CA2201592A1 - 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

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CA2201592A1
CA2201592A1 CA 2201592 CA2201592A CA2201592A1 CA 2201592 A1 CA2201592 A1 CA 2201592A1 CA 2201592 CA2201592 CA 2201592 CA 2201592 A CA2201592 A CA 2201592A CA 2201592 A1 CA2201592 A1 CA 2201592A1
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virus
gal
mice
il
tumor
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Bernard Moss
Nicholas P. Restifo
Steven A. Rosenberg
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US Department of Health and Human Services (HHS)
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The Government Of The United States Of America, Represented By The Secre Tary, Department Of Health And Human Services
Bernard Moss
Nicholas P. Restifo
Steven A. Rosenberg
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    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86Viral vectors
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52Cytokines; Lymphokines; Interferons
    • C07K14/54Interleukins [IL]
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52Cytokines; Lymphokines; Interferons
    • C07K14/54Interleukins [IL]
    • C07K14/55IL-2
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    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705Receptors; Cell surface antigens; Cell surface determinants
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    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503Immunoglobulin superfamily
    • C07K14/70532B7 molecules, e.g. CD80, CD86
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
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    • C12N2710/00MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA Viruses
    • C12N2710/00011MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA Viruses dsDNA Viruses
    • C12N2710/24011Poxviridae
    • C12N2710/24041Use of virus, viral particle or viral elements as a vector
    • C12N2710/24043Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2710/00MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA Viruses
    • C12N2710/00011MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA Viruses dsDNA Viruses
    • C12N2710/24011Poxviridae
    • C12N2710/24111Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24141Use of virus, viral particle or viral elements as a vector
    • C12N2710/24143Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Abstract

The present invention is a recombinant virus which has incorporated into its genome or portion thereof a gene encoding an antigen at a disease causing agent in combination with an immunostimulatory molecule for the purpose of stimulating an immune response against the disease causing agent. Methods of treatment of diseases such as cancer and diseases caused by pathogenic microorganisms are provided using the recombinant virus.

Description

-~ WO96/11279 ~ 2 0 1 ~ 9 2 PCT~S9~112638 1. ~

ENHANCED IMMWNE RESPONSE
BY INTRODUCTION OF CYTOKINE GENE AND/OR

RECO~BINANT VIRUS EXPRESSING SYSTEM
FIE~D OF THE lNV~NllON
The present invention relates to recombinant viral vec~or vaccines for the prevention or treatment of patllogenic 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).
BACKGROUND OF THE INVENTION
The five year survival of patients with metastatic melanoma is less than 2~ in most reported series.
Combination chemotherapy can induce objective regressions of melanoma but this treatment is rarely, if ever, curative. Tumor infiltrating lymphocytes (TIL) 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 ;mml7ne reactions against growing cancers in mice and hllm~n~. In: De Vita, Hellman and Rosenberg (eds) Important Advances in Oncology, Philadelphia, J.B. Lippincott Co., 1990, pp 19-41, Schwartzentruber et al J. Immunol 146:3674-3681, 1991).
The adoptive transfer of TIL can mediate regression in 35 to 40~ of patients with advanced melanoma and studies are underway to attempt to generate more potent TIL by genetic modification of TIL or by ;mml~n;zation with preparations cont~;n;ng tumor associated antigens. Rosenberg, J. Clin.
Oncol. 10:180-199, 1993; Rosenberg, S.A. ~. Am. Med. Assoc 268:2416-2419, 1992.
A large number of studies in experimental ~n;m~ls have ~monctrated that the cellular rather than the humoral arm of the immune response plays the major role in the elimination of murine tumors. Wunderlich, J.R. et al.

W096111279 ~ ~ G~/ ~ ~ PCT~S9Stl2638 Principles of tumor ~mmnn;ty In: DeVita et al (eds) Biolo~ic Therapy of Cancer, Philadelphia: J.B. Lippincott Co. 1991, pp 3-21. Much of this evidence was derived ~rom studies in which the adoptive transfer of T lymphocytes from ;mmlln~ ~n;m~ls could transfer resistance to tumor challenge or in some experiments the actual elimination of established cancer. Thus, most strategies for the ;mmlln;zation of patients with cancer have been directed at stimulating strong T cell immune reactions against tumor associated antigens.
Most attempts at active ;mmlln;zation against cancer antigens have involved whole tumor cells or tumor cell fragments, though it would be most desirable to ;mmlln;ze specifically against unique tumor antigens that distinguish malignant from normal cells. The molecular nature of the tumor associated antigens recognized by T
lymphocytes is poorly understood. 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.
A number of genes have been identified that encode melanoma tumor antigens recognized by TIL in the context of the HLA-A2 class I molecule. Kawakami, T. et al Proc.
Nat'l Acad. Sci. 91:3515-3519, 1994; Kawakami, Y. et al J.
Exp. Med. 180:347-352, 1994; Kawakami et al Cancer Res.
54:3124-3126, 1994. These antigens appear to be the most clinically relevant antigens responsible for mediating tumor regression in patients with advanced melanoma since the TIL used to identify these antigens were capable of mediating in vivo antitumor regression. Two such antigens, which appear to be present in virtually all fresh and cultured mel~nom~, have been called M~RT-1 (Melanoma Antigen Recognized by T Cells - 1) and gplOO.

~ Wo96/11279 22 0 1 5 ~ 2 PCT~S9~/12638 The genes encoding both of the peptides have been cloned and sequenced. The MART-l gene encodes a 118 amino acid protein of 13 kD. The gplOO gene encodes a protein identical to that recognized by monoclonal antibody H~3-45. With the exception of melanocytes and retina nonormal 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 mel~nnm~c tested Studies by others showed that the MART-l (also called Melan-A) antigen was expressed on 26 of 26 ~resh melanomas. Coulie, P.G. et al J. Exp. Med. 180:35-42, 1992. The gplOO antigen is also widely expressed in mel~n~m~. In one study, reactivity with antibody HMB-45 (reactive with gplOO) was present on 100~ of non-spindle cell type mel~nnm~s and on 62 of 67 total mel~nom~.
Wick, M.R. et al J. Cutan. Pathol. 4:201-207, 1988. In another study, 32 of 35 mel~n~m~c studied (91~) expressed gplOO (Ordonez, N.G. et al Am. J. Clin. Pathol. 4:385-390, 1988) and in a third study of 60 of 62 (97~) melanomas expressed gplOO (Gown, A.M. et al Am. J. Pathol. 123:195-203, 1986).
Of 14 separate TIL cells that were raised in the Surgery Branch, NCI from different HLA-A2 individuals, 13 of 14 recognized MART-l and 4 of 14 recognized the gplOO
antigen. Because TIL cells that recognize these determ~n~nts have been shown to be capable of mediating cancer regression in vivo, it appears that these antigens are involved in cancer regression.
Another gene coding for a hllmAn tumor specific antigen on a hllm~n melanoma was cloned by Van der Bruggen et al (Science 254:1643-1647, 1991). This antigen is coded for by a gene called MAGE-l which spans five WO96/11279 ~ ~ ~ /5 9~ PCT~S95/12638 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 A1 restricted immunod~m;n~nt 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 immunodom;n~nt peptide can confer sensitivity to lysis by a MAGE-1 specific CTL clone. MAGE-1 does not appear to be expressed in normal cells with the possible exception of testis, but is expressed on approximately half of metastatic mel~nom~, about 20~ of breast cancers as well as other selected types of cancer.
The identification of an immuno~om;n~nt peptide that represents a unique tumor antigen has opened new possibilities for ;mmlln;zation against cancer.
Substantial evidence exists in ~n;m~l models that ;mmlln;zation with immunod~m;n~nt viral peptides can induce viral specific CTL that can confer protection against viral infection. Although pure peptide alone is ineffective in stimulating T cell responses, peptides emulsified in adjuvants or complexed with lipids have been shown to prime mice against challenge with fresh virus and can induce virus specific CTL that protect mice against lethal viral inocula (Kast, W.M. et al Proc. Nat'I Acad.
Sci. U.S.A. 88:2283-2287, 1991; Deres, K. et al Nature 342:561-564, 1989; Gao, X.M. et al J. Immunol. 147:3268-3273, 1991; Aichele, P. J. Exp. Med. 171:1815-1820, 1990;
Collins, D.S. et al J. Tmmllnol 148:3336-3341, 1992). -Tmmlln;zation of mice against splenocytes coated with Listeria monocytoqenes peptide epitopes also results in the generation of Listeria specific CTL which can be expanded in culture. Adoptive transfer of these CTL can protect mice against lethal bacterial challenge (Harty, W O 96tll279 PCTrUS95/12638 J.T. et al J. Exp. Med. 175:1531-1538, 1992). Peptides representing antigenic epitopes of HIV gpl20 and gpl60 emulsified in complete Freund's adjuvant can also prime specific CT~ responses (Tak~h~h;, H. et al Proc. Nat'l Acad. Sci. ~.S.A. 85:3105-3109, 1988; Hart, M.K. et al Proc. Nat'l Açad. Sci. U.S.A. 88:9448-9452, 1991).
While ;mmlln;zation with peptides in adjuvants or complexed with lipids gives rise to T cell responses in mice, the reactions are rarely strong enough to induce T
reactive cells in primary splenocytes. The detection of sensitized lymphocytes almost invariably requires secondary in vitro stimulation.
The expression of the B7 gene family has been shown to be an important mechanism of antitumor responses in both mice and hllmAn~ It is now becoming apparent that at least two signals are required for activation of naive T-cells by antigen bearing target cells: an antigen speci~ic signal, delivered through the T-cell receptor, and an antigen independent or costimulatory signal leading to lym~hok;ne products (Hellstrom, K.E. et al. Annals NY
Acad. Sci. 690:225-230, 1993). Two important costimulatory molecule~ are B7-1, which is the ligand for T-cell surface antigens CD28 and CTLA4 (Schwartz, R.H.
Cell 71:1065-1068, 1992; Chen, ~. et al. Cell 71:1093-1102, 1992; Freeman, G.J. et al. J. Tmmllnol 143:2714-2722, 1989; Freeman, G.J. et al. J. Exp. Med. 174:625-631, 1991), and B7-2, an alternative ligand for CT~A4 (Freeman, G.J. et al. Science 262:813-960, 1995). To date, both murine B7-1 and B7-2 (Freeman, G.J. et al. J. Exp. Med.
174:625-631, 1991; Freeman, G.J. et al. Science 262:813-960, 1995) and hllm~n B7-1 and B7-2 have been described (Freemanr G.J. et al. J. Immunol 143:2714-2722, 1989;
Freeman, G.J. et al Science 262:909-911, 1993). It is unclear at this time whether the costimulatory signals provided by B7-1 and B7-2 are functionally distinct or re~lln~nt mech~n; sm~ for T-cell activation (Hathcock, K.S.

WO96/11279 ~ ~ ~/5 ~~ PCT~S95/l2638 et al. J. Exp. Med. 180:631-640, 1994). Most murine and human tumors do not express B7-1 or B7-2, implying that even when a tumor expresses a potential rejection antigen, it is unlikely to activate antitumor T-cell responses (Hellstrom, K.E. et al Annals. N.Y. Acad. Sci. 690:225-230, 1993); Hellstrom, I. Annals. N.Y. Acad. Sci. 690:24-31, 1993). In essence, anergy may result from only one signal being received by the T-cell tHellstrom, K.e. et al. Annals. N.Y. Acad. Sci. 690:225-230, 1993.
Transfection of B7 into melanoma cells was found to induce the rejection of a murine melanoma in vivo (Townsend, S.E.
et al Science 259:368-370, 1993).
Vaccinia viruses have been extensively used in hum~ans 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 ~m;n;stration~
Attempts have been made to develop vaccinia virus vectors for the prevention of other diseases.
Vaccina virus is a me-m-ber 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 ;mml~n;ty against a variety of foreign gene products and can protect against infectious diseases in several ~ntm~l models. Recombinant vaccina viruses have been used in hllm~n clinical trials as well. Cooney et al ;mmlln;zed 35 healthy HIV seronegative males with a recombinant vaccinia virus expressing the gpl60 envelope gene of HIV (Cooney, E.. The ~ancet 337:567-572, 1991). Graham et al r~n~om;zed 36 volunteers to receive either recombinant vaccinia virus cont~;n;ng the gpl60 HIV envelope protein or control vaccinia virus (Graham, B.S. et al J. Infect. Dis. 166:244-252, 1992).

2 2 0 1 5 q ~ pcT~ss~ll2638 Phase I studies using recom~binant vaccinia virus have begun in patients with metastatic melanoma using a recombinant virus expressing the p97 melanoma antigen (Estin, C.D. et al Proc. Nat'l Acad. Sci. 85:1052-1056, 1988) and a trial to use recombinant vaccinia virus expressing the hllm~n carcinoembryonic antigen in patients with advanced colorectal carcinoma is about to begin (Schlom, J. personal commlln;cation). In these trials, vaccinia virus is Ar7.m; n; stered by intradermal scarification and side effects have been m;n;mAl including local skin irritation, lymphadenopathy and transient flu-like symptoms.
Fowlpox viruses are members of the pox virus ~amily (avipox virus genes). Fowlpox virus will only replicate in avian cells and cannot replicate in hllmAn 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 ~owlpox virus expressing rabies glycoprotein has been used to protect mice, cats and dogs against live rabies virus challenge. Tmmlln;zation 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). Cararypox virus, another member of the avipox genus similar to fowlpox, was safely ~r7m; n; stered subcutaneously to 25 normal human volunteers at doses up to 10" infectious doses (Cadox, M. et al The Lancet 339:1429-1432, 1992). In a recent trial sponsored by the NIAID (Protocol 012A: A Phase I safety and immunogenicity trial of live recombinant canarypox-gpl60 MN (A~VAC VCP125 HIV-lgpl60.~N0 in HIV-1 uninfected adults) patients received recombinant canarypox virus contA;n;ng the HIV
gpl60 gene by intramuscular injection at doses up to 10 pfu with little to no toxicity (personal comml7n;cation, P.
Fast, NIAID).

