377 aacatttttggcctcgatgtcattgaaacaccagaaggagacaagatgccacaactgatt 1436
V.l : 1452 gttcaaaaggagttagatagggaagagaaggatacctacgtgatgaaagtaaaggttgaa 1511 llll II 111 II II I I I II I I II Ml MUM II II II 1 II I II I II II II I I I II III I V.8 : 1437 gttcaaaaggagttagatagggaagagaaggatacctatgtgatgaaagtaaaggttgaa 1496
V.l : 1512 gatggtggctttcctcaaagatccagtactgctattttgcaagtgagtgttactgataca 1571
I III II llll II II I III I II II MM II III I II llll I II II 1) II II II I II I III V.8 : 1497 gatggtggctttcctcaaagatccagtactgctattttgcaagtaagtgttactgataca 1556
V.l : 1572 aatgacaaccacccagtctttaaggagacagagattgaagtcagtataccagaaaatgct 1631
I Ml II II llll II II II I II I II I MM I II Ml I II I I I I I II II I I II I II II I II 1 V.8 : 1557 aatgacaaccacccagtctttaaggagacagagattgaagtcagtataccagaaaatgct 1616
V.l : 1632 cctgtaggcacttcagtgacacagctccatgccacagatgctgacataggtgaaaatgcc 1691
I I III I II I III II I III I III llll lllll I I III I I I I I I II I I I I II I I I II I I I II
V.8 : 1617 cctgtaggcacttcagtgacacagctccatgccacagatgctgacataggtgaaaatgcc 1676
V.l : 1692 aagatccacttctctttcagcaatctagtctccaacattgccaggagattatttcacctc 1751
II III II II II I I I III I I III III I II MM II llll I I I I II I I I I II I II II I II II
V.8 : 1677 aagatccacttctctttcagcaatctagtctccaacattgccaggagattatttcacctc 1736
V.l : 1752 aatgccaccactggacttatcacaatcaaagaaccactggatagggaagaaacaccaaac 1811
III llll I I II I I I I II I II II II llll II I I M III I I I I I II I I I II I I I II I I III I V.8 : 1737 aatgccaccactggacttatcacaatcaaagaaccactggatagggaagaaacaccaaac 1796
V.l : 1812 cacaagttactggttttggcaagtgatggtggattgatgccagcaagagcaatggtgctg 1871
I III II I I II M II II I I I I III lllll 111 II I III I I I I II I I I Ml I I II I I MM I V.8 : 1797 cacaagttactggttttggcaagtgatggtggattgatgccagcaagagcaatggtgctg 1856
V.l : 1872 gtaaatgttacagatgtcaatgataatgtcccatccattgacataagatacatcgtcaat 1931
I lllll I II I I I llll I II II III llll I II I I I MM II II I II II II I I II I II I I I I V.8 : 1857 gtaaatgttacagatgtcaatgataatgtcccatccattgacataagatacatcgtcaat 1916
V.l : 1932 cctgtcaatgacacagttgttctttcagaaaatattccactcaacaccaaaattgctctc 1991 II I I I II II I II I I I II II I II I I I II I I II I I I I I II I II II I I II I I II II I I II II I V.8 : 1917 cctgtcaatgacacagttgttctttcagaaaatattccactcaacaccaaaattgctctc 1976
V.l : 1992 ataactgtgacggataaggatgcggaccataatggcagggtgacatgcttcacagatcat 2051
I II llll II II III III I II I II I I I II I Ml I I II II I II I I II II II II I 11 I I I I II V.8 : 1977 ataactgtgacggataaggatgcggaccataatggcagggtgacatgcttcacagatcat 2036
V.l : 2052 gaaatccctttcagattaaggccagtattcagtaatcagttcctcctggagactgcagca 2111 lllll I II lllll II II I II I II I I II III II I II II II II Ml I II I I I I I I I llll V.8 : 2037 gaaattcctttcagattaaggccagtattcagtaatcagttcctcctggagaatgcagca 2096
V.l : 2112 tatcttgactatgagtccacaaaagaatatgccattaaattactggctgcagatgctggc 2171
I I llll II III I I I 111 II II I II II I II I llll I II I I I II I II I I I II I I I I I I I II I
V.8 : 2097 tatcttgactatgagtccacaaaagaatatgccattaaattactggctgcagatgctggc 2156
V.l : 2172 aaacctcctttgaatcagtcagcaatgctcttcatcaaagtgaaagatgaaaatgacaat 2231
II llll I lllll II I I I II I I II I I II I II II II I II I II I II I I I II I II II I II I II I
V.8 : 2157 aaacctcctttgaatcagtcagcaatgctcttcatcaaagtgaaagatgaaaatgacaat 2216
V.l : 2232 gctccagttttcacccagtctttcgtaactgtttctattcctgagaataactctcctggc 2291
I llll I I MM llll II I II I) II II II Mill II I I I I I I II I II II I I I II II II II 1 V.8 : 2217 gctccagttttcacccagtctttcgtaactgtttctattcctgagaataactctcctggc 2276
V.l : 2292 atccagttgacgaaagtaagtgcaatggatgcagacagtgggcctaatgctaagatcaat 2351
I lllll MM llll II 111 II II I I II I II I II I I II II II I I II II II I I I II II I
V.8 : 2277 atccagttgatgaaagtaagtgcaacggatgcagacagtgggcctaatgctgagatcaat 2336
V.l : 2352 tacctgctaggccctgatgctccacctgaattcagcctggattgtcgtacaggcatgctg 2411
II llll I llll MM II III I III I I II I I II I II I I I I II I I I llll II I II I I I II 1
V.8 : 2337 tacctgctaggccctgatgctccacctgaattcagcctggatcgtcgtacaggcatgctg 2396
V.l : 2412 actgtagtgaagaaactagatagagaaaaagaggataaatatttattcacaattctggca 2471
I I III II llllllll II I II II II I I I I I I M I II III II I I I I I 11 I I 11 I I I II II I I V.8 : 2397 actgtagtgaagaaactagatagagaaaaagaggataaatatttattcacaattctggca 2456
V.l : 2472 aaagataacggggtaccacccttaaccagcaatgtcacagtctttgtaagcattattgat 2531 II llll I III I I II I I II I I Ml I II I II II I I II I III II I II II I II II V.8 : 2457 aaagataatggggtaccacccttaaccagcaatgtcacagtctttgtaagcattattgat 2516
V.l : 2532 cagaatgacaatagcccagttttcactcacaatgaatacaacttctatgtcccagaaaac 2591
I I I III II III III III II I II I I I II I II II I II II I II I I I II II II I I I I I II I II V.8 : 2517 cagaatgacaatagcccagttttcactcacaatgaatacaaattctatgtcccagaaaac 2576
V.l : 2592 cttccaaggcatggtacagtaggactaatcactgtaactgatcctgattatggagacaat 2651
I II I I I I II lllll I I I II II I II I II I II II II I II II I II I II I I I I I I I I I II I II I V.8 : 2577 cttccaaggcatggtacagtaggactaatcactgtaactgatcctgattatggagacaat 2636
V.l : 2652 tctgcagttacgctctccattttagatgagaatgatgacttcaccattgattcacaaact 2711
I I II III II III II II II I II I II I II I II I Ml I II II I II I II I II II I I I II I I II I V.8 : 2637 tctgcagttacgctctccattttagatgagaatgatgacttcaccattgattcacaaact 2696
V.l : 2712 ggtgtcatccgaccaaatatttcatttgatagagaaaaacaagaatcttacactttctat 2771
I I lllll I II I III II II II I II I III II III II I II II I II II I II II I I II II I II II V.8 : 2697 ggtgtcatccgaccaaatatttcatttgatagagaaaaacaagaatcttacactttctat 2756 V.l : 2772 gtaaaggctgaggatggtggtagagtatcacgttcttcaagtgccaaagtaaccataaat 2831
I II III II II I Mill II I I I III I I III I I I II I II II Ml I II I I II I II V.8 : 2757 gtaaaggctgaggatggtggtagagtatcacgttcttcaagtgccaaagtaaccataaat 2816
V.l : 2832 gtggttgatgtcaatgacaacaaaccagttttcattgtccctccttccaactgttcttat 2891
I II Ml II Ml lllll I III II I I II I II Ml I I I II I I I II I II I lllll I I III II V.8 : 2817 gtggttgatgtcaatgacaacaaaccagttttcattgtccctccttacaactattcttat 2876
V.l : 2892 gaattggttctaccgtccactaatccaggcacagtggtctttcaggtaattgctgttgac 2951
I II I II II II I Mill I III II II I I I Mill I I I II I I I II III III II I II II Ml 11 V.8 : 2877 gaattggttctaccgtccactaatccaggcacagtggtctttcaggtaattgctgttgac 2936
