WO1997013855A1

WO1997013855A1 - Melanoma-associated protein

Info

Publication number: WO1997013855A1
Application number: PCT/EP1995/003988
Authority: WO
Inventors: Gerd Pluschke; Peter Schmid
Original assignee: Novartis Ag
Priority date: 1995-10-10
Filing date: 1995-10-10
Publication date: 1997-04-17
Also published as: EP0854919A1; AU3804995A

Abstract

This invention relates to a melanoma-associated protein (MCSP), a derivative of said protein and to means and methods for the production thereof. The invention is also directed to isolated nucleic acids coding for said melanoma-associated protein, to a method of obtaining such nucleic acid molecules, and to their expression. Furthermore, the invention is directed to uses of said protein and nucleic acid, particularly uses relating to diagnosis, prophylaxis and therapy of tumors producing the protein, such as human malignant melanoma, sarcoma and glioblastoma.

Description

Melanoma-associated Protein

During the last two decades there has been considerable interest in the biology and pathophysiology of human malignant melanoma, in part, because of the poor prognosis and increasing incidence of this disease. The fatal nature of human cutaneous melanoma, which is attributable to poor response to conventional radiation and chemotherapy, has prompted a growing interest in melanoma-associated antigens (MAAs). Such proteins are expressed on melanoma cells but not on normal skin melanocytes and include antigens that are unique for melanoma, or particular stages of melanoma progression, and others that are typical for all tumors of neuroectodermal origin. Based on proven and putative biochemical and immunological characteristics MAAs may be categorized into cell substrate-interacting glycoproteins, ion transport and binding proteins, gangliosides, and receptors for growth factors.

The category of cell substrate-interacting glycoproteins comprises several MAAs of relatively high molecular weight Up to today, murine monoclonal antibodies (mAb) raised against human melanoma cells or membrane preparations of such cells have been relied upon for identification and partial characterization of these antigens. Thus, human melanoma chondroitin sulfate proteoglycan (MCSP), also referred to as high molecular weight-melanoma associated antigen (HMW-MAA), has been identified with mAb 9.2.27 (Morgan et al., Hybridoma 1, 27-36 (1981)). MCSP is expressed on more than 90% of human melanoma tissues and cultures where 80 to 100 % of cells express MCSP at densities ranging from 1×10⁵ to 6×10⁶ binding sites per cell (Bumol and Reisfeld,

Proc. Natl. Acad. Sci. U.S.A.79, 1245-1249 (1982); Bumol et al., J. Biol. Chem. 267, 12733-12741 (1984); Mueller and Reisfeld in Encyclopedia of Human Biology (Dulbecco, ed.), pp. 957-967, Academic Press, New York (1991)).

MCSP has been reported to be a unique glycoprotein-proteoglycan complex. A 250 kDa molecule is the core glycoprotein of MCSP possessing asparagine-N-linked oligosaccharides of the high mannose type. Addition of chondroitin sulfate glycosaminolycan polysyccharide chains to serine residues of me core glycoprotein converts the 250 kDa core protein to the high molecular weight proteoglycan form. The molecular mass of the mature proteoglycan containing a full complement of chondroitin sulfate chains has been estimated at 420 to 1000 kDa (Harper and Reisfeld in "Biology of Proteoglycans" (Wight and Mecham eds.), pp. 345-366, Academic Press, Orlando (1987)). The primary structure of the core protein of the rat homologue, referred to as NG2, is known from Nishiyama et al., J. Cell Biology 114, 359-371 (1991).

Proteoglycans have been implicated in growth control, involvement in adhesion, in cell-substratum interaction and cell-cell contacts (Hardingham and Fosang, FASEB J. 6, 861-870 (1992)). Thus, MCSP is found to be expressed on the melanoma cells upper surface on microspikes, consisting of 1-2 μm structures that range up to 20 μm at the cell periphery. These peripheral structures are involved in cell-cell contacts and also form complex footpads that are in contact with the substratum (Bumol et al., 1984, supra;

Harper et al., J. Immunol. 132, 2096-2104 (1984); Garrigues et al., J. Natl. Cancer Inst.71, 259-263 (1986)). Adhesion plaques deposited along the cell membrane also expressed MCSP very well (Harper et al., supra; Harper and Reisfeld, J. Natl. Cancer Inst. 71, 259-263 (1983)). A possible role of this molecule in stabilizing cell-substratum

interactions is suggested by the finding that mAb 9.2.27, directed against both the proteoglycan and the core protein, blocks early events of melanoma cell spreading on endothelial basement membranes, while only slightly interfering with cell adhesion. Data indicating that MCSP core protein is expressed on the cell surface in two forms, either modified by the addition of chondroitin sulfate chains or chondroitin sulfate nonmodified, suggest that glycosaminoglycan (GAG) chains may not be necessary for cell surface expression of the core protein. Hence, it seems unlikely that such a modification serves as a marker to segregate molecules on the cell surface (Harper et al., J. Biol. Chem. 261, 3600-3606 (1986)). Furthermore MCSP recognized by mAb 9.2.27 is reported to act as a co-receptor for spreading and focal contact formation in association with α4β1 integrin in melanoma cells, implying a model in which MCSP communicates with α4β1 integrin by an inside-out signaling mechanism (Iida et al., Cancer Research 55, 2177-2185 (1995)).

MCSP also proved to be an effective target for radioimaging of tumors of melanoma patients (Oldham et al., J. Clin. Oncol. 2, 1235-1245 (1995)) and is currently used to target active-specific immunotherapy with antiidiotypic mAb, which bear the internal image of antigenic determinants defined by anti-MCSP mAb (Kusama et al., J. Immunol. 143, 3844-3852 (1989); Chen et al., Cancer Res.53, 112-119 (1993)). Thus, conjugation of antiidiotypic mAb with a carrier and administration with an adjuvant induced humoral anti-MCSP immunity in about 60% of immunized patients with advanced melanoma (Mittelman et al., Proc. Natl. Acad. Sci. U.S.A. 89, 466-470 (1992)). Development of this anti-MCSP immunity was found to be associated with statistically significant prolongation of survival.

Although some features of MCSP have been described in the literature as shown above, the precise structure of MCSP has not been previously established. In view of the pathological significance of high MCSP expression, particularly in metastatic lesions, there is a need for a better understanding of cellular signal transduction via MCSP as well as in the role of MCSP in the interaction with surrounding cells and tumor spreading. So far, a deeper insight into human malignant melanoma and tumor progression as well as eventual improvements in diagnosis, prophylaxis and therapy of neoplasms showing high MCSP expression has been significantly hampered by the inavailability of MCSP in a purified form and amino acid and nucleic acid sequence information. This lack of knowledge has particularly handicapped the search for human therapeutic agents capable of influencing tumor growth and metastatic spreading.

The present invention has achieved the isolation and sequencing of DNA encoding full-length human MCSP, thus providing the amino acid sequence of human MCSP and enabling the production of MCSP, e.g. by recombinant DNA techniques. Synthesis of a complete cDNA coding for the full-length protein was extemely difficult and could not be achieved by conventional methods. The present invention for the first time enables correlations between MCSP structure and function, thereby providing e.g. means for improved diagnosis, prophylaxis and therapy of a tumor characterized by MCSP expression, e.g. a melanoma, glioma or sarcoma expressing MCSP.

More specifically, the present invention relates to a purified or isolated protein designated MCSP, or a derivative thereof.

As used herein before or hereinafter, the term "purified" or "isolated" is intended to refer to a molecule of the invention in an essentially pure form, said molecule being obtainable from a natural source or by means of genetic engineering. The purified protein, DNA or RNA of the invention may be useful in ways that the protein, DNA and RNA as they naturally occur are not, such as identification of compounds selectively modulating the expression or the activity of MCSP.

In a preferred embodiment, the invention concerns a protein having the amino acid sequence set forth in SEQ ID NO:2, and particularly a mature protein having the amino acid sequence extending from the amino acid at position 1 (Ala) to the amino acid at position 2293 (Val). Hereinafter, such protein will be referred to as MCSP. The peptide comprising amino acids -29 to -1 of SEQ ID NO:2 represents the MCSP signal peptide. MCSP is found to be an integral membrane protein with a large amino-terminal ectodomain separated from a relatively short cytoplasmic tail by a single hydrophobic transmembrane region.