W O 96/11279 ~ ~ ~/ J 9~ PCTAUS95/12638 Fowlpox virus thus represents an attractive vehicle for ;mml~n;zatiQn since it can stimulate both humoral and cellular ~mmlln;ty, it can be economically produced in high titer (109 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 ;mmllnocompromised hosts.
Another considerable advantage of ~owlpox virus is that there is apparently little or no cross-reactivity with vaccinia virus and thus previously vaccinated hllm~nR
will not have pre-existing immune reactivity to ~owlpox virus proteins.
SUMM~RY OF THE lNv~NllON
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 ;mmllnQstimulatory molecules.
The present invention is also a composition comprising a recom.binant 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 ;mmllnostimulatory molecule, chemotherapeutic drug, antibiotic, antifungal drug, antiviral drug, or combination thereof.
Another aspect of the present invention is a composition 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 ;mmllnostimulatory 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 220 l 5q2 ~ WO96111279 PCT~S95112G38 .. ~.

g o a therapeutic composition and a method of treating or preventing a disease in a m~mmAl comprising A~ministering to the m~mm~l an effective amount of a recombinant virus and optionally an exogenous immunostimulatory or ;mmllnomodulator, chemotherapeutic drug antibiotic, antifungal drug, antiviral molecule, the amount effective in preventing or ameliorating the disease.
It is also an object of the present invention to provide a method of making an immune enhancing recombinant virus against a disease causing agent comprising inserting a nucleic acid sequence encoding one or more antigens or immuno~om;n~nt epitopes thereof and a nucleic acid sequence encoding one or more immunostimulatory molecules into the genome or portion thereof of a recombinant virus.
It is also an object o~ the present invention to provide an immune enhancing recombinant virus against a disease causing agent comprising an ;mmlln~ enhancing recombinant virus comprising a nucleic acid sequence encoding one or more antigens or immunod~m;nAnt epitopes thereof and a nucleic acid sequence encoding one or more ;mmllnostimulatory molecules.
It is also an object of the present invention to provide a vaccine against a disease causing agent comprising a recombinant virus cont~;n;ng a nucleic acid sequence encoding one or more antigens or immuno~om;n~nt epitopes thereof and a nucleic acid sequence encoding one or more ;mmllnostimulatory molecules. The vaccine of the present invention is able to prevent or inhibit infection or disease caused by the disease causing agent.
BRIEF DESCRIPTION OF THE DRAWINGS
These and other objects, feature and many of the attendant advantages of the invention will be better understood upon a reading of the detailed description of the invention.
Figure l shows ~-Galactosidase production after BSCl cell infection with various reco-m-binant vaccinia virus W096/11279 ~ ~ e~ 5 ~ ~ PCT~S9~/12638 ~

constructs after 24 hr (-) or 36 hr (O) 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 (O); IL-2rW O);
TNF~rW (-); IFN~rW (v); VJS6 (O). Effector: Target cells ratios are plotted versus ~ specific 51Cr 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 (~); IL-2rW O); TNF~rW (-); IFN~rW (V); VJS6 (O). Effector: Target cells ratios are plotted versus specific 51Cr 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 (O); IL-2rW O)i TNF~rW (-); IFN~rW (v); VJS6 (O). Effector: Target cells ratios are plotted versus specific 51Cr 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-CSFrVV (-); IL-2rW (O);
TNF~rW (v); IFNrrW (-); VJS6 O). Effector: Target cells ratios are plotted versus ~ specific 51Cr 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 O)i IL-2rW (3); TNF~rW (v); IFN~rW (-); VJS6 (-). Effector cells per well is plotted versus ~ specific s1Cr 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-~ WO96111279 2 2 0 1 5 9 2 PCT~S95112638 CSFrW (O); IL-2rW (-); TNF~rW (-); IFN~rW (v); VJS6 (O). Effector- target cells ratios are plotted versus speci~ic 51Cr release by targets.
~ Figure 8 shows active anti-tumor ;mml~notherapy 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 (lO0,000 I.U., I.P., BID x 3 days) (_); rW +BG +
HD IL-2 (lO0,000 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. GM-CSFrW (-); IL-2rW (-);
TNF~rW (~); IFN~rW (-); VJS6 (-); HBSS O). Days after tumor injection is plotted versus ~ surviving mice.
Figure lO shows survival data in an active tU-m-or ;mmllnotherapy model in mice with established ~-gal expressing tumor, CT 26.C25. GM-CSFrW (-); IL-2rW (-);
TNF~rW (~); IFN~rW (-); VJS6 (-); HBSS (-). Days after tumor injection is plotted versus ~ surviving mice.
Figure ll shows active treatment of established plllmo~ry metastases using variou~ recom~binant vaccinia constructs. The average number of plllmo~ry metastases is plotted versus recombinant vaccinia vector used for treatment. HB2m = Hllm~n Beta-2-microglobulin rW, Ld =
murine H-2~d MCH Class I molecule rW, NA = Neuramimidase rW, MVA = Ankara - attenuated vaccinia virus, Kb = murine H-2Kb MCH Class I molecule rW .
Figure 12 shows active treatment of established pnlm~ry metastases using various recombinant vaccinia constructs. The average number of pulmonary metastases is plotted versus recombinant vaccinia vector used for treatment. ICAM-l = Intracellular adhesion molecule-l rW, Dd = murine H2-Dd MCH Class I molecule rW, Kd =

W096111279 ~ o~/~ 9~ PCT~S9~/12638 murine H-2 Kd MHC Class I molecule rW, Ld = H-2 Ld MHC
Class I molecule rW; Kb = H-2Kb MHC Class I molecule rW;
HLA-A2.1 = Human MHC Class I molecule rW, h~2m = 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, MVA = Ankara - attenuated vaccinia virus, HBSS =
Hank's Balanced Salt Solution.
Figure 13 shows a Western blot analysis of recombinant vaccinia virus expressed murine B7-l. BS-C-l cells were infected with l, v. MCB7-l; 2, v. MCMHA and extracts were prepared 20 hours post infection. A murine specific B7-l hamster monoclonal antibody was used to detect expression of recombinant proteins. 125I protein A
(O.l ~Ci/ml) and autoradiography were used to identify bound antibody. Protein sizes were estimated using 14C
molecular weight markers (MW).
Figure 14 shows the results of non-irradiated BALB/c mice (5/group) which were injected intravenously with 5 x 105 tumor cells of CT26.WT or CT26.C25 on day #0. On day #3 mice were ;mmlln;zed intravenously with different recombinant vaccinia viruses (107 PFUs). Mice were r~n~om;zed, and euthanized on day #12. Lung were harvested and st~;ne~, and plllmo-n~ry 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 rW were involved in the following number of experiments: v. I~2/~-gal -- five;
v. MCB7-l/~-gal -- six; v. Dd/~-gal -- one; v. Kd/~-gal --one, v. Ld/~-gal -- one, v. Kb/~-gal -- one, v. HLA.A2.l/~-gal -- one, v. m~2m/~-gal -- one, v. h~2m/~-gal -- one, v.
GM-CSF/~-gal -- four, v. IFN-~/~-gal -- four, v. TNF-~/~-gal -- four; v. JS6 -- greater than five, v. MVA/~-gal ---one.
Figure 15 shows the results of non-irradiated BALB/c ~ WO96111279 2 2 0 1 5 9 ~ PCT~S95112638 .

mice (l0/group) which were injected intravenously with HBSS, v. MCB7~ -gal, or v. MCMHA/~-gal. Twenty-one days later mice were challenged intravenously with 5 x l05 - tumor cells CT26.C25 or CT26.WT. Mice were randomized, and euthanized on day #12. Lung were harvested and stained, and plllmon~ry metastases were ~n-lmerated in a blinded fashion. Duplicate experiments confirmed these results.
Figure 16 shows the results of non-irradiated BA~B/c mice (l0/group) which were injected with l05 or 107 PFUs of v. MCB7-l/~-gal, v. MCMHA/~-gal, or HBSS on day #0.
Twenty-one days later a splenectomy was performed on all ;mmlln;zed mice. 2 x 107 splenocytes from designated groups o~ ;mmlln;zed mice were adoptively transferred to similar mice (5/group) injected intravenously three days earlier with 5 x 105 tumor cells of CT26.C25 or CT26.WT.
Designated groups of mice were treated with IL-2 (l00,000 IU i.p. BID x 3 days). Mice were randomized and euthanized nine days later. Lung were harvested and st~;ne~, and plllm~n~ry 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 105 CT26.WT or CT26.C25 tumor cells on day #0. On day #3 mice were ;mmlln~zed intravenously with 107 PFUs of v.
MCB7-l/~-gal, v. MCMHA/~-gal, v. MCB7-l/NP or HBSS. On day# 3 - 6, designated groups of mice were treated with IL-2 (l00,000 IU i.p. BID x 3 days). Mice were r~n~nm;zed, and euthanized on day #12. Lung were harvested and st~;n~ and plllmo~ry metastases were ~nllm~rated 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 WO96/11279 G~ -- 9~ PCT~S9S/12638 mice (5/group) which were injected intravenously with 5 x 105 CT26.WT or CT26.C25 tumor cells on day #0. On day #6 5 mice were ;mml~ntzed intravenously with 107 PFUs of v.
MCB7~ -gal, v. MCMHA/~-gal, v. MCB7-1/NP or HBSS. On day# 6 - 9, designated groups of mice were treated with IL-2 (100,000 IU i.p. BID x 3 days). Mice were r~n~om;zed, and euthanized on day #12. Lung were harvested and stained, and plllmo~Ary metastases were ~n~lm~rated 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 o~ non-irradiated BALB/c mice (10/group) which were injected intravenously with 5 x 105 CT26.WT or CT26.C25 tumor cells on day #0.
On day #3 mice were ;mmlln;zed intravenously with 107 PFUs of v. MCB7-1/~-gal, v. MCMHA/~-gal, v. MCB7-1/NP or HBSS.
On 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 105 CT26.WT or CT26.C25 tumor cells on day #0. On day #3 mice were ;mmlln;zed intravenously with 107 PFUs of v.
MCB7-1/~-gal, v. MCB7-2/~-gal, v. MCB7-1/B7-2/~-gal, v.
MCMHA/B7-2/~-gal, v. MCMHA/~-gal, v. MCB7-1/NP, or HBSS.
On day# 3 - 6, designated groups of mice were treated with IL-2 (100,000 IU i.p. BID x 3 days). Mice were 3 r~n~om;zed, and euthanized on day #12. Lung were harvested and stained, and plllm~ry metastases were ~nllmPrated 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 21f show a FACS analysis of splenocytes isolated from normal non-depleted (Figs. 21a ~ WO96/11279 2 2 0 1 5 9 2 PCT~S9~/12638 and 21b), T~8+-depleted (Figs. 21c and 21d), or T~+-depleted (Fig~. 21e and 21f) mice. Mice were given two i.v. injections of GKl.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+, and lower left shows scatter for all cells. Figure 21a, no depletion Using fluorescein isothiocyante-labeled anti-CD4 and phycoerythrin labeled anti-CD8 antibodies, FACS
analysis was performed 1 day prior to ;mmlln;zation~ and again at day 7 to verify depletion.
Figure 2lg shows the mean number of pl]l mon~ ry metastases of CD4- and CD8- immuno depleted mice vaccinated with V.B7-1/~-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 105 CT26.C25 tumor cells. On day #3 mice were ;mmlln;zed intravenously with 107 PFUs of v.
MCB7~ -gal, v. MCMXA/~-gal, v. MCB7-1/NP or HBSS. 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 plll mon~ry metastases were enumerated in a blinded fashion. FACS analysis was performed 1 day prior to immlln;zation, and again at day 7 to verify depletion. Only data from v.B7-1/B-gal vaccination groups is shown.
Duplicate experiments confirmed these results.
Figures 22a and 22b show active ;mmllnotherapy is enhanced when exogenous rI~-2 and rW are given in concert. BALB/c mice (five per group) were challenged i.v. with 5 x 105 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 cont~;n;ng W O 96tll279 9 PCTrUS9~/12638 5 x 106 PFU of different TK- rW either expressing (VJS6) or not expressing (V69) ~-gal. Two different regimens of rIL-2 ~m; n; stration 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 105 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), 107 PFU of FPV.bg40k (rVPV), or FPV wild-type (FPVwt). rIL-2 (100,000 Cetus U, twice a day) was ~m;n;stered 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 105CT26.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 ~m;n;stered. Mice were checked daily for survival. No prolongation of survival was obtained by the 2S various treatments in mice bearing 6-day-old pnlmo~ry 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 105 tumor cells of either CT26.WT or CT26.CL25 cell lines.
Three days later they received a single i.v. injection of HBSS alone (none) or cont~;n;ng 5 x 106PFU/mouse of different rW, as indicated. On day 12 post tumor challenge, lungs were harvested and pl]lmo~ry nodules were -WO96/11279 2 2 0 1 5 9 2 PCT~S9~/12638 ~nl~mPrated 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 l05 CT26.WT (Fig. 26a) or CT26.C~25 tumor cells (Fig. 26b). After 3 days they received a single i.v. injection of plain medium (HBSS) or medium cont~;n;ng 5 x 106 PFU of rW encoding ~-gal alone (VJS6) or together with IL-2 (IL-2 rW). Twelve hours after rW, l00,000 rIL-2U were inoculated, according to the regimen described in Figs. 22a and 22b. Mice were checked twice a day for survival.
Figure 27 shows that expression of IL-2 by drW
enhances the antivaccinia CTL response. Two BALB/c mice were ;mmlln;zed with 5 x l06 PFU/mouse of different rW.
After 6 days the spleens were aseptically removed, m; ~e~
together, and tested directly in a 6-h s1Cr release assay against CT26.WT tumor cell line, either infected (CT26 vaccinia) or non;nfected (CT26.WT) during the isotope labeling with more than l0:l moi of crude l9 W
preparation. Spontaneous release of target cells never exceeded 20~. E:T cell ratio was l00:l and then 1:3 dilutions. Lytic units 30~ (L.U. 30~) indicate the number f effector cells required to obtain 30~ lysis of l0,000 target cells. h.U. 30~ were normalized for the total numiber 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 ;mmlln;zed with 5 x lo6 PFU/mouse of either VJS6 or IL-2 rW. On day 9 after tumor challenge (day 6 after vaccination) the primary cytotoxic response was evaluated in a 6-h s1Cr WO96111279 9 PCT~S95/12638 release assay against CT26.WT, CT26.WT pulsed with the ~-gal Ld-restricted peptide (CT26T + peptide), CT26.CL25, and the irrelevant target cells E22 as shown. The effects of escalating doses of tumor on the generation of the primary cytotoxic response is shown for the ;mmnn;zation with IL-2rW in nontumor-bearing mice (-), or in mice bearing 5 x 104 CT26.CL25 (Q), 1 X 105 CT26.CL25 O), 5 x 105 CT26.CL25 (O), or the highest dose, 5 x 105 of the parental (non-~-gal-expressing) CT26.WT cell line ( G ) . An additional control is the non-IL-2-expressing VJS6 virus injected in mice bearing 5 x 105 CT26.CL25 (-).
Figure 29 shows the effects of recombinant IL-l0 on the therapeutic effectiveness of ;mmlln;zation with the vaccinia virus, VJS6 + IL-l O); VJS6 alone (X).
Figure 30 shows the IL-l0 adjuvant therapy on the therapeutic effectiveness of ;mmlln;zation with various concentrations of recombinant vaccinia virus encoding the model tumor antigen.

