WO1996011279A2 - Enhanced immune response by introduction of cytokine gene and/or costimulatory molecule b7 gene in a recombinant virus expressing system - Google Patents
Enhanced immune response by introduction of cytokine gene and/or costimulatory molecule b7 gene in a recombinant virus expressing system Download PDFInfo
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- WO1996011279A2 WO1996011279A2 PCT/US1995/012638 US9512638W WO9611279A2 WO 1996011279 A2 WO1996011279 A2 WO 1996011279A2 US 9512638 W US9512638 W US 9512638W WO 9611279 A2 WO9611279 A2 WO 9611279A2
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Classifications
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/54—Interleukins [IL]
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- 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
- C07K14/52—Cytokines; Lymphokines; Interferons
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- C07K14/55—IL-2
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- 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
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- 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
- C07K14/70503—Immunoglobulin superfamily
- C07K14/70532—B7 molecules, e.g. CD80, CD86
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/24011—Poxviridae
- C12N2710/24041—Use of virus, viral particle or viral elements as a vector
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- C12N2710/00011—Details
- C12N2710/24011—Poxviridae
- C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
- C12N2710/24141—Use of virus, viral particle or viral elements as a vector
- C12N2710/24143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
Definitions
- the present invention relates to recombinant viral vector vaccines for the prevention or treatment of pathogenic diseases and cancer. More particularly, it relates to recombinant viral vector vaccines comprising a genes encoding an antigens and a gene(s) encoding an immunostimulatory molecule(s).
- Tumor infiltrating lymphocytes have been identified in patients with melanoma that appear to recognize unique cancer antigens in an MHC restricted fashion. (Rosenberg, S.A. J. Clin. Oncol. 10:180-199, 1993; Topalian, S.C. et al Tumor-infiltrating lymphocytes: Evidence for specific immune reactions against growing cancers in mice and humans. In: De Vita, Hellman and Rosenberg (eds) Important Advances in Oncology.
- T lymphocytes In contrast to antibodies that recognize epitopes on intact proteins, T cells recognize short peptide fragments (8-18 amino acids) that are presented on cell surface class I or II major histocompatibility (MHC) molecules and it is likely that tumor associated antigens are presented and recognized by T cells in this fashion.
- MHC major histocompatibility
- MART-1 Melanoma Antigen Recognized by T Cells - 1
- gplOO gplOO
- the genes encoding both of the peptides have been cloned and sequenced.
- the MART-1 gene encodes a 118 a ino acid protein of 13 kD.
- the gplOO gene encodes a protein identical to that recognized by monoclonal antibody HMB- 45. With the exception of melanocytes and retina no normal tissues express this antigen and no expression of these gene products has been seen on cancers other than melanoma. Both antigens, therefore, appear to be melanocyte lineage specific.
- the MART-l antigen was expressed on all nine tissue culture lines tested (that were established from melanomas in the Surgery Branch, NCI) and on all fresh melanomas tested. Studies by others showed that the MART-1 (also called Melan-A) antigen was expressed on 26 of 26 fresh melanomas. Coulie, P.G. et al J. Exp. Med. 180:35-42, 1992. The gplOO antigen is also widely expressed in melanomas. In one study, reactivity with antibody HMB-45 (reactive with gplOO) was present on 100% of non-spindle cell type melanomas and on 62 of 67 total melanomas. Wick, M.R. et al J. Cutan. Pathol.
- TIL cells that were raised in the Surgery Branch, NCI from different HLA-A2 individuals, 13 of 14 recognized MART-1 and 4 of 14 recognized the gplOO antigen. Because TIL cells that recognize these determinants have been shown to be capable of mediating cancer regression in vivo, it appears that these antigens are involved in cancer regression.
- MAGE-1 Another gene coding for a human tumor specific antigen on a human melanoma was cloned by Van der Bruggen et al (Science 254:1643-1647, 1991). This antigen is coded for by a gene called MAGE-1 which spans five kilobases. A 2,419 base pair coding sequence produces a predicted protein product of 26 kD.
- the MAGE antigen is HLA-A1 restricted and the nine amino acid fragment that represents the Al restricted immunodominant peptide has been defined as Glu-Ala-Asp-Pro-Thr-Gly-His-Ser-Tyr. This nine amino acid peptide is encoded by the third exon of the MAGE gene.
- Transfection of a 500 base pair fragment of this gene can confer recognition by a MAGE specific CTL clone.
- Incubation of an EBV cell line with the immunodominant peptide can confer sensitivity to lysis by a MAGE-l specific CTL clone.
- MAGE-l does not appear to be expressed in normal cells with the possible exception of testis, but is expressed on approximately half of metastatic melanomas, about 20% of breast cancers as well as other selected types of cancer.
- Peptides representing antigenic epitopes of HIV gpl20 and gpl60 emulsified in complete Freund's adjuvant can also prime specific CTL responses (Takahashi, H. et al Proc. Nat'1 Acad. Sci. U.S.A. 85:3105-3109, 1988; Hart, M.K. et al Proc. Nat'l Acad. Sci. U.S.A. 88:9448-9452. 1991) .
- B7-1 which is the ligand for T-cell surface antigens CD28 and CTLA4 (Schwartz, R.H. Cell 71:1065-1068, 1992; Chen, L. et al.
- Vaccinia viruses have been extensively used in humans and the use of a vaccina based vaccine against smallpox has led to the worldwide eradication of this disease (reviewed in reference Moss, B. Science 252:1662-1667, 1991) .
- Vaccinia viruses have the advantages of low cost, heat stability and a simple method of administration. Attempts have been made to develop vaccinia virus vectors for the prevention of other diseases.
- Vaccina virus is a member of the pox virus family of cytoplasmic DNA viruses. DNA recombination occurs during replication of pox viruses and this has been used to insert DNA into the viral genome. Recombinant vaccina virus expression vectors have been extensively described. These vectors can confer cellular immunity against a variety of foreign gene products and can protect against infectious diseases in several animal models. Recombinant vaccina viruses have been used in human clinical trials as well. Cooney et al immunized 35 healthy HIV seronegative males with a recombinant vaccinia virus expressing the gpl60 envelope gene of HIV (Cooney, E.. The Lancet 337:567-572, 1991).
- vaccinia virus is administered by intradermal scarification and side effects have been minimal including local skin irritation, lymphadenopathy and transient flu ⁇ like symptoms.
- Fowlpox viruses are members of the pox virus family (avipox virus genes) . Fowlpox virus will only replicate in avian cells and cannot replicate in human cells. It is a cytoplasmic virus that does not integrate into the host genome but is capable of expression of a large number of recombinant genes in eukaryotic cells.
- Recombinant fowlpox virus expressing rabies glycoprotein has been used to protect mice, cats and dogs against live rabies virus challenge. Immunization of chickens and turkeys with a recombinant fowlpox expressing the influenza HA antigen protected against a lethal challenge with influenza virus (Taylor, J. et al Vaccine 6:504-508, 1988). Canarypox virus, another member of the avipox genus similar to fowlpox, was safely administered subcutaneously to 25 normal human volunteers at doses up to 10" infectious doses (Cadox, M. et al The Lancet 339:1429-1432, 1992).
- Fowlpox virus thus represents an attractive vehicle for immunization since it can stimulate both humoral and cellular immunity, it can be economically produced in high titer (10 9 pfu/ml) and yet its inability to productively infect human cells substantially increases the safety of its use, compared to replicating viruses such as vaccina view, especially in immunocompromised hosts.
- fowlpox virus Another considerable advantage of fowlpox virus is that there is apparently little or no cross-reactivity with vaccinia virus and thus previously vaccinated humans will not have pre-existing immune reactivity to fowlpox virus proteins.
- the present invention is a recombinant virus comprising a viral genome or portion thereof, one or more nucleic acid sequences encoding one or more antigens of a disease causing agent and one or more nucleic acid sequences encoding one or more immunostimulatory molecules.
- the present invention is also a composition
- a composition comprising a recombinant virus comprising a viral genome or portion thereof and one or more nucleic acid sequences encoding one or more antigens of a disease causing agent and optionally an exogenous immunostimulatory molecule, chemotherapeutic drug, antibiotic, antifungal drug, antiviral drug, or combination thereof.
- compositions comprising a recombinant virus comprising a viral genome or portion thereof, one or more nucleic acid sequences encoding one or more antigens of a disease causing agent and one or more nucleic acid sequences encoding one or more immunostimulatory molecules and optionally an exogenous immunostimulatory molecule, chemotherapeutic drug, antibiotic, antifungal drug, antiviral drug, or combination thereof.
- Another aspect of the present invention is to provide a therapeutic composition and a method of treating or preventing a disease in a mammal comprising administering to the mammal an effective amount of a recombinant virus and optionally an exogenous immunostimulatory or immunomodulator, chemotherapeutic drug antibiotic, antifungal drug, antiviral molecule, the amount effective in preventing or ameliorating the disease.
- the vaccine of the present invention is able to prevent or inhibit infection or disease caused by the disease causing agent.
- Figure 1 shows j3-Galactosidase production after BSC1 cell infection with various recombinant vaccinia virus constructs after 24 hr ( ⁇ ) or 36 hr (D) incubation.
- Figure 2 shows the primary response of mice at Day 6 after injection I.V. with various recombinant vaccinia virus constructs as follows: GM-CSFrW (D) ; IL-2rW (•) ; TNF ⁇ rW ( ⁇ ) ; IFN ⁇ rW(v); VJS6 (O) . Effector: Target cells ratios are plotted versus % specific 5l Cr release by targets.
- Figure 3 shows the results from secondary cultures of mice at Day 6 after injection I.V. with various recombinant vaccinia virus constructs as follows: GM- CSFrW (D) ; IL-2rW (•) ; TNF ⁇ rW ( ⁇ ) ; IFN ⁇ rW(v); VJS6 (O) . Effector: Target cells ratios are plotted versus % specific 51 Cr release by targets.
- Figure 4 shows the results from secondary cultures of mice at Day 14 after injection I.V. with various recombinant vaccinia virus constructs as follows: GM- CSFrW (D) ; IL-2rW (•) ; TNF ⁇ rW (T) ; IFN ⁇ rW(v); VJS6 (O) . Effector: Target cells ratios are plotted versus % specific 51 Cr release by targets.
- Figure 5 shows the primary response of mice against vaccinia-infected CT26 tumor cells at Day 6 after injection S.C. with various recombinant vaccinia virus constructs as follows: GM-CSFrW (•) ; IL-2rW (D) ; TNF ⁇ rW (v); iFN ⁇ rW(v); VJS6 ( ⁇ ) . Effector: Target cells ratios are plotted versus % specific 51 Cr release by targets.
- Figure 6 shows the titration of lytic units 30% from a primary response of mice against vaccinia-infected CT26 tumor cells at Day 6 after injection S.C. with various recombinant vaccinia virus constructs as follows: GM- CSFrW (•) ; IL-2rW (D) ; TNF ⁇ rW (v) ; IFN ⁇ rW( ⁇ ) ; VJS6 ( ⁇ ) . Effector cells per well is plotted versus % specific 51 Cr release from CT26VAC target cells.
- Figure 7 shows the results from secondary cultures of mice at Day 14 after injection S.C. with various recombinant vaccinia virus constructs as follows: GM- CSFrW (D) ; IL-2rW (•) ; TNF ⁇ rW ( ⁇ ) ; IFN ⁇ rW(v); VJS6 (0) . Effector: target cells ratios are plotted versus % specific 3, Cr release by targets.
- Figure 8 shows active anti-tumor immunotherapy in mice using exogenous IL-2 together with recombinant vaccinia virus expressing a tumor associated antigen.
- Treatment groups included HBSS (—) ; rW+BG ( ); rW-BG +
- HD IL-2 (100,000 I.U., I.P., BID x 3 days) (•) ; rW+BG + HD IL-2 (100,000 I.U., I.P., BID x 3 days) (- - - -); rW + BG + LD IL-2 (15,000 I.U., BID x 3 days (-) .
- Survival time (days) is plotted versus proportion of surviving mice.
- Figure 9 shows survival data in an active-tumor immunotherapy model in mice with an established non- transduced tumor, CT26.
- Days after tumor injection is plotted versus % surviving mice.
- Figure 10 shows survival data in an active tumor immunotherapy model in mice with established j ⁇ -gal expressing tumor, CT 26.C25. GM-CSFrW (A) ; IL-2rW ( ⁇ ) ;. TNF ⁇ rW ($ ); IFN ⁇ rW( ) ; VJS6 ( ⁇ ) ; HBSS (•) . Days after tumor injection is plotted versus % surviving mice.
- Figure 11 shows active treatment of established pulmonary metastases using various recombinant vaccinia constructs. The average number of pulmonary metastases is plotted versus recombinant vaccinia vector used for treatment.
- HB2m Human Beta-2-microglobulin rW
- Ld murine H-2L d MCH Class I molecule rW
- NA Neuramimidase rW
- MVA Ankara - attenuated vaccinia virus
- Kb murine H-2K b MCH Class I molecule rW.
- FIG. 12 shows active treatment of established pulmonary metastases using various recombinant vaccinia constructs. The average number of pulmonary metastases is plotted versus recombinant vaccinia vector used for treatment.