V.l : 2952 aatgacactggcatgaatgcagaggttcgttacagcattgtaggaggaaacacaagagat 3011
Ml II I II I II llll lllll I II Ml I I II I I II II Ml I II II I II I I III II V.8 : 2937 aatgacactggcatgaatgcagaggttcgttacagcattgtaggaggaaacacaagagat 2996
V.l : 3012 ctgtttgcaatcgaccaagaaacaggcaacataacattgatggagaaatgtgatgttaca 3071
I II I I I I II II MM III I lllll II I Mill II Ml I I I II III I II II I I II I II I II V.8 : 2997 ctgtttgcaatcgaccaagaaacaggcaacataacattgatggagaaatgtgatgttaca 3056
V.l : 3072 gaccttggtttacacagagtgttggtcaaagctaatgacttaggacagcctgattctctc 3131
I III I III I II lllll II Mlllll I I II III I I I II I I I II II I II I I lllll V.8 : 3057 gaccttggtttacacagagtgttggtcaaagctaatgacttaggacagcctgattctctc 3116
V.l : 3132 ttcagtgttgtaattgtcaatctgttcgtgaatgagtcggtgaccaatgctacactgatt 3191
III I I I II II I lllll I II II llll I II II II I I I II I I II Ml II I I I I II I lllll I V.8 : 3117 ttcagtgttgtaattgtcaatctgttcgtgaatgagtcagtgaccaatgctacactgatt 3176
V.l : 3192 aatgaactggtgcgcaaaagcactgaagcaccagtgaccccaaatactgagatagctgat 3251
I I I IM II II llll II II I II I II I I llll I I I II I I II II I II II II I I I I I lllll I
V.8 : 3177 aatgaactggtgcgcaaaagcattgaagcaccagtgaccccaaatactgagatagctgat 3236
V.l : 3252 gtatcctcaccaactagtgactatgtcaagatcctggttgcagctgttgctggcaccata 3311
II III II I I I I llll I I II II II II II I I II III I I I II I I I II II III I II I II llll I
V.8 : 3237 gtatcctcaccaactagtgactatgtcaagatcctggttgcagctgttgctggcaccata 3296
V.l : 3312 actgtcgttgtagttattttcatcactgctgtagtaagatgtcgccaggcaccacacctt 3371
I III II II I II Mlllll II II I II I I Ml II II I II II I II II II II Ml Ml II III I V.8 : 3297 actgtcgttgtagttattttcatcactgctgtagtaagatgtcgccaggcaccacacctt 3356
V.l : 3372 aaggctgctcagaaaaacaagcagaattctgaatgggctaccccaaacccagaaaacagg 3431
I II I 1 III II III I II III llll II MM II II I II I I I II I II II II I I II I III II I
V.8 : 3357 aaggctgctcagaaaaacatgcagaattctgaatgggctaccccaaacccagaaaacagg 3416
V.l : 3432 cagatgataatgatgaagaaaaagaaaaagaagaagaagcattcccctaagaacttgctg 3491
II I II llll II MM II I I II II II I II III I I III I I I II I II II II I I II I I lllll
V.8 : 3417 cagatgataatgatgaagaaaaagaaaaagaagaagaagcattcccctaagaacctgctg 3476
V.l : 3492 cttaattttgtcactattgaagaaactaaggcagatgatgttgacagtgatggaaacaga 3551 I I II II III II II llll I I III II I II I I II II I I II II II 111 I II III II I V.8 : 3477 cttaatgttgtcactattgaagaaactaaggcagatgatgttgacagtgatggaaacaga 3536
V.l : 3552 gtcacactagaccttcctattgatctagaagagcaaacaatgggaaagtacaattgggta 3611
III I I II I II II II I I III II Ml I I I III II I I I II II I I II II llll I II I I llll I I
V.8 : 3537 gtcacactagaccttcctattgatctagaagagcaaacaatgggaaagtacaattgggta 3596 V.l : 3612 actacacctactactttcaagcccgacagccctgatttggcccgacactacaaatctgcc 3671
I II I I I II I I I II II I II llll I II II II II I I I I II I I II II I II I II I I II I II II I V.8 : 3597 actacacctactactttcaagcctgacagccctgatttggcccgacactacaaatctgcc 3656
V.l : 3672 tctccacagcctgccttccaaattcagcctgaaactcccctgaattcgaagcaccacatc 3731
I I I II I II I II I I II III MM lllll I I I I I II II I I II II II I I II I Ml II I III 1 V.8 : 3657 tctccacagcctgccttccaaattcagcctgaaactcccctgaatttgaagcaccacatc 3716
V.l : 3732 atccaagaactgcctctcgataacacctttgtggcctgtgactctatctccaagtgttcc 3791
I III I I I II II I II II I I III II III II II II I I I I II I III II I I II II I I I llllll V.8 : 3717 atccaagaactgcctctcgataacacctttgtggcctgtgactctatctccaattgttcc 3776
V.l : 3792 tcaagcagttcagatccctacagcgtttctgactgtggctatccagtgacgaccttcgag 3851
I II I II I I I I I I I II I II llll lllll II II I II II I II II I II I I I II I V.8 : 3777 tcaagcagttcagatccctacagcgtttctgactgtggctatccagtgacaaccttcgag 3836
V.l : 3852 gtacctgtgtccgtacacaccagaccg 3878
I II I I I llll I I II II I I III II III I V.8 : 3837 gtacctgtgtccgtacacaccagaccg 3863
Score = 1567 bits (815), Expect = O.OIdentities = 829/836 (99%) Strand = Plus / Plus
V.l : 3 ggtggtccagtacctccaaagatatggaatacactcctgaaatatcctgaaaactttttt 62
II I II I II II I I I I II I II llll II II I I II II I I I I II I I II II I I I I II I I I I) III V.8 : 1 ggtggtccagtacctccaaagatatggaatacactcctgaaatatcctgaaacctttttt 60
V.l : 63 ttttcagaatcctttaataagcagttatgtcaatctgaaagttgcttacttgtactttat 122
I II I I I I MM I I II II I II II I I Mlllll I I II II I I II I II I II II I I I I II I II II V.8 : 61 ttttcagaatcctttaataagcagttatgtcaatctgaaagttgcttacttgtactttat 120
V.l : 123 attaatagctattcttgtttttcttatccaaagaaaaatcctctaatccccttttcacat 182
I II II I I II I II I I II I II I I II lllll III II II I I II II I I II I I I II I II I II II II
V.8 : 121 attaatagctattcttgtttttcttatccaaagaaaaatcctctaatccccttttcacat 180
V.l : 183 gatagttgttaccatgtttaggcattagtcacatcaacccctctcctctcccaaacttct 242
II I I II II I I I II I II I II llll III I I I III II II II I II II I I II I II I II II llll V.8 : 181 gatagttgttaccatgtttaggcgttagtcacatcaacccctctcctctcccaaacttct 240
V.l : 243 cttcttcaaatcaaactttattagtccctcctttataatgattccttgcctcgttttatc 302
I II I II II II II I I I I I II III I II II II Ml I I I I I II II II I I I I II II I II II I II V.8 : 241 cttcttcaaatcaaactttattagtccctcctttataatgattccttgcctccttttatc 300
V.l : 303 cagatcaattttttttcactttgatgcccagagctgaagaaatggactactgtataaatt 362
I II II II 1 II II I llll II III I I I II II III I II II I II I II II I I II II I II II III V.8 : 301 cagatcaattttttttcactttgatgcccagagctgaagaaatggactattgtataaatt 360
V.l : 363 attcattgccaagagaataattgcattttaaacccatattataacaaagaataatgatta 422
I I I II II I I I I I I II II I llll II III I III II II II I II I I II I I I II II I II I II II V.8 : 361 attcattgccaagagaataattgcattttaaacccatgttataacaaagaataatgatta 420
V.l : 423 tattttgtgatttgtaacaaataccctttattttcccttaactattgaattaaatatttt 482
I I I II I I I I II I I II II I I II I II III I I I I II II I I I I II I I I I I I I II I II II I II II V.8 : 421 tattttgtgatttgtaacaaataccctttattttcccttaactattgaattaaatatttt 480 V.l : 483 aattatttgtattctctttaactatcttggtatattaaagtattatcttttatatattta 542
MIMMMMI MIIMMM MMMMMMMM Mlllll I lllll V.8 : 481 aattatttgtattctctttaactatcttggtatattaaagtattatcttttatatattta 540
V.l 543 tcaatggtggacacttttataggtactctgtgtcatttttgatactgtaggtatcttatt 602 II III III II I II II Ml I I 1 I I III I I II II I II Mill II II I I I I II II I 11 II I II V.8 541 tcaatggtggacacttttataggtactctgtgtcatttttgatactgtaggtatcttatt 600