Included within the scope of " isolated MCSP" or "a protein of the invention", as these terms are understood herein, is any deglycosylated, unglycosylated or glycosylated form of the protein having the amino acid sequence set forth in SEQ ID NO:2, a splice variant encoded by mRNA generated by alternative splicing of a primary MCSP-encoding transcript, and an amino acid mutant of the protein of SEQ ID NO:2.

Additionally, the invention concerns an in vitro generated covalent or aggregative derivative of a protein of the invention.

According to the invention, purified MCSP is essentially free of all naturally occurring substances with which it is typically found in human tissue. For example, MCSP produced by recombinant means will be free of those contaminants typically found in its in vivo physiological milieu. Purified MCSP also encompasses a protein according to the invention in recombinant cell culture.

A beforementioned protein of the invention, or a derivative thereof, displays a biological profile which is qualitatively essentially identical to the profile characteristic of native MCSP, or at least a cross-section of said MCSP- profile. The biological profile in vitro and in vivo includes antigenicity, ligand binding and signal transduction. The biological profile of a protein of the invention, or a particular biological activity thereof, may be evaluated in a suitable assay employing said protein in a purified form or a host cell producing MCSP. In any case, a protein of the invention bears at least one immune epitope in common with MCSP, or mimics such epitope. Such protein is referred to as immunological equivalent of MCSP. A protein which bears at least one immune epitope in common with MCSP comprises at least eight to about eleven consecutive amino acids of SEQ ID NO:2 and is capable of cross-reacting with an antibody which is specific for native MCSP. Thus, a protein of the invention is capable of competing with native MCSP for binding to an anti-MCSP antibody, e.g. such antibody raised against melanoma cells or a membranous fraction thereof. Examples of such antibodies are mAb 9.2.27 (Bumol and Reisfeld, Proc. Natl. Acad. Sci. U.S.A. 1245-1249 (1982)), mAb 225.28 (European Patent No. 0 380607, ATCC accession no. HB 10141) and mAb 763.74 (Giacomini et al., J. Immunol. 135, 696 (1985)).

MCSP acts as a cell surface receptor for human type VI collagen. Therefore, an assay suitable for determining the ligand binding activity of a protein of the invention is an assay determining the interaction between said protein of the invention and collagen VI. Such an assay is known in the art (see e.g. Stallcup et al., J. Cell Biol. 111, 3177-3188 (1990); Nishiyama and Stallcup, Mol. Biol. Cell 4, 1097-1108 (1993)) and comprises contacting a cell producing a protein of the invention with collagen VI and assessing the binding to collagen VI to said cell as compared to a suitable negative control, e.g. by

immunofluorescent staining. For example, mammalian cells which do not produce endogenous MCSP, or a homologue thereof, but are capable of secreting type VI collagen, such as B28 rat neural cells or U251MG human glioma cells, are transfected with a DNA coding for a membrane-bound protein of the invention. The transfected cells producing said protein of the invention on the cell-surface are assayed for the ability of the protein of the invention to anchor collagen VI to the cell surface. Alternatively, a ligand binding assay may be performed using purified collagen VI, advantageously attached to a solid phase, and an isolated protein of the invention.

Cell surface MCSP is capable of modifying the function and/or activity of α4β1 integrin (Iida et al., J. Cell Biol. 118, 431-444 (1992)). Hence, this ability of a protein of the invention may be tested in a conventional cell adhesion assay (see e.g. Iida et al., J. Cell Biol. 118, 431-444 (1992)) using suitable cells transfected with a DNA encoding a protein of the invention and producing said protein on the cell surface. Signal transduction of a protein of the invention may be studied by evaluating the collaboration between said protein of the invention and α4β1 integrin in the modulation of cell spreading and focal contact adhesion according to methods available in the art, e.g. the method described by Iida et al. (Cancer Research 55, 2177-2185 (1995)). A glycosylated form of MCSP according to the invention is e.g. MCSP having a native (human) glycosylation pattern, e.g. a MCSP glycoprotein comprising asparagine N-linked oligosaccharides of the high mannose type, or a MCSP proteoglycan containing a partial or full complement of chondroitin sulfate chains, or a glycosylation variant having a glycosylation pattern which is different from that found for native MCSP. A

nonglycosylated form of MCSP according to the invention may be obtained by

deglycosylation of a glycosylated form of MCSP, e.g. by enzymatic removal of the glycosyl residues, or by expression of a nucleic acid encoding a protein of the invention in suitable prokaryotic cells.

According to the invention, an amino acid mutant (mutein) may be a substitutional, insertional or deletional variant of a protein with the amino acid sequence set forth in SEQ ID NO:2. Contrary to a naturally occurring allelic or interspecies variant, such a mutant is characterized by the predetermined nature of the variation. Substitutions, deletions and insertions may be combined to arrive at an amino acid mutant of the invention.

For example, a substitutional amino acid mutant is any polypeptide having an amino acid sequence substantially identical to the sequence set forth in SEQ ID NO:2, in which one or more residues have been conservatively substituted with a functionally-similar amino acid residue and which is capable of mimicking an MCSP epitope as described herein before. Conservative substitutions include e.g. the substitution of one non-polar (hydrophobic) residue, such as methionine, valine, leucine, isoleucine for another, substitution of one polar (hydrophilic) residue for another, such as between glycine and serine, between arginine and lysine, and between glutamine and asparagine. Substitutional or deletional mutagenesis may be employed to eliminate O- or N-linked glycosylation sites from MCSP. MCSP has 15 potential N-linked glycosylation sites, which are characterized by the occurrence of the acceptor amino acid asparagine (Asn) in the tripepetide sequence Asn-X-Thr(Ser), wherein X can be any of the twenty naturally occurring L-amino acids except possibly aspartic acid (Asp) (Hubbard and Ivatt, Ann. Rev. Biochem. 50, 555-583 (1981)). Potential O-linked glycosylation sites in the MCSP sequence are characterized in that a serine residue precedes a glycine residue. Such Ser/Gly pairs are located at positions 51/52, 178/179, 570/571, 966/967, 1020/1021, 1067/1068, 1131/1132, 1309/1310, 1355/1356, 1475/1476 and 1872/1873 in SEQ ID NO: 2. Deletions of cysteine or other labile amino acid residues may also be desirable, for example to increase the oxidative stability of a protein of the invention. As defined herein, a deletional amino acid mutant of MCSP also includes a fragment of mature full-length MCSP consisting of eight or more contiguous amino acids, i.e. eight to 2292 contiguous amino acids, of SEQ ID NO:2. According to the invention, such MCSP fragment is a preferred embodiment of a deletional mutant. Preferred are fragments of mature MCSP comprising from about ten to about hundred, particularly, from about ten to about fifty contiguous amino acids of SEQ ID NO:2. A major class of deletional mutants are those involving the transmembrane and/or cytoplasmic region of MCSP. The transmembrane domain succeeds the N-terminal extracellular domain and essentially consists of about 25 amino acids. Extending from about residue 2193 (Met) to about amino acid residue 2217 (Leu) in SEQ ID NO:2, this highly hydrophobic domain has the proper size to span the lipid bilayer of the cellular membrane. The cytoplasmic domain of MCSP follows the transmembrane domain and is the C-terminal sequence of amino acid residues approximately commencing at position 2218 (Arg) in SEQ ID NO: 2. Deletion or substitution of either or both of the cytoplasmic and transmembrane domains will facilitate recovery of a recombinant protein of the invention by reducing its cellular or membrane lipid affinity and improving its solubility in water or buffers so that detergents will not be required to maintain the protein in aqueous solution. An example of a deletional mutant involving the transmembrane and the cytoplasmic region of MCSP is the MCSP fragment with the sequence extending from amino acid 1 (Met) to amino acid 1593 (Val) in SEQ ID NO:2. Such deletional mutant and fragments thereof consisting of at least eight, particularly from about ten to about fifty consecutive amino acids of SEQ ID NO:2 are particularly preferred.

Preferred proteins of the invention are mature MCSP having the amino acid sequence set forth in SEQ ID NO:2 in a glycosylated or non-glycosylated form, and a deletional variant thereof, which is a fragment of MCSP as defined above.