- ICAM-1 Intracellular adhesion molecule-1 rW
- D d murine H2-D d MCH Class I molecule rW
- K d murine H-2 K d MHC Class I molecule rW
- L d H-2 L d MHC Class I molecule rW
- K b H-2K b MHC Class I molecule rW
- HLA-A2.1 Human MHC Class I molecule rW
- hjSjin Human Beta-2-microglobulin rW
- NA neuramimidase rW
- GM-CSF granulocyte, monocyte colony stimulatory factor rW
- IFN ⁇ interferon gamma rW
- TNF ⁇ tumor necrosis factor alpha rW
- Figure 13 shows a Western blot analysis of recombinant vaccinia virus expressed murine B7-1.
- BS-C-1 cells were infected with 1, v. MCB7-1; 2, v. MCMHA and extracts were prepared 20 hours post infection.
- a murine specific B7-1 hamster monoclonal antibody was used to detect expression of recombinant proteins.
- 125 I protein A (0.1 ⁇ Ci/ml) and autoradiography were used to identify bound antibody. Protein sizes were estimated using 14 C molecular weight markers (MW) .
- Figure 14 shows the results of non-irradiated BALB/c mice (5/group) which were injected intravenously with 5 x 10 5 tumor cells of CT26.WT or CT26.C25 on day #0.
- mice were immunized intravenously with different recombinant vaccinia viruses (10 7 PFUs) .
- Mice were randomized, and euthanized on day #12. Lung were harvested and stained, and pulmonary metastases were enumerated in a blinded fashion. All mice injected with CT26.WT had 500 metastases (data not shown).
- This graph represents a compilation of data drawn from several separate experiments. The individual r were involved in the following number of experiments: v. IL2/0-gal -- five; v.
- Figure 15 shows the results of non-irradiated BALB/c mice (10/group) which were injected intravenously with HBSS, v. MCB7-l//S-gal, or v. MCMHA/0-gal. Twenty-one days later mice were challenged intravenously with 5 x 10 J tumor cells CT26.C25 or CT26.WT. Mice were randomized, and euthanized on day #12. Lung were harvested and stained, and pulmonary metastases were enumerated in a blinded fashion. Duplicate experiments confirmed these results.
- Figure 16 shows the results of non-irradiated BALB/c mice (10/group) which were injected with 10 5 or 10 7 PFUs of v. MCB7-l/j8-gal, v. MCMHA/j8-gal, or HBSS on day #0. Twenty-one days later a splenectomy was performed on all immunized mice. 2 x 10 7 splenocytes from designated groups of immunized mice were adoptively transferred to similar mice (5/group) injected intravenously three days earlier with 5 x 10 s tumor cells of CT26.C25 or CT26.WT. Designated groups of mice were treated with IL-2 (100,000 IU i.p. BID x 3 days) .
- IL-2 100,000 IU i.p. BID x 3 days
- mice were randomized and euthanized nine days later. Lung were harvested and stained, and pulmonary metastases were enumerated in a blinded fashion. All mice inoculated with CT26.WT had large tumor burdens (data not shown) . Duplicate experiments confirmed these results.
- Figure 17 shows the results of non-irradiated BALB/c mice (5/group) which were injected intravenously with 5 x 10 5 CT26.WT or CT26.C25 tumor cells on day #0.
- mice were immunized intravenously with 10 7 PFUs of v. MCB7-l/j8-gal, v. MCMHA/jS-gal, v. MCB7-1/NP or HBSS.
- day# 3 - 6 designated groups of mice were treated with IL-2 (100,000 IU i.p. BID x 3 days). Mice were randomized, and euthanized on day #12. Lung were harvested and stained, and pulmonary metastases were enumerated in a blinded fashion. All mice inoculated with CT26.WT had large tumor burdens (data not shown). Four separate experiments confirmed these results.
- Figure 18 shows the results of non-irradiated BALB/c mice (5/group) which were injected intravenously with 5 x 10 s CT26.WT or CT26.C25 tumor cells on day #0.
- mice were immunized intravenously with 10 7 PFUs of v. MCB7-l/3-gal, v. MCMHA/S-gal, v. MCB7-1/NP or HBSS.
- designated groups of mice were treated with IL-2 (100,000 IU i.p. BID x 3 days). Mice were randomized, and euthanized on day #12. Lung were harvested and stained, and pulmonary metastases were enumerated in a blinded fashion. All mice inoculated with CT26.WT had large tumor burdens (data not shown). Duplicate experiments confirmed these results.
- Figure 19 shows the results of non-irradiated BALB/c mice (10/group) which were injected intravenously with 5 x 10 s CT26.WT or CT26.C25 tumor cells on day #0.
- mice were immunized intravenously with 10 7 PFUs of v. MCB7-l/j8-gal, v. MCMHA/ / S-gal, v. MCB7-1/NP or HBSS.
- day# 3 - 6 designated groups of mice were treated with IL-2 (100,000 IU i.p. BID x 3 days). All mice inoculated with CT26.WT were dead by day # 41 (data not shown) . Survival was followed daily and events recorded as deaths.
- Figure 20 shows the results of non-irradiated BALB/c mice (5/group) which were injected intravenously with 5 x 10 s CT26.WT or CT26.C25 tumor cells on day #0.
- mice were immunized intravenously with 10 7 PFUs of v. MCB7-l/0-gal, v. MCB7-2/3-gal, v. MCB7-l/B7-2/3-gal, v. MCMHA/B7-2/j3-gal, v. MCMHA//3-gal, v. MCB7-1/NP, or HBSS.
- day# 3 - 6 designated groups of mice were treated with IL-2 (100,000 IU i.p. BID x 3 days).
- mice were randomized, and euthanized on day #12. Lung were harvested and stained, and pulmonary metastases were enumerated in a blinded fashion. All mice inoculated with CT26.WT had large tumor burdens (data not shown) . Duplicate experiments confirmed these results.
- Figures 21a through 2If show a FACS analysis of splenocytes isolated from normal non-depleted (Figs. 21a and 21b), T CD * + -depleted (Figs. 21c and 21d) , or T d .,. - depleted (Figs. 21e and 21f) mice.
- Mice were given two i.v. injections of GK1.5 at 100 mg/ml or of empirically determined levels of 2.43 monoclonal antibodies 48 hours prior to receiving tumor challenge, and again 6 days later.
- Upper left hand panel in each square displays percent CD8+ cells
- upper right panel shows percent CD4+CD8+ cells
- lower right shows percent CD4+
- lower left shows scatter for all cells.
- Figure 21g shows the mean number of pulmonary metastases of CD4- and CD8- immuno depleted mice vaccinated with V.B7-l/3-gal.
- Prior to tumor challenge non-irradiated BALB/c mice (5/group) were injected with anti-CD4 and anti-CD8 monoclonal antibodies (GKI.5 (anti- CD4) and 2.43 (anti-CD8) .
- On day#0 mice were injected intravenously with 5 x 10 5 CT26.C25 tumor cells.
- mice were immunized intravenously with 10 7 PFUs of v. MCB7-l/j8-gal, v. MCMHA/0-gal, v. MCB7-1/NP or HBSS.
- mice On day #6, the mice were again injected with anti-CD4 and anti-CD8 monoclonal antibodies. Mice were randomized, and euthanized on day #12. Lung were harvested and stained, and pulmonary metastases were, enumerated in a blinded fashion. FACS analysis was performed 1 day prior to immunization, and again at day 7 to verify depletion. Only data from v.B7-l/B-gal vaccination groups is shown. Duplicate experiments confirmed these results.
- Figures 22a and 22b show active immunotherapy is enhanced when exogenous rIL-2 and rW are given in concert.
- BALB/c mice (five per group) were challenged i.v. with 5 x 10 s CT26.WT (Fig. 22a) or CT26.CL25 tumor cells (Fig. 22b) . After 3 days they received a single i.v. injection of medium alone (HBSS) or medium containing 5 x 10 6 PFU of different TK " rW either expressing (VJS6) or not expressing (V69) /3-gal.
- Two different regimens of rIL-2 administration were started 12 h after rW injection: high dose (HD, 100,000 Cetus U, twice a day, i.p. for 3 days) or low dose (LD, 15,000 Cetus U, twice a day, i.p. for 5 days) . Mice were checked twice a day for survival.
- Figures 23a and 23b show that exogenous rIL-2 enhances the function of rFPV.
- BALB/c mice (five per group) were inoculated i.v. with 5 x 10 5 CT26.WT (Fig. 23a) or CT26.CL25 tumor cells (Fig. 23b).
- On day 3 after tumor injection they received a single i.v. injection of the following viruses: no virus (HBSS alone), 10 7 PFU of FPV.bg40k (rVPV) , or FPV wild-type (FPVwt) .
- rIL-2 (100,000 Cetus U, twice a day) was administered i.p. starting 12 h after FPV injection and continued for 3 days. Mice were checked twice a day for survival.
- Figure 24 shows exogenous rIL-2 plus rW is therapeutic in the more advanced 6-day tumor model.
- BALB/c mice (five per group) were inoculated i.v., with 10 5 CT26.CL25 tumor cells.
- Six days after tumor injection they received the same treatments described in Figures 22a and 22b with the exception that only the highest dose of rIL-2 was administered.
- Mice were checked daily for survival. No prolongation of survival was obtained by the various treatments in mice bearing 6-day-old pulmonary metastases of CT26.WT tumor (data not shown) .
- Figures 25a and 25b show a drW expressing IL-2, but not GM-CSF, IFN- ⁇ , or TNF- ⁇ , significantly reduces the number of pulmonary metastases in a 3-day model.
- Five BALB/c mice per group were injected i.v. with 5 x 10 s tumor cells of either CT26.WT or CT26.CL25 cell lines.
- On day 12 post tumor challenge lungs were harvested and pulmonary nodules were enumerated in a blind fashion. An independent repeat of this experiment gave identical results.
- Figures 26a and 26b show the function of a drW expressing IL-2 is further enhanced when additional exogenous IL-2 is provided.
- BALB/c mice (five per group) were challenged i.v. with 5 x 10 s CT26.WT (Fig. 26a) or CT26.CL25 tumor cells (Fig. 26b). After 3 days they received a single i.v. injection of plain medium (HBSS) or medium containing 5 x 10 6 PFU of rW encoding /S-gal alone (VJS6) or together with IL-2 (IL-2 rW) . Twelve hours after rW, 100,000 rIL-2U were inoculated, according to the regimen described in Figs. 22a and 22b. Mice were checked twice a day for survival.
- HBSS plain medium
- VJS6 medium containing 5 x 10 6 PFU of rW encoding /S-gal alone
- IL-2 rW IL-2 rW
- Figure 27 shows that expression of IL-2 by drW enhances the antivaccinia CTL response.
- Two BALB/c mice were immunized with 5 x 10 6 PFU/mouse of different rW. After 6 days the spleens were aseptically removed, mixed together, and tested directly in a 6-h 5, Cr release assay against CT26.WT tumor cell line, either infected (CT26 vaccinia) or noninfected (CT26.WT) during the isotope labeling with more than 10:1 moi of crude 19 W preparation. Spontaneous release of target cells never exceeded 20%. E:T cell ratio was 100:1 and then 1:3 dilutions. Lytic units 30% (L.U. 30%) indicate the number of effector cells required to obtain 30% lysis of 10,000 target cells. L.U. 30% were normalized for the total number of cells recovered for each spleen and expressed as total L.U./spleen.
- Figure 28 shows that the presence of tumor cells specifically enhance the CTL response elicited by IL-2 rW in a dose-dependent manner.
- BALB/c mice were injected with HBSS alone or with varying doses of CT26.WT or CT26.CT25 as specified. After 3 days, mice were immunized with 5 x 10 6 PFU/mouse of either VJS6 or IL-2 rW.
- the primary cytotoxic response was evaluated in a 6-h Cr release assay against CT26.WT, CT26.WT pulsed with the ⁇ - gal L d -restricted peptide (CT26T + peptide), CT26.CL25, and the irrelevant target cells E22 as shown.
- Figure 29 shows the effects of recombinant IL-10 on the therapeutic effectiveness of immunization with the vaccinia virus, VJS6 + IL-1 (•) ; VJS6 alone (X) .
- Figure 30 shows the IL-10 adjuvant therapy on the therapeutic effectiveness of immunization with various concentrations of recombinant vaccinia virus encoding the model tumor antigen.
- the present invention is a novel recombinant virus expressing an antigen(s) from a disease causing agent and an immunostimulatory molecule(s).
- the novel recombinant virus is capable of eliciting or upregulating an immune response in a mammal to T-dependent antigens for the purpose of preventing or treating a disease.
- the novel recombinant virus of the present invention is particularly important in upregulating cell-mediated immunity.
- Cell-mediated immunity is crucial to resistant to diseases caused by cancer and pathogenic microorganism, particularly viruses and other intracellular microorganisms.
- the recombinant virus has incorporated into its genome or portion thereof a gene encoding an antigen from a disease causing agent and one or more genes encoding one or more immunostimulatory molecules.
- a host cell infected with the recombinant virus expresses both the antigen(s) from a disease causing agent and expresses the immunostimulatory molecule(s) .
- the antigen may be expressed at the cell surface of the infected host cell.
- the immunostimulatory molecule may be expressed at the cell surface or may be actively secreted by the host cell.
- the expression of both the antigen and the immunostimulatory molecule provides the necessary MHC restricted peptide to specific T cells and the appropriate signal to the T cell to aid in antigen recognition and proliferation or clonal expansion of antigen specific T cells.
- the overall result is an upregulation of the immune system.