V.l : 603 tcatttatctttattcttaatgtacgaattcataatatttgattcagaacaaatttatca 662
MM MM II I II II I II II I II II II II II I I I I I II II II I III I I II I V.8 : 601 tcatttatctttattcttaatgtacgaattcataatatttgattcagaacagatttatca 660
V.l 663 ctaattaacagagtgtcaattatgctaacatctcatttactgattttaatttaaaacagt 722 Ml llll III II I I II I I I II I Ml II I III II I II II II I III I II I I II Ml I I I Ml V.8 661 ctaattaacagagtgtcaattatgctaacatctcatttactgattttaatttaaaacagt 720
V.l : 723 ttttgttaacatgcatgtttagggttggcttcttaataatttcttcttcctcttctctct 782
II Ml I) I I I I III I II I I I II I III II llll II I II II I II I III I I I II I II I 111 II V.8 : 721 ttttgttaacatgcatgtttagggttggcttcttaataatttcttcttcctcttctctct 780
V.l : 783 ctcctcttcttttggtcagtgttgtgcgggttaatacaacaaactgtaacaagtgt 838
II II llll M I I I II I II II I II llll I I I II II II II I I I II I I II I II II I I I
V.8 : 781 ctcctcttcttttggtcagtgttgtgcgggttaatacaacaaactgtcacaagtgt 836
Table LΙV(g). Peptide sequences of protein coded by 109P1D4 v.8 (SEQ ID NO: 276)
MFRVGFLIIS SSSSLSPLLL VSWRVNTTN CHKCLLSGTY IFAVLLVCW FHSGAQEKNY 60 TIREEIPENV LIGNLLKDLN LSLIPNKSLT TTMQFKLVYK TGDVPLIRIE EDTGEIFTTG 120 ARIDREKLCA GIPRDEHCFY EVEVAILPDE IFRLVKIRFL IEDINDNAPL FPATVINISI 180 PENSAINSKY TLPAAVDPDV GINGVQNYEL IKSQNIFGLD VIETPEGDKM PQLIVQKELD 240 REEKDTYVMK VKVEDGGFPQ RSSTAILQVS VTDTNDNHPV FKETEIEVSI PENAPVGTSV 300 TQLHATDADI GENAKIHFSF SNLVSNIARR LFHLNATTGL ITIKEPLDRE ETPNHKLLVL 360 ASDGGLMPAR AMVLVNVTDV NDNVPSIDIR YIVNPVNDTV VLSENIPLNT KIALITVTDK 420 DADHNGRVTC FTDHEIPFRL RPVFSNQFLL ENAAYLDYES TKEYAIKLLA ADAGKPPLNQ 480 SAMLFIKVKD ENDNAPVFTQ SFVTVSIPEN NSPGIQLMKV SATDADSGPN AEINYLLGPD 540 APPEFSLDRR TGMLTWKKL DREKEDKYLF TILAKDNGVP PLTSNVTVFV SIIDQNDNSP 600 VFTHNEYKFY VPENLPRHGT VGLITVTDPD YGDNSAVTLS ILDENDDFTI DSQTGVIRPN 660 ISFDREKQES YTFYVKAEDG GRVSRSSSAK VTINWDVND NKPVFIVPPY NYSYELVLPS 720 TNPGTWFQV lAVDNDTGMN AEVRYSIVGG NTRDLFAIDQ ETGNITLMEK CDVTDLGLHR 780 VLVKANDLGQ PDSLFSWIV NLFVNESVTN ATLINELVRK SIEAPVTPNT EIADVSSPTS 840 DYVKILVAAV AGTITWWI FITAWRCRQ APHLKAAQKN MQNSEWATPN PENRQMIMMK 900 KKKKKKKHSP KNLLLNWTI EETKADDVDS DGNRVTLDLP IDLEEQTMGK YNWVTTPTTF 960 KPDSPDLARH YKSASPQPAF QIQPETPLNL KHHIIQELPL DNTFVACDSI SNCSSSSSDP 1020 YSVSDCGYPV TTFEVPVSVH TRPSQRRVTF HLPEGSQESS SDGGLGDHDA GSLTSTSHGL 1080 PLGYPQEEYF DRATPSNRTE GDGNSDPEST FIPGLKKEIT VQPTVEEASD NCTQECLIYG 1140 HSDACWMPAS LDHSSSSQAQ ASALCHSPPL SQASTQHHSP PVTQTIVLCH SPPVTQTIAL 1200 CHSPPPIQVS ALHHSPPLVQ GTALHHSPPS AQASALCYSP PLAQAAAISH SSSLPQVIAL 1260 HRSQAQSSVS LQQGWVQGAN GLCSVDQGVQ GSATSQFYTM SERLHPSDDS IKVIPLTTFA 1320 PRQQARPSRG DSPIMETHPL 1340
Table LV(g). Amino acid sequence alignment of 109P1 D4 v.1 (SEQ ID NO: 277) and 109P1 D4 v.8 (SEQ ID NO: 278)
Score = 1961 bits (5081), Expect = O.OIdentities = 992/1009 (98%), Positives = 995/1009 (98%)
V.l 3 LLSGTYIFAVLLACWFHSGAQEKNYTIREEMPENVLIGDLLKDLNLSLIPNKSLTTAMQ 62
LLSGTYIFAVLL CWFHSGAQEKNYTIREE+PENVLIG+LLKDLNLSLIPNKSLTT MQ V.8 35 SGTYIFAVLLVCWFHSGAQEKNYTIREEIPENVLIGNLLKD N S IPNKS TTTMQ 94 V.l 63 FKLVYKTGDVPLIRIEEDTGEIFTTGARIDREKLCAGIPRDEHCFYEVEVAILPDEIFRL 122
FKLVYKTGDVPLIRIEEDTGEIFTTGARIDREKLCAGIPRDEHCFYEVEVAILPDEIFRL V.8 95 FKLVYKTGDVPLIRIEEDTGEIFTTGARIDREKLCAGIPRDEHCFYEVEVAILPDEIFR 154 123 VKIRFLIEDINDNAPLFPATVINISIPENSAINSKYTLPAAVDPDVGINGVQNYE IKSQ 182
VKIRF IEDINDNAPLFPATVINISIPENSAINSKYT PAAVDPDVGINGVQNYELIKSQ 155 VKIRF IEDINDNAPLFPATVINISIPENSAINSKYTLPAAVDPDVGINGVQNYE IKSQ 214
183 NIFGLDVIETPEGDKMPQLIVQKELDREEKDTYVMKVKVEDGGFPQRSSTAILQVSVTDT 242
NIFG DVIETPEGDKMPQLIVQKELDREEKDTYVMKVKVEDGGFPQRSSTAILQVSVTDT 215 NIFGLDVIETPEGDKMPQLIVQKELDREEKDTYVMKVKVEDGGFPQRSSTAILQVSVTDT 274
243 NDNHPVFKETEIEVSIPENAPVGTSVTQLHATDADIGENAKIHFSFSNLVSNIARRLFHL 302
NDNHPVFKETEIEVSIPENAPVGTSVTQLHATDADIGENAKIHFSFSNLVSNIARR FHL 275 NDNHPVFKETEIEVSIPENAPVGTSVTQLHATDADIGENAKIHFSFSNLVSNIARRLFHL 334
.1 303 NATTGLITIKEPLDREETPNHKLLVLASDGGMPARAMVLVNVTDVNDNVPSIDIRYIVN 362
NATTG ITIKEPLDREETPNHKL VLASDGGLMPARAMVLVNVTDVNDNVPSIDIRYIVN 335 NATTGLITIKEPLDREETPNHKLLVLASDGG MPARAMVLVVTDVNDNVPSIDIRYIVN 394
363 PVNDTWLSENIPLNTKIALITVTDKDADHNGRVTCFTDHEIPFRLRPVFSNQF LETAA 422
PVNDTWLSENIPLNTKIALITVTDKDADHNGRVTCFTDHEIPFRLRPVFSNQFL E AA 395 PVNDTW SENIPLNTKIA ITVTDKDADHNGRVTCFTDHEIPFRLRPVFSNQF ENAA 454
423 YLDYESTKEYAIKLLAADAGKPPLNQSAM FIKVKDENDNAPVFTQSFVTVSIPENNSPG 482
YLDYESTKEYAIK LAADAGKPPLNQSAMLFIKVKDENDNAPVFTQSFVTVSIPENNSPG 455 YLDYESTKEYAIKLLAADAGKPPLNQSAMLFIKVKDENDNAPVFTQSFVTVSIPENNSPG 514
483 IQLTKVSAMDADSGPNAKINY LGPDAPPEFSLDCRTGMLTWKKLDREKEDKYLFTILA 542
IQL KVSA DADSGPNA+INYLLGPDAPPEFS D RTGMLTWKKLDREKEDKYLFTI A 515 IQ KVSATDADSGPNAEINYLLGPDAPPEFSLDRRTGMLTWKKLDREKEDKYLFTILA 574
543 KDNGVPPLTSNVTVFVSIIDQNDNSPVFTHNEYNFYVPENLPRHGTVGLITVTDPDYGDN 602
KDNGVPPLTSNVTVFVSIIDQNDNSPVFTHNEY FYVPENLPRHGTVGLITVTDPDYGDN 575 KDNGVPPLTSNVTVFVS1IDQNDNSPVFTHNEYKFYVPENLPRHGTVGLITVTDPDYGDN 634
603 SAVTLSILDENDDFTIDSQTGVIRPNISFDREKQESYTFYVKAEDGGRVSRSSSAKVTIN 662
SAVT SILDENDDFTIDSQTGVIRPNISFDREKQESYTFYVKAEDGGRVSRSSSAKVTIN 635 SAVT SILDENDDFTIDSQTGVIRPNISFDREKQESYTFYVKAEDGGRVSRSSSAKVTIN 694
663 WDVNDNKPVFIVPPSNCSYELVLPSTNPGTWFQVIAVDNDTGMNAEVRYSIVGGNTRD 722
VVDVNDNKPVFIVPP N S.YELVLPSTNPGTWFQVIAVDNDTGMNAEVRYSIVGGNTRD 695 WDV DNKPVFIVPPYNYSYELVLPSTNPGTWFQVIAVDNDTG NAEVRYSIVGGNTRD 754
723 LFAIDQETGNITLMEKCDVTDLGLHRVLVKANDLGQPDSLFSVVIVNLFVNESVTNATLI 782
LFAIDQETGNITLMEKCDVTDLGLHRVLVKANDLGQPDSLFSWIVNLFVNESVTNAT I 755 LFAIDQETGNITLMEKCDVTDLGLHRV VKAND GQPDS FSWIVNLFVNESVTNATLI 814
783 NELVRKSTEAPVTPNTEIADVSSPTSDYVKILVAAVAGTITWWIFITAVVRCRQAPH 842
NE VRKS EAPVTPNTEIADVSSPTSDYVKILVAAVAGTITWWIFITAWRCRQAPHL 815 NELVRKSIEAPVTPNTEIADVSSPTSDYVKILVAAVAGTITWWIFITAWRCRQAPHL 874
843 KAAQKNKQNSEWATPNPENRQ IMMKKKKKKKKHSPKNLLLNFVTIEETKADDVDSDGNR 902
KAAQKN QNSEWATPNPENRQMIMMKKKKKKKKHSPKNLLLN VTIEETKADDVDSDGNR 875 KAAQKNMQNSEWATPNPENRQMIMMKKKKKKKKHSPKN LLNWTIEETKADDVDSDGNR 934
903 VTLD PIDLEEQTMGKYNWVTTPTTFKPDSPDLARHYKSASPQPAFQIQPETPLNSKHHI 962
VTLDLPIDLEEQT GKYN VTTPTTFKPDSPDLARHYKSASPQPAFQIQPETPLN KHHI 935 VTLDLPIDLEEQTMGKYN VTTPTTFKPDSPD ARHYKSASPQPAFQIQPETP N KHHI 994
963 IQELP DNTFVACDSISKCSSSSSDPYSVSDCGYPVTTFEVPVSVHTRP 1011
IQE PLDNTFVACDSIS CSSSSSDPYSVSDCGYPVTTFEVPVSVHTRP 995 IQELPLDNTFVACDSISNCSSSSSDPYSVSDCGYPVTTFEVPVSVHTRP 1043