DETAILED DESCRIPTION OF THE lNV~NllON
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 m~mm~l 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 ;mmnn;ty.
Cell-mediated ;mmlln;ty is crucial to resistant to diseases caused by cancer and pathogenic microorg~n;~m, 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 ;mmllnostimulatory molecules. A host cell infected ~ WO96/11279 2 2 0 1 5 9 2 PCT~S95/12638 o ~ 19 -with the recombinant virus expresses both the antigen(s) from a disease causing agent and expresses the ;mmllnostimulatory 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 ;mmllnostimulatory 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 ;mmlln~ system. In a preferred embodiment the upregulation of the ;mmllne response is an increase in antigen specific cy~o~oxic iymphocy~es whic~1 are able LO K111 OL inhibit the growth of a disease causing agent or a cell infected with a disease causing agent.
In one e-m-bodiment~ the recombinant virus comprises the virus genome or portions thereof, the nucleic acid sequence encoding an antigen from a pathogenic microorg~n;sm and one or more nucleic acid sequences encoding one or more ;mmllnostimulatory molecules.
In another embodiment, the recombinant virus comprises the virus genome or portions therecf, the nucleic acid sequence encoding a tumor associated antigen, and one or more nucleic acid sequences encoding one or more ;mmllnostimulatory molecules.
In one embodiment the recombinant viruses have been constructed and co-express model tumor antigens together with cytokines (TNF-~, IFN-~, GM-CSF, IL-lO and IL-2), restriction elements (class l ~- ch~ ; n~ and ~2m), and co-stimulatory and accessory molecules (B7-l, B7-2 and ICAM-l and the like) alone and in a variety of combinations.
Simultaneous production of an ;mmllnostimulatory molecule and the model TAA at the site of virus replication/infection (in any case, the site of TAA

W O 96/11279 , ~ ~ ~7/ J ~ ~ PCT~US9~/12638 production) enhances the generation of specific effectors.
Dependent upon ~he specific ;mmnnostimulatory molecules, different mechanisms might be responsible for the enhanced ;mmllnogenicity: augmentation of help signal (IL-2), recruitment of professional APC (GM-CSF), increase in CTL
frequency (IL-2), effect on antigen processing pathway and MHC expression (IFN~ and TNF~) and the like. The co-expression of a model antigen together with at least one ;mml~nQstimUlatory molecule is effective in an active immunotherapy model.
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 ;mmllnostimulatory molecule for the added benefit of upregulating an immune response against the antigen.
In some cases it may be beneficial to make a recombinant virus comprising more than one antigen of interest for the purpose of having a multivalent vaccine.
For example, 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.
In one embodiment, the recombinant virus comprises the vaccinia virus genome or portions thereof, the nucleic acid sequence encoding MART-1 and the nucleic acid sequence encoding the ;mml~nostimulatory molecule, B 7.1 alone or in combination with the nucleic acid sequence encoding the ;mmllnostimulatory molecule, B7.2.
In another embodiment, the recombinant virus comprises the fowlpox virus genome or portions thereof, the nucleic acid sequence encoding M~RT-1, the nucleic acid sequence encoding MAGE-1, and the nucleic acid sequence encoding the ;mmllnostimulatory molecule, IL-2, alone or in combination with the nucleic acid sequence ~ WO 96/11279 ~ 2 0 1 5 ~ 2 PCTlUS9~i112638 O ~ 21 ~
encoding the immunostimulatory molecule, B7.1. In a speciic embodiment the recombinant virus i5 McB7-1, McB71-1/NP, McB7-1~-gal, McB7-1/ova, McB7-1/PIA, McB7-1/B7-2, McMHA, McMHA/NP, McMHA/~-gal, McMHA/ova, 5~ McMHA/PIA, McMHA/B7-2, McB7-2, McB7-2/~-gal, IL-2(~-gal)rW, GM-CSF(~-gal)rVV, IFN~(~-gal)rW, IL-10(~-gal) rW, and TNF~(~-gal)rW.
The insertion of costimulatory molecules and/or cytokine genes in recombinant vaccinia virus (rW genome cont~;n;ng the model TAA) is beneficial in treatment of established metastases.
The present invention encompassed a composition comprising a recombinant virus cont~;n;ng a viral genome or portion thereof, one or more nucleic acid sequences lS encoding one or more antigens from one or more disease causing agents and one or more nucleic acid sequences encoding one or more ;mmllnostimulatory molecules. The compositions of the present invention may also comprise an exogenous ;mmllnostimulatory molecule or combinations o~
immunostimulatory molecules and/or may comprise a chemotherpeutic drug, antibiotic, antifungal drug, antiviral drug and the like and combinations thereof.
In one embodiment, the composition contains a recombinant virus which has incorporated into its genome 2 or portion thereof a gene encoding a tumor associated antigen and exogenous I~-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 Vectors `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. In one embodiment the virus vector WO96/11279 ~ ~/J ~ æ PCT~S95112638 has a tropism for a specific cell type in the m~mm~l. In another embodiment, the virus vector of the present invention is able to infect professional antigen presenting cells such as dendritic cells and macrophages.
In yet another embodiment of the present invention, the virus vector is able to infect any cell in the m~mm~l 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.
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).
A general strategy for construction of vaccinia virus expression vectors have been described (Smith and Moss Bio Techniques Nov/Dec, p. 306-312, 1984; U.S. Patent No.

4,738,846). Briefly, the first step in formation of recombinant viruses expressing heterologous DNA is the construction of a ch;m~ric gene cont~;n;ng 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.
Importantly, additional ATG triplets between the transcriptional and translational start sites should be eliminated so that fusion polypeptides or incorrect reading frames are avoided. Generally, the promoters are included in DNA fragments 200-300 base pairs in length, but smaller fragments have also proved functional. After their assembly in plasmid vectors, the ch;m~riC genes are wos61ll279 220 ~ 59~ PCT~S9~tl2638 ~.

inserted into the virus genome by homologous recombination in ~ivo. This method i9 necessary since the great size of vaccinia DNA makes in vi tro construction o~ recombinant molecules impractical. To facilitate homologous 5 recombination, the chim~ric gene is first flanked by vaccinia DNA taken from a non-essential region of the virus genome. The resulting plasmid, called a recombination vector, is trans~ected into vaccinia virus-infected cells, whereupon homologous recombination results 10 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 determ; n~S the site of insertion. Only nonessential 15 regions can be used and several of these have now been identified. A cnmmonl y 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 20 alternatively incorporated into the TK region of recombinant vaccinia virus vector. Likewise, a gene encoding an ;mmllnostimulatory molecule may be incorporated into the HIND F13L region or the TK region of recombinant vaccinia virus vector.
Sutter and Moss (Proc. Nat'l. Acad. Sci U.S.A~
25 89:10847-10851, 1992) and Sut~ter et al. (Virology 1994) disclose the construction and use as a vector, the non-replicating recombinant Ankara virus (MVA, modi~ied vaccinia Ankara) which may be used as a viral vector in the present invention.
Baxby and Paoletti (Vaccine 10:8-9, 1992) 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 ~or use in the present WO96/11279 ~ 9~ PCT~S95/12638 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, t~rm~n~tion 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. It will be understood by one skilled in the art the correct combination of required or preferred expression control elements will depend on the host system chosen. It will further be understood that the expression vector should contain additional elements necessary for the transfer and subsequent replication of the expression vector cont~1n~ng 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.
Disease Causing Aqents 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 immuno~om;n~nt epitope on the antigen which is crucial in immune recognition and ultimate elimination or control of the disease causing agent in a m~mm~1, é
- 25 ~
sometimes referred to in the art as a protective antigen.
The mAmmAlian immlln~ system must come in contact with the antigen or ;mmllno~om;n~nt 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 immuno~omlnAnt epitopes on the antigens and one or more nucleic acid sequences encoding one or more ;mmllnostimulatory molecules for the purpose of enhancing ;mmlln~ response against the disease causing agent.
Such disease causing agents include but are not limited to cancer and pathogenic microorganisms or mAmmAls~ ~AmmAls include but are not limited to hllmAn~, primates, rats, mice, guinea pigs, rabbits, horses, cows, sheep, pigs, goats and the like. Cancers of mAmm~ls which may be treated using the recombinant virus of the present invention include but are not limited to melanoma, metastases, adenocarcinoma, thymoma, lymph~mA, 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.
The term melanoma includes, but is not limited to, melanomas, metastatic mel~n~mA~, melanomas derived from either melanocytes or melanocytes related nevus cells, melanocarc;nomA~, melanoepitheliomas, melanosarcomas, melanoma i situ, superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma, acral lentiginous melanoma, invasive melanoma or familial atypical mole and melanoma (FAM-M) syndrome. Such melAnom~ in m~mmAls may be caused by, chromosomal abnormalities, degenerative growth and developmental disorders, mitogenic agents, ultraviolet radiation (W), viral infections, WO96111279 -- ~ ~ /~ 9~ PCT~S95/12638 inappropriate tissue expression of a gene, alterations in expression of a gene, and presentation on a cell, or carcinogenic agents.
The aforementioned cancers can be assessed or treated 5 ~ by methods described in the present application. In the case of cancer, 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 ;mmllnostimulatory molecules. The antigen associated with the cancer may be expressed on the surface of a cancer cell or may be an internal antigen. In one embodiment the antigen associated with the cancer is a tumor associated antigen (TAA) or portion thereof.
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. 180:347-352, 1994), MAGE-1, MAGE-3, GP-100, (Kawakami et al. Proc. Nat'l. Acad. Sci. U.S.A. 91:6458-6462, 1994), CEA, TRP-1, P-15, and tyrosinase (Brichard et al. ~. Exp.
Med. 178:489, 1993) and the like.
The nucleotide sequence of the MAGE-3 gene is disclosed in Gaugler et al. J. Exp. Med. 179:921-930, 1994. MAGE-3 is expressed on many tumors of several types, such as melanoma, head and neck squamous cell carc~nom~, 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. The cloning of MART-1 is disclosed in KawaKami et al J. Exp. Med. 180:347-352, 1994 and Serial No. 08/231,565 filed April 22, 1994. The full length MART-1 nucleic acid sequence can be isolated from the pCRII plasmid by digestion with HINDII and XhoI
restriction enzymes. This 1.6kb nucleic acid sequence or WO96111279 2 2 0 1 5 9 2 PCT~S95/12638 portions thereof can then be incorporated into the genome ~ of the recombinant viruses described herein along with an immunostimulatory gene or genes.
- In another embodiment the TAAs are CA-l9-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).
The present invention is in no way limited to the genes encoding the above listed TAAs. Other TA~s may be 1 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 m~mm~l S and include viruses such as HIV (GP-120, pl7, GP-160 antigens), influenza (NP, HA antigen), herpes simplex (HSVdD antigen), hllm~n 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. In a preferred embodiment the pathogenic microorganism is an intracellular organism.