A derivative of a protein of the invention is a covalent or aggregative conjugate of said protein with another chemical moiety, said derivative displaying essentially the same biological profile as the underivatized protein of the invention.

An exemplary covalent conjugate according to the invention is a conjugate of a protein of the invention with another protein or peptide, such as a protein comprising a protein of the invention, particularly an MCSP fragment, and a carrier protein suitable for enhancing the in vivo antigenicity of said protein of the invention. A covalent conjugate of the invention further includes a protein of the invention labelled with a detectable group, e.g. a protein of the invention which is radiolabelled, covalently bound to a rare earth chelate or biotin, or conjugated to a fluorescent moiety.

An aggregative derivative of a protein of the invention is e.g. an adsorption complex of said protein with a cell membrane.

A protein of the invention is obtainable from a natural source, e.g. by isolation from human cells or human tissue expressing MCSP, such as human melanoma tissue, or, preferably, by chemical synthesis or recombinant DNA techniques. Also, a combination of these techniques may be used to obtain a protein of the invention.

Based on the amino acid sequence information provided in SEQ ID NO:2 chemical synthesis of a protein of the invention is performed according to conventional methods known in the art In general, those methods comprise the sequential addition of one or more amino acid residues to a growing (poly)peptide chain. If required, potentially reactive groups, e.g. free amino or carboxy groups, are protected by a suitable, selectively removable protecting group. Chemical synthesis may be particularly advantageous for fragments of MCSP having no more than about 100, and usually no more than about 20 to 40, amino acid residues.

The invention also provides a method for preparing a protein of the invention, said method being characterized in that suitable host cells producing the protein of the invention are multiplied in vitro or in vivo. Preferably, the host cells are transformed or transfected with a hybrid vector comprising an expression cassette comprising a promoter and a DNA sequence coding for a protein of the invention which DNA is controlled by said promoter. Subsequently, the protein of the invention may be recovered. Recovery comprises e.g. isolating the protein of the invention from the host cells or isolating the host cells comprising the protein, e.g. from the culture broth.

Suitable host cells include eukaryotic cells, e.g. animal cells, plant cells and fungi, and prokaryotic cells, such as gram-positive and gram-negative bacteria, e.g. E. coli.

As used herein, in vitro means ex vivo, thus including e.g. cell culture and tissue culture conditions.

An amino acid mutant, as defined hereinbefore, may be produced e.g. from a DNA encoding a protein of SEQ ID NO:2, which DNA has been subjected to site-specific in vitro mutagenesis resulting e.g. in an addition, exchange and/or deletion of one or more amino acids. While the site for introducing an amino acid variation is predetermined, the mutation per se need not be predetermined, but random mutagenesis may be performed at the target codon or region. For example, substitutional, deletional and insertional variants are prepared by recombinant methods and screened for immuno-crossreactivity with the native forms of the protein of the invention. Alternatively, mutants of the invention may be prepared by chemical synthesis using methods routinely employed in the art

A transmembrane and/or cytoplasmic deleted or substituted amino acid mutant of the invention can be produced directly in recombinant cell culture or as a fusion with a signal sequence, preferably a host-homologous signal. For example, in constructing a procaryotic expression vector, the transmembrane and the cytoplasmic domains are deleted in favor of the bacterial alkaline phosphatase, or heat stable enterotoxin II leaders, and for yeast the domains are substituted by yeast invertase, alpha factor or acid phosphatase leaders. In mammalian cell expression the transmembrane and the cytoplasmic domains may be replaced with a mammalian cell viral secretory leader. The advantage of a variant lacking both the transmembrane and the cytoplasmic region is that it is capable of being secreted into the culture medium.

A protein of the invention may also be derivatized in vitro according to conventional methods known in the art

A protein of the invention, or a derivative thereof, may be used, for example, as immunogen, e.g. to raise MCSP specific immunoreagents, as immunoreagent in a drug or ligand screening assay, or in a purification method, such as affinity purification of a binding ligand. A protein of the invention, or a derivative thereof, suitable for in vivo administration and capable of competing with endogenous MCSP for an endogenous ligand, e.g. collagen VI, is envisaged as therapeutic agent.

The invention also relates to the use of a protein of the invention, or a derivative thereof, for the generation of a monoclonal or polyclonal antibody, which specifically binds to MCSP. Such anti-MCSP antibody is intended to include immune sera. Particularly useful for this purpose is a MCSP fragment consisting of at least eight or more, preferably eight to about twenty, consecutive amino acids of MCSP of SEQ ID NO:2. The antibodies raised against a protein of the invention may react with a non-glycosylated or glycosylated form of MCSP, or both.

Monoclonal and particularly polyclonal anti-MCSP antibodies generated against a protein of the invention may be employed as immunoreagents to detect tumor-associated MCSP expression, e.g. in the diagnosis of human malignant melanoma or in the monitoring of

melanoma progression and treatment For example, such antibodies are suitable for MCSP detection in fixed paraffin embedded melanoma lesions. The antibodies are produced in a mammal, e.g. mouse, rat goat or rabbit according to methods well-established in the art

For the generation of anti-MCSP antibodies to be used as immunoreagents it is

advantageous, if the protein of the invention used as antigen does not comprise a

glycosylation site or any part of the transmembrane domain of MCSP. Particularly useful as immunoreagent are antibodies raised against a peptide of the invention which

represents a single MCSP determinant

The invention also relates to the use of a suitably immunogenic protein of the invention, or a suitably immunogenic derivative thereof, as a vaccine, and to a method of vaccinating a human, comprising administration of a suitably immunogenic protein of the invention, or a suitably immunogenic covalent conjugate thereof, to said human. Such a method is intended to also refer to a method of inducing an anti-tumor response in a human comprising administration of a suitably immunogenic protein of the invention, or a suitably immunogenic derivative thereof. A suitably immunogenic protein of the invention, or a suitably immunogenic derivative thereof, is capable of inducing an anti-MCSP response in vivo. A vaccine according to the invention is applicable in the prophylactic and therapeutic treatment of patients having a disposition for or suffering from an MCSP-expressing tumor. As mentioned above, MCSP is a suitable target for active immunotherapy of melanoma, because it is expressed by a high percentage of melanoma lesions involved in metastatic spreading. Thus, a suitably immunogenic protein of the invention, or a derivative thereof, is e.g. a useful agent for the control, treatment or adjuvant treatment of a MCSP-expressing tumor, e.g. melanoma. More specifically, a suitably immunogenic protein of the invention, or a derivative thereof, can be successfully employed e.g. to cause tumor regression and/or prevent tumor recurrence of early stage melanoma patients remaining at risk for metastatic disease after surgery for the primary lesion. Such protein or derivative can be "tailor-made" to bear or mimic a specific determinant of MCSP.

Preferred for use as a vaccine is recombinant MCSP of SEQ ID NO:2, or a fragment thereof consisting of at least eight or more, preferably from eight to about fifty, consecutive amino acids of SEQ ID NO:2. Advantageously, a peptide to be used as vaccine lacks a glycosylation site and consists of eight to about fourty contiguous amino acids of the extracellular domain of MCSP. Said N-terminal domain consists of about 2192 amino acids and extends from the amino acid residue at position 1 to approximately the amino acid residue at position 2192 in SEQ ID NO:2. The preferred limitation on fragment size is primarily due to the size and purity limitations on synthetic polypeptide imposed by current technologies. Particularly preferred for use as a vaccine are the fragments accentuated above.

Inducement of an appropriate T-cell dependent (memory) response on vivo administration may demand enhancement of the immunogenicity of the protein of the invention, e.g. by conjugation of said protein to a carrier protein, the presence of an adjuvans or expression by vehicles suitable for life vaccination, such as viruses, bacteria or autologous antigen presenting cells. The induction of an anti-MCSP immune response following vaccination may be analyzed according to methods known in the art, e.g. by determination of the anti-MCSP antibody litre in a body fluid of a vaccinated patient e.g. serum, by means of an enzyme-linked immunoabsorbent-type assay (ELISA).