Table Lll(h). Nucleotide sequence of transcript variant 109P1 D4 v.9 (SEQ ID NO: 279) cccctttctc cccctctgtt aagtccctcc ccctcgccat tcaaaagggc tggctcggca 60 ctggctcctt gcagtcggcg aactgtctgg gcgggaggag ccgtgagcag tagctgcact 120 cagctgcccg cgcggcaaag aggaaggcaa gccaaacaga gtgcgcagag tggcagtgcc 180 agcggcgaca caggcagcac aggcagcccg ggctgcctga atagcctcag aaacaacctc 240 agcgactccg gctgctctgc ggactgcgag ctgtggcggt agagcccgct acagcagtcg 300 cagtctccgt ggagcgggcg gaagcctttt ttctcccttt cgtttacctc ttcattctac 360 tctaaaggca tcgttattag gaaaatcctg ttgtgaataa gaaggattcc acagatcaca 420 taccagagcg gttttgcctc agctgctctc aactttgtaa tcttgtgaag aagctgacaa 480 gcttggctga ttgcagtgca ctatgaggac tgaatgacag tgggttttaa ttcagatatt 540 tcaagtgttg tgcgggttaa tacaacaaac tgtcacaagt gtttgttgtc cgggacgtac 600 attttcgcgg tcctgctagt atgcgtggtg ttccactctg gcgcccagga gaaaaactac 660 accatccgag aagaaattcc agaaaacgtc ctgataggca acttgttgaa agaccttaac 720 ttgtcgctga ttccaaacaa gtccttgaca actactatgc agttcaagct agtgtacaag 780 accggagatg tgccactgat tcgaattgaa gaggatactg gtgagatctt cactaccggc 840 gctcgcattg atcgtgagaa attatgtgct ggtatcccaa gggatgagca ttgcttttat 900 gaagtggagg ttgccatttt gccggatgaa atatttagac tggttaagat acgttttctg 960 atagaagata taaatgataa tgcaccattg ttcccagcaa cagttatcaa catatcaatt 1020 ccagagaact cggctataaa ctctaaatat actctcccag cggctgttga tcctgacgta 1080 ggcataaacg gagttcaaaa ctacgaacta attaagagtc aaaacatttt tggcctcgat 1140 gtcattgaaa caccagaagg agacaagatg ccacaactga ttgttcaaaa ggagttagat 1200 agggaagaga aggataccta tgtgatgaaa gtaaaggttg aagatggtgg ctttcctcaa 1260 agatccagta ctgctatttt gcaagtaagt gttactgata caaatgacaa ccacccagtc 1320 tttaaggaga cagagattga agtcagtata ccagaaaatg ctcctgtagg cacttcagtg 1380 acacagctcc atgccacaga tgctgacata ggtgaaaatg ccaagatcca cttctctttc 1440 agcaatctag tctccaacat tgccaggaga ttatttcacc tcaatgccac cactggactt 1500 atcacaatca aagaaccact ggatagggaa gaaacaccaa accacaagtt actggttttg 1560 gcaagtgatg gtggattgat gccagcaaga gcaatggtgc tggtaaatgt tacagatgtc 1620 aatgataatg tcccatccat tgacataaga tacatcgtca atcctgtcaa tgacacagtt 1680 gttctttcag aaaatattcc actcaacacc aaaattgctc tcataactgt gacggataag 1740 gatgcggacc ataatggcag ggtgacatgc ttcacagatc atgaaattcc tttcagatta 1800 aggccagtat tcagtaatca gttcctcctg gagaatgcag catatcttga ctatgagtcc 1860 acaaaagaat atgccattaa attactggct gcagatgctg gcaaacctcc tttgaatcag 1920 tcagcaatgc tcttcatcaa agtgaaagat gaaaatgaca atgctccagt tttcacccag 1980 tctttcgtaa ctgtttctat tcctgagaat aactctcctg gcatccagtt gatgaaagta 2040 agtgcaacgg atgcagacag tgggcctaat gctgagatca attacctgct aggccctgat 2100 gctccacctg aattcagcct ggatcgtcgt acaggcatgc tgactgtagt gaagaaacta 2160 gatagagaaa aagaggataa atatttattc acaattctgg caaaagataa tggggtacca 2220 cccttaacca gcaatgtcac agtctttgta agcattattg atcagaatga caatagccca 2280 gttttcactc acaatgaata caaattctat gtcccagaaa accttccaag gcatggtaca 2340 gtaggactaa tcactgtaac tgatcctgat tatggagaca attctgcagt tacgctctcc 2400 attttagatg agaatgatga cttcaccatt gattcacaaa ctggtgtcat ccgaccaaat 2460 atttcatttg atagagaaaa acaagaatct tacactttct atgtaaaggc tgaggatggt 2520 ggtagagtat cacgttcttc aagtgccaaa gtaaccataa atgtggttga tgtcaatgac 2580 aacaaaccag ttttcattgt ccctccttac aactattctt atgaattggt tctaccgtcc 2640 actaatccag gcacagtggt ctttcaggta attgctgttg acaatgacac tggcatgaat 2700 gcagaggttc gttacagcat tgtaggagga aacacaagag atctgtttgc aatcgaccaa 2760 gaaacaggca acataacatt gatggagaaa tgtgatgtta cagaccttgg tttacacaga 2820 gtgttggtca aagctaatga cttaggacag cctgattctc tcttcagtgt tgtaattgtc 2880 aatctgttcg tgaatgagtc agtgaccaat gctacactga ttaatgaact ggtgcgcaaa 2940 agcattgaag caccagtgac cccaaatact gagatagctg atgtatcctc accaactagt 3000 gactatgtca agatcctggt tgcagctgtt gctggcacca taactgtcgt tgtagttatt 3060 ttcatcactg ctgtagtaag atgtcgccag gcaccacacc ttaaggctgc tcagaaaaac 3120 atgcagaatt ctgaatgggc taccccaaac ccagaaaaca ggcagatgat aatgatgaag 3180 aaaaagaaaa agaagaagaa gcattcccct aagaacctgc tgcttaatgt tgtcactatt 3240 gaagaaacta aggcagatga tgttgacagt gatggaaaca gagtcacact agaccttcct 3300 attgatctag aagagcaaac aatgggaaag tacaattggg taactacacc tactactttc 3360 aagcctgaca gccctgattt ggcccgacac tacaaatctg cctctccaca gcctgccttc 3420 caaattcagc ctgaaactcc cctgaatttg aagcaccaca tcatccaaga actgcctctc 3480 gataacacct ttgtggcctg tgactctatc tccaattgtt cctcaagcag ttcagatccc 3540 tacagcgttt ctgactgtgg ctatccagtg acaaccttcg aggtacctgt gtccgtacac 3600 accagaccga ctgattccag gacatgaact attgaaatct gcagtgagat gtaactttct 3660 aggaacaaca aaattccatt ccccttccaa aaaatttcaa tgattgtgat ttcaaaatta 3720 ggctaagatc attaattttg taatctagat ttcccattat aaaagcaagc aaaaatcatc 3780 ttaaaaatga tgtcctagtg aaccttgtgc tttctttagc tgtaatctgg caatggaaat 3840 ttaaaattta tggaagagac agtgcagcgc aataacagag tactctcatg ctgtttctct 3900 gtttgctctg aatcaacagc catgatgtaa tataaggctg tcttggtgta tacacttatg 3960 gttaatatat cagtcatgaa acatgcaatt acttgccctg tctgattgtt gaataattaa 4020 aacattatct ccaggagttt ggaagtgagc tgaactagcc aaactactct ctgaaaggta 4080 tccagggcaa gagacatttt taagacccca aacaaacaaa aaacaaaacc aaaacactct 4140 ggttcagtgt tttgaaaata ttgactaaca taatattgct gagaaaatca tttttattac 4200 ccaccactct gcttaaaagt tgagtgggcc gggcgcggtg gctcacgcct gtaattccag 4260 cactttggga ggccgaggcg ggtggatcac gaggtcagga tattgagacc atcctggcta 4320 acatggtgaa accccatctc cactaaaaat acaaaaaatt agctgggcgt ggtggcgggc 4380 gcctgtagtc ccagctactc gggaggctga ggcaggagaa tggcgtgaac ccgggaggcg 4440 gagcttgcag tgagccgaga tggcgccact gcactccagc ctgggtgaca gagcaagact 4500 ctgtctcaaa aagaaaaaaa tgttcagtga tagaaaataa ttttactagg tttttatgtt 4560 gattgtactc atgctgttcc actcctttta attattaaaa agttattttt ggctgggtgt 4620 ggtggctcat acctgtaatc ccagcacttt gggaggccga ggcgggtgga tcacctgagg 4680 tcaggagttc aagaccagtc tggccaacat 4710
Table Llll(h). Nucleotide sequence alignment of 109P1D4 v.1 (SEQ ID NO: 280) and 109P1D4 v.9 (SEQ ID NO: 281)
Score = 5664 bits (2946), Expect = O.OIdentities = 3000/3027 (99%) Strand = Plus / Plus
V.l : 852 ttgttgtccgggacgtacattttcgcggtcctgctagcatgcgtggtgttccactctggc 911
I I II II I II I I I II I II I II III III II II II I I II I II I II II I II II II II II III I V.9 : 583 ttgttgtccgggacgtacattttcgcggtcctgctagtatgcgtggtgttccactctggc 642
V.l : 912 gcccaggagaaaaactacaccatccgagaagaaatgccagaaaacgtcctgataggcgac 971
I II I I I I II I I I II II I MUM II II llll I I II II I I II I I I II II II II II II II V.9 : 643 gcccaggagaaaaactacaccatccgagaagaaattccagaaaacgtcctgataggcaac 702