Tmmllnostimulatory Molecules: Costimulation/Accessory Molecules and Cytokines The gene from 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.
Examples of costimulation molecules include but are not limited to B7-l, B7-2, ICAM-l, ICAM-2, LFA-l, LFA-3, CD72 and the like. Examples of cytokines encompassed by the present invention include but are not limited to IL-2, IL-WO96111279 ~ ~ ~ ~ PCT~S95/12638 1, IL-3 through I~-9, IL-ll, IL-13 through I~-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. Examples of ch~mokines encompassed by the present invention include but are not limited to CTAP III, ENA-78, GR0, I-309, PF-4, IP-10, LD-78, MBSA, MIP-l~, MIP-lB and the like.
IL-2 construct 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).
In one embodiment 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.
Also encompassed in the present invention is a rh;meric gene containing a pox virus promotor region linked to the coding segment of one or more foreign genes encoding an antigen(s) from a disease causing agent and the coding segment of one or more foreign genes encoding an immunostimulatory molecule(s). The chimeric genes then incorporated into the pox virus genome by homologous recombination in cells that have transfected with a plasmid vector containing the ch;meric gene and infected with the pox virus.
In a one embodiment the IL-2 construct of the present invention comprises the recombinant vaccinia cont~; n; ng the IL-2 gene and the P synthetic late promotor and an antigen in the TK region from a disease causing agent in the F13~ region of the vaccinia virus genome promotor.
B7 Construct Co-stimulatory molecules of the B7 family (namely WO96/11279 2 2 0 1 5 9 2 PCT~S95/12638 B7.1, B7.2, and possibly B7.3) 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. No significant amplification of the ;mmllne response against a given antigen occurs without co-stimulation (June et al.
(Immunoloqy Today 15:321-331, 1994); Chen et al.
(Immunology Today 14:483-486); Townsend et al. (Science 259:368-370)). Freeman et al. (J. TmmllnQl 143:2714-2722, 1989) report cloning and sequencing of B7.1 gene. Azuma et al. (Nature 366:76-79, 1993) report cloning and sequencing B7.2 gene.
In one embodiment the B7.1 gene was inserted into the Hind F13L region of the vaccinia virus, with the ~-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.
In another embodiment 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.
In the method of treatment, the ~ministration of the recombinant virus of the invention may be for either W O 96/11279 ~ ~ PCTrUS9~112638 llprophylactic" or "therapeutic" purpose. When provided prophylactically, the recombinant virus of the present invention is provided in advance of any symptom. The prophylactic A~mi n; stration of the recombinant virus serves to prevent or ameliorate any subsequent infection or disease. When provided therapeutically, the recombinant virus is provided at (or shortly after) the onset of a symptom of infection or disease. Thus 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.
The genetic definition of 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 ;mmllne response in terms of prevention in patient with an increased risk of cancer development (preventive ~mmlln;zation)l prevention of disease recurrence after primary surgery (anti-metastatic vaccination), or as a tool to ~pAn~ the number of CTL i vivo, thus improving their effectiveness in eradication of diffuse tumors (treatment of established disease).
Finally, 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).
The term "unit dose" as it pertains to the inoculum refers to physically discrete units suitable as unitary dosages for mAmmA1s, each unit contA;n;ng a predetermined a quantity of recombinant virus calculated to produce the desired ;mmllnQgenic 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 ;mml]nologic effect to 22~ 1 5~2 WO96/11279 PCT~S95/12638 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 phArm~ceutical 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 A~m; n; stered at least once. Subsequent doses may be A~m; n; stered as indicated.
In providing a mAmmAl with the recombinant virus of the present invention, preferably a hl~m~n, 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, tU-mor burden and the like.
In general, it is desirable to provide the recipient with a dosage of recombinant virus in the range of from about 105 to about lo10 plaque forming units/mg mAmm~Al, although a lower or higher dose may be A~m; n; stered.
The recombinant viral vector can be introduced into a mAmm~l 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. Examples of methods for ~m; n; stering the viral vector into mAmmAls include, but are not limited to, exposure of cells to the recombinant virus ex vivo, or injection of the recombinant virus into the affected tissue or intravenous S.C., I.D.
or I.M. A~m; n; stration of the virus. Alternatively the recombinant viral vector or com~bination of recombinant viral vectors may be ~m; n; stered locally by direct injection into the cancerous lesion or topical application in a ph~rm~ceutically acceptable carrier. The quantity of recombinant viral vector, carrying the nucleic acid WO96/11279 ~/~9~ PCT~S95/12638 sequence of one or more TAAs to be A~m;n;stered is based on the titer of virus particles. A preferred range of the immunogen to be ~m;n;stered is 105 to l01 virus particles per m~mm~l, preferably a human.
After ;mml~n;zation the efficacy of the vaccine can be assessed by production of antibodies or ;mml~ne 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 m~mm~l to be ;mml~n;zed is already afflicted with cancer or metastatic cancer the vaccine can be ~m;n;stered in conjunction with other therapeutic treatments.
In one method of treatment, 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. By combination therapy is meant that the recombinant virus cont~;n;ng 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 ~m;n;stered to the patient in combination with other exogenous ;mmnnomodulators or ;mmllnostimUlatory molecules, chemotherapeutic drugs, antibiotics, antifungal drugs, antiviral drugs and the like alone or in combination thereof. In one embodiment the combination therapy includes a recombinant virus and exogenous I~2. In another embodiment the combination therapy includes a recombinant virus encoding one or more WO 96/11279 ~ 2 0 1 5 9 2 PCTIUS9~/12638 exogenous antigens and exogenous I~-10. Examples oî other exogenously added agents include exogenous I~-2, I~-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 ;mmlln;zation with the recombinant virus of the present invention. The antibody has speci~icity for and reacts or binds with the antigen of interest. In this e-m-bodiment of the invention the antibodies are monoclonal or polyclonal in origin.
Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact iIr~nunoglobulin molecules or those portions of an ;mmllnoglobulin molecule that contain the antigen binding site, including those portions of ;mmnnoglobulin 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 o~ Rodent and ~llm~n 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. 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.
In one embodiment the antibodies of this invention are used in immunoassa~s to detect the novel antigen of interest in biological samples.
In one embodiment, the MART-1 antibodies of this 35 invention generated by ;mm~ln;zation with recombinant WO96/11279 ~ PCT~S9~/12638 vaccinia virus expressing MART-l and B7.l are used to assess the presence of the MART-l antigen from a tissue biopsy of a m~mm~l afflicted with melanoma using immunocytochemistry. Such assessment o~ the delineation of the MART-l antigen in a diseased tissue can be used to prognose the progression of the disease in a m~mm~l afflicted with the disease or the efficacy o~
immunotherapy. Conventional methods for ;mmnno~;stochemistry are described in (Harlow and Lane (eds) (1988) In "Antibodies A ~aboratory ~ml~l ", Cold Spinning Harbor Press, Cold Spring Harbor, New York;
Ausbel et al. (eds) (1987). In Current Protocols In Molecular Biology, ~ohn Wiley and Sons (New York, New York).
In another embodiment the antibodies of the present invention are used for immunotherapy. The antibodies of the present invention may be used in adoptive ; mmllnotherapy.
In providing a patient with the antibodies or antigen binding fragments to a recipient m~mm~l, preferably a hllm~n, the dosage of administered antibodies or antigen binding fragments will vary depending upon such factors as the mAmm~l's age, weight, height, sex, general medical condition, previous medical condition and the like.
In general, it is desirable to provide the recipient with a dosage of antibodies or antigen-binding fragments which is in the range of from about l mg/Kg to about lO
mg/Kg body weight of the m~mm~l, although a lower or higher dose may be ~m; n; stered.
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.

WO96/11279 2 2 0 I S 9 2 PCT/US9~;112638 Example 1 Construction and Characterization of Recombinant vaccinia Viruses Expressing Murine B7 (mB7) and Additional Foreign Proteins Various methods for inserting foreign genes into a 5 vaccinia virus expression vector are known in the art (Smith, G.L. et al, Vaccinia Virus Expression Vectors:
Construction, Properties and Applications Bio Techniques Nov/Dec:306-311, 1984; Flexner, C. et al. Expression o~
~llmi:ln Interleukin-1 by live recombinant vaccinia virus.
Vaccines 87, Cold Spring, Harbor Lab., pp 380-383; U.S.
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. For control viruses, the mB7 gene was replaced by the measles HA (mHA) gene.
Construction and Characterization of Vaccinia Recombinant Viruses Expressinq mB7-1 mB7-2 and m~.
The construction of initial recombinants expressing the above genes employed the plaque formation selection system in which the foreign gene i5 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) - 30 is also situated within the flanking regions. The mB7-1, Mb7-2 and mHA were individually cloned adjacent to E/L
within pRB21. 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 WO96111279 ~d ~/~ - PCT~S95/12638 were carried out according to st~n~rd published procedures. Reco-m-binant viruses were readily identified according to their plaque positive phenotype, in contrast to the plaque deficient non-recombinants.
e Partially plaque purified recombinants from each S transfection were consequently characterized using antibodies specific for the individual foreign protein expressed. This was carried out by direct immuno-st~;n;ng of plaques on unfixed monolayers of BS-C-l 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-l cells (African Green monkey kidney cell line American Type Culture Collection (ATCC Accession No. CCL 26) followed by plaquing on STO
cells e-mbryonic 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-l 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 80C. A single aliquot from each batch was thawed, sonicated and titred on BSC-l cells. Infected monolayers were ;mmllnostained to determine continued expression of foreign proteins.
The B7-l recombinant (v.MCB7.l) was further characterized in a FACS analysis to illustrate surface expression and its ability to bind CTLA4-Ig (B7-l T-cell receptor) and by Western blot analysis using the anti-B7-l antibody.
The B7-l recombinant has been shown to be attenuated compared to wild-type WR, in a mouse LDs assay.
Conqtruction and Characterization of Vaccina Recombinants ~201592 WO96/11279 PCT~S95112638 o mB7-1 , mB7-2 or m~a Co-expre~sing Additional Genes ~ Additional genes were inserted into the TK region o~
the above recombinants. The following is a list of recombinants constructed and characterized (the first part of the code indicates the gene situated in the Fl3L loci under control of the E/L promoter and latter abbreviation denotes genes in the TK region):
Code Genes Expressed and Promoters McB7-l mB7-l, P. E/L

McB71-l/NP mB7-l, P. E/L
Influenza NP, P7.5K

McB7-l~-gal mB7.l, P.E/L
B-gal. P.7.5K
McB7-l/ova mB7-l, P.E/L
Ovalbumin, Pll . K
B-gal, llk McB7-l/PlA mB7-l, P.E/L
Murine PlA, P7.5K
~-gal. P.llk McB7-l/B7-2 mB7-l, P.E/L
B-gal, P.llk McMHA Measle Hemaggluttin, P.E./L

McMHA/NP
McMHA/~-gal McMHA/ova TK inserted, details as McMHA/PlA above McMHA/B7-2 mB7-2, 7.5K
B-gal. P.llk McB7-2 B7-2. P.E./L

WO96/11279 ~ S9~ PCT~S9~/12638 o McB7-2/~-gal B7-2, P.E./L
B-gal. P.llk A recombinant vaccinia virus cont~ln;ng 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 ~-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.
Example 2 Recombinant Fowlpox Virus Expressing A TAA. ~acZ and B7.1 A recombinant fowlpox virus (rFPV), which is replication incompetent in m~mm~lian cells, is constructed that expres~ the model TAA, lacZ encoding ~-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. Hu-m--. Retroviruses, 7:991, 1991). Foreign sequences are inserted into FPV by homologous recombination as previously described (Jenkins et al. 1991). Recombinant ~owlpox 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. ~, Virol. 60:436, 1986), inserted into the BAM HI J
region of the FPV genome.

~ WO96/11279 2 2 0 1 5 9 2 PCT~Sg~/12638 o Example 3 Recombinant Modified Vaccinia Ankara (MVA) Ex~ressinq the TAA lac Z and IC-2 ~ Modified Vaccinia Ankara (MVA) strain is used as the expre8sion vector. MVA was derived from vaccinia virus (WT) by over 570 serial passages in chicken embryo fibroblast cells (CEF) (Mayr, A. et al. Infection 3:6-14, 1975). The resulting MNA strain lost the capacity to productively infect m~mm~l ian cells (Altenburger, W. et al. Arch. Virol 105:15-27, 1989; Meyer, H. et al. J. Gen.
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-lS 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 P11 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 ~-gal and B7.1 upon infection of nonpermissive human cells is detected by methods described herein.
Exa~lple 4 Effect Of Inoculation Of Various Recombinant Vaccinia Virus Constructs On Primary And Secondary Responses A~ainst Vaccinia Or ~-Galactosidase Materials and Methods S BALB/c (H-2d) 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 .
In all these constructs the mouse model tumor W O 96/11279 ~ ~ o~f ~ ~ ~ PCTrUS95tl2638 ~
~. I

associated antigen, ~-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~ he~ic~ e promoter (Davidson et al Nucleic Acid Research 18 (No. 14):4285-5286, 1991). All these rW were generated by recombination in the TK region of vaccinia using the plasmid pMJ601 described in Davison et al Nucleic Acids Research 18 (No. 14):4285-5286, 1991.

In this construct, cont~;n;ng the E6 protein from Human Papilloma Virus (HPV) a pE/L promoter (the "synthetic superpromoter") is placed just upstream the p7.5 promoter controlling the ~-Gal expression and oriented in the same direction.
Control V~c~ne Control vaccinia (crude 19, NP- W) does not expressing ~-Galactosidase.
Primary in vivo Responses and SecQn~ry in vitro Responses Female BALB/c mice, 8-12 weeks old, were obtained ~rom the ~n; m~l1 Production Colonies, Frederick Cancer Research Facility, NIH, Frederick, MD. Primary lymphocyte populations were generated by injecting BALB/c mice i.v.
or subcutaneously with 106-107 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 ~-gal expressing'and control targets in a six hour s~Cr release assay.
Secondary in vitro effector populations were generated by harvesting the spleens of mice 21 days after ;mmlln;zation with recombinant virus and culturing single cell suspensions of splencytes in T-75 flasks (Nunc, Denmark) at a density of 5.0 x 106 splenocytes/ml. with 1 ~g/ml of antigenic peptide in a total volume of 30 ml of culture medium consisting of RPMI 1640 with 10~ fetal calf serum (both from Biofluids) that contained 0.1 mM non-essential ~ WO96/11279 2 2 0 1 5 ~ ~ PCT~S95/12638 amino acids, 1.0mM sodium pyruvate (both from Biofluids) ~ and 5 x 10-5 M 2-mercaptoethanol (GIBCOjBRL, Rockville, MD) in the absence of IL-2. Six days later splenocytes were - harvested and washed in culture medium before testing in a 5~ 5ICr release assay.
Cr relea~e assay Six-hour s1Cr release assays were performed as previously described (Restifo, N. et al. J. Exp. Med.
177:265, 1993). Briefly, 1 x 106 target cells were incubated with 200mCi Na5lCrO4(5lCr) for ninety minutes.
Peptide pulsed targets were incubated with 1 ~g/ml (which is roughly l~M) of antigenic peptide (for ~-gal =
TPHPARIGL peptide) during labeling as previously described (Restifo, N. et al. J. Immunol 147:1453, 1991). Target cells were then mixed with effector cells for six hours at the effector to target (E:T) ratios indicated. The amount of s~Cr released was determ;ned by ~-counting and the present specific lysis was calculated from triplicate samples as follows: [(experimental cpm - spontaneous cpm)/(m~;m~l cpm - spontaneous cpm)] x 100.
In vivo protectlon and treatment studies For in vivo protection studies, mice were ;mmlln;zed with recombinant virus 21 days before a subcutaneous challenge with 104 tumor cells or an intravenous challenge with 5 x 105 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 ~m; nt stered tumor were euthanized on day 12 and randomized before counting lung metastases in a blinded fashion as previously described (2).
For in vivo treatment studies, un-irradiated BALB/c W O 96/11279 ~ ~ / ~ ~ ~ PCTrUS9~/12638 mice were challenged with either 105 or 5 x 105 tumor cells intravenously in order to establish pulmonary metastases.
Mice were subsequently vaccinated with 107 PFU of the designated recombinant virus intravenously or subcutaneously on days three or six. Mice receiving intravenously ~m; n; stered tumor were euthanized on day 12 and randomized before counting lung metastases in a blinded fashion.
The following targets were used in each assay:
CT26 = murine H-2d adenocarcinoma CT26 gal = murine H-2d adenocarcinoma expressing ~-Gal. CT26 gal also expresses higher levels o~ class I MHC
molecules.
E22 = murine H-2d thymoma expressing ~-Gal.
CT26 + peptide = CT26 pulsed with TPHPARIGL (876-884) Ld -restricted ~-gal peptide 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.

Example 5 Cytokine Secretion After Infection Of BSC-l Cells With Various 25 Recombinant Vaccinia Virus Constructs Duplicate wells o~ 105 BSCl in 1 ml of 2.5~ fetal calf serum (FCS) medium RPMI 1640 with antibiotics (24 well plates) were infected with VJS6, rW-IL-2, rVV- GM-CSF, rW-IFN~ or rW-TNF~. At the end of incubation at 27C 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, IFN~; IL-2 and TNF~.