As carrier protein component a suitably immunogenic conjugate of the invention may

comprise any carrier protein useful in humans, e.g a non-toxic, nonpyrogenic, water

soluble, pharmaceutically acceptable carrier protein, preferably such a protein having

exposed amino groups. Suitable as a carrier protein component is any proteinaceous

molecule containing highly immunogenic promiscuous T-cell epitopes which will bind to a broad range of polymorphic HLA class I and class II gene products, e.g. a microbial,

particularly a bacterial, protein, polypeptide or oligopeptide.

Particularly preferred is a covalent conjugate of the invention comprising an above

captioned MCSP fragment and a carrier protein component obtainable from a bacterial

toxin, e.g. tetanus toxin (see e.g. B. Bizzini in Bacterial Vaccines, Academic Press, 1984:

Tetanus, pp. 38-68) or diphteria toxin, which protein component is devoid of toxin

activity, but retains the antigenic properties particular to the toxin, e.g. potent

immunogenicity, e.g. a mutant diphteria toxin devoid of toxin activity (Uchida et al.,

J. Pharma Biol. Chem. 218, 3838-3844 (1973)). Such non-toxic carrier protein component is obtainable e.g. by detoxification of the toxin, e.g. in case of tetanus toxin, or by

mutation, e.g. in case of diphteria toxin.

Diphteria toxin is obtainable from culture supernatants of Corynebacterium diphteriae

PW8 according to the method disclosed by R.K. Holmes, Infect. Immun. 12, 1392 (1975). The preferred carrier protein to form a covalent conjugate of the invention is the mutant diphteria toxin CRM197. CRM197 is an atoxic protein which crossreacts immunologically with diphteria toxin and is obtainable fromm culture supernatants of C. diphteriae C7 (R.K.. Holmes, supra; U.S. Patent No. 4,925,792, which are incorporated herein by reference). The CRM197 protein has the same molecular weight as the diphteric toxin and is composed of a fragment B which is identical as to its function and structure to those of the toxin, and of a fragment A, which is nontoxic and differs from the original fragment by one amino acid.

The carrier protein may be covalently attached to a protein of the invention involving a functional group thereof. The coupling reaction is performed according to methods known in the art in such a way that protein aggregation is avoided. Alternatively, the

MCSP-carrier protein conjugate may be produced as a fusion protein by recombinant means.

The invention also relates to an immunogen for use in a mammal comprising a suitably immunogenic protein of the invention, preferably an above specified preferred peptide of the invention, or a derivative thereof. Preferred is such immunogen comprising in a single protein such peptide according to the invention and a carrier protein, as described above.

The invention also concerns pharmaceutical compositions comprising a protein according to the invention. In particular, the invention relates to a pharmaceutical composition comprising an above-specified peptide of the invention in a suitably immunogenic form, e.g. an above-specified preferred peptide of the invention covalently attached to an appropriate carrier protein, as decribed above. The pharmaceutical compositions comprise, for example, a therapeutically effective amount of a protein of the invention in a suitably immunogenic form together or in admixture with pharmaceutically acceptable, inorganic or organic, solid or liquid carriers. Preferred are pharmaceutical compositions additionally comprising an adjuvant i.e. an agent further increasing the immune response. Possible adjuvants are Freund's complete adjuvant (emulsion of mineral oil, water, and

mycobacterial extracts), Freund's incomplete adjuvant (emulsion of water and oil only), mineral gels, e.g. aluminium hydroxide gels, surface active substances such as

lysolecithin, polyanions, peptides, BCG (Bacillus Calmette-Guerin), etc.. Particularly preferred are pharmaceutical compositions comprising a suitably immunogenic conjugate of the invention and MF59 (international patent application WO 90/14837) as adjuvant, and, optionally, N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2- [1,2-dipalmitoyl-sn-glycero-3-(hydroxyphosphoryloxy]ethylamide (MTP-PE; international patent application WO 90/14837).

Preferred are pharmaceutical compositions for parenteral application. Compositions for intramuscular, subcutaneous or intravenous application are e.g. isotonic aqueous solutions or suspensions, optionally prepared shortly before use from lyophilized or concentrated preparations. The pharmaceutical compositions may be sterilized and contain adjuvants e.g. for conserving, stabilizing, wetting, emulsifying or solubilizing the ingredients, salts for the regulation of the osmotic pressure, buffer and/or compounds regulating the viscosity, e.g. sodium carboxycellulose, dextran, polyvinylpyrrolidine or gelatine. They are prepared by methods known in the art, e.g. by conventional mixing, dissolving or lyoprtilizing, and contain from approximately 0.01% to approximately 50% of active ingredients. The compositions for injections are processed, filled into ampoules or vials, and sealed under aseptic conditions according to methods known in the art For example, owing to the solubility in aqueous solutions a conjugate of the invention may be formulated as a "two vial system" with an above adjuvant e.g. MF59-0.

Preferred is a pharmaceutical composition comprising an above-captioned conjugate of the invention suitable for intramuscular administration in a depot formulation together with an adjuvant

Also preferred is a pharmaceutical composition comprising a suitably immunogenic protein of the invention, preferably a conjugate of the invention, which is appropriate for mucosal application (H.F. Staats et al., Current Opinion in Immunology 6, 572-583 (1994)), or a stabilized pharmaceutical composition that can be swallowed for oral immunization.

The specific mode of administration and the dosage will be selected by the attending physician taking into account the particulars of the patient, state and type of the disease to be treated, and the like.

Furthermore, a protein of the invention, or a derivative thereof, can be used for the qualitative and quantitative determination of antibodies directed against MCSP. This is especially useful for the detection of an anti-MCSP immune response induced by an anti-melanoma vaccine, such as an antiidiotypic antibody bearing me internal image of MCSP, particularly such antibody disclosed in European Patent Application EP-A-0428 485, melanoma cells, or membraneous fractions thereof, melanoma cell lysates, or a suitably immunogenic protein of the invention. For instance, a protein of the invention, or a derivative thereof according to the invention, can be used in any of the known immunoassays which rely on the binding interaction between the idiotopes of the anti-MCSP antibody and said protein of the invention. Examples of such assays are radio-, enzyme, fluorescence, chemiluminescence, immunoprecipitation, latex agglutination, and hemagglutination immunoassays.

This invention also concerns test kits for the qualitative and quantitative determination of antibodies directed against MCSP comprising a protein of the invention and/or derivatives thereof and, optionally, adjuncts.

In a further aspect the present invention relates to a nucleic acid (DNA, RNA) comprising an isolated, preferably recombinant nucleic acid (DNA, RNA) coding for a protein of the invention, or a fragment of such a nucleic acid consisting of at least 14 nucleotides.

According to the invention, isolated MCSP-encoding nucleic acid of the invention includes a MCSP-encoding nucleic acid present in other than in the form or setting in which it is found in nature, thus embracing such nucleic acid in ordinarily MCSP expressing cells, where the nucleic acid is in a chromosomal location different from that of natural cells or is otherwise flanked by a different DNA sequence than that found in nature. The MCSP gene maps to human chromosome 15.

In particular, the invention provides a purified or isolated DNA molecule encoding a protein of the invention, or a fragment of such DNA suitable for use as a screening probe as specified hereinafter. By definition, such a DNA comprises a coding single stranded DNA, a double stranded DNA consisting of said coding DNA and complementary DNA thereto, or this complementary (single stranded) DNA itself. Preferred is a DNA coding for an above protein of the invention herein identified as being preferred, or a fragment of said DNA.

Preferred is a DNA coding for the mature protein having the amino acid sequence set forth in SEQ ID NO:1 , particularly a DNA having substantially the nucleotide sequence set forth in SEQ ID NO:1, or a DNA coding for a fragment of said protein consisting of at least 14 consecutive amino acids of the amino acid sequence set forth in SEQ ID NO:1 excluding the DNA with the sequence extending from bp 4867 to bp 7898 in SEQ ID NO:1 and the DNA with the sequence extending from bp 4858 to 5357 in SEQ ID NO:1 , respectively. Particularly preferred is a DNA coding for mature MCSP set forth in SEQ ID NO:1 , or coding for a MCSP fragment which is accentuated above, e.g a DNA coding for the MCSP fragment extending from amino acid 1 to amino acid 1593 in SEQ ID NO:1 , or a portion of said fragment

It is envisaged that a nucleic acid of the invention can be readily modified by nucleotide substitution, nucleotide deletion, nucleotide insertion or inversion of a nucleotide stretch, and any combination thereof. Such modified sequences can be used to produce a mutein having an amino acid sequence differing from the sequence of MCSP found in nature. Mutagenesis may be predetermined (site-specific) or random. A mutation which is not a silent mutation must not place sequences out of reading frames and preferably will not create complementary regions that could hybridize to produce secondary mRNA structures such as loops or hairpins.