V.l : 972 ttgttgaaagaccttaacttgtcgctgattccaaacaagtccttgacaactgctatgcag 1031
I I II II I 11 I I II II II I I II II II II I 11 I II I II I III II I II I II II I I I II MM V.9 : 703 ttgttgaaagaccttaacttgtcgctgattccaaacaagtccttgacaactactatgcag 762
V.l : 1032 ttcaagctagtgtacaagaccggagatgtgccactgattcgaattgaagaggatactggt 1091
I II I I I I I I I I I llll I II III Ml II III II I II I II I I II I II II II II II II II I II V.9 : 763 ttcaagctagtgtacaagaccggagatgtgccactgattcgaattgaagaggatactggt 822
V.l : 1092 gagatcttcactactggcgctcgcattgatcgtgagaaattatgtgctggtatcccaagg 1151
I 11 II I I I I I I I II II II I I II II I I II I I II I I II lllll I llll II llll I V.9 : 823 gagatcttcactaccggcgctcgcattgatcgtgagaaattatgtgctggtatcccaagg 882
V.l : 1152 gatgagcattgcttttatgaagtggaggttgccattttgccggatgaaatatttagactg 1211
I I 1 I II II II 1 III I I III Mill II II III II III I 1 II I II II I II II II lllll I I 1 V.9 : 883 gatgagcattgcttttatgaagtggaggttgccattttgccggatgaaatatttagactg 942
V.l : 1212 gttaagatacgttttctgatagaagatataaatgataatgcaccattgttcccagcaaca 1271
III I I I I I II I III II II II II III II II I II II I II I I III II II II II II II I I MM V.9 : 943 gttaagatacgttttctgatagaagatataaatgataatgcaccattgttcccagcaaca 1002
V.l : 1272 gttatcaacatatcaattccagagaactcggctataaactctaaatatactctcccagcg 1331
I II II I I I I I I II III I Mill III II III I I I II I II I I II II I II lllll Mlllll I V.9 : 1003 gttatcaacatatcaattccagagaactcggctataaactctaaatatactctcccagcg 1062
V.l : 1332 gctgttgatcctgacgtaggaataaacggagttcaaaactacgaactaattaagagtcaa 1391
I II Ml II I I I III III II I lllll Ml II I I II II Ml I llll II II II II II llll I V.9 : 1063 gctgttgatcctgacgtaggcataaacggagttcaaaactacgaactaattaagagtcaa 1122
V.l : 1392 aacatttttggcctcgatgtcattgaaacaccagaaggagacaagatgccacaactgatt 1451
I I I I II II I I II II I II II II III II II I I II II II I II II II I II II II III II II I II V.9 : 1123 aacatttttggcctcgatgtcattgaaacaccagaaggagacaagatgccacaactgatt 1182
V.l : 1452 gttcaaaaggagttagatagggaagagaaggatacctacgtgatgaaagtaaaggttgaa 1511 III IMMMMMMMMMM V.9 : 1183 gttcaaaaggagttagatagggaagagaaggatacctatgtgatgaaagtaaaggttgaa 1242
V.l : 1512 gatggtggctttcctcaaagatccagtactgctattttgcaagtgagtgttactgataca 1571 llll llll I II II I I I II II I II II lllll I I II I I 11 II III I II II III II
V.9 : 1243 gatggtggctttcctcaaagatccagtactgctattttgcaagtaagtgttactgataca 1302
V.l : 1572 aatgacaaccacccagtctttaaggagacagagattgaagtcagtataccagaaaatgct 1631
II II II II I II II I I I II II I lllll II I II II II II II I I M III I II I I II I I III II
V.9 : 1303 aatgacaaccacccagtctttaaggagacagagattgaagtcagtataccagaaaatgct 1362
V.l : 1632 cctgtaggcacttcagtgacacagctccatgccacagatgctgacataggtgaaaatgcc 1691 llll II II II III I I I II II I Mill II II llll Ml I I I I II llll I II II II
V.9 : 1363 cctgtaggcacttcagtgacacagctccatgccacagatgctgacataggtgaaaatgcc 1422
V.l : 1692 aagatccacttctctttcagcaatctagtctccaacattgccaggagattatttcacctc 1751
I II I II I Mill I I II III II I II II II 111 II I I II II I I II I II I I
V.9 : 1423 aagatccacttctctttcagcaatctagtctccaacattgccaggagattatttcacctc 1482
V.l : 1752 aatgccaccactggacttatcacaatcaaagaaccactggatagggaagaaacaccaaac 1811 lllll I II llll I I I I I II I I I llll II II I II II II III I II I III III I II 1
V.9 : 1483 aatgccaccactggacttatcacaatcaaagaaccactggatagggaagaaacaccaaac 1542
V.l : 1812 cacaagttactggttttggcaagtgatggtggattgatgccagcaagagcaatggtgctg 1871
I I II II II I II II I I I II II 1 Mil II 11 II II II I I I I I II II II I II I II II I II 111
V.9 : 1543 cacaagttactggttttggcaagtgatggtggattgatgccagcaagagcaatggtgctg 1602
V.l : 1872 gtaaatgttacagatgtcaatgataatgtcccatccattgacataagatacatcgtcaat 1931
I II I II II II 111 I I I II II I lllll MM II II I II II II III I II II II II I I lllll
V.9 : 1603 gtaaatgttacagatgtcaatgataatgtcccatccattgacataagatacatcgtcaat 1662
V.l : 1932 cctgtcaatgacacagttgttctttcagaaaatattccactcaacaccaaaattgctctc 1991
II 11 I I I II II II I II I III I II I I II I II II II Ml II I I II I Ml II II 111
V.9 : 1663 cctgtcaatgacacagttgttctttcagaaaatattccactcaacaccaaaattgctctc 1722
V.l : 1992 ataactgtgacggataaggatgcggaccataatggcagggtgacatgcttcacagatcat 2051
III I II II II II I I I I II II I lllll II II Ml II I I II I I III I II I I II II I I lllll
V.9 : 1723 ataactgtgacggataaggatgcggaccataatggcagggtgacatgcttcacagatcat 1782
V.l : 2052 gaaatccctttcagattaaggccagtattcagtaatcagttcctcctggagactgcagca 2111
II I II I I II II I II II II I I I III I II II III II III II I II I II II I I 111
V.9 : 1783 gaaattcctttcagattaaggccagtattcagtaatcagttcctcctggagaatgcagca 1842
V.l : 2112 tatcttgactatgagtccacaaaagaatatgccattaaattactggctgcagatgctggc 2171
I II I I II I III II I I I II I I II III I llll I II II II I I II I Ml III I II II I II III I
V.9 : 1843 tatcttgactatgagtccacaaaagaatatgccattaaattactggctgcagatgctggc 1902
V.l : 2172 aaacctcctttgaatcagtcagcaatgctcttcatcaaagtgaaagatgaaaatgacaat 2231
II III I II II II I I I II II I III II I II II II II I II I II I II I II I II II III
V.9 : 1903 aaacctcctttgaatcagtcagcaatgctcttcatcaaagtgaaagatgaaaatgacaat 1962
V.l : 2232 gctccagttttcacccagtctttcgtaactgtttctattcctgagaataactctcctggc 2291
I I I I Ml I II I I I I I I III I I I III II MM 11 II I II I I II II II II I II II I II III I
V.9 : 1963 gctccagttttcacccagtctttcgtaactgtttctattcctgagaataactctcctggc 2022
V.l : 2292 atccagttgacgaaagtaagtgcaatggatgcagacagtgggcctaatgctaagatcaat 2351 IIIIMMM MIMMMIMM I I I II II I I I I I M II I I I M I 1 II V.9 : 2023 atccagttgatgaaagtaagtgcaacggatgcagacagtgggcctaatgctgagatcaat 2082
V.l : 2352 tacctgctaggccctgatgctccacctgaattcagcctggattgtcgtacaggcatgctg 2411
II I I I I Ml I I I I I llll III III II II I I II II I I III I II I Mlllll I 11
V.9 : 2083 tacctgctaggccctgatgctccacctgaattcagcctggatcgtcgtacaggcatgctg 2142
V.l : 2412 actgtagtgaagaaactagatagagaaaaagaggataaatatttattcacaattctggca 2471
III I II II II II I I I II III lllll III I II lllll I llll I I I llll 111 I II II I III
V.9 : 2143 actgtagtgaagaaactagatagagaaaaagaggataaatatttattcacaattctggca 2202
V.l : 2472 aaagataacggggtaccacccttaaccagcaatgtcacagtctttgtaagcattattgat 2531 I I I I I llll I lllll MM II llll II II III II 111 I II I I II II lllll V.9 : 2203 aaagataatggggtaccacccttaaccagcaatgtcacagtctttgtaagcattattgat 2262