~ WO96/11279 2 2 0 1 5 9 2 PCT~S95/12638 The results show that high concentrations of each ~ cytokine were detected. The highest concentration of cytokine produced was I~-2 from BSC-l cells infected with rW-IL2 (Table l).
s WO 96111279I `~ ,~/S9,~ PCT/US95/12638 Table 1 DETECTION OF CYTOKINES SECRETED A~;TER -INFECTION OF BSCl CELLS WITH DIFFERENT rVV
MOI 12h M[OI 36h rvv 1:1 0.1:10.01:1 1111~ 1:1 0.1:1 0.01:1 ' GM-CSF d I ' kit VJS6 C 15.6 < 15.6 < 15.6 - < 15.6 < 15.6 < 15.6 IL-2 < 15.6 < 15.6 22 - < 15.6 < 15.6 < 15.6 ¦ GMCSF >250 >250>250 s.lXl05 >250 >250 >250 4.2x106 IFN~y < 15.6 < 15.6 < 15.6 - < 15.6 < 15.6 < 15.6 TNF~Y < 15.6 < 15.6 < 15.6 - < 15.6 < 15.6 IFN~ d - kit VJS6 <47 <47 <47 ~ <47 <47 <47 IL-2 < 47 < 47 < 47 - < 47 < 47 < 47 GMCSF < 47 < 47 < 47 - < 47 < 47 < 47 ¦ IFN~y > 12000 9000 1000 56000 > 12000 > 12000 > 12000 > 67000 TNF~ <47 <47 <47 ~ <47 <47 <47 II~2'' ' kit VJS6 <34 <34 <34 - <34 <34 <34 IL,2 > 850 > 850 > 8502X106 > 850 > 850 > 850 3.9x106 GMCSF < 34 < 34 50 - 54 63 110 IFN~y <34 121 135 - 94 97 132 TNFcY ~ 34 < 34 < 34 - < 34 < 34 < 34 TNFa~ 'e-' kit VJS6 <30.2 <30.2 <30.2 - <30.2 <30.2 <30.2 IL-2 <30.2 <30.2 <30.2 - <30.2 <30.2 <30.2 GMCSF <30.2 <30.2 <30.2 - <30.2 <30.2 <30.2 IFN~y <30.2 <30.2 <30.2 - <30.2 <30.2 <30.2 ¦ TNF~ 2050 310 295 2050 >2450 >2450 >2450 135000 a. s~ --- from 1: 1 MOI (mlll~iplirity of il f~ivily) infected cells, that were expected to exceed the highest point of the standard curve, were serially diluted (1:10 dilution) to calculate the precise cytokine c~,..c~
b. Only one of the two wells was positive.

WO96111279 2 2 0 1 5 9 2 PCT~S95/12638 Example 6 ~ ~-Galactosidase Production After BSCl Cell I~fection With Various Vaccinia Virus Constructs One Xl05 BSCl cells were in~ected with l:l MOI of 5either rW-VJS6, rW-IL2, rW -GM-CSF- rW-TNF~ or rW-IFN~. At this MOI the cytopathic effect was less pronounced at time end o~ incubation, and presumably no leakage of the enzyme had occurred. Pelleted cells were subject to three freeze-thaw rounds to release the cytoplasm content, cellular debris was removed and the supernatant used for detection of galactosidase activity on the substrate O-nitrophenyl-~-D-galactopyranoside (ONPG) using a Promega Kit No. B2000 (Promega, Madison, Wisconsin). One unit of galactosidase hydrolyze l~M ONPG
per minute at pH 7.5 at 37C. Duplicate wells were run on 15each sample. The results from two different experiments are shown in Figure l. The control vaccinia, NP- W, did not express ~-gal. VJS6 samples were serially diluted to fit within the depicted range. The results show a l log difference in the ~-Gal enzymatic activity of VJS6 20compared to the other recombinant vaccinia virus. The results as shown in Figure l parallel with the kinetics of blue st~;n;ng with X-gal in plaque assays (data not shown) .
Example 7 Primary Response Of Mice At Day 6 After Injection I.V. With Various Vaccinia Virus Constructs 5 BALB/c mice were injected intravenously (I.V.) with 5xl06 plaque forming units (pfu) of the following recombinant vaccinia virus: VJS6, I~2-rW, GM-CSF-rW, - 30TNF~-rW OR IFN~-rW . At day 6, 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 35and GM-CSF produced by rW during infection notably WO96111279 ~ ~ PCT~S95112638 enhanced the primary CTL response against vaccinia determ;n~nts. The spleen cells also gave a weak primary response toward ~-gal.
Example 8 Secondary Cultures From Mice Infected I.V. With Various Recombinant Vaccinia Virus Constructs 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 6xl08 splenocytes/ml in complete medium containing l ~g/ml TPHPARIGC peptide as indicated in Example 4. The results showed that CTL ~rom VJS6, IL2-rW
and GM-CSF-rW vaccinated mice could not be restimulated with an ~-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-~-gal immune response of CTLs.
Example 9 Primary Response Of Mice At Day 6 After Injection S.C. With Various Recombinant Vaccinia Virus Constructs 5 BA~B/c mice were injected subcutaneously (S.C.) with 5xlO6 pfu of the following recombinant vaccinia virus: VJS6, I~2-rW, GM-CSF-rW, TNF~-rW or IFN~-rW.
At day 6, 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.
Figure 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, -~ W O 96/11279 2 2 0 1 5 q 2 PCTrUS9~112638 `

higher doses and/or different time intervals is expected to increase the magnitude of the primary reRponse against vaccinia using the subcutaneous route of inoculation.
The lytic units (L.U.) 30~ was determined by the 5. 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. The L.U./spleen (x103) of each rW is as follows: Gm-CSF=
124.3; VJS6 - 14.9; IFN-~ - 5.06; TNF~ = 2.88 and I~-2 =
96.2.
Example 10 Secondary Cultures From Mice Infected S.C. With Various Recombinant Vaccinia Virus Constructs Mice were infected with various recombinant vaccinia virus constructs as described in Example 4. Splenocytes were harvested at day 14 after inoculation with rW.
Secondary cultures were generated by 6 days incubation of 6X108 splenocytes/ml in complete medium cont~;n;ng 1 ~g/ml 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.
Example 11 Treatment Of Established Pl]lm~Ary Metastases With Recombinant Vaccinia Viruses Secreting Different Cytokines The three-day pl]lmonAry 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 5xlOs tumor cells (CT26 or CT26 ~ gal). Three days later they received an I.V. injection of 5x105 PFU of VJS6, I~-2-rW, GM-CSF-rW, IFN~-rW, TNF~-rW or Hanks' Balanced Salt Solution ~HBSS) (control mice). The mice were rAn~nm;zed, and the lungs were harvested after 12 days. The number of W096/11279 ~ ~ /J ~ ~ PCT~S9S/12638 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.
Table 2 DAY 3 TREATMENT OF EST~RT l.~Fn PULMONARY METASTASES WlTH
RECOMBINANT VACCINIA V~USES SECRETING DIFFERENT CYTOKINES
CT26 CT26 ,Bgal rVV l.c average # .~ 'e5/ mour.e average # .... t~
1 0 ~ mouse none~ > 500 > 500 x 5 > 500 > 500 x 5 VJS6 >500 >500 x 5 186162, 116, 115, 362, 175 IL-2 rVV 452.4500 x 4, Z62 11.413, 2, 24, 12, 7 GM-CSF rVV 470.2500 x 4, 351 373.6500 x 2, 405, 283, 179 IFN~y rVV >500 >500 x 5 232.8280, 97, 190, 260, 337 TNFcY rVV >500 ~500 x 5 361.2>500 x 2, 389, 258, 159 ~ control rnice were injected witn HBSS alone ~m~le 12 Treatment Of Established plll moTl~3ry Metastases With Reco-m~binant Vaccinia Virus Secreting Different Cytokines Plus Exogenous IL2 Mice were injected with tumor cells and rW as indicated in Example 4. Treatment with exogenous rIh-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.
Moderate doses of IL2 (produced by rW or exogenously administered together with rW) were effective in treatment of 3 day old metastases induced by CT26 ~- gal.
(Table 3) -o Table 3 DAY 3 TREATMENT OF EST~RT l.~Fn PULMONARY METASTASES WlTEI
RECOMBINANT VACCINIA VIRUSES PRODUCING DIFFERENT CYTOE~[NES PLUS
~iXOGENOUS II~2 CT26 CT26,Bgal rVV tlG ' ' average # " ~ ~, ,t~_.. ,/ mouse average # "" ~.t,.l~.c/
t~- ~ r 5 ~ " ~ :tl. cr e mouse none~ >500 >500 x 5 >500b >500 x 5 VJS6 >500 >500 x 5 405.2 >500 x 3, 267, IL-2 rVV 437.4 500 x 3~ 127 4c 184, 7, 126, 91, 267, 420 229 rI~2 >500 >500 x 5 >500 >500 x 5 rIL-2 + VJS6 406.4 >500 x 3, 20.8' 43, 6, 0, 52, 3 298, 234 GM-CSF rVV 413.2 >500 x 3, 500 500 x 5 163, 403 y rVV 447.6 >500 x 4, 238 412.2 500 x 4, 66 ' control rnice were injected with HBSS alone b all mice in thi~ group died between days 11 and 12 (before lung harvest) c p value between the data in the fra~ne is 0.07 Bxample 13 Active Immunotherapy Using Exogenous IL-2 Together With rW
Ex~ressing A Tumor Associated Antigen A recombinant vaccinia virus expressing a Tumor Associated Antigen (TAA) was constructed as described in Example 1.
Mice were injected with 5 x 105 of CT-26 expressing ~-gal via an I.V. route of ~m; n; stration. Three days later the mice were injected with rW-TAA-~-gal, alone or in co-m-bination with exogenous IL2. [High dose (HD) exogenous IL-2 (100,000 I.U., I.P., BID x 3d) or low dose - 30 (IL) IL-2 (15,000 I.U., I.P., BID x 3d)] was ~m;n;stered as indicated and then r~n~nm;zed.
The number of mice surviving and survival time in days was monitored. Those mice receiving rW-TAA plus exogenous IL2 survived. (Figure 8) WO96/11279 ~ e~/~ 9~2 PCT~59S/12638 Example 14 Figure 9 shows the survival of mice with non-~-gal expressing tumor established after immunotherapy with different recombinant vaccinia vectors. Mice were injected with 5 x 105 CT 26 tumor cells (non-~-gal) via I.V. route of ~m;n;stration. On day three they were injected with 103 pfu of the following rW which also cont~;n~ the ~-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 l0 shows survival in mice with an established ~-gal expressing tumor, CT26.25 after immunotherapy with different recombinant vaccinia vectors. Mice were injected with 5 x l05 CT26.25 (~-gal expressing) via I.V.
route of ~m;n;stration~ On day three they were injected with 103 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 ~-gal gene) compared to treatment with any other rW.
Example 15 Active Treatment Of Established plllmo~ry Metastases Using B7 - rW Construct All of the following recombinant Vaccinia vectors 2~ were constructed by placing both the cytokine gene and the TA~ 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 105 tumor cells were injected into mice. Three days later 106-107 pfu of each vector was injected I.V. Lungs were harvested on day 12.
The results as depicted in Figure ll and 12 showed that the rW vectors encoding ~-gal and the -W 0 96/11279 2 2 0 1 5 9 2 PCTrUS9~/12638 immunostimulatory molecule, B7.1, I~-2 or ICAM-l profoundly inhibited p1l1mo~ry metastases in an antigen specific m~nner.
The primary response of the mice on Day 9 after injection with tumor cells was det~rm;n~d. The results are depicted in Table 4. The percent lysis of 5lCr labeled target tumor cells is recorded. A significant primary immune response was seen with multiple recombinant vaccinia vectors, most notably B7.lrW, which correlates with the in vivo treatment response.
Table 4 Primary Response of Mice 6 Days After Injection I.V. With Various Vaccinia virus Constructs Tumor cells rVV (pfu) Effector CT26 CT26/P C25 CT26/V69 lS Construct Target B7.1 lOQ:l -3.86* 82.99 81.45 21.69 33:1 -4.86 91.86 69.01 14.49 11:1 -6.10 61.64 71.75 7.33 37:1 -8.61 53.99 54.22 2.19 1.2:1 -9.53 39.93 40.87 3.25 04:1 -16.57 27.37 26.74 -0.15 B7.1 106 10 0: 1 2.75 68.18 5270 70.75 33:1 -2.05 84.93 66.76 82.89 11:1 -2.85 41.27 73.12 56.05 37:1 -3.76 41.93 350.06 42.19 12:1 -8.05 26.24 31.70 44.97 0.4:1 -8.52 20.50 35.49 37.13 NA 107** 100:1 4.04 79.02 69.65 14.06 33:1 1.70 72.66 82.36 10.65 37:1 -1.01 51.75 63.41 7.53 11:1 -2.75 35.65 42.33 0.37 12:1 -4.88 34.98 28.49 -11.67 - 30 0.4:1 -6.57 6.14 20.02 -13.66 NA 106 100: 1 27.40 88.40 121.19 31.48 33:1 13.47 74.16 112.42 1.41 11:1 0.53 41.16 97.33 -4.07 3.7:1 -0.35 8.95 86.27 1.58 1.2:1 -0.43 -2.05 69.52 371 04:1 -4.06 -2.59 40.13 -4.37 WO 96111279 ~ PCT~S95/12638 o MVA 108 100:1 12.76 27.43 65.08 12.10 33:1 8.52 17.62 61.83 8.37 11:1 3.45 2.95 49.38 4.34 37:1 0.06 5.76 28.12 3.13 1.2:1 -1.30 2.43 28.05 -3.60 0.4:1 -4.26 -0.61 16.78 -7.65 MVA 107 100: 1 17.54 71.07 46.31 0.14 33:1 13.81 70.97 37.10 1.10 11:1 6.36 44.59 35.00 -0.30 3.7:1 -2.29 17.29 28.58 -6.94 1.2:1 -3.54 2.82 27.91 -0.76 0.4:1 -17.53 -6.63 19.66 -9.87 Kb 107 100: 1 24.83 20.58 33.37 34.47 33:1 20.74 15.35 19.42 34.75 11:1 12.61 -10.25 9.73 27.77 37:1 9.82 -11.37 8.51 17.51 1.2:1 -5.98 -18.23 8.58 17.01 0.4:1 -10.88 -29.04 5.55 9.10 Kb 106 100:1 11.60 28.14 42.96 -0.32 33:1 -2.37 16.79 21.60 -1.60 11:1 -7.83 16.12 -1.67 -7.08 37:1 -12.70 8.51 -3.28 -11.12 1.2:1 -18.17 6.15 -11.68 -9.36 0.4:1 -24.72 -4.83 -17.02 -14.28 Ld 107 100:1 -9.69 86.06 92.89 -5.1 33: 1 - 32.65 33.05 53.22 - 8.54 11:1 -39.51 35.26 46.63 -19.29 37:1 -42.44 15.50 24.28 -17.91 1.2:1 -47.50 2.17 12.71 -17.45 0.4: 1 -51.77 -1.65 9.44 -30.42 Ld 106 100:1 28.10 56.29 30.10 16.62 33: 1 16.09 41.69 26.49 - 5.75 11:1 -1.22 24.49 24.50 -17.06 37: 1 - 3.46 24.04 18.81 -21.05 1.2:1 -9.65 8.65 13.16 -20.82 0.4:1 -17.10 2.42 8.34 -27.53 VJS6 107 100: 1 29.38 33.59 73.98 22.03 33: 1 24.69 31.48 72.14 24.43 11:1 20.17 27.87 61.82 13.66 37:1 16.83 23.47 57.46 7.24 ~ WO96/11279 22 0 1 5 9 2 PCT~S95/12638 .