Given the guidance of the present invention, a nucleic acid of the invention is obtainable according to methods well known in the art The present invention further relates to a process for the preparation of such nucleic acid.

For example, a DNA of the invention is obtainable by chemical synthesis, by recombinant DNA technology or by polymerase chain reaction (PCR). A suitable method for preparing a DNA of the invention may e.g. comprise the synthesis of a number of oligonucleotides, their amplification by PCR methods, and their splicing to give the desired DNA sequence.

Preparation of a DNA of the invention, or a fragment thereof by recombinant DNA technology may involve screening of a suitable cDNA or genomic library. A suitable library is commercially available, e.g. a library employed in the Examples, or can be prepared from human melanoma tissue samples, cell lines and the like. After screening the library, e.g. with a DNA including substantially the entire MCSP coding region or a suitable oligonucleotide (probe) based on a said DNA, positive clones are identified by detecting a hybridization signal; the identified clones are characterized by restriction enzyme mapping and/or DNA sequence analysis, and then examined, e.g. by comparison with the sequences set forth herein, to ascertain whether they include DNA encoding complete MCSP (i.e., if they include translation initiation and termination codons). If the selected clones are incomplete, they may be used to rescreen the same or a different library to obtain overlapping clones. If the library is genomic, then the overlapping clones may include exons and introns. If the library is a cDNA library, then the overlapping clones will include an open reading frame. In both instances, complete clones may be identified by comparison with the DNA sequences and deduced amino acid sequence provided herein.

In order to detect the presence or any abnormality of endogenous MCSP genetic screening may be carried out using a nucleotide sequence of the invention as hybridization probe. Also, based on the nucleic acid sequences provided herein antisense-type therapeutic agents may be designed.

In addition to being useful for the production of an above mentioned recombinant protein of the invention, a nucleic acid of the invention is useful as probe, thus e.g. enabling those skilled in the art to identify and/or isolate nucleic acid encoding MCSP or a novel non-human homologue thereof. Such probe according to the invention may be unlabeled or labeled with a chemical moiety suitable for ready detection. As a screening probe, there may be employed a DNA or RNA comprising substantially the entire coding region of MCSP, or a suitable oligonucleotide probe based on said DNA. A suitable oligonucleotide probe (for screening involving hybridization) includes a single stranded DNA or RNA that has a sequence of nucleotides that comprises at least 14, preferably at least about 20 to 30, contiguous bases that are the same as (or complementary to) any 14 or more contiguous bases set forth in SEQ ID NO:1. The nucleic acid sequences selected as probes should be of sufficient length and sufficiently unambiguous so that false positive results are minimized. Examplary probes are the oligonucleotides with the sequences set forth in SEQ ID NOs. 14 to 19.

For example, a method suitable for identifying a nucleic acid encoding MCSP, or a novel non-human homologue thereof, comprises contacting a sample comprising MCSP candidate DNA or RNA with a nucleic acid probe described above, and identifying nucleic acid(s) which hybridize (s) to that probe. In particular, nucleic acid according to the invention is useful e.g. in a method for determining the presence of MCSP-mRNA, said method comprising hybridizing the DNA (or RNA) encoding (or complementary to) a protein of the invention, or a fragment of said DNA, to test sample nucleic acid and determining the presence of the desired mRNA, or amplifying, e.g. by PCR, MCSP-RNA using MCSP specific oligonucleotide primers derivable from a nucleic acid sequence provided herein. This method may be employed in tumor diagnosis, e.g. for localization of MCSP mRNA in a tumor, particularly primary melanomas or metastatic lesions of malignant melanoma. For example, specific in situ hybridization signals for MCSP mRNA expression obtained with antisense RNA probes according to the invention are clearly associated with cells obtained from a metastatic lesion of malignant melanoma. Only non-specific background signals are found with tumor infiltrating cells. Hybridization with sense control RNA yields only non-specific background signals.

The DNA encoding a protein of the invention can be incorporated into vectors for further manipulation. Furthermore, the invention concerns a recombinant DNA which is a hybrid vector comprising at least one of the above mentioned DNAs.

A hybrid vector of the invention comprises an origin of replication or an autonomously replicating sequence, one or more dominant marker sequences and, optionally, expression control sequences, signal sequences and additional restriction sites.

Preferably, the hybrid vector of the invention comprises an above described nucleic acid insert operably linked to an expression control sequence, in particular those described hereinafter.

Vectors typically perform two functions in collaboration with compatible host cells. One function is to facilitate the cloning of the nucleic acid that encodes the protein of the invention, i.e. to produce usable quantities of the nucleic acid (cloning vectors). The other function is to provide for replication and expression of the gene constructs in a suitable host either by maintenance as an extrachromosomal element or by integration into the host chromosome (expression vectors). A cloning vector comprises the DNAs as described above, an origin of replication or an autonomously replicating sequence, selectable marker sequences, and optionally, signal sequences and additional restriction sites. An expression vector additionally comprises expression control sequences essential for the transcription and translation of the DNA of the invention. Thus an expression vector refers to a recombinant DNA construct such as a plasmid, a phage, recombinant virus or other vector that upon introduction into a suitable host cell, results in expression of the cloned DNA. Suitable expression vectors are well known in the art and include those that are replicable in eukaryotic and/or prokaryotic cells.

Most expression vectors are capable of replication in at least one class of organisms but can be transfected into another organism for expression. For example, a vector is cloned in E. coli and then the same vector is transfected into yeast or mammalian cells even though it is not capable of replicating independently of the host cell chromosome. DNA may also be amplified by insertion into the host genome. However, the recovery of genomic DNA encoding MCSP is more complex than that of exogenously replicated vector because restriction enzyme digestion is required to excise MCSP DNA. DNA can be amplified by PCR and be directly transfected into the host cells without any replication component

Advantageously, expression and cloning vectors according to the invention contain a selection gene also referred to as selectable marker. This gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will not survive in the culture medium. Typical selection genes encode proteins that confer resistance to antibiotics and other toxins, e.g. ampicillin, neomycin, methotrexate or tetracycline, complement auxotrophic deficiencies, or supply critical nutrients not available from complex media.

Since the amplification of the vectors is conveniently done in E. coli, an E. coli genetic marker and an E. coli origin of replication are advantageously included. These can be obtained from E. coli plasmids, such as pBR322, Bluescript vector or a pUC plasmid.

Suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up MCSP nucleic acid, such as dihydrofolate reductase (DHFR, methotrexate resistance), thymidine kinase, or genes confering resistance to G418 or hygromycin. The mammalian cell transfectants are placed under selection pressure which only those transfectants are uniquely adapted to survive which have taken up and are expressing the marker.

Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to MCSP nucleic acid. Such promoter may be inducible or constitutive. The promoter is operably linked to DNA encoding a protein of the invention by removing the promoter from the source DNA by restriction enzyme digestion and inserting the isolated promoter sequence into the vector.

Promoters suitable for use with prokaryotic hosts include, for example, the β-lactamase and lactose promoter systems, alkaline phosphatase, a tryptophan (trp) promoter system and hybrid promoters such as the tac promoter. Their nucleotide sequences have been published, thereby enabling the skilled worker operably to ligate them to DNA encoding a protein of the invention, using linkers or adaptors to supply any required restriction sites. Promoters for use in bacterial systems will also generally contain a Shine-Delgarno sequence operably linked to the DNA encoding MCSP.

MCSP gene transcription from vectors in mammalian host cells may be controlled by promoters compatible with the host cell systems, e.g. promoters derived from the genomes of viruses. Suitable plasmids for expression of the protein of the invention in eukaryotic host cells, particularly mammalian cells, are e.g. cytomegalovirus (CMV)

promoter-containing vectors, RSV promoter-containing vectors and SV40 promoter-containing vectors and MMTV LTR promoter-containing vectors. Depending on the nature of their regulation, promoters may be constitutive or regulatable by experimental conditions.