- the upregulation of the immune response is an increase in antigen specific cytotoxic lymphocytes which are able to kill or inhibit the growth of a disease causing agent or a cell infected with a disease causing agent.
- the recombinant virus comprises the virus genome or portions thereof, the nucleic acid sequence encoding an antigen from a pathogenic microorganism and one or more nucleic acid sequences encoding one or more immunostimulatory molecules.
- the recombinant virus comprises the virus genome or portions thereof, the nucleic acid sequence encoding a tumor associated antigen, and one or more nucleic acid sequences encoding one or more immunostimulatory molecules.
- the recombinant viruses have been constructed and co-express model tumor antigens together with cytokines (TNF- ⁇ , IFN- ⁇ , GM-CSF, IL-10 and IL-2), restriction elements (class 1 ⁇ -chains and ⁇ gti) , and co ⁇ stimulatory and accessory molecules (B7-1, B7-2 and ICAM-1 and the like) alone and in a variety of combinations.
- cytokines TNF- ⁇ , IFN- ⁇ , GM-CSF, IL-10 and IL-2
- restriction elements class 1 ⁇ -chains and ⁇ gti
- co ⁇ stimulatory and accessory molecules B7-1, B7-2 and ICAM-1 and the like
- IL-2 help signal
- GM-CSF professional APC
- IL-2 increase in CTL frequency
- IFN ⁇ and TNF ⁇ effect on antigen processing pathway and MHC expression
- the present invention also encompasses a recombinant virus comprising the virus genome or portion thereof, the nucleic acid sequence encoding the antigen of interest and more than one nucleic acid sequences encoding more than one immunostimulatory molecule for the added benefit of upregulating an immune response against the antigen.
- the recombinant virus may comprise the virus genome or portions thereof, the nucleic acid sequence encoding GP120 (from HIV) , the nucleic acid sequence encoding Hep B surface antigen and one or more immunostimulatory molecules.
- the recombinant virus comprises the vaccinia virus genome or portions thereof, the nucleic acid sequence encoding MART-l and the nucleic acid sequence encoding the immunostimulatory molecule, B 7.1 alone or in combination with the nucleic acid sequence encoding the immunostimulatory molecule, B7.2.
- the recombinant virus comprises the fowlpox virus genome or portions thereof, the nucleic acid sequence encoding MART-1, the nucleic acid sequence encoding MAGE-l, and the nucleic acid sequence encoding the immunostimulatory molecule, IL-2, alone or in combination with the nucleic acid sequence encoding the immunostimulatory molecule, B7.1.
- the recombinant virus is McB7-l, MCB71-1/NP, McB7-10-gal, McB7-l/ova, MCB7-1/PIA, McB7- 1/B7-2, McMHA, McMHA/NP, McMHA//S-gal, McMHA/ova, McMHA/PIA, MCMHA/B7-2, McB7-2, McB7-2/j8-gal, IL-2(S- gal)rW, GM-CSF(S-gal)rW, IFN ⁇ (0-gal)rW, IL-10(0-gal) rW, and TNF ⁇ (3-gal)rW.
- rW genome containing the model TAA The insertion of costimulatory molecules and/or cytokine genes in recombinant vaccinia virus (rW genome containing the model TAA) is beneficial in treatment of established metastases.
- the present invention encompassed a composition
- a composition comprising a recombinant virus containing a viral genome or portion thereof, one or more nucleic acid sequences encoding one or more antigens from one or more disease causing agents and one or more nucleic acid sequences encoding one or more immunostimulatory molecules.
- the compositions of the present invention may also comprise an exogenous immunostimulatory molecule or combinations of immunostimulatory molecules and/or may comprise a chemotherpeutic drug, antibiotic, antifungal drug, antiviral drug and the like and combinations thereof.
- the composition contains a recombinant virus which has incorporated into its genome or portion thereof a gene encoding a tumor associated antigen and exogenous IL-2. In another embodiment, the composition contains a recombinant virus which as incorporated into its genome or portion thereof a gene encoding a tumor associated antigen in combination with exogenous IL-10.
- Virus that may be used in the present invention are those in which a portion of the genome can be deleted to introduce new genes without destroying infectivity of the virus.
- the virus vector of the present invention is a nonpathogenic virus.
- the virus vector has a tropism for a specific cell type in the mammal.
- the virus vector of the present invention is able to infect professional antigen presenting cells such as dendritic cells and macrophages.
- the virus vector is able to infect any cell in the mammal.
- the virus vector may also infect tumor cells.
- the virus of the present invention include but is not limited to Poxvirus such as vaccinia virus, fowlpox virus and a highly attenuated vaccinia virus (Ankara or (MVA) ) , retrovirus, adenovirus, baculovirus and the like.
- Poxvirus such as vaccinia virus, fowlpox virus and a highly attenuated vaccinia virus (Ankara or (MVA)
- retrovirus adenovirus
- baculovirus baculovirus
- the vaccinia virus genome is known in the art. It is composed of a HIND F13L region, TK region, and an HA region.
- the recombinant vaccinia virus has been used in the art to incorporate an exogenous gene for expression of the exogenous gene product (Perkus et al. Science 229:981- 984, 1985; Kaufman et al. Int. J. Cancer 48:900-907, 1991; Moss Science 252:1662, 1991).
- the first step in formation of recombinant viruses expressing heterologous DNA is the construction of a chimeric gene containing a vaccinia promoter fused to the protein coding sequences of the foreign gene.
- the chimeric gene is assembled in a plasmid vector and engineered so that the transcriptional start site of the promoter is positioned close to the translational initiation codon of the foreign gene.
- additional ATG triplets between the transcriptional and translational start sites should be eliminated so that fusion polypeptides or incorrect reading frames are avoided.
- the promoters are included in DNA fragments 200-300 base pairs in length, but smaller fragments have also proved functional.
- the chimeric genes are inserted into the virus genome by homologous recombination in vivo. This method is necessary since the great size of vaccinia DNA makes in vitro construction of recombinant molecules impractical.
- the chimeric gene is first flanked by vaccinia DNA taken from a non-essential region of the virus genome.
- the resulting plasmid is transfected into vaccinia virus- infected cells, whereupon homologous recombination results in site-specific insertion of the chimeric gene into the virus genome.
- Recombinant genomes are replicated and packaged into infectious progeny virus within the infected cells.
- the nature of the flanking DNA is important since this determines the site of insertion. Only nonessential regions can be used and several of these have now been identified.
- a commonly used locus is the vaccinia TK gene since recombinant viruses are consequently TK.
- a gene encoding an antigen of a disease causing agent may be incorporated into the HIND F13L region or alternatively incorporated into the TK region of recombinant vaccinia virus vector.
- a gene encoding an immunostimulatory molecule may be incorporated into the HIND F13L region or the TK region of recombinant vaccinia virus vector.
- Baxby and Paoletti disclose the construction and use as a vector, of the non- replicating poxvirus, including canarypox virus, fowlpox virus and other avian species which may be used as a viral vector in the present invention.
- Expression vectors suitable for use in the present invention comprise at least one expression control element operationally linked to the nucleic acid sequence.
- the expression control elements are inserted in the vector to control and regulate the expression of the nucleic acid sequence.
- Examples of expression control elements includes, but is not limited to, lac system, operator and promoter regions of phage lambda, yeast promoters and promoters derived from polyoma, adenovirus, retrovirus or SV40.
- Additional preferred or required operational elements include, but are not limited to, leader sequence, termination codons, polyadenylation signals and any other sequences necessary or preferred for the appropriate transcription and subsequent translation of the nucleic acid sequence in the host system.
- the expression vector should contain additional elements necessary for the transfer and subsequent replication of the expression vector containing the nucleic acid sequence in the host system. Examples of such elements include, but are not limited to, origins of replication and selectable markers. It will further be understood by one skilled in the art that such vectors are easily constructed using conventional methods (Ausubel et al., (1987) in "Current Protocols in Molecular Biology", John Wiley and Sons, New York, New York) or commercially available.
- the recombinant virus of the present invention is effective in treating or preventing disease caused by disease causing agents or a disease state.
- Each disease causing agent or disease state has associated with it an antigen or immunodominant epitope on the antigen which is crucial in immune recognition and ultimate elimination or control of the disease causing agent in a mammal, sometimes referred to in the art as a protective antigen.
- the mammalian immune system must come in contact with the antigen or immunodominant epitope on the antigen in order to mount a humoral and/or cellular immune response against the associated disease causing agent.
- the recombinant virus of the present invention comprises the one or more nucleic acid sequences encoding one or more isolated antigens or immunodominant epitopes on the antigens and one or more nucleic acid sequences encoding one or more immunostimulatory molecules for the purpose of enhancing immune response against the disease causing agent.
- Such disease causing agents include but are not limited to cancer and pathogenic microorganisms or mammals.
- Mammals include but are not limited to humans, primates, rats, mice, guinea pigs, rabbits, horses, cows, sheep, pigs, goats and the like.
- Cancers of mammals which may be treated using the recombinant virus of the present invention include but are not limited to melanoma, metastases, adenocarcinoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, colon cancer, non-Hodgkins lymphoma, Hodgkins lymphoma, leukemias, uterine cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, bladder cancer, kidney cancer, pancreatic cancer and the like.
- melanoma includes, but is not limited to, melanomas, metastatic melanomas, melanomas derived from either melanocytes or melanocytes related nevus cells, melanocarcinomas, melanoepitheliomas, melanosarcomas, melanoma in situ, superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma, acral lentiginous melanoma, invasive melanoma or familial atypical mole and melanoma (FAM-M) syndrome.
- Such melanomas in mammals may be caused by, chromosomal abnormalities, degenerative growth and developmental disorders, mitogenic agents, ultraviolet radiation (UV) , viral infections, inappropriate tissue expression of a gene, alterations in expression of a gene, and presentation on a cell, or carcinogenic agents.
- UV ultraviolet radiation
- a gene encoding an antigen associated with the cancer is incorporated into the recombinant virus genome or portion thereof along with a gene encoding one or more immunostimulatory molecules.
- the antigen associated with the cancer may be expressed on the surface of a cancer cell or may be an internal antigen.
- the antigen associated with the cancer is a tumor associated antigen (TAA) or portion thereof.
- TAA tumor associated antigen
- Examples of TAA that may be used in the present invention include but are not limited to melanoma TAAs which include but are not limited to MART-1 (Kawakami et al. J. Exp. Med.
- MAGE-3 The nucleotide sequence of the MAGE-3 gene is disclosed in Gaugler et al. Jj_ Exp. Med. 179:921-930, 1994. MAGE-3 is expressed on many tumors of several types, such as melanoma, head and neck squamous cell carcinomas, lung carcinoma and breast carcinoma but not in normal tissues except for testes.
- the approximately 1.6 Kilobase (kb) cDNA of MART-1 was cloned into a vector and the resulting plasmid, deposited with the American Type Culture Collection (ATCC) 12301 Parklawn Drive, Rockville, MD 20852 USA on April 14, 1994, and given ATCC Deposit Number 75738.
- ATCC American Type Culture Collection
- MART-1 nucleic acid sequence can be isolated from the pCRII plasmid by digestion with HINDU and Xhol restriction enzymes. This 1.6kb nucleic acid sequence or portions thereof can then be incorporated into the genome of the recombinant viruses described herein along with an immunostimulatory gene or genes.
- the TAAs are CA-19-A (pancreatic cancer) , CA-125 (ovarian cancer) , PSA (prostate cancer) , erb-2 (breast cancer, CA-171A) and the like (Boon et al. Ann. Rev. Immunol 12:337, 1994).
- TAAs are in no way limited to the genes encoding the above listed TAAs.
- Other TAAs may be identified, isolated and cloned by methods known in the art such as those disclosed in U.S. Patent No. 4,514,506.
- Genes encoding an antigen of a disease causing agent in which the agent is a pathogenic microorganism in mammals include viruses such as HIV (GP-120, pl7, GP- 160 antigens) , influenza (NP, HA antigen) , herpes simplex (HSVdD antigen) , human papilloma virus, equine encephalitis virus, hepatitis (Hep B Surface Antigen) feline leukemia virus, canine distemper, rabies virus, and the like.
- Pathogenic bacteria include but are not limited to Chlamydia, Mycobacteria, Legioniella and the like.
- Pathogenic protozoans include but are not limited to malaria, Babesia, Schistosoma, Toxiplasma, Toxocara canis. and the like.
- Pathogenic yeast include Aspergillus, invasive Candida, and the like.
- the pathogenic microorganism is an intracellular organism.
- costimulation/accessory molecule and/or gene encoding an a cytokine in combination with a gene encoding an antigen from a disease causing agent is incorporated into the genome of a recombinant virus.
- costimulation molecules include but are not limited to B7-1, B7-2, ICAM-1, ICAM-2, LFA-1, LFA-3, CD72 and the like.
- cytokines encompassed by the present invention include but are not limited to IL-2, IL- 1, IL-3 through IL-9, IL-11, IL-13 through IL-15, G-CSF, M-CSF, GM-CSF, TNF ⁇ , IFN ⁇ , IFN , IL-10, IL-12, Regulated upon activation, normal T expressed and presumably secrected cytokine (RANTES) , and the like.
- chemokines encompassed by the present invention include but are not limited to CTAP III, ENA-78, GRO, 1-309, PF-4, IP-10, LD-78, MBSA, MlP-l ⁇ , MIP-1B and the like.
- the IL-2 gene of the present invention was made made as disclosed by Taniguchi et al (Structure and expression of a cloned cDNA for human interleukin-2 Nature 302:305, 1983) .