o 1.2:1 8.44 17.93 51.60 4.11 0.4:1 4.01 19.12 33.13 -5.46 VJS6 106 100: 1 1 . 53 63.88 63.8 45.57 33:1 -23.69 35.15 36.13 39.89 ~ 11:1 -27.32 29.65 27.61 29.29 37:1 -41.54 21.54 21.57 24.26 1.2:1 -45.02 18.21 16.21 21.06 0.4:1 -52.85 14.18 8.78 18.50 WB2m 107 100:1 3.50 11.12 23.52 4.69 33:1 0.32 11.95 10.52 3.46 11:1 -10.09 5.10 7.51 -2.53 37:1 -21.06 0.22 1.10 -2.51 1.2:1 -28.28 -3.99 -3.04 -4.26 0.4:1 -30.42 -7.84 -11.03 -7.36 WB2m 106 100: 1 25.09 16.18 22.86 1.54 33:1 25.61 6.04 19.48 -2.55 11:1 22.77 -1.80 14.43 -11.26 37:1 9.18 -8.25 11.40 -19.36 1.2:1 3.90 -19.60 10.69 -24.29 0.4:1 -13.93 -28.67 -1.28 -29.35 HBSS 100:1 6.00 17.33 6.73 -4.50 33:1 0.30 14.46 6.58 -9.50 11:1 -10.32 9.65 -9.60 -5.68 37:1 -14.34 1.66 -17.73 -13.69 1.2:1 -23.70 -9.03 -30.78 -17.72 0.4:1 -29.28 -12.56 -38.08 -17.75 * ~ lysis of 5ICr labeled tumor cells ** NA = neuraminidase recombinant viral vector (rW), MVA
= Ankara non-replicating vaccinia virus rW, Kb = murine H-2Kb MHC Class I molecule rW, Ld = murine H-2Ld MHC
Class I molecule rW, WB2m = murine Beta 2-microglobulin rW, HBSS = Hank's Balanced Salt Solution The secondary response of the mice is depicted in Table 5. The results indicate a secondary immune response - 30 seen by both VJS6 and B7.1 against the relevant target.

WO96111279 ~ /S9~ PCT~S95/12638 Table 5 Secondary Response of Mice 6 days After I.V. In~ection with Various Vaccinia Virus Constructs Tumor cells rVV (pfu) Effector CT26 CT26/P C25 CT26/V69 Construct Target HBSS 100:1 30.26* 45.93 37.05 29.95 33:1 15.83 13.38 10.05 18.24 11:1 2.59 5.88 4.37 11.39 37:1 0.83 5.64 -1.68 -1.00 1.2:1 0.01 2.14 -2.71 4.12 0.4:1 1.66 6.71 8.61 -0.11 VJS6 107 100:1 19.40 72.31 78.94 28.42 33:1 21.52 83.69 63.82 18.04 11:1 10.84 62.35 61.87 16.80 37:1 7.91 44.31 52.99 4.75 1.2:1 10.24 21.03 18.30 5.72 0.1:1 4.59 14.14 11.92 10.07 VJS6 106 ~00:1 18.55 48.89 38.21 15.27 33:1 11.33 41.52 34.52 8.25 11:1 8.06 26.63 33.00 1.17 37:1 2.57 16.54 29.26 -2.23 1.2:1 7.86 14.96 13.57 0.56 0.4:1 2.05 3.65 9.47 -8.17 Ld 107** 100: 1 0 . 11 30.56 25.05 -4.91 33:1 -2.78 25.03 26.24 -0.48 11:1 2.49 16.01 11.97 -3.99 37:1 -1.02 7.17 7.28 3.50 1.2:1 0.52 9.57 4.36 4.06 0.4:1 3.10 6.23 2.35 3.29 Ld 106 100: 1 18.65 25.02 14.24 11.70 33:1 0.63 11.23 7.51 11.36 11:1 -0.35 7.47 10.24 2.76 37:1 -0.47 1.38 -0.00 3.32 1.2:1 -3.19 6.35 5.89 6.16 0.4:1 4.08 5.70 2.86 4.04 B7.1 107 100: 1 - 10 . 62 83.64 69.56 -3.17 33:1 0.20 63.61 59.63 0.23 11:1 -6.10 40.58 61.77 3.02 37:1 -1.91 22.29 22.86 -2.5 1.2:1 -1.38 8.0 10.25 0.22 ~ WO96/11279 22 0 1 5 9 2 PCT~S95/12638 0.4:1 4.73 8.39 9.49 2.22 B7.1 106 100:1 30.59 59.22 61.29 32.13 33:1 31.24 74.4 53.99 23.29 11:1 13.92 41.68 49.63 22.94 37:1 10.66 31.02 34.86 5.63 S 1.2:1 4.87 33.61 6.45 3.73 0.4:1 8.78 7.66 3.98 7.16 MVA 100:1 28.16 18.13 25.09 30.30 33:1 33.76 28.62 27.99 20.10 11:1 28.69 12.63 9.75 9.13 37:1 6.55 -0.43 12.79 2.78 1.2:1 8.08 4.47 1.63 -0.45 0.4:1 5.68 -1.30 1.21 4.60 MVA 107 100 :1 22.77 32.85 28.05 27.07 33:1 10.46 28.94 30.49 8.80 11:1 5.48 24.20 22.32 -3.73 37:1 0.49 11.9 12.82 4.36 1.2:1 -0.95 5.29 13.32 6.3 0.4:1 4.75 -0.73 14.69 9.59 * ~ lysis of s~Cr labeled tumor cells ** NA = neuraminidase recombinant viral vecot (rW), MVA
= Ankara non-replicating vaccinia virus rW, Kb = murine H-2K6 MHC Class I molecule rW, Ld = murine H-2Ld MHC
Class I molecule rW, WB2m = murine Beta 2-microglobulin rW, HBSS = Hank's Balanced Salt Solution Example 16 Clinical Protocol For Phase I/II Trials In Patients With Metastatic Melanoma and Metastatic Breast Cancer of Tmmlln;zation with a Recombinant Vaccinia Virus ExDressing the M~GE-1 Peptide and B7.1 In this protocol patients with advanced melanoma and advanced breast cancer are ;mmlln;zed against MAGE-1, an ;mmllng~gm;nAnt peptide from a cancer antigen in - 30 combination with costimulatory/accessory molecule B7-1.
~atlent Eliqibility Among other criteria, patients must have evidence of measurable or evaluable metastatic melanoma or breast cancer that has failed stAn~Ard effective therapy.
Patients must have tumors that express the MAGE-1 antigen WO96/11279 ~ ~/~ ~ PCT~S95/12638 as evidenced by PCR or Northern Blot analysis of tumor cell RNA.

Treatment 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 ;mmlln;zation with lo8 plaque forming units/ml every four weeks for a total of three doses. Patients are evaluated for toxicity. When three patients in each category have been ~ollowed for at least two weeks after the first ;mmnn;zation without achieving grade 3 or 4 toxicity not easily reversible by stAn~Ard measures then the dose in that plaque category will be escalated to lO9 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.
If any patient receiving the lo8 pfu/ml dose achieves grade 3 or 4 toxicity not easily reversible by stAn~Ard measures, then an additional three patients will be treated at that dose. If a second patient develops grade 3 or 4 toxicity, not easily managed by stAn~rd procedures, then the dose will not be escalated in that category.

Vaccine preparation. 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 hllm~n~ The recombinant virus has the gene coding for the MAGE-l nine amino acid m;n;mAl det~rm;nAnt plus the B7.l sequence inserted into the viral thymidine kinase gene-~ WO96111279 PCT~S95/12638 `

o The recombinant virus is produced in a Food and Drug ~m; n; stration approved facility for the manufacture of GMP grade clinical material.
Vaccination procedure. The recombinant vaccinia virus will be supplied as a sterile, frozen suspension and will be approximately diluted to either 10~ or 109 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 o~ the needle into vaccine. The needle will pick up a drop of vaccine in the space between the two points.
c) Tv~ir.g the s~mLe biFurcated reedle, use the multiple pressure technique by pressing the needle through the vaccine drop on the subject's skin. Fifteen needle pressures will be ~m; n; stered.
d) Alternative upper extremities will be used for subsequent vaccinations.
Patients will be observed for 24 hours after ;mmlln;zation. Body temperature will be measured at 12 hours and at 24 hours and any adverse reactions noted.

Pos~ vaccination evaluation On day 14 after vaccination, patients will be seen and the following tests obtained:
a) Complete history and physical ~;n~tion~
including any symptoms and progress of reaction from the vaccination.
b) Photograph of the vaccination site.
c) Complete blood count with differential count.
- d) Platelet counts.
e) Acute care, hepatic, mineral and thyroid blood rh~m; ~try panel.
f) Serum and lymphocytes stored as performed WO96/11279 ~ ~ ~ ~ PCT~S9~/12638 pretreatment.
Patients will be evaluated at 14 days following the second vaccination and similar procedures will be performed prior to and following the third and final vaccination on day 56.
Patients will have a complete restaging of all sites of disease with appropriate physical ~m; n~tion and x-rays and nuclear medicine studies at the time of one and two months following the final vaccination.

Immunologic studies TmmllnologiC 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.
Assessment of response 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.

~ W 0 96/11279 ~ 2 0 1 5 92 PCTrUS9~112638 ., Minor responses are defined as 25-49~ decrease in the - sum of the products of the perpendicular diameters o~ all measurable lesions with no appearance of new lesions and - no increase in any lesions.
Any patient with less than a partial response will be considered a non-responder.
The appearance of new lesions or greater than 25 increase in the product of perpendicular diameters of prior lesions following a partial or complete response will be considered as a relapse.
Similar protocols will be followed for the evaluation of other recombinant virus vaccines against cancer.
Vaccines to be tested include but are not limited to Recombinant Vaccinia virus encoding MaRT-l and IL-2, Recombinant Vaccinia virus encoding MART-1 and B7.1, Recombinant Vaccinia virus encoding GP100 and I~-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.

Example 17 Use of Lymphocytes Sensitized To Immunogenic Peptides Derived From Melanoma Antigens For Therapeutically Treatinq ~Amm~ls A~flicted With Melanoma T-lymphocytes presensitized to the melanoma antigen may be effective in therapeutically treating mAmmAls afflicted with melanom~. T-lymphocytes are isolated ~rom 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 1 to lOmg/ml.
T-lymphocytes exposed to the antigen will be A~m;nistered WO96111279 ~9~ PCT~S95/12638 to the m~mm~l, preferably a human at about l09-lO12 lymphocytes per m~mm~ 1 . The lymphocytes may be ~m; n;stered either intravenously, intraperitoneally or intralesionally. This treatment may be ~ml n;stered concurrently with other therapeutic treatments such as cytokines, radiotherapy, surgical excision of melanoma lesions and chemotherapeutic drugs, adoptive T lymphocyte therapy.
Example 18 Active Immunotherapy with Recombinant Vaccinia Virus Co-expressing B7-l, B7-2 or Both and a Model Tumor Antigen Mediates Regression of Established Pulmonary Metastases Materials and Methods Animals Female BALB/c (H-2d) mice were obtained from Frederick Cancer Research Center (Frederick, MD). All mice were used at 6 - 8 weeks of age.
Preparation of Cell Lines CT26 is an N-nitroso-N-methylurethrane induced BALB/c (H-2d) 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 ~. Tmmllnol 154(9):4685-4692, 1995. Briefly, a plasmid donated by A.D. Miller cont~;n;ng the gene for ~-galactosidase and a neomycin resistance marker were used to construct the ~ZSN
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 ~-galactosidase were screened by X-gal st~;n;ng and in 51Cr release assays with anti-~-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

~` WO96/11279 ~ 5~2 PCT~S9~/12638 molecule H-2 Ld. BS-C-1 or HeLa S3 cells (ATCC, American Tissue Typing Collection, Rockville, MD CCL 16) were used to expand and titer all viruses. Cell lines were maint~n~A in RPMI 1640, 10~ heat inactivated FCS
(Biofluids, Rockville, MD), 0.03~ L-glut~m;n~, 100 ~g/ml streptomycin, 100 ~g/ml penicillin and 50 ~g/ml gentamicin sulfate (NIH Media Center). CT26.CL25 was maintained in media cont~;n;ng 400 or 800 ~g/ml G418 (GIBCO, Grand Island, N.Y.).
Construction and Characterization of Recombinant Va~
viruses Construction and characterization of recombinant vaccinia viruses containing murine B7-1( 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. G~rm~;n, 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 VV
synthetic early/late promoter. The gene of interest was inserted into the Hind III F region of W. Methods used for the production and selection o~ rW in which the VV
thymidine kinase gene was utilized as an insertion site, were similar to those described previously by Earl, P.L.
et al. In: Current Protocols in Molecular Biol. Ausubil et al (eds) Greene Publishing Assoc. and Wiley Interscience, 1991, 16.18.1-16.18.10 unless otherwise stated. The v.MCB7-1/~-gal and v.MCMHA/~-gal were constructed by homologous recombination of the plasmid pSC65~ (a modification of pSC65 in which the E. 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/~-gal was constructed with both the B7-2 gene and the E. coli lac Z gene under transcriptional WO96/11279 ~9~ PCT~S95/12638(~

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/~- -gal was constructed by homologous recombination of the plasmid pSC65~ (modified as stated previously to create the v. MCB7-2/~-gal construct) with the v. MCB7-1.
Table 6. Construction of recombinant vaccinia viruses.