Transcription of a DNA encoding a protein according to the invention by higher eukaryotes may be increased by inserting an enhancer sequence into the vector.

Construction of vectors according to the invention employs conventional ligation techniques. The various DNA segments of the vector DNA are operatively linked, i.e. they are contiguous and placed into a functional relationship to each other. Isolated plasmids or DNA fragments are cleaved, tailored, and religated in the form desired to generate the plasmids required. If desired, analysis to confirm correct sequences in the constructed plasmids is performed in a manner known in the art Suitable methods for constructing expression vectors, preparing in vitro transcripts, introducing DNA into host cells, and performing analyses for assessing MCSP expression and function are known to those skilled in the art Gene presence, amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting, e.g. to quantitate the transcription of mRNA, dot blotting (DNA or RNA analysis), in situ hybridization, using an appropriately labelled probe based on a sequence provided herein, binding assays, immunodetection and functional assays.

The invention further provides host cells capable of producing a protein of the invention and including heterologous (foreign) DNA encoding said protein.

The nucleic acids of the invention can be expressed in a wide variety of host cells, e.g. those mentioned above, that are transformed or transfected with an appropriate expression vector. A protein of the invention may also be expressed as a fusion protein. Recombinant cells can then be cultured under conditions whereby the protein (s) encoded by the DNA of the invention is (are) expressed.

Suitable prokaryotes include eubacteria, such as Gram-negative or Gram-prositive organisms, such as E. coli, e.g. E. coli K-12 strains, DH5α and HB 101, or Bacilli.

Further host cells suitable for MCSP encoding vectors include eukaryotic microbes such as filamentous fungi or yeast e.g. Saccharomyces cerevisiae. Higher eukaryotic cells include insect amphebian and vertebrate cells, particularly mammalian cells. In recent years propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. The host cells referred to in this application comprise cells in in vitro culture as well as cells that are within a host animal. Advantageously, a host cell of the invention does not produce endogenous MCSP or a homologue thereof.

Stably transfected mammalian cells may be prepared by transfecting cells with an expression vector having a selectable marker gene, and growing the transfected cells under conditions selective for cells expressing the marker gene. To prepare transient

transfectants, mammalian cells are transfected with a reporter gene to monitor transfection efficiency.

Host cells are transfected or transformed with the above-captioned expression or cloning vectors of this invention and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. Heterologous DNA may be introduced into host cells by any method known in the art, such as transfection with a vector encoding a heterologous DNA by the calcium phosphate coprecipitation technique, by electroporation or by lipofectin-mediated. Numerous methods of transfection are known to the skilled worker in the field. Successful transfection is generally recognized when any indication of the operation of this vector occurs in the host cell. Transformation is achieved using standard techniques appropriate to the particular host cells used. (See, e.g. Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor

Laboratory Press).

A DNA of the invention may also be expressed in a non-human transgenic animal, particularly a transgenic warm-blooded animal, and in non-human transgenic tumor cells. Methods for producing a transgenic animal, including mouse, rat, rabbit, sheep and pig, are known in the art and are disclosed, for example, by Hammer et al. (Nature 315, 680-683, (1985)). For instance, an expression unit including a DNA of the invention coding for MCSP together with appropriately positioned expression control sequences, is introduced into pronuclei of fertilized eggs, or in tumor cells. Introduction may be achieved, e.g. by microinjection. Integration of the injected DNA is detected, e.g. by blot analysis of DNA from suitable tissue samples. It is preferred that the DNA be incorporated into the germ line of the animal, so that it is passed to the animal's progeny. Transgenic tumor cells are introduced into a suitable animal.

Furthermore, a knock-out animal may be developed by introducing a mutation in the endogenous MCSP-homologue, thereby generating an animal, which does not express the functional MCSP-homologue gene anymore. For example, in a rat the NG2 gene may be knocked out A mutated or nonmutated MCSP gene is introduced into the knock-out animal. Expression of human counterpart MCSP on a homologous gene knock-out background has the unique advantage of excluding differences in efficacies of a potential drug on the given protein (in this case MCSP) caused by species-specific sequence differences in said protein.

In a further aspect the invention relates to an assay for identifying a compound which is capable of interacting with MCSP, comprising contacting cells containing a heterologous DNA encoding a protein of the invention and producing said protein with at least one compound to be tested for its ability to interact with MCSP, and analysing cells for a difference in ligand binding or signal transduction. Suitable analysing methods are known in the art, or may be readily designed based on the known methods and the guidelines provided herein. Preferably, the heterologous DNA comprises substantially the entire coding region. The result obtained in such assay is compared to an assay suitable as a negative control.

Assay methods generally require comparison to various controls. A change in MCSP activity or function is said to be induced by a test compound if such an effect does not occur in the absence of the test compound. An effect of a test compound on a protein of the invention is said to be mediated by said protein if this effect is not observed in cells which do not produce said protein.

For example, by interacting with MCSP compounds may affect melanoma cell growth and spreading, cell-adhesion including cell-substratum interaction and cell-cell contact and MCSP related signal transduction, thus being potential anti-tumor drugs. An assay as described above is suitable to identify a compound which is capable of inhibiting the binding of collagen VI to MCSP.

The invention particularly relates to the specific embodiments (proteins, nucleic acids, methods for the preparation and uses thereof) as described in the Examples which serve to illustrate the present invention, but should not be construed as limitation thereof.

Example 1: Isolation of MCSP cDNA

A radiolabeled approximately 4.0 kb cDNA encoding the carboxyl-terminus of the rat NG2 transcript (G11, cf. Fig. 2 in Nishiyama et al., J. Cell Biol. 114, 359-371 (1991), is used to screen a λgt11 human melanoma cDNA library prepared from RNA extracted from the M21 human melanoma cell line (Clontech Laboratories, San Francisco, CA) for MCSP candidate cDNA clones. An initial screen of recombinant phages containing dT-primed cDNA yields several NG2 reactive reactive clones.

Recombinant phages (5×10⁵) are plated at a density of 4× 10⁴ plaques per 150 mm petri dish and propagated for 12 hrs at 37°C in the Y 1090r E. coli host strain. Phage DNA is transferred to nitrocellulose filters, denatured in 0.5 N NaOH, 1.5 mM NaCl and subsequently neutralized in 0.5 M Tris-HCl, pH 8.0, 1.5 M NaCl. Non-specific nucleic acid binding sites are blocked in a prehybridization medium consisting of 2xSSC (SSC: 150 mM sodium chloride, 15 mM sodium citrate) and 50 μg/ml denatured, sheared salmon sperm DNA at 55°C for 2 hrs. Hybridization reactions are performed under the same conditions for 8 to 12 hrs in the presence of 10 ng/ml of G11 NG2 cDNA fragment (supra) radiolabeled with ³²P adCTP to a specific activity of 4×10⁸ cpm/μg by random priming (Sam brook et al, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989)). Following hybridization, the filters are washed in 2xSSC at 55°C and exposed to XAR film (Eastman Kodak) for autoradiography. This screen yields seven NG2 reactive clones, including one clone designated λM3.1, containing 3.1 kb of cDNA. Nucleotide sequence analysis of this isolate by the chain termination method of Sanger et al. (Sambrook et al., supra) indicates an open reading frame encoding 700 carboxyl-terminal amino acid residues with approximately 79% homology to the NG2 protein.

The 3.1 kb isolate of the λM 3.1 clone (supra) is utilized as probe in Northern analysis of polyadenylated RNA extracted from human melanoma cell lines expressing the mAb 9.2.27-reactive antigen. RNA gel blotting and hybridization is done by size-fractionation of 2 μg of polyadenylated RNA on 1.2% formaldehyde-agarose gels and transferred onto nytran. Prehybridization and hybridization conditions are as described above. Transcript sizes are estimated by comparison with size standards (Gibco-Bethesda Research

Laboratories). Hybridization to an 8.0 kb transcript present in M21 melanoma cells (Bumol et al., J. Biol. Chem. 267, 12733-12741 (1991)) and UAC 903 melanoma cells is observed, said transcript being similar in size to the NG2 transcript. The UAC903 cell line expresses approximately five-fold more MCSP transcript than the M21 cell line, an observation that is consistent with a similar elevation in the level of MCSP core protein produced in these cell lines. The cDNA fail to hybridize to RNA from the mAb

9.2.27-unreactive RAJI lymbhoblastoid cell line (Dierich et al., J. Immunol. 112, 1766-1773 (1974)).