- the entire IL-2 gene as disclosed in Taniguchi et al is incorporated into the TK gene sequence of vaccinia virus.
- the promotor sequence for the IL-2 construct of the present invention is made up of the P synthetic late promotor as disclosed in Davidson et al Nucleic Acid Research 18 (No. 14) :4285-4286, 1991.
- the chimeric genes then incorporated into the pox virus genome by homologous recombination in cells that have transfected with a plasmid vector containing the chimeric gene and infected with the pox virus.
- the IL-2 construct of the present invention comprises the recombinant vaccinia containing the IL-2 gene and the P synthetic late promotor and an antigen in the TK region from a disease causing agent in the F13L region of the vaccinia virus genome promotor.
- B7 Construct Co-stimulatory molecules of the B7 family represent a more recently discovered, but important group of molecules.
- B7.1 and B7.2 are both member of the Ig gene superfamily. These molecules are present on macrophages, dendritic cells, monocytes, i.e., antigen presenting cells (APCs) . If a lymphocyte encounters an antigen alone, with co- stimulation by B7.1, it will respond with either anergy, or apoptosis (programmed cell death) ; if the co ⁇ stimulatory signal is provided it will respond with clonal expansion against the target antigen.
- APCs antigen presenting cells
- the B7.l gene was inserted into the Hind F13L region of the vaccinia virus, with the /S-gal placed in the TK region.
- the construct for B7.2 and B7.1/B7.2 in conjunction with a tumor antigen are prepared in the same fashion as B7.1.
- the B7 gene is inserted into the TK region of vaccinia virus and the gene encoding ⁇ - gal inserted in the Hind F13L region of the vaccinia virus.
- the IFN ⁇ construct, TNF ⁇ construct, GM-CSF construct and ICAM-1 construct were constructed as disclosed in Davidson et al Nucleic Acid Research 18 (No. 14) :4285- 4286, 1991.
- the present invention also encompasses methods of treatment or prevention of a disease caused by the disease causing agents disclosed here.
- the administration of the recombinant virus of the invention may be for either "prophylactic" or "therapeutic" purpose.
- the recombinant virus of the present invention is provided in advance of any symptom.
- the prophylactic administration of the recombinant virus serves to prevent or ameliorate any subsequent infection or disease.
- the recombinant virus is provided at (or shortly after) the onset of a symptom of infection or disease.
- the present invention may be provided either prior to the anticipated exposure to a disease causing agent or after the initiation of the infection or disease.
- tumor-specific antigens allows for the development of targeted antigen-specific vaccines for cancer therapy.
- Insertion of a tumor antigen gene in the genome of viruses in combination with a immunostimulatory molecule is a powerful system to elicit a specific immune response in terms of prevention in patient with an increased risk of cancer development (preventive immunization) , prevention of disease recurrence after primary surgery (anti-metastatic vaccination) , or as a tool to expand the number of CTL in vivo, thus improving their effectiveness in eradication of diffuse tumors (treatment of established disease) .
- recombinant viruses of the present invention can elicit an immune response in patient that is enhanced ex vivo prior to being transferred back to the tumor bearer (adoptive immunotherapy) .
- unit dose refers to physically discrete units suitable as unitary dosages for mammals, each unit containing a predetermined a quantity of recombinant virus calculated to produce the desired immunogenic effect in association with the required diluent.
- the specifications for the novel unit dose of an inoculum of this invention are dictated by and are dependent upon the unique characteristics of the recombinant virus and the particular immunologic effect to be achieved.
- the inoculum is typically prepared as a solution in tolerable (acceptable) diluent such as saline, phosphate- buffered saline or other physiologically tolerable diluent and the like to form an aqueous pharmaceutical composition.
- tolerable (acceptable) diluent such as saline, phosphate- buffered saline or other physiologically tolerable diluent and the like to form an aqueous pharmaceutical composition.
- the route of inoculation may be intravenous (I.V.), intramuscular (I.M.), subcutaneous (S.C), intradermal (I.D.) and the like, which results in eliciting a protective response against the disease causing agent.
- the dose is administered at least once. Subsequent doses may be administered as indicated.
- the dosage of administered recombinant virus will vary depending upon such factors as the mammal's age, weight, height, sex, general medical condition, previous medical history, disease progression, tumor burden and the like.
- a dosage of recombinant virus in the range of from about 10 s to about 10 10 plaque forming units/mg mammal, although a lower or higher dose may be administered.
- the recombinant viral vector can be introduced into a mammal either prior to any evidence of cancers such as melanoma or to mediate regression of the disease in a mammal afflicted with a cancer such as melanoma.
- methods for administering the viral vector into mammals include, but are not limited to, exposure of cells to the recombinant virus gx vivo, or injection of the recombinant virus into the affected tissue or intravenous S.C, I.D. or I.M. administration of the virus.
- the recombinant viral vector or combination of recombinant viral vectors may be administered locally by direct injection into the cancerous lesion or topical application in a pharmaceutically acceptable carrier.
- the quantity of recombinant viral vector, carrying the nucleic acid sequence of one or more TAAs to be administered is based on the titer of virus particles.
- a preferred range of the immunogen to be administered is 10 s to 10 10 virus particles per mammal, preferably a human.
- the efficacy of the vaccine can be assessed by production of antibodies or immune cells that recognize the antigen, as assessed by specific lytic activity or specific cytokine production or by tumor regression.
- One skilled in the art would know the conventional methods to assess the aforementioned parameters. If the mammal to be immunized is already afflicted with cancer or metastatic cancer the vaccine can be administered in conjunction with other therapeutic treatments.
- autologous cytotoxic lymphocytes or tumor infiltrating lymphocytes may be removed from the patient with cancer as disclosed in U.S. Patent No. 5,126,132 and U.S. Patent No. 4,690,915.
- the lymphocytes are grown in culture and antigen specific lymphocytes expanded by culturing in the presence of the recombinant virus of the present invention.
- the antigen specific lymphocytes are then reinfused back into the patient.
- the present invention also encompasses combination therapy.
- combination therapy is meant that the recombinant virus containing one or more genes encoding one or more antigens associated with one or more disease agents and one or more genes encoding one or more immunostimulatory molecules is administered to the patient in combination with other exogenous immunomodulators or immunostimulatory molecules, chemotherapeutic drugs, antibiotics, antifungal drugs, antiviral drugs and the like alone or in combination thereof.
- the combination therapy includes a recombinant virus and exogenous IL2.
- the combination therapy includes a recombinant virus encoding one or more exogenous antigens and exogenous IL-10.
- exogenously added agents examples include exogenous IL-2, IL-6, IL- 12, GM-CSF, interferon, IL-10, tumor necrosis factor, RANTES (Promega, G5661) , cyclophosphamide, and cisplatin, gancyclovir, amphotericin B and the like.
- This invention further comprises an antibody or antibodies elicited by immunization with the recombinant virus of the present invention.
- the antibody has specificity for and reacts or binds with the antigen of interest.
- the antibodies are monoclonal or polyclonal in origin.
- Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules or those portions of an immunoglobulin molecule that contain the antigen binding site, including those portions of immunoglobulin molecules known in the art as F(ab), F(ab'); F(ab ' ) 2 and F(v) .
- Polyclonal or monoclonal antibodies may be produced by methods known in the art. (Kohler and Milstein (1975) Nature 256, 495-497; Campbell “Monoclonal Antibody Technology, the Production and Characterization of Rodent and Human Hybridomas” in Burdon et al. (eds.) (1985) "Laboratory Techniques in Biochemistry and Molecular Biology," Volume 13, Elsevier Science Publishers, Amsterdam) .
- the antibodies or antigen binding fragments may also be produced by genetic engineering.
- the technology for expression of both heavy and light chain genes in E_j_ coli is the subject of the PCT patent applications: publication number WO 901443, WO 901443 and WO 9014424 and in Huse et al. (1989) Science 246:1275- 1281.
- the antibodies of this invention are used in immunoassays to detect the novel antigen of interest in biological samples.
- the MART-1 antibodies of this invention generated by immunization with recombinant vaccinia virus expressing MART-l and B7.1 are used to assess the presence of the MART-1 antigen from a tissue biopsy of a mammal afflicted with melanoma using immunocytochemistry.
- Such assessment of the delineation of the MART-1 antigen in a diseased tissue can be used to prognose the progression of the disease in a mammal afflicted with the disease or the efficacy of immunotherapy.
- Conventional methods for immunohistochemistry are described in (Harlow and Lane (eds) (1988) In “Antibodies A Laboratory Manual", Cold Spinning Harbor Press, Cold Spring Harbor, New York; Ausbel et al. (eds) (1987). In Current Protocols In Molecular Biology, John Wiley and Sons (New York, New York) .
- the antibodies of the present invention are used for immunotherapy.
- the antibodies of the present invention may be used in adoptive immunotherapy.
- the dosage of administered antibodies or antigen binding fragments will vary depending upon such factors as the mammal's age, weight, height, sex, general medical condition, previous medical condition and the like.
- a dosage of antibodies or antigen-binding fragments which is in the range of from about 1 mg/Kg to about 10 mg/Kg body weight of the mammal, although a lower or higher dose may be administered.
- the antibodies or antigen-binding fragments of the present invention are intended to be provided to the recipient subject in an amount sufficient to prevent, lessen or attenuate the severity, extent or duration of the disease or infection.
- Patent No. 4,738,846 These as well as other techniques may be employed to construct the recombinant virus of the present invention.
- Recombinant vaccinia viruses expressing mB7 and additional extrinsic proteins were constructed. The mB7 and additional foreign genes were inserted (by homologous recombination) into the vaccinia VP37 (Hind III F) and TK (Hind III J) genes.
- the mB7 gene was replaced by the measles HA (mHA) gene.
- mHA measles HA
- the construction of initial recombinants expressing the above genes employed the plaque formation selection system in which the foreign gene is directed into the vaccinia F13L (VP37) loci. Briefly, the foreign gene is cloned into a vaccinia transfer plasmid (pRB21) adjacent to the synthetic early late promoter (E/L) and flanked by DNA homologous to the F13L region in vaccinia. A full- length F13L gene (under control of its authentic promoter) is also situated within the flanking regions. The mB7-l, Mb7-2 and mHA were individually cloned adjacent to E/L within pRB2l.
- a virus that has a plaque deficient phenotype (due to deletion of the F13L plaque forming gene) was used for transfection and the proceeding homologous recombination of plasmid DNA. Transfections were carried out according to standard published procedures. Recombinant viruses were readily identified according to their plaque positive phenotype, in contrast to the plaque deficient non-recombinants.
- Partially plaque purified recombinants from each transfection were consequently characterized using antibodies specific for the individual foreign protein expressed. This was carried out by direct immuno-staining of plaques on unfixed monolayers of BS-C-1 cells. As cells were unfixed this also indicated that the target proteins were expressed on the cell surface. For the majority of studies rat anti-mB7-l and B7-2 MAbs (Pharmagen) were used to identify surface expression of these proteins.
- the vaccinia recombinants were plaque purified at least 4 times on BS-C-1 cells (African Green monkey kidney cell line American Type Culture Collection (ATCC Accession No. CCL 26) followed by plaquing on STO cells embryonic mouse fibroblast, Accession No.
- ATCC/CRL 1503 in the presence of 6-thioguanine (TG) (Sigma Chemical Co.) which selects against unstable single cross-over recombinants.
- Recombinants were again plaque purified on BS-C-1 cells at least once before stocks were grown in HeLa Spinner cells. The virus was semi-purified by ultracentrifugation through a 36% sucrose cushion. Aliquots were prepared and stored at 80°C A single aliquot from each batch was thawed, sonicated and titred on BSC-1 cells. Infected monolayers were immunostained to determine continued expression of foreign proteins.
- TG 6-thioguanine
- the B7-1 recombinant (v.MCB7.l) was further characterized in a FACS analysis to illustrate surface expression and its ability to bind CTLA4-Ig (B7-1 T-cell receptor) and by Western blot analysis using the anti-B7-l antibody.
- the B7-1 recombinant has been shown to be attenuated compared to wild-type WR, in a mouse LD 50 assay. Construction and Characterization of Vaccina Recombinants mB7-l , H-B7-2 or mHa Co-expressing Additional Genes
- McMHA/ova TK inserted, details as McMHA/P1A above
- a recombinant vaccinia virus containing the genes for B7.1 and the tumor associated antigen, ⁇ -gal was deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852 USA under Accession No. VR2485 according to terms of the Budapest Treaty.
- a recombinant vaccinia virus containing the genes for IL-2 and 3-gal was deposited with ATCC under Accession No. VR2486.
- Sucrose cushion purified stocks of all characterized recombinant viruses have been prepared.
- the above method is also employed to construct other recombinant virus using the other previously mentioned viruses such as fowlpox virus in combination with other foreign antigens and immunostimulatory molecules.
- a recombinant fowlpox virus which is replication incompetent in mammalian cells, is constructed that express the model TAA, lacZ encoding /3-gal, and the immunostimulatory molecule, B7.1 under the influence of the 40K vaccinia virus early/late promotor.
- the POXVAC-TC (Schering Corp.) strain of FPV is used.
- FPV is propagated on primary chick embryo dermal cultures (Jenkins, S. et al. AIDS Res. Hum. Retroviruses ⁇ 7:991, 1991) . Foreign sequences are inserted into FPV by homologous recombination as previously described (Jenkins et al. 1991).