V.l : 2532 cagaatgacaatagcccagttttcactcacaatgaatacaacttctatgtcccagaaaac 2591 llll I Ml I I I I I llll I II Mill MM II II II I I III I I II I II I II I I 1 II I II I V.9 : 2263 cagaatgacaatagcccagttttcactcacaatgaatacaaattctatgtcccagaaaac 2322
V.l : 2592 cttccaaggcatggtacagtaggactaatcactgtaactgatcctgattatggagacaat 2651
II I I III II 111 I I III I I llll II I I II I II II I 11 II III II II II II I V.9 : 2323 cttccaaggcatggtacagtaggactaatcactgtaactgatcctgattatggagacaat 2382
V.l : 2652 tctgcagttacgctctccattttagatgagaatgatgacttcaccattgattcacaaact 2711
I I I I I II II II I I I II Mlllll II MM I III II 11 I 11 II I II 1 II II I I Ml I MM V.9 : 2383 tctgcagttacgctctccattttagatgagaatgatgacttcaccattgattcacaaact 2442
V.l : 2712 ggtgtcatccgaccaaatatttcatttgatagagaaaaacaagaatcttacactttctat 2771
I I I II I III I I I 1 MM I llll III II II llll II II I II I I 111 I III II lllll I Ml V.9 : 2443 ggtgtcatccgaccaaatatttcatttgatagagaaaaacaagaatcttacactttctat 2502
V.l : 2772 gtaaaggctgaggatggtggtagagtatcacgttcttcaagtgccaaagtaaccataaat 2831
I I I II I I II II I 1 II II II III III II I llll I I I II I II I I I II II III I III II I II I
V.9 : 2503 gtaaaggctgaggatggtggtagagtatcacgttcttcaagtgccaaagtaaccataaat 2562
V.l : 2832 gtggttgatgtcaatgacaacaaaccagttttcattgtccctccttccaactgttcttat 2891
II I II I I II 111 I llll II Mill II I I III II I I I II lllll Mlllll
V.9 : 2563 gtggttgatgtcaatgacaacaaaccagttttcattgtccctccttacaactattcttat 2622
V.l : 2892 gaattggttctaccgtccactaatccaggcacagtggtctttcaggtaattgctgttgac 2951
I 1 llll I I I 11 II II II II MM I llll III I III I Ml llll I I II I II I III I II III V.9 : 2623 gaattggttctaccgtccactaatccaggcacagtggtctttcaggtaattgctgttgac 2682
V.l : 2952 aatgacactggcatgaatgcagaggttcgttacagcattgtaggaggaaacacaagagat 3011
1 II I II II I I 1 I I II II II Mill II II III I I 1 II I II I II II I I II II I MM lllll V.9 : 2683 aatgacactggcatgaatgcagaggttcgttacagcattgtaggaggaaacacaagagat 2742
V.l : 3012 ctgtttgcaatcgaccaagaaacaggcaacataacattgatggagaaatgtgatgttaca 3071
I 1 I I II II I II I llll I Mlllll II MM II I I I II I II I I III I MM I I III lllll V.9 : 2743 ctgtttgcaatcgaccaagaaacaggcaacataacattgatggagaaatgtgatgttaca 2802
V.l : 3072 gaccttggtttacacagagtgttggtcaaagctaatgacttaggacagcctgattctctc 3131
I I I I II II I I I I III I I II lllll II I III II I II II I II I II II I Ml II llll lllll V.9 : 2803 gaccttggtttacacagagtgttggtcaaagctaatgacttaggacagcctgattctctc 2862 V.l : 3132 ttcagtgttgtaattgtcaatctgttcgtgaatgagtcggtgaccaatgctacactgatt 3191
II I I I I II I II III Mlllll III III llll III Ml I I 111 I llll I II III
V.9 : 2863 ttcagtgttgtaattgtcaatctgttcgtgaatgagtcagtgaccaatgctacactgatt 2922
V.l : 3192 aatgaactggtgcgcaaaagcactgaagcaccagtgaccccaaatactgagatagctgat 3251
Ml ill I I I II I II II II I II I II I I II I II I I II II I II II II I II II V.9 : 2923 aatgaactggtgcgcaaaagcattgaagcaccagtgaccccaaatactgagatagctgat 2982
V.l : 3252 gtatcctcaccaactagtgactatgtcaagatcctggttgcagctgttgctggcaccata 3311
III II I I I I I lllll lllll II II II II 111 I I I II I II II lllll III II III
V.9 : 2983 gtatcctcaccaactagtgactatgtcaagatcctggttgcagctgttgctggcaccata 3042
V.l : 3312 actgtcgttgtagttattttcatcactgctgtagtaagatgtcgccaggcaccacacctt 3371
I I I II I I I I I III I II llll llll llll MM MM I I I II I III II lllll II llll II
V.9 : 3043 actgtcgttgtagttattttcatcactgctgtagtaagatgtcgccaggcaccacacctt 3102
V.l : 3372 aaggctgctcagaaaaacaagcagaattctgaatgggctaccccaaacccagaaaacagg 3431
II I I I I I I I I I II I lllll I Ml III 111 I III II I I I II I II Ml II II II Ml II II
V.9 : 3103 aaggctgctcagaaaaacatgcagaattctgaatgggctaccccaaacccagaaaacagg 3162
V.l : 3432 cagatgataatgatgaagaaaaagaaaaagaagaagaagcattcccctaagaacttgctg 3491
II 1 I 1 III II II II I Mill Ml III II I Mill I II llll I MM II llll II Mill V.9 : 3163 cagatgataatgatgaagaaaaagaaaaagaagaagaagcattcccctaagaacctgctg 3222
V.l : 3492 cttaattttgtcactattgaagaaactaaggcagatgatgttgacagtgatggaaacaga 3551 llllll I I I I II Ml III MM II 11 II II I II II III II II II 111 MM V.9 : 3223 cttaatgttgtcactattgaagaaactaaggcagatgatgttgacagtgatggaaacaga 3282
V.l : 3552 gtcacactagaccttcctattgatctagaagagcaaacaatgggaaagtacaattgggta 3611
I I II I II I II 11 II III IM II I I II II 11 I 1 III I I IM II II II II I II III
V.9 : 3283 gtcacactagaccttcctattgatctagaagagcaaacaatgggaaagtacaattgggta 3342
V.l : 3612 actacacctactactttcaagcccgacagccctgatttggcccgacactacaaatctgcc 3671
II I I II I I I I II I II I II II I Ml 11 II II II II I II I MM llll II III
V.9 : 3343 actacacctactactttcaagcctgacagccctgatttggcccgacactacaaatctgcc 3402
V.l : 3672 tctccacagcctgccttccaaattcagcctgaaactcccctgaattcgaagcaccacatc 3731
I I II II I I I II II I MM I MM I II II I I III I MM II 111 III I V.9 : 3403 tctccacagcctgccttccaaattcagcctgaaactcccctgaatttgaagcaccacatc 3462
V.l : 3732 atccaagaactgcctctcgataacacctttgtggcctgtgactctatctccaagtgttcc 3791
I I I I I II II III II llll II III II III I I Ml II I II II I I II II II lllll II II II V.9 : 3463 atccaagaactgcctctcgataacacctttgtggcctgtgactctatctccaattgttcc 3522
V.l : 3792 tcaagcagttcagatccctacagcgtttctgactgtggctatccagtgacgaccttcgag 3851
I I I I I I I I I I II lllll II llll II II II I I II Ml I I I llll I llll II Mlllll II V.9 : 3523 tcaagcagttcagatccctacagcgtttctgactgtggctatccagtgacaaccttcgag 3582
V.l : 3852 gtacctgtgtccgtacacaccagaccg 3878
I I II II I I I llll Ml II Ml V.9 : 3583 gtacctgtgtccgtacacaccagaccg 3609
Table LΙV(h). Peptide sequences ofprotein coded by 109P1D4v.9 (SEQ ID NO: 282)
MTVGFNSDIS SVVRVNTTNC HKCLLSGTYI FAVLLVCWF HSGAQEKNYT IREEIPENVL 60
IGNLLKDLNL SLIPNKSLTT TMQFKLVYKT GDVPLIRIEE DTGEIFTTGA RIDREKLCAG 120
IPRDEHCFYE VEVAILPDEI FRLVKIRFLI EDINDNAPLF PATVINISIP ENSAINSKYT 180 LPAAVDPDVG INGVQNYELI KSQNIFGLDV IETPEGDKMP QLIVQKELDR EEKDTYVMKV 240
KVEDGGFPQR SSTAILQVSV TDTNDNHPVF KETEIEVSIP ENAPVGTSVT QLHATDADIG 300
ENAKIHFSFS NLVSNIARRL FHLNATTGLI TIKEPLDREE TPNHKLLVLA SDGGLMPARA 360
MVLVNVTDVN DNVPSIDIRY IVNPVNDTW LSENIPLNTK lALITVTDKD ADHNGRVTCF 420
TDHEIPFRLR PVFSNQFLLE NAAYLDYEST KEYAIKLLAA DAGKPPLNQS AMLFIKVKDE 480
NDNAPVFTQS FVTVSIPENN SPGIQLMKVS ATDADSGPNA EINYLLGPDA PPEFSLDRRT 540
GMLTWKKLD REKEDKYLFT ILAKDNGVPP LTSNVTVFVS IIDQNDNSPV FTHNEYKFYV 600
PENLPRHGTV GLITVTDPDY GDNSAVTLSI LDENDDFTID SQTGVIRPNI SFDREKQESY 660
TFYVKAEDGG RVSRSSSAKV TINWDVNDN KPVFIVPPYN YSYELVLPST NPGTWFQVI 720
AVDNDTGMNA EVRYSIVGGN TRDLFAIDQE TGNITLMEKC DVTDLGLHRV LVKANDLGQP 780