A 500 bp fragment from the 5' end of λM3.1 (extending from bp 4858 to 5357 in SEQ ID NO:1) is employed as a probe to screen 5 × 10⁵ clones from two independent λgt11 cDNA libraries derived from the human melanoma cell line M21 for overlapping cDNA clones extending further upstream. Exhaustive screening of four independent melanoma cell cDNA libraries with λM3.1 fails to identify clones extending further upstream of the 5' end of λM3.1.

The impossibility to obtain a complete cDNA strand is probably due to MCSP RNA secondary structure. Therefore, a strategy is developed that allows to obtain small overlapping cDNA clones. cDNA synthesis for sequence determination of the entire coding sequence of MCSP core protein is accomplished by polymerase chain reaction (PCR) using a wide range of different primers (see Table 1) and a variety of different RNA denaturation conditions to secure that suitable conditions for each individual portion are present in a sample. Thus, cDNA fragments are obtained which are suitable for PCR amplification.

Experimental details for the generation of PCR-amplified cDNA clones are as follows: RNA is prepared from A375-Met human melanoma cells (Kozlowski et al.,

J. Natl. Cancer Inst 72, 913-917 (1984); cell line A 357 is obtainable from the American Type Culture Collection (ATCC) under accession no. ATCC CRL 1619) by the acid guanidinium thiocyanate phenol-chloroform method (Chomzynski and Sacchi, Anal. Biochem. 162, 156 (1987)). Polyadenylated RNA is obtained using a Qiagen Oligotex mRNA preparation kit (Diagen, Hilden, Germany). First strand cDNA is prepared with an M-MuLV Reverse Transcriptase Kit (Life Technologies, Gaithersburg, MD) and either MCSP sequence-specific oligonucleotides, oligo (dT) or random primers in parallel samples. Generally, the best results are obtained with MCSP sequence-specific oligonucleotides (cf. Table 1, infra). On several occasions, the primary products are reamplified with a nested 5' primer to improve specificity. PCR amplifications are done with the primers indicated in Table 2 applying standard protocols (Rolfs et al., PCR:

Clinical Diagnostics and Research, Springer Verlag, Berlin (1992)). Taq, Pwo (Boehringer Mannheim, Germany) or Pfu DNA polymerase (Stratagene, La Jolla, CA) are applied as thermostable polymerases. Anchored PCR is performed after dG tailing of cDNA

(Pluschke et al., Eur. J. Immunol. 21, 2749-2754 (1991)). Prior to tailing, first strand cDNA is treated with RNAse H (Life Technologies) and purified with a Glass MAX DNA isolation spin cartridge system (Life Technologies). cDNA is dG-tailed using the

Deoxynucleotidyl Terminal Transferase kit (Life Technologies). Oligonucleotides are obtained from Microsynth (Windisch, Switzerland). PCR amplification products of the expected size are isolated from 1% agarose gels and are introduced into plasmids pBluescript KS (Stratagene) or pGEM-1 (Promega, Madison, WI) either by blunt-end cloning into the Hinc II site (Table 1) or by directional cloning (Table 2). Double-stranded plasmid DNAs are sequenced directly with the Sequenase kit (US Biochemical,

Cleveland, OH). Sequence data are processed with the aid of a GCG Wisconsin Software Package (Genetics Comuting Group, Madison, WI).

In a first step, anchored PCR is used to obtain a clone designated an44 (Table 1) , that extends 5' of λM3.1. After dG tailing of cDNA, oligo dC is applied as 5' primer and a sequence corresponding to the 5' end of λM3.1 is used as 3' anti-sense primer in this amplification. Subsequently, a series of seven additional cDNA clones are generated by PCR to cover the entire MCSP coding sequence (see Table 1, infra). Anchored PCR with oligo dC 5' primers are applied for the amplification of three of these overlapping clones (an2, an38 and an1; Table 1), while conventional PCR using 5' sense primers that correspond to rat NG2 sequences are employed for the generation of the remaining four clones (ra23, ra4, ra1 and ra25; Table 1). Taq is used as DNA polymerase. Sequences of the 3' anti-sense primers used for the generation of these PCR clones are complementary to the 5' end of the respective previous clones. In five cases (cf. Table 1), the primary products are reamplified with a nested 5' primer to improve specificity. Nucleotide sequences derived from this first series of PCR clones and from the λgt11 clone are reconfirmed by analyzing a second set of independently derived overlapping PCR clones (Table 2). Discrepancies which are probably caused by mistakes introduced by PCR amplification (Keohavong and Thilly, Proc. Natl. Acad. Sci. USA 86, 9253 (1989)), are resolved by further analyzing independently-derived PCR clones (Table 2).

The complete coding sequence of the MCSP core protein and the deduced amino acid sequence are shown in SEQ ID NO:1. An open reading frame coding for 2322 amino acids is found. The 3'untranslated region consists of 926 nucleotides. The first 29 amino acids (amino acids -29 to -1; SEQ ID NO:1) represent a putative signal sequence, which is only 48% identical with that of NG2. The subsequent stretch of 18 amino acids is 89% identical. A hydrophobic segment of 25 consecutive amino acid residues near the carboxy terminus (amino acid residues 2193-2217, SEQ ID NO:2) is followed by several basic arginine and lysine residues and thus meets the criteria for a transmembrane domain. Thus, the deduced amino acid sequence of the MCSP core protein predicts an integral membrane protein comprising a large extracellular domain separated from a relatively short cytoplasmic tail (75 amino acids) by a single hydrophobic transmembrane region of 25 amino acids.

The large extracellular domain of MCSP spanning 2192 amino acids can be roughly divided into three structural domains: an amino- terminal domain (amino acids 1-611, SEQ ID NO:1) containing eight cysteines and three serine/glycine pairs; a cysteine-free, a serine/glycine rich domain (amino acids 612-1561, SEQ ID NO:1) including seven such potential attachment sites for glycosaminoglycans; and a third structural domain (amino acids 1562 to 2192, SEQ ID NO:1) with only two cysteines and one serine/glycine pair.

The first structural domain (amino acids 1 to 611), which is approximately 82 % structurally homologous to the corresponding domain in the rat NG2 proteoglycan, contains three of the 15 potential N-linked glycosylation sites. This domain also appears to have a compact configuration, since it contains eight of the ten cysteines of the entire ectodomain, i.e. four potential disulfide bridges in a region spanned by 611 amino acids.

A key feature of the second structural domain of the MCSP ectodomain (amino acids 612 to 1561; SEQ ID NO:1) is its lack of cysteines in a region spanning 950 amino acids; however, this domain contains seven of the eleven serine/glycine pairs of the MCSP extracellular domain, which can serve as potential chondroitin sulfate attachment sites; however, the signal sequence SerGlyXGly for glycosaminoglycans (GAG) occurs only once (amino acids 1308-1340, SEQ ID NO:1). Six of the 15 potential N-linked MCSP glycosylation sites are found in this domain, which is 79% structurally homologous with its counterpart in the rat NG2 proteoglycan.

The third structural domain of MCSP encompassing 630 amino acids (amino acids 1562 to 2192, SEQ ID NO:1) is approximately 75% homologous in structure with the

corresponding domain of NG2. This domain consists of two cysteines, separated by 105 amino acids and likely forms a disulfide bridge. The domain has only one potential GAG attachment site indicated by one serine/glycine pair and contains six of the 15 potential N-linked glycosylation sites of the MCSP ectodomain. This is in contrast to the corresponding NG2 domain that features eight cysteines, one serine/glycine pair and five of its 11 potential N-linked glycosylation sites. The major difference between the deduced sequences of NG2 and MCSP is evident between amino acid residues 2043 and 2091. Thus, for NG2, it is reported that a cluster of six cysteine residues is present in this region (Nishiyama et al., supra, Fig. 3). An alignment of the MCSP and NG2 gene portion encoding for this region reveals three additional bases in the MCSP sequence, which are not found in the NG2 gene. The first additional base in position 6128 (SEQ ID NO:1) causes a difference in the reading frame, which continues after the second additional base in position 6244, but is resolved after the third additional base in position 6273. The three additional bases are found both in the λgt11 (??) cDNA clone and in several independent PCR clones derived from the human melanoma cell lines M21 and A375-Met,

respectively.