- Recombinant fowlpox contains the E. coli lacZ gene and the B7.1 gene under the control of the vaccinia virus 40K promotor (designed H6 in Rosel, J. et al. J L. Virol. 60:436, 1986), inserted into the BAM HI J region of the FPV genome.
- Example 3
- MVA Modified Vaccinia Ankara
- WT vaccinia virus
- CEF chicken embryo fibroblast cells
- the resulting MVA strain lost the capacity to productively infect mammalian cells (Altenburger, W. et al. Arch. Virol 105:15-27, 1989; Meyer, H. et al. J. Ge . Virol 72:1031-1038, 1991).
- the expression of late, as well as early, viral genes is unimpaired in human cells despite the inability of MVA to produce infectious progeny (Sutter and Moss, Proc. Natl. Acad. Sci. USA 89:10847- 10851, 1992) .
- An insertion plasmid is constructed with the lac Z gene and the B7.1 gene under the control of the vaccinia virus late promotor Pll to allow homologous recombination at the site of a naturally occurring 3500-base-pair deletion within the MVA genome.
- MVA recombinants are isolated and propagated in permissive avian cells as described in Sutter and Moss 1992 and Sutter et al. Virology 1994.
- the expression of 3-gal and B7.1 upon infection of nonpermissive human cells is detected by methods described herein.
- mice per group were inoculated i.v. or s.c. with different doses the following vaccinia recombinant (rW) : IL-2 rW; GM-CSF-rW; TNF ⁇ -rW; IFN -rW.
- rW vaccinia recombinant
- IL-2 rW IL-2 rW
- GM-CSF-rW GM-CSF-rW
- TNF ⁇ -rW TNF ⁇ -rW
- IFN -rW vaccinia recombinant
- mice model tumor associated antigen, /3-Gal was under the control of the p7.5-kD (E/L) promoter (Cahran, M.A. et al J. Virol. 54 (No. 1) :30-37, 1995) while the cytokine production was driven by p ⁇ u ⁇ c * promoter (Davidson et al Nucleic Acid Research 18 (No.
- a pE/L promoter (the "synthetic superpromoter") is placed just upstream the p7.5 promoter controlling the /3-Gal expression and oriented in the same direction.
- Control vaccinia (crude 19, NP-W) does not expressing /3-Galactosidase.
- mice Female BALB/c mice, 8-12 weeks old, were obtained from the Animal Production Colonies, Frederick Cancer Research Facility, NIH, Frederick, MD. Primary lymphocyte populations were generated by injecting BALB/c mice i.v. or subcutaneously with 10 6 -10 7 plaque forming units (pfu) of recombinant virus (2 doses in some cases) . To assay for primary in vivo responses spleens were harvested on day six, dispersed into a single cell suspension and tested for their ability to lyse jS-gal expressing and control targets in a six hour sl Cr release assay.
- pfu plaque forming units
- mice 21 days after immunization with recombinant virus were culturing single cell suspensions of splencytes in T-75 flasks (Nunc, Denmark) at a density of 5.0 x 10 6 splenocytes/ml.
- the amount of 51 Cr released was determined by -counting and the present specific lysis was calculated from triplicate samples as follows: [(experimental cpm - spontaneous cpm) / (maximal cpm - spontaneous cpm)] x 100.
- mice were immunized with recombinant virus 21 days before a subcutaneous challenge with 10 4 tumor cells or an intravenous challenge with 5 x 10 5 tumor cells, as previously described (31) . After tumor challenge all mice were randomized. Mice receiving subcutaneous tumor were measured twice a week. When tumors developed, they all grew progressively and were lethal. Mice were euthanized, however, when they were moribund. All mice that appear as long term survivors had no palpable tumor. Mice receiving intravenously administered tumor were euthanized on day 12 and randomized before counting lung metastases in a blinded fashion as previously described (2) .
- mice un-irradiated BALB/c mice were challenged with either 10 5 or 5 x 10 5 tumor cells intravenously in order to establish pulmonary metastases. Mice were subsequently vaccinated with 10 7 PFU of the designated recombinant virus intravenously or subcutaneously on days three or six. Mice receiving intravenously administered tumor were euthanized on day 12 and randomized before counting lung metastases in a blinded fashion.
- CT26 murine H-2 d adenocarcinoma
- CT26 gal murine H-2 d adenocarcinoma expressing ⁇ - Gal. CT26 gal also expresses higher levels of class I MHC molecules.
- E22 murine H-2 d thymoma expressing /3-Gal.
- CT26-vac CT26 infected with crude 19 vaccinia
- Lytic Units 30% were calculated for the anti-vaccinia cytotoxic response. Lytic units 30% (L.U. 30) indicate the number of effector cells necessary to give a 30% lysis of 10,000 target cells.
- Duplicate wells of 10 s BSC1 in 1 ml of 2.5% fetal calf serum (FCS) medium RPMI 1640 with antibiotics were infected with VJS6, rW-IL-2, rW-GM- CSF, rW-IFN ⁇ or rW-TNF ⁇ . At the end of incubation at 27°C for 24 hours, supernatants were removed, centrifuged, and the concentration of cytokine determined as shown in Table 1. Values are expressed in pg/ml.
- the concentration of each cytokine was determined using commercially available detection kits for GM-CSF, IFN7; IL-2 and TNF ⁇ . The results show that high concentrations of each cytokine were detected. The highest concentration of cytokine produced was IL-2 from BSC-1 cells infected with rW-IL2 (Table 1) .
- VJS6 ⁇ 15.6 ⁇ 15.6 ⁇ 15.6 - ⁇ 15.6 ⁇ 15.6 ⁇ 15.6 -
- GMCSF >250 >250 >250 5.1X10 5 >250 >250 >250 4.2X10 6
- VJS6 ⁇ 30.2 ⁇ 30.2 ⁇ 30.2 - ⁇ 30.2 ⁇ 30.2 ⁇ 30.2 -
- mice 5 BALB/c mice were injected intravenously (I.V.) with 5xl0 6 plaque forming units (pfu) of the following recombinant vaccinia virus: VJS6, IL2-rW, GM-CSF-rW, TNF ⁇ -rW OR IFN7-rW.
- VJS6, IL2-rW plaque forming units
- GM-CSF-rW GM-CSF-rW
- TNF ⁇ -rW TNF ⁇ -rW OR IFN7-rW.
- the spleens were removed and tested against the following target cells as described in Example 4: CT26, CT26 peptide, CT26 gal, E22, and CT26 vac.
- Figure 2 shows the peak primary CTL response.
- IL-2 and GM-CSF produced by rW during infection notably o enhanced the primary CTL response against vaccinia determinants.
- the spleen cells also gave a weak primary response toward 3-gal.
- mice were infected with various recombinant vaccinia virus constructs as described in Example 4. Splenocytes were harvested at day 6 or day 14 after inoculation with the rW. Secondary cultures were generated by 6 days incubation of 6xl0 8 splenocytes/ml in complete medium containing 1 ⁇ g/ml TPHPARIGC peptide as indicated in Example 4. The results showed that CTL from VJS6, IL2-rW and GM-CSF-rW vaccinated mice could not be restimulated with an /8-gal peptide 6 days after viral injection (Figure 3) .
- the TNF-rW response of CTLs was characterized by the induction of a long-lasting non-specific cytotoxicity ( Figures 3 and 4) .
- the GM-CSF production had a negative effect on the anti-/3-gal immune response of CTLs.
- mice 5 BALB/c mice were injected subcutaneously (S.C.) with 5xl0 6 pfu of the following recombinant vaccinia virus: VJS6, IL2-rW, GM-CSF-rW, TNF ⁇ -rW or IFN7-rW.
- VJS6, IL2-rW the following recombinant vaccinia virus
- GM-CSF-rW the following recombinant vaccinia virus
- TNF ⁇ -rW TNF ⁇ -rW or IFN7-rW.
- the spleens were removed and tested against the following target cell as described in Example 4: CT26, CT26 peptide, CT26 gal, E22 and CT26 vac.
- FIG. 5 shows in all cases, each cytokine helped to increase the response to vaccinia in comparison to VJS6 using the CT26 target, with the IL-2-rW treatment being the highest. No lysis was obtained with the other target cells. This indicates that the kinetics of the immune response induced by S.C. inoculation may be different or delayed in comparison to I.V. inoculation. However, higher doses and/or different time intervals is expected to increase the magnitude of the primary response against vaccinia using the subcutaneous route of inoculation.
- the lytic units (L.U.) 30% was determined by the number of effector cells necessary to give 30% lysis of 10,000 target cells using the CT26-vac cells as targets, Figure 6 shows the results of this titration.
- mice were infected with various recombinant vaccinia virus constructs as described in Example 4. Splenocytes were harvested at day 14 after inoculation with rW.
- TPHPARIGC peptide as indicated in Example 4.
- the results as depicted in Figure 7 show a high level of non-specific response to each target cell regardless of the effector cell.
- mice The three-day pulmonary metastases mouse model was used to evaluate the efficacy of treatment using rW. Nontreated mice normally succumb to the metastases in 11- 14 days using this model system.
- mice were injected I.V. on day 0 with 5x10 s tumor cells (CT26 or CT26 ⁇ gal) . Three days later they received an I.V. injection of 5x10 s PFU of VJS6, IL-2-rW, GM-CSF-rW, IFN ⁇ -rW, TNF7-rW or Hanks' Balanced Salt Solution (HBSS) (control mice) . The mice were randomized, and the lungs were harvested after 12 days. The number of metastases was determined by counting in a blinded fashion.
- Table 2 shows that moderate doses of IL2 produced by the rW was effective in treatment of 3 day old metastases induced by CT26 ⁇ gal.
- VJS6 >500 >500 x 5 186 162, 116, 115,
- TNF ⁇ rVV >500 > 500 5 361.2 > 500 x 2, 389, 258, 159
- mice were injected with tumor cells and rW as indicated in Example 4. Treatment with exogenous rIL-2 (15,000 cetus units, twice a day, I.P.) was started 6 h after rW injection and protracted for 5 days. Lungs were harvested after 12 days.
- a recombinant vaccinia virus expressing a Tumor Associated Antigen (TAA) was constructed as described in Example 1.
- mice were injected with 5 x 10 5 of CT-26 expressing /3-gal via an I.V. route of administration. Three days later the mice were injected with rW-TAA-/3-gal, alone or in combination with exogenous IL2. [High dose (HD) exogenous IL-2 (100,000 I.U., I.P., BID x 3d) or low dose (IL) IL-2 (15,000 I.U., I.P., BID x 3d)] was administered as indicated and then randomized.
- High dose (HD) exogenous IL-2 100,000 I.U., I.P., BID x 3d
- IL low dose
- Figure 9 shows the survival of mice with non-/3-gal expressing tumor established after immunotherapy with different recombinant vaccinia vectors.
- Mice were injected with 5 x 10 5 CT 26 tumor cells (non-/S-gal) via I.V. route of administration. On day three they were injected with 10 3 pfu of the following rW which also contained the jS-gal gene: IL-2rW, TNF ⁇ -rW, VJS6, GM- CSFrW. The number of mice surviving and survival time in days was monitored. No difference in survival was observed in any of the treatment groups.
- Figure 10 shows survival in mice with an established
- mice were injected with 5 x 10 s CT26.25 (/3-gal expressing) via I.V. route of administration. On day three they were injected with 10 3 pfu of the rW described above. The results show that a clear survival advantage was conferred on those mice treated with the IL-2rW (also containing the jS-gal gene) compared to treatment with any other rW.
- Vaccinia vectors were constructed by placing both the cytokine gene and the TAA gene in the TK region of the vaccinia vector genome. Promotors varied based on the plasmid used for recombination. The majority of the constructs used the P7.5K promotor.
- the three-day pulmonary metastases model was used as described in Example 4. Five x 10 5 tumor cells were injected into mice. Three days later 10 6 -10 7 pfu of each vector was injected I.V. Lungs were harvested on day 12. The results as depicted in Figure 11 and 12 showed that the rW vectors encoding ⁇ -gal and the immunostimulatory molecule, B7.1, IL-2 or ICAM-1 profoundly inhibited pulmonary metastases in an antigen specific manner.
- mice on Day 9 after injection with tumor cells was determined. The results are depicted in Table 4. The percent lysis of 51 Cr labeled target tumor cells is recorded. A significant primary immune response was seen with multiple recombinant vaccinia vectors, most notably B7.1rW, which correlates with the in vivo treatment response.
- mice The secondary response of the mice is depicted in Table 5. The results indicate a secondary immune response seen by both VJS6 and B7.1 against the relevant target. Table 5
- VJS6 10 6 100:1 18.55 48.89 38.21 15.27
- NA neuraminidase recombinant viral vecot (rW)
- MVA Ankara non-replicating vaccinia virus rW
- Kb murine H-2K 6 MHC Class I molecule rW
- Ld murine H-2L d MHC Class I molecule rW
- WB ⁇ murine Beta 2-microglobulin rW
- HBSS Hank's Balanced Salt Solution
- patients must have evidence of measurable or evaluable metastatic melanoma or breast cancer that has failed standard effective therapy. Patients must have tumors that express the MAGE-l antigen as evidenced by PCR or Northern Blot analysis of tumor cell RNA.
- Melanoma and breast cancer patients are divided into two categories each, those who have previously been vaccinated against smallpox and those who have not.