Recombinant VP37 Thymidine Vaccinia Virus Kinase Code Promote Gene Promote Gene r r v.MCB7-1/~-gal S.E/L B7-1 7.5e ~-gal v.MCMHA~-gal S.E/L MHA 7.5.e ~-gal v.MCB7-1fNP S.E/L B7-1 7.5e NP
B7-2, 15v. MCB7-2/~gal 7.5e ~-gal B7-2, v. MCB7-l~B7- S.E/L B7-2 7.5e ~-gal 2/~-gal B7-2, S.E/L MXA 7.5e ~-gal v.MCMHA/B7-2~B-gal MHA= measle hemagglutinin gene, B7-1 = murine B7-1, B7-2 =
murine B7-2, NP = influenza nucleoprotein, ~-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 Viroloqy 160:336-345, 1987 cont~;n;ng the influenza nucleoprotein (NP) was recombined with v.MCB7-1 to yield v. MCB7-1/NP. This recom~binant virus was selected on the basis of a TK negative phenotype. Plaques were then analyzed for expression of NP by ;mmllnost~;n;ng (Sutter, G. et al Vaccine 12:1032-1040, 1994) and subsequently plaque purified under agar overlay four times before sucrose cushion purified viral stocks were prepared (Earl, P.L. et al Current Protocols in Mol. Biol., ibid (Table ~I WO96/11279 22 0 1 5 9 2 PCT/US95112638 6.).
Preparati~n of rW expressing the influenza A/PR/8/34 nucleoprotein (NP) was previously described (Smith, G.L.
et al virolQgy 160:336-345, 1987). In the control construct, v. JS6, the E.coli lac Z gene was under the control of the early VV 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-Barr~II site of a vaccinia expression vector, pM~601, which contains the b-galactosidase gene under the control of W
early p 7.5k promoter. The other cytokines (GM-CSF, IFN-g, and TNF-a) were inserted into the wild type W genome with similar procedure. The techniques used in preparation o~ the v. HI.A.A2.1/,~-gal (O'Neil, B.H. et al J. Immunol.
151:1410-1418, 1993) v. h~lm~n and murine ~2-microglobulin/,~-gal, (O'Neil, B.H. et al J. Immunol.
151:1410-1418, 1993) and all the murine MHC I (Ld, Dd, Kd, and Kb)/,B-gal (Restifo, N.P. et al J. Exp. Med. 177:265-272, 1993), has been previously described.

Analysi~ of Recombinant Proteins and Quantitation of ,~-galacto~3ida~e E~pre~sion BS-C-1 monolayers were infected with 10 infectious units of recombinant virus per cell. After 2G hours infected cells were harvested in solubilization buffer (0.06M Tris-HCl (pH 6.8), 3~ SDS, 5~ -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 m~mps for 1 hour.
Membranes were initially incubated ~or 1 hour in a blocking solutior (PBS-B) consisting of PBS cont~;n,ng non-fat milk (w/v) and 0.2~ Tween 20. 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 ~ilters for 3 hours at room temperature.

WO96/11279 ~ 9~ PCT~S9~/12638 Filters were subsequently washed three times in PBS
conta;n;ng 0.2~ Tween 20. Bound primary antibodies were detected by incubating filters, after washing with l25I-protein A (Amersham) at a concentration of 0.1~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-l 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.
In Vivo Experimen~s In vivo protection studies utilized BALB/c mice which were ;mml7n;zed intravenously with either Hanks's balanced salt ~olution (HBSS) (Biofluids, Inc., Rockville, MD), v.MCB7-1/b-gal, or v.MCMHA/b-gal (107plaque forming units (PFU)). Twenty-one days following this initial ~mmlln;zation, the mice were intravenously challenged with 5 x 105 tumor cells of CT26.WT or CT26.C25. Twelve days following tumor challenge, mice were r~n~om;zed, euthanized, and lung metastases were enumerated in a blinded fashion.
In vivo adoptive transfer studies involved intravenous ;mmlln;zation of BA~B/c mice with either HBSS, v.MCB7-1/b-gal or v.MC MHA/b-gal . The rW was ~m;n;stered at either 105or 107 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 107 lymphocytes were adoptively transferred to mice who had ~ i W 0 96~11279 22 0 1 5 q 2 PCTAUS95/12638 .

o been injected intravenously with 5 x 105 tumor cells of ~ either CT26.WT or CT26.C25. Designated groups of mice received adjuvant treatment with six doses of IL-2 ~m; n; stered intra-peritoneal at a dose of 100,000 Cetus units two times per day for three days. Nine days after adoptive transfer of primed splenocytes, the mice were r~n~m;zed, euthanized, and lung metastases counted in a blinded fashion.
In vivo active treatment studies involved non-irradiated BALB/c mice which were inoculated intravenously with 5 x 105 tumor cells of either CT26.WT
or CT26.C25. All mice were r~n~om;zed and subsequently vaccinated with HBSS, or 105or 107PFUs of the designated rW three or six days later. Designated groups of mice received adjuvant treatment with six doses of I~-2 ~m;n;stered intra-peritoneal at 100,000 Cetus units two times per day for three days. Mice were r~n~om;zed 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.

T cell S2lbset Depletions Purified culture supernatants of anti-CD4 monoclonal antibody GKI.5 (TIB 207; American Tissue Culture Collection) and ascitic fluid of hybridoma 2.43 (anti-CD8) (TIB 210: American Tissue Culture Collection) were diluted in HBSS prior to use in vivo.. For in vivo depletion BA~B/c mice were given 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. Using fluorescein isothiocyante-labeled anti-CD4 and phycoerythrin labeled anti-CD8 antibodies, FACS analysis was performed 1 day prior to ;mmlln;zation~ and again at day 7 to verify depletion.

WO96111279 ~ ~ 9~ PCT~S95/12638l ~ilhole Organ X-gal StAin;n~
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 st~;n;ng 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.

Stati sti cal An;~lysis Data from in vivo protection, adoptive, and active treatment studies was analyzed using a two-sided student's t test for unpaired samples. Errors bars represent the standard deviation within each group.
Survival was analyzed with st~n~rd Kaplan-Meier survival curves (Kaplan, E.L. J. Am. Stat. Assoc 53:457-481, 1958).
All p values presented are two-sided.
~xample 19 Characterization of Murine B7-1 and E.coli ~-qal Genes in Cells Infected by Vaccinia Viruses Following preparation of the rWs, and prior to their use in in vivo experiments, the the in vitro expression of foreign protein in infected cells was evaluated by either immunohistochemical st~;n;ng, western blot analysis, or both. Western blot analysis of murine B7-1 expression of virally infected cells illustrated the previously reported diffuse st~;n;ng pattern typical of highly glycosylated proteins (Fig. 13) (Freeman, G.J. et al J.
Ex~. Med. 174:625-631, 1991). Molecular weight markers estimate W expressed B7-1 to be approximately 55-65 k Da in size. The authenticity of rW expressed murine B7-1 ~ was ~urther veri~ied by the binding o~ CTLA4-Ig to v.
MCB7-1 infected cells. Expression of rW murine B7-2 was verified by ;mmllnoh;stochemical st~;n;ng of infected cells using a murine specific B7-2 antibody (PharMingen, CA).
Expression of the TAA, ~-galactosidase, was similar for v.
MCB7-1/~-gal and the control recombinant v. MCMHA/~-gal, in contrast v. MCB7-1/NP showed no signs of enzyme expression (data not shown).

Example 20 Initial Screen: Ability of Tmmlln;zation with Recombinant Vaccinia Virus to Mediate Regression of Established Tumor It was det~rm;n~ whether combining a TAA with one or several of these immunomodulatory molecules would enhance the antigen specific ;mml~ne response, as measured by the most stringent test, i.e., the ability to mediate regression o~ established disease. The virus, v. JS6, expressing the TAA alone, was used as control to measure either the positive or negative contribution of the ;mmllnomodulatory molecule in the vaccination vector.
The ability of a single inoculation of twelve double rW s, which expressed the genes encoding both an ;mmllnomodulatory molecule and the ~-gal model tumor antigen in the same virus, to effect regression of pnlmo~ry metastases in tumor-bearing mice was assayed.
The double rW s were constructed to include the b-galactosidase gene within the thymidine kinase (TK) region of the W genome for all vectors evaluated. The position of the ;mmllnomodulatory molecule for most vectors is also within the TK region of the W genome under the same ~ promoter as the TAA. However, in the case o~ the B7-1 vector, the gene for B7-1 is placed within the Hind III F
region as described above and shown in Table 6.
The double rW s studied were v.MCB7.1 (Hind III F

WO96/11279 ~ ~ ~5~ PCT~S95112638 region of W genome)/b-gal (TK region of W genome), v.
human b2-microglobulin (TK)/b-gal(TK) {3154}, v. murine b2- -microglobulin (TK)/b-gal(TK) (O'Neil, B.H. et al J.
Immunol. 154:1410-1418, 1993), v. murine MHC I Ld (TK)/b-gal (TK) (Restifo, N.P. J. Exp. Med. 177:265-272, 1993), v. MHC I Kd (TK)/b-gal (TK) (Restifo, N.P. J. Exp. Med.
177:265-272, 1993), v. MHC I Dd (TK)/b-gal (TK), v. MHC I
(allogeneic) Kb (TK)/b-gal (TK) (Restifo, N.P. J. Exp.
Med. 177:265-272, 1993), v. human leukocyte antigen (xenogeneic) H~A-~ (TK)/b-gal (TK) (O'Neil, B.H. et al ~. Immunol. 154:1410-1418, 1993), v. murine interleukin -2 (I~-2) (TK)/b-gal (TK), v. murine granulocyte-macrophage colony stimulating factor (GMCSF) (TK)/b-gal (TK), v.
interferon-gamma (IFN-g) (TK)/b-gal (TK) (Bronte et al., in press), and v. tumor necrosis factor-alpha (TNF-a) (TK)/b-gal (TK). The v. JS6, expressing only b-gal in the TK region (described above), and a non-replicating W, modified vaccinia Ankara (MVA) (Sutter, G. et al Proc.
Nat'l Acad. Sci. U.S.A. 89:10847-10851, 1992), also only expressing b-gal, were used as controls.
Using the in vivo active treatment protocol outlined above, mice bearing three day pnlmQ~ry metastases were ;mmlln;zed with a single dose of the selected rW. In each experiment, no therapeutic response was seen with any rW
against the parental tumor, CT26.WT, thus documenting the specificity of the response. Figure 14 displays the results of our initial screening experiment in which mice bearing three-day CT26.C25 tumors were vaccinated with rW (107 PFUs).
When compared with the HBSS vaccinated control mice, only v. IL-2/b-gal and v.MCB7-1/b-gal m~ ted both a specific and significant reduction in the number of plllmo~ry metastases (p c 0.006 and pc 0.002, re~pectively). The ability of v. JS6, the single rW
expressing b-gal alone, to me~;~te significant tumor regression was variable from experiment to experiment.

22û1 5~2 ~, WO96/11279 PCT~S9~112638 (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 re~uired for a successful CTL response. Treatment with v. IL-2/b-gal and v. MCB7-l/b-gal resulted in a lO and 5 fold further reduction in the number of metastases when compared to the best results obtained with v. JS6. These results suggest that vaccination with an ;mmllno~nm;nAnt TAA alone is capable of priming a cytotoxic antitumor ;mmllne response which can then be further enhanced by the simultaneous expression of unique immunomodulatory molecules.
Although all double rW cont~;ne~ the gene for the TAA, b-gal, only v. IL-2/b-gal and v. MCB7-l/b-gal were able to mediate significant reductions in the number of plllmonAry metastases. Insufficient b-gal expression is unlikely to account for the therapeutic failure of the ineffective double rW s. When tested, b-gal expression, at least between the individual cytokine producing rW, did not significantly different (data not shown). The potential for the gene encoding for the ;mmllnomndulatory molecule within the double rW to exert an immunosuppressive effect on the generation of a TAA
cytotoxic response, or directly inhibit TAA protein expression was not evaluated, and cannot be excluded. The possibility that the failure of the GM-CSF, IFN-~, or TNF-~ producing rWs is due to immunosuppresive effects of local cytokine production was not further explored.
Tumor regression following vaccination with v. MCB7-l/b-gal has not been previously reported. However, the therapeutic result obtained with this rW supports the notion that costimulatory molecule expression is necessary for optimal T cell activation. Of note, a single vaccination with v. Dd/b-gal at a lower viral inoculate (106 PFUs) was also capable of mediating a WO96111279 PCT~S95112638 ~ -significant reduction in the number of pnlmo~ry metastases (p < 0.005). This suggests the existence of a Dd epitope for the b-gal protein heretofore unknown.
Based on the findings of this screen, and other reports of anti-tumor effects with both B7-1 transfectants (Guinan, E.C. Blood 84:3261-3282, 1994) and v. B7-1 oncolysates (Hodge, J.W. et al Cancer Res. 54:5552-5555, 1994), a more extensively evaluate the in vivo effects of v. MCB7-1/b-gal on the cell-mediated antitumor immune response was conducted. Interestingly, the generation of CTL following B7-1 transfection is an IL-2 dependent phenomenon, since the addition of IL-2 blocking antibody during the induction phase blunts generation of a cytotoxic response (Harding, F.A. et al J. Exp. Med.
177:1791-1796, 1993).
Example 21 Protection Against Subsequent Tumor Challenge by Tmmlln;zation with v. MCB7-1/~-gal Although the identification of murine and human TA~s 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 ;mmlln;ty following vaccination with a rW expressing either the TAA alone, or in combination with B7-1 was evaluated. The rVV strategy utilized in this current study has a distinct advantage over B7 transfections studies (in addition to the ease of ~m;n;stration), 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. Whereas the protective ;mmlln;ty ~mo~trated in the transfection studies is interpreted as a specific CTL response against an undefined TA~, the unique reactivity of the CTL in this model is easily evaluable.