Example 2: Localization of MCSP-Encoding mRNA in Melanoma Lesions by In Situ

Hybridization

Three cDNA fragments corresponding to different regions of the MCSP core protein coding sequence are used as riboprobe templates for in situ hybridization experiments. Clones 11, ra23 and an44 (cf. Table 1) carry MCSP core protein inserts of 559 bp

(3756-4314 in pGEM-1), 811 pb (1431-2241 in pBluescript KS) and 677 bp (42113-4889 in pGEM-1), respectively. Sense and anti-sense RNA probes are labeled according to the instructions of the manufacturer (RNA Transkription Kit, Boehringer) with αS³⁵-UTP (more than 400 Ci/mmol, Amersham) to a specific activity of more than 10⁹ dpm/μg. Labeled riboprobes are extracted with phenol/chloroform and free nucleotides are removed by passage over a Sephadex G50 column. RNA is precipitated in 2.2 M ammonium acetate in 77% ethanol overnight at -20°C and is resuspended to an

approximate activity of 250,000 cpm/μl (5x concentrated stock solution) in 50% v/v dionized formamide containing 20 mM dithiothreithol and stored at -70°C.

Biopsies of melanoma skin metastases, primary melanomas and benign nevi are fixed in 4% paraformaldehyde in phosphate buffered salt solution (PBS) and then embedded in paraffin. Paraffin sections (8 μm) are placed on 3-aminoprophyltriethoxysilane-treated slides, which bind sections covalently to the glass surface and prevent loss of sections during experimental procedures. Paraffin sections are deparaffinized in xylene and absolute ethanol and air dried. Following rehydration with ethanol solutions of decreasing concentrations, sections are postfixed with 4% paraformaldehyde in PBS for 5 min, rinsed in PBS and water and depurinated for 20 min with 0.2 N HCl at room temperature. These sections are then treated for 30 min with 2xSSC(0.3 M NaCl, 0.03 M Na-citrate, pH 7.0) at 70°C, dehydrated with increasing ethanol solutions and finally air dried.

Pre-hybridization is performed at 54°C for 3 hrs in a solution of 50% v/v deionized formamide, 10% w/v dextransulfate, 0.3 M NaCl, 10 mM Tris, 10 mM sodium phosphate pH 6.8, 20 mM dithiothreitol, 0.2×Denhardts reagent 0.1 mg/ml Escherichia coli RNA and 0.5 μM non-radioactive αS-UTP. Hybridization is done overnight in the same solution, supplemented with 5×10⁴ cpm/μl αS³⁵-UTP-labeled RNA probe in a humified chamber at 54°C. Slides are washed in the hybridization solution lacking dextransulfate, RNA and non-radioactive UTP, but containing 50% v/v deionized formamide and 10 mM dithiothreitol at 55°C, two times for 1 hr, and equilibrated for 15 min in a buffer solution consisting of 0.5 M NaCl, 10 mM Tris, 1 mM EDTA, 10 mM dithiothreitol, pH 7.5.

Sections are then treated with 50 μg/ml RNase A in equilibration buffer for 30 min at 37°C to remove non-specifically bound probe. This is followed by washing in 2xSSC for 1 hr and then in 0.1xSSC for 1 hr at 37°C. Slides are sequentially dehydrated in 65%, 85% and 95% (v/v) ethanol solutions containing 300 mM ammonium acetate and in absolute ethanol before being air dried. Sections are coated with a 1.2 dilution of Ilford K5 photoemulsion, air dried and exposed for 12 days in a light safe box containing silica gel at 4°C. The slides are then placed into D19 developer (Kodak), fixed in 30% sodium thiosulfate and stained with Haematoxylin and Eosin. The pattern of hybridization signals on autoradiographed sections is analyzed with a photomicroscope and brightfield/darkfield illuminations.

All three probes of the MCSP coding sequence reveal comparable results. Biopsies from melanoma skin metastases that react strongly with MCSP-specific antibodies mAb 9.2.27 (Morgan et al., Hybridoma 1, 27-36 (1981)) or 763.74 (Giacomini et al., J. Immunol. 135, 696 (1985)) show abundant hybridization signals in cancer cells with all three anti-sense RNA probes and only background hybridization with the control sense RNA probes. Some hybridization is also detected in samples of benign nevi and normal epidermis, which exhibit no abundant staining with MCSP-specific antibodies.

Claims

1. Isolated protein designated melanoma-associated chondroitin sulfate proteoglycan (MCSP) and having substantially the amino acid sequence of the mature protein as set forth in SEQ ID NO:2 or an amino acid mutant of said protein excluding the deletional amino acid mutant with the amino acid sequence extending from the amino acid at position 1594 to the amino acid at position 2293 in SEQ ID NO:2, or a derivative of said protein or mutant, particularly such protein, amino acid mutant, or derivative thereof, which is in a suitably immunogenic form.

2. A method for preparing the protein, mutant or derivative according to claim 1 comprising isolation from a natural source, chemical synthesis and/or recombinant DNA technology.

3. A method for the generation of an antibody which specifically binds to MCSP, said method comprising the step of administering to a mammal a protein or mutant, or a derivative thereof, according to claim 1.

4. A pharmaceutical composition comprising a protein or mutant, or a derivative thereof, according to claim 1, and a pharmaceutically acceptable carrier.

5. Nucleic acid comprising an isolated nucleic acid coding for a protein according to claim 1, preferably such nucleic acid which is a DNA.

6. A method for identifying a nucleic acid encoding MCSP, or a novel non-human homologue thereof, comprising contacting a sample comprising candidate DNA or RNA with a nucleic acid comprising at least 14 contiguous bases that are the same as (or complementary to) any 14 or more contiguous bases set forth in SEQ ID NO: 1, and identifying nucleic acid(s) which hybridize (s) to said probe.

7. A host cell capable of producing the protein, amino acid mutant, or derivative thereof according to claim 1, and containing a heterologous nucleic acid coding for said protein or mutant, or derivative thereof.

8. The protein, mutant or derivative according to claim 1 for use in the prophylactical or therapeutical treatment of the human body, particularly for use in the control or (adjuvant) treatment of a MCSP-expressing tumor, such as melanoma, sarcoma and glioblastoma.

9. Use of the protein, mutant or derivative according to claim 1 , or a composition comprising said protein, mutant or derivative for the manufacture of a medicament, particularly a medicament suitable for the control or (adjuvant) treatment of a

MCSP-expressing tumor, such as melanoma, sarcoma and glioblastoma.

10. A method for identifying a compound which is capable of interacting with the protein, amino acid mutant, or derivative thereof according to claim 1, comprising contacting said protein, mutant or derivative, or a composition of matter comprising said protein, mutant, or derivative, with at least one compound to be tested for its ability to interact with said protein, mutant, or derivative, wherein a change of the biological activity of the protein, mutant, or derivative is indicative of the interaction.

11. Nucleic acid according to claim 5, wherein the isolated nucleic acid is a DNA having substantially the nucleotide sequence set forth in SEQ ID NO: 1 , or a fragment thereof excluding the fragment with the sequence extending from bp 4867 to bp 7898 in SEQ ID NO:1 and the DNA with the sequence extending from bp 4858 to 5357 in SEQ ID NO:1, respectively..

12. Nucleic acid according to claim 5, which is a hybrid vector.

13. The amino acid mutant according to claim 1, which is a deletional amino acid mutant, or a derivative thereof.

14. A method of vaccinating a human in need thereof comprising the step of administering to said human a protein or mutant, or a derivative thereof, according to claim 1.

15. A test kit for the qualitative or quantitative determination of anti-MCSP antibodies comprising a protein or mutant, or a derivative thereof, according to claim 1.