- Three patients in each category receive intradermal immunization with 10* plaque forming units/ml every four weeks for a total of three doses.
- Patients are evaluated for toxicity.
- the dose in that plaque category will be escalated to 10 9 plaque forming units/ml intradermally every four weeks for a total of three doses.
- Fifteen patients in each category will be treated at this dose and carefully evaluation of toxicity, immunologic effects and therapeutic efficacy will be evaluated.
- the recombinant vaccinia virus used in this protocol is the Wyeth vaccinia virus derived from the New York City Board of Health strain. This is an attenuated virus with an extensive history of previous use in humans.
- the recombinant virus has the gene coding for the MAGE-l nine amino acid minimal determinant plus the B7.1 sequence inserted into the viral thymidine kinase gene.
- the recombinant virus is produced in a Food and Drug Administration approved facility for the manufacture of GMP grade clinical material.
- the recombinant vaccinia virus will be supplied as a sterile, frozen suspension and will be approximately diluted to either 10* or 10 9 plaque forming units/ml with phosphate buffered saline.
- Vaccination will be performed in the deltoid muscle area.
- the method of vaccination is summarized as follows: a) Open the bifurcated needle by catching the butt- end of the needle and gently pulling the point end free. b) Dip the bifurcated point of the needle into vaccine. The needle will pick up a drop of vaccine in the space between the two points. c) Using the same bifurcated needle, use the multiple pressure technique by pressing the needle through the vaccine drop on the subject's skin. Fifteen needle pressures will be administered. d) Alternative upper extremities will be used for subsequent vaccinations.
- Body temperature will be measured at 12 hours and at 24 hours and any adverse reactions noted.
- Immunologic assessment will be made of the patient's response to vaccinia antigens as well as to the MAGE-l antigen. a) All serum samples will be tested for anti- vaccinia antibody by ELISA. b) All cryopreserved lymphocytes will be tested for response to MAGE-l antigen using limiting dilution analysis of precursor CTL frequency using the method of Coulie, P et al International Journal of Cancer 50:289- 297, 1992. c) Patients with easily accessible disease may have biopsy under local anesthesia of accessible tumor to study the histopathologic nature of the tumor as well as the isolation of tumor infiltrating lymphocytes for in vitro growth. Tumor infiltrating lymphocytes will be tested for specific reactivity and specific cytokine release against MAGE associated antigens.
- a complete response is defined as the disappearance of all clinical evidence of disease that lasts at least four weeks.
- a partial response is a 50% or greater decrease in the sum of the products of the perpendicular diameter of all measurable lesions for at least four weeks with no appearance of new lesions or increase in any lesions.
- Minor responses are defined as 25-49% decrease in the sum of the products of the perpendicular diameters of all measurable lesions with no appearance of new lesions and no increase in any lesions.
- Vaccines to be tested include but are not limited to Recombinant Vaccinia virus encoding MART-1 and IL-2, Recombinant Vaccinia virus encoding MART-1 and B7.1, Recombinant Vaccinia virus encoding GP100 and IL-2, Recombinant vaccinia virus encoding GP100 and B7.1, Recombinant fowlpox virus encoding MART-1 and IL2, recombinant fowlpox virus encoding MART and B7.1 and the like with the appropriate modifications depending on the antigen and virus used.
- T-lymphocytes presensitized to the melanoma antigen may be effective in therapeutically treating mammals afflicted with melanoma.
- T-lymphocytes are isolated from peripheral blood or melanoma tumor suspensions and cultured in vitro (Kawakami, Y. et al. (1988) J. Exp. Med. 168:2183-2191).
- the T-lymphocytes are exposed to cells infected with the recombinant virus expressing a melanoma associated antigen and IL2 and/or B7.1 for a period of about to 1-16 hours at a concentration of l to lOmg/ml.
- T-lymphocytes exposed to the antigen will be administered to the mammal, preferably a human at about 10 9 -10 12 lymphocytes per mammal.
- the lymphocytes may be administered either intravenously, intraperitoneally or intralesionally. This treatment may be administered concurrently with other therapeutic treatments such as cytokines, radiotherapy, surgical excision of melanoma lesions and chemotherapeutic drugs, adoptive T lymphocyte therapy.
- mice Female BALB/c (H-2 d ) mice were obtained from
- CT26 is an N-nitroso-N-methylurethrane induced BALB/c (H-2 d ) undifferentiated colon carcinoma as supplied by D. Pardoll (Baltimore, MD) .
- CT26 was cloned to produce a wild type parental tumor line, CT26.WT.
- the gene for lac Z was stably transfected into CT26.WT as previously described herein and in Wang, M. et al J. Immunol. 154(9) :4685-4692, 1995. Briefly, a plasmid donated by
- A.D. Miller containing the gene for ⁇ -galactosidase and a neomycin resistance marker were used to construct the LZSN amphotropic retrovirus which was used to transduce CT26.WT.
- Transductants were selected in G418 media, and subcloned by limiting dilution analysis at 0.3 cells/well. Subclones that expressed S-galactosidase were screened by X-gal staining and in s, Cr release assays with anti-3- galactosidase effectors.
- the subclone CT26.CL25 was selected for use in all studies because of its stable expression of both ⁇ -galactosidase and the class I molecule H-2 L d .
- BS-C-1 or HeLa S 3 cells were used to expand and titer all viruses.
- Cell lines were maintained in RPMI 1640, 10% heat inactivated FCS (Biofluids, Rockville, MD) , 0.03% L-glutamine, 100 ⁇ g/ml streptomycin, 100 ⁇ g/ml penicillin and 50 ⁇ g/ml gentamicin sulfate (NIH Media Center).
- CT26.CL25 was maintained in media containing 400 or 800 ⁇ g/ml G418 (GIBCO, Grand Island, N.Y.) .
- vaccinia viruses containing murine B7-l( V.MCB7-1) , B7-2 (v. MCB7-2), B7-1 and B7-2 (v.B7-1/B7-2) and measles hemagglutinin (HA) (v.MCMHA) is as follows. Briefly, the B7-1 gene (a gift from Dr. R. Germain, NIH) and the measles HA gene (a gift from Dr. S. Rozenblatt, Tel Aviv University, Israel) were cloned into the transfer plasmid pRB12 such that they were under control of the W synthetic early/late promoter. The gene of interest was inserted into the Hind III F region of W.
- coli lac Z gene is under transcriptional control of the early function of the W p 7.5k promoter and flanked by W derived TK DNA) with V.MCB7-1 and v.MCMHA respectively.
- the v. MCB7-2/3-gal was constructed with both the B7-2 gene and the E. coli lac Z gene under transcriptional control of the early function of the W p 7.5k promoter and flanked by W derived TK DNA.
- the v. MCB7-1/B7-2/ / 3- gal was constructed by homologous recombination of the plasmid pSC65 ⁇ (modified as stated previously to create the v. MCB7-2//3-gal construct) with the v. MCB7-1.
- MHA measle hemagglutinin gene
- B7-1 murine B7-1
- B7-2 murine B7-2
- NP influenza nucleoprotein
- /J-gal E.coli ⁇ -galactosidase gene
- S.E/L vaccinia synthetic early late promoter
- 7.5e early only function of the vaccinia 7.5k promoter.
- Recombinant viruses were simultaneously selected for their TK negative phenotype and ability to express ⁇ - galactosidase.
- Transfer plasmid pGS69 (Smith, G.L. et al Virology 160:336-345, 1987 containing the influenza nucleoprotein (NP) was recombined with V.MCB7-1 to yield v. MCB7-1/NP.
- This recombinant virus was selected on the basis of a TK negative phenotype. Plaques were then analyzed for expression of NP by immunostaining (Sutter, G.
- rW expressing the influenza A/PR/8/34 nucleoprotein (NP) was previously described (Smith, G.L. et al Virology 160:336-345, 1987).
- v. JS6 the E.coli lac Z gene was under the control of the early W p 7.5k promoter from plasmid pSC65.
- Murine IL-2 cDNA was amplified by polymerase chain reaction from pBMGNeomIL-2 and ligated into the Sma I- BamHI site of a vaccinia expression vector, pMJ601, which contains the b-galactosidase gene under the control of W early p 7.5k promoter.
- cytokines GM-CSF, IFN- g, and TNF-a
- GM-CSF, IFN- g, and TNF-a were inserted into the wild type W genome with similar procedure.
- the techniques used in preparation of the v. HLA.A2.1//3-gal (O'Neil, B.H. et al J. Immunol. 151:1410-1418, 1993) v. human and murine /3 2 - microglobulin//3-gal, (O'Neil, B.H. et al J. Immunol. 151:1410-1418, 1993) and all the murine MHC I (L d , D d , K d , and K b ) //3-gal (Restifo, N.P. et al J. Exp. Med. 177:265- 272, 1993), has been previously described.
- BS-C-1 monolayers were infected with 10 infectious units of recombinant virus per cell. After 20 hours infected cells were harvested in solubilization buffer (0.06M Tris-HCl (pH 6.8), 3% SDS, 5% /3-mercaptoethanol, 10% glycerol and 0.002% bromophenol blue). Proteins were resolved on a 10% polyacrylamide gel and subsequently transferred to a nitrocellulose membrane using a Bio-Rad Transblott Mini cell (Bio-Rad) at 250 mAmps for 1 hour. Membranes were initially incubated for 1 hour in a blocking solution (PBS-B) consisting of PBS containing 3% non-fat milk (w/v) and 0.2% Tween 20.
- PBS-B blocking solution
- a murine specific B7-1 hamster antibody (supplied by Dr. H. Reiser, Dana- Farber Cancer Inst, Boston) was diluted in PBS-B and incubated with filters for 3 hours at room temperature. Filters were subsequently washed three times in PBS containing 0.2% Tween 20. Bound primary antibodies were detected by incubating filters, after washing with 125 I- protein A (Amersham) at a concentration of O.l ⁇ Ci/ml in PBS-B. Filters were washed 4 times dried and exposed to X-ray film (Kodak) . Protein sizes were estimated using 14C molecular weight markers (Amersham) (Fig. 13) .
- Recombinant viruses were analyzed for /?-galactosidase expression using an enzyme assay kit (Promega, WI) . Briefly, monolayers of BS-C-1 cells were infected at a multiplicity of infection (MOI) of 10. Approximately 20 hours post infection cells were washed with PBS and harvested with lysis buffer. Quantitation of enzyme expression was determined by incubating cell extracts with ⁇ -galactosidase substrate and buffer solution as described in the manufactureres protocol.
- MOI multiplicity of infection
- mice which were immunized intravenously with either Hanks's balanced salt solution (HBSS) (Biofluids, Inc., Rockville, MD) , v.MCB7-l/b-gal, or v.MCMHA/b-gal (10 7 plaque forming units (PFU) ) .
- HBSS Hanks's balanced salt solution
- v.MCB7-l/b-gal v.MCMHA/b-gal
- PFU plaque forming units
- mice In vivo adoptive transfer studies involved intravenous immunization of BALB/c mice with either HBSS, v.MCB7-l/b-gal or v.MC MHA/b-gal .
- the rW was administered at either 10 5 or 10 7 PFUs. After twenty-one days, these mice were euthanized, and splenectomized. Following splenectomy, the spleens were morselized, passed through a Nytex membrane, and suspended in HBSS. Lymphocytes were counted on a hemocytometer, and 2 x 10 7 lymphocytes were adoptively transferred to mice who had been injected intravenously with 5 x 10 s tumor cells of either CT26.WT or CT26.C25.
- mice received adjuvant treatment with six doses of IL-2 administered intra-peritoneal at a dose of 100,000 Cetus units two times per day for three days.
- IL-2 administered intra-peritoneal at a dose of 100,000 Cetus units two times per day for three days.
- mice were randomized, euthanized, and lung metastases counted in a blinded fashion.
- mice In vivo active treatment studies involved non- irradiated BALB/c mice which were inoculated intravenously with 5 x 10 5 tumor cells of either CT26.WT or CT26.C25. All mice were randomized and subsequently vaccinated with HBSS, or 10 5 or l0 7 PFUs of the designated rW three or six days later. Designated groups of mice received adjuvant treatment with six doses of IL-2 administered intra-peritoneal at 100,000 Cetus units two times per day for three days. Mice were randomized and euthanized on day twelve, and lung metastases were counted in a blinded fashion. Identically treated groups of mice bearing three day tumor burdens were also followed long term to assess the effect of vaccination on survival.
- Lungs from tumor-bearing mice were removed and inflated with PBS (Biofluids) prior to X-gal staining. Lungs were fixed in a solution containing 2% formaldehyde (v/v) , 0.2% gluteraldehyde (v/v) in PBS for 45 minutes and washed with PBS three times. After washing, the lungs were stained with X-gal solution for 12 hours at 37° C. X-gal solution for whole organs was prepared by combining the following: 0.02% (v/v) NP-40, 0.01% (w/v) sodium deoxycolate, 1 mg/ml X-gal, 5 mM potassium ferrocyanide, 2 mM MgC12 in PBS. After staining in X-gal solution, the lungs were rinsed briefly with 3% (v/v) dimethyl sulfate, and then with PBS and stored at 4° C in 0.02% (w/v) sodium azide in PBS.
- PBS Bioflu
- rW murine B7-2 was verified by immunohistochemical staining of infected cells using a murine specific B7-2 antibody (PharMingen, CA) .