DSLFSWIVN LFVNESVTNA TLINELVRKS IEAPVTPNTE IADVSSPTSD YVKILVAAVA 840
GTITV VIF ITAWRCRQA PHLKAAQKNM QNSEWATPNP ENRQMIMMKK KKKKKKHSPK 900
NLLLNWTIE ETKADDVDSD GNRVTLDLPI DLEEQTMGKY NWVTTPTTFK PDSPDLARHY 960
KSASPQPAFQ IQPETPLNLK HHIIQELPLD NTFVACDSIS NCSSSSSDPY SVSDCGYPVT 1020
TFEVPVSVHT RPTDSRT 1037
Table LV(h). Amino acid sequence alignment of 109P1D4 v.1 (SEQ ID NO: 283) and 109P1D4v.9 (SEQ ID NO: 284)
Score = 1961 bits (5081), Expect = O.OIdentities = 992/1009 (98%), Positives = 995/1009 (98%)
V. l 3 LLSGTYIFAVLLACVVFHSGAQEKNYT1REEMPENVLIGDLLKDLNLSLIPNKSLTTAMQ 62
LLSGTYIFAVLL CWFHSGAQEKNYTIREE+PENVLIG+ LKD NLSLIPNKSLTT MQ V. 9 24 LSGTYIFAVL VCVVFHSGAQEKNYTIREEIPENVLIGNLLKDLNLSL1PNKSLTTTMQ 83 V . l 63 FKLVYKTGDVPLIRIEEDTGEIFTTGARIDREK CAGIPRDEHCFYEVEVAI PDEIFRL 122
FKVYKTGDVP IRIEEDTGEIFTTGARIDREKLCAGIPRDEHCFYEVEVAI PDEIFR V . 9 84 FKLVYKTGDVPLIRIEEDTGEIFTTGARIDREK CAGIPRDEHCFYEVEVAILPDEIFRL 143 V . l 123 VKIRFLIEDINDNAP FPATVINISIPENSAINSKYTLPAAVDPDVGINGVQNYELIKSQ 182
VKIRFLIEDINDNAPLFPATVINISIPENSAINSKYT PAAVDPDVGINGVQNYE IKSQ V. 9 144 VKIRFLIEDINDNAPLFPATVINISIPENSAINSKYT PAAVDPDVGINGVQNYE IKSQ 203 V. l 183 NIFGLDVIETPEGDKMPQLIVQKELDREEKDTYVMKVKVEDGGFPQRSSTAILQVSVTDT 242
NIFGLDVIETPEGDKMPQLIVQKELDREEKDTYVMKVKVEDGGFPQRSSTAI QVSVTDT V . 9 204 NIFGLDVIETPEGDKMPQLIVQ ELDREEKDTYVMKVKVEDGGFPQRSSTAILQVSVTDT 263 V . l 243 NDNHPVFKETEIEVSIPENAPVGTSVTQLHATDADIGENAKIHFSFSNLVSN1ARR FH 302
NDNHPVFKETEIEVSIPENAPVGTSVTQLHATDADIGENAKIHFSFSNLVSNIARRLFHL V . 9 264 NDNHPVFKETEIEVSIPENAPVGTSVTQLHATDADIGENAKIHFSFSNLVSNIARRLFHL 323 V . l 303 NATTGLITIKEPLDREETPNHKL VLASDGGLMPARAMVLVNVTDV DNVPSIDIRYIVN 362
NATTGLITIKEP DREETPNHKLLV ASDGGLMPARAMV VNVTDVNDNVPSIDIRYIVN V . 9 324 NATTGLITIKEPLDREETPNHKLLVLASDGGLMPARAMVVNVTDVNDNVPSIDIRYIV 383 V. l 363 PVNDTWLSENIPLNTKIALITVTDKDADHNGRVTCFTDHEIPFRLRPVFSNQFLLETAA 422
PVNDTW SENIPLNTKIALITVTDKDADHNGRVTCFTDHEIPFRLRPVFSNQFLLE AA V . 9 384 PV DTW SENIPLNTKIALITVTDKDADHNGRVTCFTDHEIPFRLRPVFSNQF ENAA 443 V . l 423 Y DYESTKEYAIKL AADAGKPP NQSA FIKVKDENDNAPVFTQSFVTVSIPENNSPG 482
YLDYESTKEYAIKLLAADAGKPPLNQSAMLFIKVKDENDNAPVFTQSFVTVSIPENNSPG V . 9 444 Y DYESTKEYAIKL AADAGKPPLNQSAMLFIKVKDENDNAPVFTQSFVTVSIPENNSPG 503 . l 483 IQLTKVSAMDADSGPNAKINYLLGPDAPPEFSLDCRTGMLTVVKK DREKEDKY FTILA 542
IQL KVSA DADSGPNA+INYLLGPDAPPEFSLD RTGMLTWKKLDREKEDKYLFTILA V. 9 504 IQLMKVSATDADSGPNAEINYLLGPDAPPEFS DRRTGMLTWKKLDREKEDKY FTILA 563 . l 543 KDNGVPPLTSNVTVFVSIIDQNDNSPVFTHNEYNFYVPENLPRHGTVGLITVTDPDYGDN 602
KDNGVPP TSNVTVFVSIIDQNDNSPVFTHNEY FYVPEN PRHGTVGLITVTDPDYGDN V . 9 564 KDNGVPPLTSNVTVFVSIIDQNDNSPVFTHNEYKFYVPENLPRHGTVGLITVTDPDYGDN 623 . l 603 SAVT SILDENDDFTIDSQTGVIRPN1SFDREKQESYTFYVKAEDGGRVSRSSSAKVTIN 662
SAVTLSILDENDDFTIDSQTGVIRPNISFDREKQESYTFYVKAEDGGRVSRSSSAKVTIN V. 9 624 SAVTLSILDENDDFTIDSQTGVIRPNISFDREKQESYTFYVKAEDGGRVSRSSSAKVTIN 683 V. l 663 WDVNDNKPVFIVPPSNCSYELVLPSTNPGTWFQVIAVDNDTGMNAEVRYSIVGGNTRD 722
WDVNDNKPVFIVPP N SYELVLPSTNPGTWFQVIAVDNDTGMAEVRYSIVGGNTRD V . 9 684 WDVNDNKPVFIVPPYNYSYELVLPSTNPGTWFQVIAVDNDTGMNAEVRYSIVGGNTRD 743 V.l 723 LFAIDQETGNITLMEKCDVTDLGLHRVLVKANDLGQPDSLFSWIVN FVNESVTNATLI 782
LFAIDQETGNITLMEKCDVTDLGLHRVLVKANDLGQPDS FSWIVNLFVNESVTNATLI V.9 744 LFAIDQETGNITLMEKCDVTDLGLHRVLVKAND GQPDSLFSWIVN FVNESVTNATLI 803 V.l 783 NELVRKSTEAPVTPNTEIADVSSPTSDYVKILVAAVAGTITWWIFITAWRCRQAPH 842
NELVRKS EAPVTPNTEIADVSSPTSDYVKILVAAVAGTITWWIFITAWRCRQAPHL V.9 804 NELVRKSIEAPVTPNTEIADVSSPTSDYV ILVAAVAGTITWWIFITAWRCRQAPHL 863 V.l 843 KAAQKNKQNSEWATPNPENRQMI MKKKKKKKKHSPKNLLLNFVTIEETKADDVDSDGNR 902
KAAQKN QNSE ATPNPENRQMI MKKKKKKKKHSPKNLLLN VTIEETKADDVDSDGNR V.9 864 KAAQKNMQNSEWATPNPENRQMIMMKKKKKKKKHSPKNL LNWTIEETKADDVDSDGNR 923 .l 903 VTLDLPID EEQTMGKYN VTTPTTFKPDSPD ARHYKSASPQPAFQIQPETPLNSKHHI 962
VTLDLPIDLEEQTMGKYN VTTPTTFKPDSPDLARHYKSASPQPAFQIQPETPLN KHHI V.9 924 VTLDLPIDLEEQTMGKYNWVTTPTTFKPDSPDLARHYKSASPQPAFQIQPETPLN KHHI 983 V.l 963 IQELPLDNTFVACDSISKCSSSSSDPYSVSDCGYPVTTFEVPVSVHTRP 1011
IQELPLDNTFVACDSIS CSSSSSDPYSVSDCGYPVTTFEVPVSVHTRP V.9 984 IQE PLDNTFVACDSISNCSSSSSDPYSVSDCGYPVTTFEVPVSVHTRP 1032

Claims

CLAIMS:
1. A composition that comprises: a) a peptide of eight, nine, ten, or eleven contiguous amino acids of a protein of Figure 2; b) a peptide of Tables VIII-XXI; c) a peptide of Tables XXII to XLV; or, d) a peptide of Tables XLVI to XLIX.
2. A composition of claim 1, which elicits an immune response.
3. A protein of claim 2 that is at least 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% homologous or identical to an entire amino acid sequence shown in Figure 2.
4. A protein of claim 2, which is bound by an antibody that specifically binds to a protein of Figure 2.
5. A composition of claim 2 wherein the composition comprises a cytotoxic T cell (CTL) polypeptide epitope or an analog thereof, from the amino acid sequence of a protein of Figure 2.
6. A composition of claim 5 further limited by a proviso that the epitope is not an entire amino acid sequence of Figure 2.
7. A composition of claim 2 further limited by a proviso that the polypeptide is not an entire amino acid sequence of a protein of Figure 2.
8. A composition of claim 2 that comprises an antibody polypeptide epitope from an amino acid sequence of Figure 2.
9. A composition of claim 8 further limited by a proviso that the epitope is not an entire amino acid sequence of Figure 2.
10. A composition of claim 8 wherein the antibody epitope comprises a peptide region of at least 5 amino acids of Figure 2 in any whole number increment up to the end of said peptide, wherein the epitope comprises an amino acid position selected from: a) an amino acid position having a value greater than 0.5 in the Hydrophilicity profile of Figure 5, b) an amino acid position having a value less than 0.5 in the Hydropathicity profile of Figure 6; c) an amino acid position having a value greater than 0.5 in the Percent Accessible Residues profile of Figure 7; d) an amino acid position having a value greater than 0.5 in the Average Flexibility profile of Figure 8; e) an amino acid position having a value greater than 0.5 in the Beta-turn profile of Figure 9; f) a combination of at least two of a) through e); g) a combination of at least three of a) through e); h) a combination of at least four of a) through e); or i) a combination of five of a) through e).
11. A polynucleotide that encodes a protein of claim 1.
12. A polynucleotide of claim 11 that comprises a nucleic acid molecule set forth in Figure 2.
13. A polynucleotide of claim 12 further limited by a proviso that the encoded protein is not an entire amino acid sequence of Figure 2.
14. A composition comprising a polynucleotide that is fully complementary to a polynucleotide of claim 11.
15. An 109P1 D4 siRNA composition that comprises siRNA (double stranded RNA) that corresponds to the nucleic acid ORF sequence of the 109P1D4 protein or a subsequence thereof; wherein the subsequence is 19, 20, 21, 22, 23, 24, or 25 contiguous RNA nucleotides in length and contains sequences that are complementary and non- complementary to at least a portion of the mRNA coding sequence.
16. A polynucleotide of claim 13 that further comprises an additional nucleotide sequence that encodes an additional peptide of claim 1.
17. A method of generating a mammalian immune response directed to a protein of Figure 2, the method comprising: exposing cells of the mammal's immune system to a portion of a) a 109P1D4-related protein and/or b) a nucleotide sequence that encodes said protein, whereby an immune response is generated to said protein.
18. A method of generating an immune response of claim 17, said method comprising: providing a 109P1 D4-related protein that comprises at least one T cell or at least one B cell epitope; and, contacting the epitope with a mammalian immune system T cell or B cell respectively, whereby the T cell or B cell is activated.
19. A method of claim 18 wherein the immune system cell is a B cell, whereby the activated B cell generates antibodies that specifically bind to the 109P1D4-related protein.
20. A method of claim 18 wherein the immune system cell is a T cell that is a cytotoxic T cell (CTL), whereby the activated CTL kills an autologous cell that expresses the 109P1D4-related protein.
21. A method of claim 18 wherein the immune system cell is a T cell that is a helper T cell (HTL), whereby the activated HTL secretes cytokines that facilitate the cytotoxic activity of a cytotoxic T cell (CTL) or the antibody-producing activity of a B cell. corresponds to the nucleic acid ORF sequence of the 109P1D4 protein or a subsequence thereof; wherein the subsequence is 19, 20, 21, 22, 23, 24, or 25 contiguous RNA nucleotides in length and contains sequences that are complementary and non-complementary to at least a portion of the mRNA coding sequence.
30. A composition of claim 28, further comprising a physiologically acceptable carrier.
31. A pharmaceutical composition that comprises the composition of claim 28 in a human unit dose form.
32. A composition of claim 28 wherein the substance comprises an antibody or fragment thereof that specifically binds to a protein of Figure 2.
33. An antibody or fragment thereof of claim 32, which is monoclonal.
34. An antibody of claim 32, which is a human antibody, a humanized antibody or a chimeric antibody.
35. A non-human transgenic animal that produces an antibody of claim 32.
36. A hybridoma that produces an antibody of claim 33.
37. A composition of claim 28 wherein the substance reduces or inhibits the viability, growth or reproduction status of a cell that expresses a protein of Figure 2.
38. A composition of claim 28 wherein the substance increases or enhances the viability, growth or reproduction status of a cell that expresses a protein of Figure 2.
39. A composition of claim 28 wherein the substance is selected from the group comprising: a) an antibody or fragment thereof, either of which immunospecifically binds to a protein of Figure 2; b) a polynucleotide that encodes an antibody or fragment thereof, either of which immunospecifically binds to a protein of Figure 2; c) a ribozyme that cleaves a polynucleotide having a 109P1 D4 coding sequence, or a nucleic acid molecule that encodes the ribozyme; and, a physiologically acceptable carrier; and d) human T cells, wherein said T cells specifically recognize a 109P1 D4 peptide subsequence in the context of a particular HLA molecule; e) a protein of Figure 2, or a fragment of a protein of Figure 2; f) a nucleotide encoding a protein of Figure 2, or a nucleotide encoding a fragment of a protein of Figure 2; g) a peptide of eight, nine, ten, or eleven contiguous amino acids of a protein of Figure 2; h) a peptide of Tables VIII-XXI; i) a peptide of Tables XXII to XLV; j) a peptide of Tables XLVI to XLIX; k) an antibody polypeptide epitope from an amino acid sequence of Figure 2;
I) a polynucleotide that encodes an antibody polypeptide epitope from an amino acid sequence of Figure 2; or m) an 109P1 D4 siRNA composition that comprises siRNA (double stranded RNA) that corresponds to the nucleic acid ORF sequence of the 109P1D4 protein or a subsequence thereof; wherein the subsequence is 19, 20, 21, 22, 23, 24, or 25 contiguous RNA nucleotides in length and contains sequences that are complementary and non-complementary to at least a portion of the mRNA coding sequence.
40. A method of inhibiting viability, growth or reproduction status of cancer cells that express a protein of Figure 2, the method comprising: administering to the cells the composition of claim 28, thereby inhibiting the viability, growth or reproduction status of said cells.
41. The method of claim 40, wherein the composition comprises an antibody or fragment thereof, either of which specifically bind to a 109P1D4-related protein.
42. The method of claim 40, wherein the composition comprises (i) a 109P1 D4-related protein or, (ii) a polynucleotide comprising a coding sequence for a 109P1D4-related protein or comprising a polynucleotide complementary to a coding sequence for a 109P1D4-related protein.
43. The method of claim 40, wherein the composition comprises a ribozyme that cleaves a polynucleotide that encodes a protein of Figure 2.
44. The method of claim 40, wherein the composition comprises human T cells to said cancer cells, wherein said T cells specifically recognize a peptide subsequence of a protein of Figure 2 while the subsequence is in the context of the particular HLA molecule.
45. The method of claim 40, wherein the composition comprises a vector that delivers a nucleotide that encodes a single chain monoclonal antibody, whereby the encoded single chain antibody is expressed intracellularly within cancer cells that express a protein of Figure 2.
46. A method of delivering an agent to a cell that expresses a protein of Figure 2, said method comprising: providing the agent conjugated to an antibody or fragment thereof of claim 32; and, exposing the cell to the antibody-agent or fragment-agent conjugate.
47. A method of inhibiting viability, growth or reproduction status of cancer cells that express a protein of Figure 2, the method comprising: administering to the cells the composition of claim 28, thereby inhibiting the viability, growth or reproduction status of said cells.
48. A method of targeting information for preventing or treating a cancer of a tissue listed in Table I to a subject in need thereof, which comprises: detecting the presence or absence of the expression of a polynucleotide associated with a cancer of a tissue listed in Table I in a sample from a subject, wherein the expression of the polynucleotide is selected from the group consisting of:
(a) a nucleotide sequence in Figure 2;
(b) a nucleotide sequence which encodes a polypeptide encoded by a nucleotide sequence in Figure 2;
(c) a nucleotide sequence which encodes a polypeptide that is 90% or more identical to the amino acid sequence encoded by a nucleotide sequence in Figure 2; directing information for preventing or treating the cancer of a tissue listed in Table I to a subject in need thereof based upon the presence or absence of the expression of the polynucleotide in the sample.
49. The method of claim 48, wherein the information comprises a description of detection procedure or treatment for a cancer of a tissue listed in Table I.
50. A method for identifying a candidate molecule that modulates cell proliferation, which comprises:
(a) introducing a test molecule to a system which comprises a nucleic acid comprising a nucleotide sequence selected from the group consisting of:
(i) the nucleotide sequence of SEQ ID NO:1;
(ii) a nucleotide sequence which encodes a polypeptide consisting of the amino acid sequence set forth in Figure 3;
(iii) a nucleotide sequence which encodes a polypeptide that is 90% or more identical to the amino acid sequence set forth in Figure 3; and
(iv) a fragment of a nucleotide sequence of (i), (ii), or (iii); or introducing a test molecule to a system which comprises a protein encoded by a nucleotide sequence of (i), (ii), (iii), or (iv); and
(b) determining the presence or absence of an interaction between the test molecule and the nucleotide sequence or protein, whereby the presence of an interaction between the test molecule and the nucleotide sequence or protein identifies the test molecule as a candidate molecule that modulates cell proliferation.
51. The method of claim 50, wherein the system is an animal.
52. The method of claim 50, wherein the system is a cell.
53. The method of claim 50, wherein the test molecule comprises an antibody or antibody fragment that specifically binds the protein encoded by the nucleotide sequence of (i), (ii), (iii), or (iv).
54. A method for treating a cancer of a tissue listed in Table I in a subject, which comprises administering a candidate molecule identified by the method of claim 50 to a subject in need thereof, whereby the candidate molecule treats a cancer of a tissue listed in Table I in the subject.
55. A method for identifying a candidate therapeutic for treating a cancer of a tissue listed in Table I, which comprises: (a) introducing a test molecule to a system which comprises a nucleic acid comprising a nucleotide sequence selected from the group consisting of:
(i) the nucleotide sequence of SEQ ID NO:1;
(ii) a nucleotide sequence which encodes a polypeptide consisting of the amino acid sequence set forth in Figure 3;
(iii) a nucleotide sequence which encodes a polypeptide that is 90% or more identical to the amino acid sequence set forth in Figure 3; and
(iv) a fragment of a nucleotide sequence of (i), (ii), or (iii); or introducing a test molecule to a system which comprises a protein encoded by a nucleotide sequence of (i), (ii), (iii), or (iv); and
(b) determining the presence or absence of an interaction between the test molecule and the nucleotide sequence or protein, whereby the presence of an interaction between the test molecule and the nucleotide sequence or protein identifies the test molecule as a candidate therapeutic for treating a cancer of a tissue listed in Table I.
56. The method of claim 55, wherein the system is an animal.
57. The method of claim 55, wherein the system is a cell.
58. The method of claim 55, wherein the test molecule comprises an antibody or antibody fragment that specifically binds the protein encoded by the nucleotide sequence of (i), (ii), (iii), or (iv).
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AU2008212020B2 (en) 2012-05-24
EP1622571A4 (en) 2012-05-02
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