- Expression of the TAA, j8-galactosidase was similar for v. MCB7-l/jS-gal and the control recombinant v. MCMHA/ ⁇ -gal, in contrast v. MCB7-1/NP showed no signs of enzyme expression (data not shown) .
- the ability of a single inoculation of twelve double rWs, which expressed the genes encoding both an immunomodulatory molecule and the /3-gal model tumor antigen in the same virus, to effect regression of pulmonary metastases in tumor-bearing mice was assayed.
- the double rWs were constructed to include the b- galactosidase gene within the thymidine kinase (TK) region of the W genome for all vectors evaluated.
- TK thymidine kinase
- the position of the immunomodulatory molecule for most vectors is also within the TK region of the W genome under the same promoter as the TAA.
- the gene for B7-1 is placed within the Hind III F region as described above and shown in Table 6.
- the double rWs studied were V.MCB7.1 (Hind III F region of W genome)/b-gal (TK region of W genome), v. human b 2 -microglobulin (TK)/b-gal (TK) ⁇ 3154 ⁇ , v. murine b 2 - microglobulin (TK) /b-gal (TK) (O'Neil, B.H. et al J L_ Immunol. 154:1410-1418, 1993), v. murine MHC I L d (TK) /b- gal (TK) (Restifo, N.P. J. EXP. Med. 177:265-272, 1993), v.
- TK TK/b-gal
- TK TK/b-gal
- v. JS6 expressing only b-gal in the TK region (described above)
- MVA modified vaccinia Ankara
- v. IL-2/b-gal and v.MCB7-l/b-gal mediated both a specific and significant reduction in the number of pulmonary metastases (p ⁇ 0.006 and p ⁇ 0.002, respectively) .
- the ability of v. JS6, the single rW expressing b-gal alone, to mediate significant tumor regression was variable from experiment to experiment. (Data not shown) .
- the inability of v. MVA/b-gal, a non- replicating W which produces high levels of b-gal, to mediate any regression may indicate that prolonged TAA expression (as would occur with a replicating W) is required for a successful CTL response.
- IL-2/b-gal and v. MCB7-l/b-gal resulted in a 10 and 5 fold further reduction in the number of metastases when compared to the best results obtained with v. JS6.
- TAAs and TAA specific CTLs confirm that an immunocompetent host can generate a significant cytotoxic T cell response against autologous neoplasms; the immunogenicity of these TAA is generally weak allowing for the progressive growth of tumors.
- Protective immunity following vaccination with a rW expressing either the TAA alone, or in combination with B7-1 was evaluated.
- the rW strategy utilized in this current study has a distinct advantage over B7 transfections studies (in addition to the ease of administration) , since the specificity of the immune response against a defined model TAA can be measured by comparing the response in the transduced tumor cell line CT26.C25, with the parental tumor, CT26.WT.
- mice were initially vaccinated with either v. MCB7-l/b-gal, or a control virus, v. MCMHA/b-gal.
- the v. MCMHA/b-gal vector expressed an irrelevant protein, measles HA, which is a similarly sized and glycosylated molecule like B7-1, as well as the model TAA to allow for evaluation of the contribution of B7-1 to the response generated.
- Mice immunized with both v. MCB7-l/b-gal and v. MCMHA/b-gal were protected against tumor challenge with CT26.CL25, but not the wild type tumor, CT26.WT (p ⁇ 0.0002 and p ⁇ 0.0014, respectively) (Fig. 15.).
- Tumorigenicity is reflective of the state of T cell activation.
- vaccination a rW coexpressing B7-1 and a model TAA would result in the generation of a primary TAA CTL response which could be used in the adoptive therapy of 0 tumor-bearing syngeneic host.
- the therapeutic advantage gained by immunizing against a known TAA to elicit CTLs reactive against both autologous or allogeneic tumors are numerous. Confirmation of the specificity of the CTL response was demonstrated in the initial observation that 5 no reduction in the number of pulmonary metastasis was noted after the adoptive transfer of any group of primed lymphocytes to syngeneic mice bearing CT26.WT tumors.
- mice bearing CT26.C25 tumors and inoculated with HBSS primed splenocytes showed no therapeutic benefit, however the addition of adjuvant IL-2 to similarly treated mice resulted in a significant reduction in the number of pulmonary metastases (p ⁇ .0054) (Fig. 16.).
- CT26.C25 tumor-bearing mice treated with v. MCB7-l/b-gal rW splenocytes at two doses (10 s and 10 7 PFUS) showed a significant reduction in tumor burden (p ⁇ 0.007 and p ⁇ 0.007, respectively).
- adjuvant IL-2 treatment resulted in a 16.5 and 12.5 fold further reduction in the number of pulmonary metastases in this group.
- the addition of adjuvant IL-2 to the v. MCMHA/b-gal treatment group resulted in a 2 fold reduction in the number of pulmonary metastases in the group treated with v. MCMHA//B-gal primed splenocytes at 10 5 PFU.
- Vaccination with a rW expressing only the model TAA was capable of priming a specific immune response which mediated tumor regression on adoptive transfer.
- Vaccination with a rW containing both the costimulatory molecule, B7-1, and the model TAA increased the therapeutic response by up to 150 fold.
- the increased therapeutic benefit mediated by v. MCB7-/b-gal primed lymphocytes may represent either or both a quantitative increase in the precursor frequency of TAA specific CTL in the adoptively transferred population, or a qualitative increase in the cytotoxic potential of the transferred cells mediated by B7/CD28 costimulatory signals.
- MCB7-l/b-gal would provide any MHC class I expressing cell the required elements necessary to mediate a CTL response, without the need for APCs or CD4+ T helper cells (Dohring, C. et al Int. J. Cancer 57:754- 759, 1994; Wu, Y. et al Curr. Biol. 4:499-505, 1994). Infection with v. MCB7-l/b-gal would also provide T cells with the capacity for autonomous stimulation leading to both a proliferative response to antigen and increased cytokine production (Dohring, C. et al ibid; Azuma, M. et al J. EXP. Med. 177:845-850, 1993).
- binding of B7/CD28 costimulates IL-2 mRNA accumulation (Linsley, P.S. et al J. Exp. Med. 173:721-730, 1991) important for T cells proliferative response, as well as IFN- ⁇ , important in Thl induction, as well as IL-4 important in Th2 induction (Walter, H. et al. Eur. Cytokine Netw. 5:13-21, 1994) .
- naive T cells exhibit suboptimal proliferation and decreased stability of mRNA for several cytokines important to the T cell proliferative and differentiation response (Linsley, P.S.
- the mechanism responsible for this response may be due to either or both a qualitative increase in antigen specific T cell precursors mediated by B7/CD28 (or CTLA-4) interaction, or B7/CD28 (or CTLA-4) mediated differentiation of T cell effector populations.
- the simplest explanation for the therapeutic response seen with IL-2 alone is that is represents a lymphokine activated killer cell (LAK) response mediating non-specific cytotoxicity.
- LAK lymphokine activated killer cell
- IL-2 treatment was therapeutic only in the mice bearing CT26.C25 tumors, which expressed the model TAA. Therefore, a more likely explanation for results seen with IL-2 alone and as an adjuvant, is an IL-2 mediated proliferation of antigen-specific CTL.
- v. MCB7-l/b-gal Treatment of established disease remains the ultimate goal of immunotherapy. If lack of costimulation is a principal means through which tumors evade immune surveillance in this model system, then vaccination with a v. MCB7-l/b-gal should generate an active and specific therapeutic immune response in animals bearing established tumor.
- tumor-bearing mice were immunized with a single injection of rWs, with designated groups receiving adjuvant treatment with IL-2 as outlined above.
- v. MCB7-l/b-gal and v. MCMHA/b-gal v. MCB7-1/NP was used in these studies. The v.
- MCB7-1/NP co-expresses the B7-1 protein with an irrelevant protein (influenza nucleoprotein, NP) not expressed on the CT26.C25, and allows for assessment of the role of B7-1 expression alone. No significant tumor regression was seen following rW vaccination in mice bearing CT26.WT tumors.
- mice bearing CT26.C25 tumors immunization with v. MCB7-l/b-gal mediated a significant reduction in the number of metastases (p ⁇ 0.006) (Fig. 17.).
- the high therapeutic effect precluded evaluation of the effect of adjuvant IL-2.
- mice immunized with v. MCMHA/b-gal no significant tumor regression (p ⁇ 0.36).
- a significant decrease in the number of pulmonary metastases for mice immunized with v. MCMHA/b-gal at 10 7 PFU was seen only once (p ⁇ 0.009).
- IL-2 administration did not significantly improve upon tumor regression in v. MCMHA/b- gal treated mice.
- mice immunized with v. MCB7-l/b-gal showed a 170 fold and 117 fold greater reduction in the number of pulmonary metastases than mice in the v. MCMHA/b-gal and v. MCB7- l/NP immunization groups, respectively.
- Exogenous IL-2 alone did not mediate further tumor regression. This data implies that expression of the TAA and the costimulatory molecule are both necessary and sufficient to effect maximal regression of tumor in this active treatment model and suggests a role for B7-1 in both the induction and effector phase of the CTL response.
- MCB7-1/NP had no significant reduction in the number of pulmonary metastases (p ⁇ 0.12, and p ⁇ 0.22, respectively) . Again, adjuvant IL-2 did not improve this result. It is concluded therefore, that a rW co-expressing both the costimulatory molecule, B7-1, and the TAA, b-gal, are required to mediate regression of both large and small tumor burdens in this model system. Consistent with the previous data disclosed herein and in Wang et al J. Immunol.
- CT26.C25 tumor bearing mice demonstrated varying degrees of survival among vaccination groups (Figure 19) .
- Mice vaccinated with v. MCMHA/b-gal showed a significant prolongation of survival with 50% of mice alive at day 27, and the last mouse dying at day # 46 (p ⁇ 0.0002)
- Adjuvant IL-2 did not statistically improve upon this result.
- 50 % were alive at 70 days, and 30% continue to be long term survivors at over 100 days (p ⁇ 0.0001).
- Adjuvant IL-2 therapy for this group added no statistically significant benefit to survival, though a trend was suggested with 50% still alive at day # 88, and 40% alive at 100 days follow- up.
- optimal T cell activation require two signals, once delivered by the MHC/TCR interaction and a second from the B7-1/CD28 or CTLA-4 interaction. It was evaluated whether a rW coexpressing B7-2 (v. MCB7-2//3- gal) , or both B7-1 and B7-2 (v. MCB7-l/B7-2/ / 3-gal) , with a TAA would be 3. more potent vaccine to stimulate TAA specific CTL response.
- mice were initially inoculated with 5 x 10 s tumor cells of CT26.WT or CT26.C25. Mice bearing three day tumors were then immunized with a single injection of rW, with designated groups receiving adjuvant treatment with IL-2 as outlined above.
- an additional rW was constructed v. MHA/B7-2/b-gal, which contained the MHA gene in the Hind III region of the W genome, and both B7-2 and the E. coli lac Z gene in the TK region. This was created to control for the presence of the B7-1 gene in the v. MCB7-l/B7-2//3-gal construct (Fig. 20.). No significant tumor regression was seen following rW vaccination in mice bearing CT26.WT tumors.
- MCMHA/B7-2/j3- gal also mediated significant reduction in the number of metastases (p ⁇ 0.0018), which did not differ statistically from either v. MCB7-2/,3-gal or v. MCB7-1/B7- 2/j8-gal.
- Adjuvant IL-2 did not statistically improve upon these results.
- B7-1 may be a more potent costimulator of antitumor immunity than B7-2. That is, rW expressing B7-1 alone mediated a 10 fold greater reduction than a rW expressing B7-2 alone, or both B7-1 and B7-2. Similarly, rW expressing both B7-1 and B7-2 was three times as potent as the vector containing the irrelevant protein, MHA together with B7-2.
- CD8+ depletion would significantly reduce the precursor frequency of responding cytotoxic cells (3% remained in this study) , the increased number of cells capable of presenting antigen and costimulating may blunt the effect of the depletion.
- the incompleteness of the CD8+ depletion may be a simple explanation for the observed results.
- CD4+ depletion completely abrogated any tumor regression following vaccination. Whether this phenomenon is mediated simply by a reduction in IL-2 production normally provided by CD4+ T helper cells, or represents a depletion of either Thl or Th2 cells important in mediating the antitumor response is unclear.
- CT26 is an N-nitroso-N-metylurethane-indiced BALB/c (H-2 d ) undifferentiated colon carcinoma generously supplied by D. Pardoll (Baltimore, MD) .
- CT26 was cloned to produce a wild-type parental tumor line, CT26.WT.
- the gene for lacZ was stably transfected into CT26.WT as described previously (Wang et al, J. Immunol. 154:4685, 1995) . Briefly, a plasmid donated from A.D.
- rW All the rW used in this study were originated by insertion of the foreign genes into the W thymidine kinase (TK) gene by homologous recombination, resulting in the generation of TK-negative progeny as described (Chakrabarti et al, Mol. Cell Biol. 5:3403, 1985).
- the recombinant stocks were produced by using the TK-human osteosarcoma 143/B cell line (ATCC, CRL8303) . From these stocks, rW were propagated in BSC-1 cells and used as crude cell lysate. The BSC-1 cell line was also utilized to determine virus concentration by plaque titration.
- Control CR19 W wild-type vaccinia was kindly provided by J. Yewdell and J. Bennink (NIAID, NIH, Bethesda, MD) .
- Murine IL-2 cDNA was amplified by PCR from pBMGNomIL-2 and ligated into the Smal-Ba HI site of a vaccinia expression vector, pMJ601 (a gift from B. Moss) , which contains the (3-gal gene under the control of the p 75 early vaccinia promoter (Whitman, E.D. et al, Surgery 116:183, 1994).
- the other cytokines (GM-CSF, IFN- ⁇ , and TNF- ⁇ ) were inserted into the wtW genome with a similar procedure. The cytokines produced after infection with the rW have been confirmed for their bioactivity.
- FPV.wt The POXVAX-TC strain of FPV was used in these studies and is designated FPV.wt.
- Foreign sequences were inserted into FPV by homologous recombination as described by Jenkins, S.L. et al, AIDS Res. Hum. Retroviruses 7:991, 1991.
- FPV.bg40K is a recombinant that contains the E. coli lacZ gene under the control of the W 40k promoter, placed in the Ba HI J region of the FPV genome.
- Splenocytes were resuspended in complete media including RPMI 1640 with 10% FCS (Biofluids, Rockville, MD) , 0.1 mM nonessential amino acids, 1 mM sodium pyruvate (Biofluids) and 5 x 10 "s ⁇ M2-ME (Life Technologies, Inc., Rockville, MD) , and used in 6-h 51 CR release assays as described elsewhere (Restifo, N.P. et al J. Immunol. 147:1453, 1991) . To summarize, 2 x 10 6 target cells were treated with 200 mCi Na s, Cr0 4 ( 51 Cr) for 90 min.
- Cytokine production was determined by ELISA.
- the ELISA kits for detection of murine GM-CSF, IFN- , TNF- ⁇ , and IL-2 from Endogen were used according to the manufacturer's instructions.
- IL-2 concentrations were sometimes given in Cetus units, 1 Cetus unit/ml corresponding to 600 pg/ml of IL-2.
- mice were immunized with virus (5 x 10 6 to 10 7 PFUs) 3 or 6 days after i.v. challenge with tumor cells (10 5 to 5 x 10 s ) to establish pulmonary metastases. All animals were randomized before receiving virus. Treatment with IL-2 was initiated 12 h after immunization; 6 doses of high dose IL-2 (100,000 Cetus units/injection) or 10 doses of low dose IL-2 (15,000 Cetus units/injection) were administered to selected groups of mice. Mice were killed on day 12 and lung metastases were enumerated in a blind fashion. Identically treated groups of mice were followed for survival.
- Lungs from mice treated with i.v. tumor challenge as described above were inflated with PBS (Biofluids) before X-gal staining. Lungs were fixed in a solution containing 2% formaldehyde (v/v), 0.2% glutaraldehyde (v/v) in PBS for 45 min, washed in PBS three times, and stained in X- gal solution for 12 h at 37°C.
- X-gal solution for whole organs was prepared by combining the following: 0.02% (v/v) Nonidet P-40, 0.01% (w/v) sodium deoxycholate, 1 mg/ml X-gal, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM MgCl 2 in PBS. After staining in X-gal solution, the lungs were rinsed briefly with 3% (v/v) dimethyl sulfate, and then with PBS. The stained lungs were stored at 4°C in 0.02% (w/v) sodium azide in PBS (Lin, W.C. et al, Cancer Res. 50:2808, 1990).
- the Wilcoxon-Mann-Whitney U test was used to examine the null hypothesis of identify of ranks between two sets of data.
- Kaplan-Meier plots and Mantel-Haenszel test were used to compare survival of mice belonging to different treatment groups.
- VJS6 rW- encoding /3-gal
- VJS6 >500 >5O0 x 5 405.2 >500 x 3, 267. 259
- mice Five BALB/c mice per each treatment group were injected i.v. with 0.5 ml of HBSS containing 5 x 10 s tumor cells of either CT26.WT or CT26.CL25. Three days later they received a single i.v. injection of 5 x 10* PFU of ⁇ - gal expressing rW, VJS6. Treatment with exogenous rIL-2 (15,000 Cetus U, twice a day, i.p.) was started 6 h after rW inoculation and continued for 5 days. Lungs were harvested on day 12 after tumor inoculation and pulmonary metastases were counted in a blind fashion. b Control mice were injected with HBSS alone. c All the mice in this group died between days 11 and 12 (before lung harvest) .
- VJS6 exerted a marginal effect on mouse survival, consistent with a partial reduction of the number of metastases observed in some experiments (see below) .
- a clear improvement of survival was obtained when exogenous rIL-2 was administed with the /3-gal-encoding rW but not with the NP-expressing rW (Fig. 22b) .
- There was no significant difference in the survival of mice receiving the "high dose” or "low dose” rIL-2 regimens (p 0.231). Improved survival of mice bearing the parental cell line was not observed in any treatment group (Fig. 22a) .
- the therapeutic advantage of exogenous rIL-2 was not limited to fully replication-competent viruses because a prolongation of survival was also observed with rFPV encoding /3-gal but not with the wild-type virus (Figs. 23a and 23b).
- the effect of a single i.v. injection of 10 7 PFU of the recombinant FPV.bg40k, expressing ⁇ -gal under the control of the 40k vaccinia promoter, on the survival of mice bearing the /3-gal-positive tumor was limited and consistent with a partial reduction in the number of pulmonary metastases.
- daily inoculation of high IL-2 doses significantly increased the therapeutic effect, resulting in 40% of mice still surviving 2 mo after treatment.
- Example 28 Example 28
- the large vaccinia genome can host up to 25 kb of exogenous DNA, allowing the expression of different heterologous proteins in addition to the model TAA (Moss, B. Curr. Opin. Genet. Dev. 3:86, 1993; Mackett, M. et al. Proc. Natl Acad. Sci. USA 79:7415, 1982).
- the results obtained with exogenous rIL-2 prompted us to investigate the effect of cytokine production at the site of virus infection and replication.
- a drW co-encoding the model TAA 3-gal and IL-2 was previously described (Flexner, C. et al, Vaccine 8:17, 1990).
- a new set of dr W in which cytokine production was driven by a powerful synthetic promoter was constructed.
- the murine cytokines included in this study were the following: IL-2, GM-CSF, IFN-7, and TNF- ⁇ . Because the same plasmid was used to construct the various TKdrW, a similar level of /3-gal enzymatic activity was detected after infection of the BSC-1 cell line (data not shown) . Supernatants from the same infected cultures were harvested at various times and tested for the presence of the different cytokines.
- Table 9 shows that specific and elevated cytokine production was detected only in supernatants from BSC-1 cells infected with the relevant viruses. For example, approximately 40,000 U IL-2/ml were released during 36 h in supernatant from BSC-1 at a moi of 1. IL-2 was barely detectable in all the other supernatants. Comparable levels of IL-2 production in murine and human tumor cell lines infected with the same rW were recently reported (Whitman, E.D. Surgery 116:183, 1994). No cytokine was present in supernatants from cell cultures infected with the VJS6 virus expressing the E6 protein from HPV16 in addition to /3-gal. Table 9
- IL-2 enhances the primary CTL response against W and heterologously expressed /3-gal
- VJS6-immunized splenocytes against the CT26.WT target cells infected with the crude 19 W can be assumed to be the baseline for the primary response elicited by a TK " W, because the viruses presented in Figure 27 were all generated through recombination in the TK region of the vaccinia genome.
- the virus-driven cytokine production resulted in either an increase (IL-2 and GM-CSF rW) or in a suppression (TNF- ⁇ and IFN-7) of the primary cytotoxic response to vaccinia Ags.
- mice were mock inoculated (HBSS) or injected with different doses (5 x 10 5 , 10 , 5 x 10 4 ) of either the parental CT26.WT or the /3-gal-positive CT26.CL25 cell line. After 3 days mice received a single i.v. dose of different rW, and their spleens, collected after 6 days from virus immunization, were tested for primary CTL activity.
- Figure 28 summarizes the results of one experiment representative of three others.
- immunization of normal BALB/c mice with IL-2 drW produced a small but consistent primary response against the 3-gal peptide-pulsed CT26.WT and the transduced CT26.CL25 target cells; this response was augmented in a dose-dependent fashion in mice bearing an increasing number of /8-gal-positive pulmonary metastases as a consequence of an increased inoculum of tumor cells.
- the specific anti-/3-gal response in mice injected with the highest dose of CT26.WT tumor cells was not comparably increased (in Fig.
- mice inoculated with the highest number of CT26.WT cells were shown) and was characterized by an elevated level of nonspecific cytotoxicity against the E22 target cells, a / 3-gal- positive line expressing a different restriction element (H-2 b ) .
- H-2 b restriction element
- the same enhancement of primary response was obtained with exogenous IL-2 and VJS6 (data not shown) .
- Tumor cells devoid of the / 8-gal marker protein are easily detected by a simple assay, facilitating the recognition of antigenic modulation occurring in vivo.
- Lungs from mice found dead during the survival follow-up were inflated, fixed, and exposed to an X-gal solution allowing the staining of the whole organ (Lin. W.C. et al, Cancer Res. 50:2808, 1990).
- mice The effects of recombinant IL-10 on the therapeutic effectiveness of replicating and non-replicating poxyiruses
- BALB/c mice (5-10/group) were injected intravenously with 5 x 10 5 CT26-J-gal transfectants (CT-26.C.25) then randomized.
- mice were treated with a recombinant vaccinia virus (designated VJS6) that encodes the model tumor antigen (full-length / 8-galactosidase) at 10 6 and 10 7 PFU either alone or in combination with O.l ⁇ g of IL-10 I.P. per day for 7 days starting 12 hrs after the virus was given.
- VJS6 recombinant vaccinia virus
- the source of IL-10 was human recombinant IL-10 obtained from Biological Response Modifiers Program (Frederick, MD) .
- a non-replicating recombinant fowlpox virus that also encodes the model tumor antigen (full-length ⁇ -galactosidase) at 10 6 and 10 7 PFU either alone or in combination with O.l ⁇ g of IL-10 I.P. per day for 7 days starting 12 hrs after the virus was given.
- Pulmonary metastases were enumerated on day 12 in a coded, blinded fashion. The data is provided in Table 10.
- mice were injected intravenously with 5 x 10 s CT26- / 8-gal transfectants (CT-26.C.25) then randomized. Three days later, mice were treated with number of PFU listed on the x-axis of a recombinant vaccinia virus (designated VJS6) that encodes the model tumor antigen-full-length ⁇ - galactosidase either alone or in combination with O.l ⁇ g of IL-10 per day for 7 days starting 12 hrs after the virus was given. Pulmonary metastases were enumerated on day 12 in a coded, blinded fashion. Animals receiving no treatment or IL-10 alone had an average of 250 pulmonary metastases (Figure 29) .
- VJS6 recombinant vaccinia virus
- mice were injected I.V. with 5 x 10 5 CT26-/S-gal transfectants. Three days later mice were treated with 10 ⁇ 10 2 , 10 3 , 10 4 , 10 s , 10 6 , or 10 7 PFU of VJS6 either alone or in combination with O.l ⁇ g of IL-10 per day for 7 days starting 12 hrs after the virus was given. Pulmonary metastases were enumerated on day 12 as above. Animals receiving no treatment or IL-10 alone had an average of 250 pulmonary metastases (Figure 30) .
Abstract
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Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1994016716A1 (en) * | 1993-01-21 | 1994-08-04 | Virogenetics Corporation | Recombinant virus immunotherapy |
WO1994024267A1 (en) * | 1993-04-20 | 1994-10-27 | Robinson, William, S. | Methods and materials for treatment of individuals infected with intracellular infectious agents |
US7122163B2 (en) * | 2000-06-12 | 2006-10-17 | Japan Science And Technology Agency | Rare-earth oxide or sulfide of nano size and process for producing the same through photochemical reaction |
-
1995
- 1995-10-02 AU AU39982/95A patent/AU712714B2/en not_active Expired
- 1995-10-02 EP EP95938706A patent/EP0789774A2/en not_active Ceased
- 1995-10-02 CA CA002201592A patent/CA2201592A1/en not_active Abandoned
- 1995-10-02 WO PCT/US1995/012638 patent/WO1996011279A2/en not_active Application Discontinuation
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1994016716A1 (en) * | 1993-01-21 | 1994-08-04 | Virogenetics Corporation | Recombinant virus immunotherapy |
WO1994024267A1 (en) * | 1993-04-20 | 1994-10-27 | Robinson, William, S. | Methods and materials for treatment of individuals infected with intracellular infectious agents |
US7122163B2 (en) * | 2000-06-12 | 2006-10-17 | Japan Science And Technology Agency | Rare-earth oxide or sulfide of nano size and process for producing the same through photochemical reaction |
Non-Patent Citations (2)
Title |
---|
DATABASE WPI Section Ch, Week 8837 Derwent Publications Ltd., London, GB; Class B04, AN 88-264270 & US,A,7 122 163 (MOSS B ET AL) , 2 August 1988 * |
IMMUNOLOGY AND CELL BIOLOGY, vol. 68, 1990 CARLTON, AU, pages 113-117, J.RUBY ET AL. 'Response of monkeys to vaccination with recombinant vaccinia virus which coexpress HIV gp160 and human interleukin-2' * |
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Also Published As
Publication number | Publication date |
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WO1996011279A3 (en) | 1996-05-17 |
AU712714B2 (en) | 1999-11-11 |
EP0789774A2 (en) | 1997-08-20 |
AU3998295A (en) | 1996-05-02 |
CA2201592A1 (en) | 1996-04-18 |
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