~ W O 96/11279 PCTrUS95/12638 ~ ~ ,.

Naive mice were initially vaccinated with either v.
~ MCB7-1/b-gal, or a control virus, v. MCMHA/b-gal. The v.
MCMXA/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 7mmlln;zed with both v. MCB7-1/b-gal and v. MCMHA/b-gal were protected against tumor challenge with CT26.CL25, but not the wild type tumor, CT26.WT (p c 0.0002 and p ~ 0.0014, respectively) (Fig. 15.). These results confirm previous reports that vaccination with a rW expressing a unique TAA alone can confer protection to subsequent challenge with a TAA-expressing tumor.
(Kantor, J. et al ~. Nat'l Cancer Inst. 84:1084-1091, 1992; Estin, C.D. et al Proc. Nat'l Acad. Sci. U.S.A.
85:1052-1056, 1988). The completeness of the protection by both viruses precluded a more thorough evaluation o$
the impact of the B7-1 gene in this situation. In this model system, b-gal protein expression by both v. MCMHA/b-gal and v. MCB7-1/b-gal was sufficient to exploit intracellular antigen processing mechanisms leading to protective ;mmlln;ty.
Example 22 Primary Adoptive Trans~er o~ v. MCB7-1/~-gal Primed Splenocytes to Tumor-Bearing Synqeneic Mice Tumorigenicity is reflective of the state of T cell activation. In the present invention it was hypothesized that 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 o~
r 30 tumor-bearing syngeneic host. The therapeutic advantage gained by ;mmnn;zing 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 ~mo~ctrated in the initial observation that no reduction in the number of pulmonary metastasis was WO96/11279 ~~ PCT~S95/12638 noted after the adoptive trans~er of any group of primed lymphocytes to ~yngeneic 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 plllmon~ry metastases (p <.0054) (Fig. 16.). CT26.C25 tumor-bearing mice treated with v. MCB7-l/b-gal rW
splenocytes at two doses (lOs and 107 PFUS) showed a significant reduction in tumor burden (p ~ 0.007 and p c 0.007, respectively). Again, adjuvant IL-2 treatment resulted in a 16.5 and 12.5 fold further reduction in the number of pulmonary metastases in this group.
Significant tumor regression was also seen in mice receiving splenocytes primed with v. MCMHA/b-gal at lO5 PFU, but not at 107 PFU (p ~ 0.0052, and p c 0.094, respectively). The addition of adjuvant IL-2 to the v.
MCMHA/b-gal treatment group resulted in a 2 fold reduction in the number of plllmo~ry metastases in the group treated with v. MCMHA//B-gal primed splenocytes at 105 PFU.
Vaccination with a rVV expressing only the model TA~, was capable of priming a specific ;mml1n~ response which m~ ted tumor regression on adoptive transfer.
Vaccination with a rVV cont~;n;ng both the costimulatory molecule, B7-l, 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.
Infection with v. 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-~20 1 59~
~! wo 96/11279 PCTAUS95/12638 i -O
759, 1994; Wu, Y. et al Curr. Biol. 4:499-505, 1994).
- Infection with v. MCB7-1/b-gal would also provide T cells with the capacity for autonomous stimulation leading to P both a proliferative response to antigen and increased 5 ~ cytokine production tDohring, C. et al ibid; Azu-mal M. et al J. Exp. Med. 177:845-850, 1993). Notably, binding of B7/CD28 costimulates I~-2 mRNA accumulation (~insley, 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). In the absence of an antigen non-specific costimulatory signal delivered by interaction between B7-CD28, 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. et al Annu. Rev. Immunol. 11:191-212, 1993; Harding, F.A. et al J. Exp. Med, 177:1791-1796, 1993). Coculture of CD8+ T cells with B7+, but not B7-tumors results in potent cytotoxic response, and 20 proliferation (Harding, F.A. et al J. Exp. Med. 177:1791-1796, 1993), which are inhibited by both anti-CD28 Fab fragments, and interestingly by anti-IL-2 antibodies. The results ~mnn~trate that direct activation of CT~ by rW
which provide appropriate costimulation and antigen 2~ specificity results in improved therapeutic response upon the adoptive ;mmllnotherapy of primed splenocytes to syngeneic tumor-bearing mice. The mechanism responsible for this response may be due to either or both a qualitative increase in antigen specific T cell precursors r 30 mediated by B7/CD28 (or CThA-4) interaction, or B7/CD28 (or CT~A-4) mP~;~ted differentiation of T cell effector populations.
The adjuvant benefit received by adjuvant IL-2 alone upon the adoptive transfer of splenocytes in these studies confirms prior reports documenting direct IL-2 antitumor WO96/11279 ~ ~ /S9~ PCT~S95/12638l~
G

responsiveness. The simplest explanation for the therapeutic response seen with IL-2 alone is that is represents a lymphokine activated killer cell ~hAK) response mediating non-specific cytotoxicity. However, Ih-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 Ih-2 alone and as an adjuvant, is an IL-2 mediated proliferation of antigen-specific CTL.
Example 23 Active Immunotherapy of Established pnlmo~ry Me~astases with v. MCB7-1/~-gal Treatment of established disease r~m~; n~ the ultimate goal of ;mml~nstherapy. If lack of costimulation is a principal means through which tumors evade ;mmllne surveillance in this model system, then vaccination with a v. MCB7-1/b-gal should generate an active and specific therapeutic immune response in ~n;m~ls bearing established tumor. To test this hypothesis, tumor-bearing mice were ;mmnn;zed with a single injection of rWs, with designated groups receiving adjuvant treatment with Ih-2 as outlined above. In addition to v. MCB7-1/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.
In mice bearing CT26.C25 tumors, ;mmlln;zation with v.
MCB7-1/b-gal mediated a significant reduction in the number of metasta~es (p c 0.006) (Fig. 17.). The high therapeutic effect precluded evaluation of the effect of adjuvant Ih-2. In the same experiment, mice ;mmnn;zed with v. MCMHA/b-gal mediated no significant tumor regression (p ~ 0.36). In four separate experiments a W 0 96/11279 22 0 1 5 9 2 PCTrUS9~112638 significant decrease in the number of pl~lmo~ry metastases - for mice ;mmlln;zed with v. MCMHA/b-gal at 107PFU was seen only once (p c 0.009). IL-2 ~m;n;stration did not significantly improve upon tumor regression in v. MCMHA/b-gal treated mice. In the same and subsequent experiments, v. MCB7-1/NP had no effect on tumor regression. Overall, mice ;mmlln;zed with v. MCB7-1/b-gal showed a 170 ~old and 117 fold greater reduction in the number of pl]lmon~ry metastases than mice in the v. MCMHA/b-gal and v. MCB7-1/NP ;mmlln;zation groups, re~pectively. Exogenous I~-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 ef~ect m~;m~l regression of tumor in this active treatment model and suggests a role for B7-1 in both the induction and effector phase of the CT~ response. This conclusion is further supported by the finding that a vaccination vector combining b-gal with an irrelevant protein did not reliably reduce tumor burden. (Note in one of four experiments, a significant reduction was observed, although to a much lesser degree than that seen in the v.
MCB7-1/b-gal ;mmnn;zation group.) The presence of B7-1 alone was insufficient to effect any significant regression, which is consistent with reports that the mere expression of B7-1 on tissue without a new or foreign antigen does not result in tissue specific auto;mmlln;ty (Guerder, S. et al Tmmlln;ty 1:155-166, 1994; Harlan, D.M.
et al Proc. Nat'l Acad. Sci. U.S.A. 91:3137-3141, 1994).
The results from four separate active ;mmllnotherapy studies of mice ;mmnn;zed with 107 PFUs o~ rW were e 30 normalized to the HBSS group for each individual experiment, and the data pooled and analyzed as percent residual disease (data not shown). Tmmnn;zation with v.
MCB7-1/b-gal group showed an almost 20 fold reduction in the tumor burden compared to HBSS control, with only 5.2 of tumor r~m~;n;ng. Adjuvant I~-2 treatment did not W 096tll279 ~ S ~ ~ PCTrU~95/12638 improve upon this result, though a trend was seen in this group and the v. MCMHA/b-gal group. Tmml-n;zation with v.
MCMHA/b-gal mediated a nearly 2 fold reduction in the number of metastases with 40-55 ~ of tumor burden rPm~;n;ng, while the v. MCB7-1/NP mediated no mp~n;ngf reduction with 83-93~ of tumor burden rPm~;n;ng. This cumulative data confirms the finding seen in the individual active immunotherapy studies.
In order to evaluate whether active ;mmllnotherapy results with v. MCB7-1/b-gal were limited to the treatment of small tumor burdens, the same active treatment experiment in mice bearing six day metastases as repeated.
A six day tumor burden is macroscopically visible with individual metastases measuring up to 2.5 millimeters.
Again, no significant tumor regression was seen following rW vaccination in mice bearing C T26.W T tumors. Figure 18 demonstrates that in the six day model, a single ;mmlln;zation with v. MCB7-1/b-gal mp~;~ted a significant reduction in the number of plllmon~ry metastases in mice bearing CT26.25 tumors (p ~ 0.008). Mice ;mmlln;zed with either v. MCMHA/b-gal or v. MCB7-1/NP had no significant reduction in the number of plllmoI~ry metastases (p c O.12, and p c 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 mP~;~te 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. 154 (9) : 4685-4692, 1995, ;mmlln;zation with a rW containing only the ;mmllno~om;n~nt TAA, b-gal, was capable of inducing a cytotoxic 1mmllnP
response resulting in a reduction in the number of plllmon~ry metastases when small tumor burdens are present, but has no efficacy against a larger tumor burden.
Tmmlln;zation with a rW expressing the costimulatory molecule alone without the TA~, i.e., v. MCB7-1/NP, was 2201 5~2 ~ WO96/11279 PCT~S95/12638 r 77 ~
unable to prime an immune response and mediate tumor - regression in either situation.
A reduction ~n tumor burden is frequently o~trated for several adjuvant interventions, but rarely translates into a therapeutic benefit in terms of prolonged survival. In a parallel experiment to the three day active treatment protocol described above, ten mice per group were followed daily for survival and events were recorded as either deaths, or severely moribund condition requiring euth~n~ia~ In this study,all mice inoculated with CT26.WT were dead by day 43 with 50~ dead in all groups by day 32.
CT26.C25 tumor bearing mice ~mo~trated varying degrees of survival among vaccination groups (Figure l9).
Mice receiving HBSS, IL-2 alone, and v. MCB7-l/NP alone (with or without adjuvant I~-2), were dead by day 39. Mice vaccinated with v. MCMHA/b-gal showed a significant prolongation o~ survival with 50~ o~ 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. In the v. MCB7-l/b-gal vaccinated mice 50 ~ were alive at 70 days, and 30~ continue to be long term survivors at over l00 days (p c 0.000l). Adjuvant I~-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 l00 days follow-up .
The regression of plllmo~ry metastases seen following ;mmlln;zation with the v. MCB7-l/ b-gal thus translated into prolonged survival for most ~n;m~ls treated. Two mice who were long-term survivors in the v. MCB7-l/b-gal treated group, subse~uently died and their lungs were subjected to whole organ X-gal st~;n;ng. One of the mice showed no evidence of X-gal StA; n;ng, despite bulky tumor metastases. The second mouse showed moderate positivity for X-gal st~;n;ng~ with some metastases exhibiting no s9~
WO96111279 PCT~S95/12638 staining. Although the outgrowth of b-gal negative tumors as the cause of death in these long term survivors can not be concluded, it is a likely possibility.
Example 24 Active Immunotherapy of Established nllmnn~ry Metastases with v. B7-2/~-qal and v. MCB7-l/B7-2/~-gal As stated earlier, optimal T cell activation require two signals, once delivered by the MHC/TCR interaction and a second from the B7-l/CD28 or CThA-4 interaction. It was evaluated whether a rW coexpressing B7-2 (v. MCB7-2/~-gal), or both B7-l and B7-2 (v. MCB7-l/B7-2/~-gal), with a TAA would be a more potent vaccine to stimulate TAA
specific CTL response.
Mice were initially inoculated with 5 x lOs tumor cells of CT26.WT or CT26.C25. Mice bearing three day tumors were then ;mmlln;zed with a single injection of rW, with designated groups receiving adjuvant treatment with I~-2 as outlined above. In addition to the rW described, 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-l gene in the v. MCB7-l/B7-2/~-gal construct (Fig. 20.). No significant tumor regression was seen following rW vaccination in mice bearing CT26.WT tumors.
As seen in previous active ;mmllnotherapy experiments with mice bearing three-day CT26.C25 tumors, vaccination with v. MCB7-l/~-gal resulted in a significant reduction in the number of plllmo~Ary metastases (p ~ 0.0014), while v. MCMHA/~-gal mediated only a m;n;m~l reduction (p cO.054), and v. B7-l/NP mediated no reduction (p ~ 0.13).
Vaccination with both v. MCB7-2/~-gal and v. MCB7-l/B7-2/~-gal resulted in a significant reduction in the number of pnl m~ry metastases (p c 0.0018 and p ~ 0.0018, respectively). Similarly, treatment with v. MCMHA/B7-2/~-gal also m~ ted significant reduction in the number of WO96/11279 2 ~ O 1 5 q 2 PCT~S95/12638 t metastases (p ~ 0.0018), which did not differ - statistically from either v. MCB7-2/~-gal or v. MCB7-1/B7-2/~-gal. Adjuvant IL-2 did not statistically improve upon these results.
No statistical difference between rW expressing either B7-1, B7-2, or both could be demonstrated. O~
note, however, there is some suggestion that B7-1 may be a more potent costimulator of antitumor ;mml~n;ty than B7-2.
That is, rW expressing B7-1 alone m~ ted 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 cont~'n'ng the irrelevant protein, MHA together with B7-2.
However, whereas the B7-1 and B7-2 molecules are within different regions of the W genome, under independent promoters, whether these findings are reflective of an increased potency ~or B7-1 as a costimulator, or merely reflective of variable expression will require further analysis.
Example 25 Effect of In Vivo T cell Subset Depletions on the Ability of v. MCB7-1/~-gal to Mediate Tumor Reqression of Established Disease Adoptive transfer and active immunotherapy studies demonstrated a specificity of re~ponse suggesting that the cell-m~ ted arm of the immune system, i.e., T-lymphocytes, were responsible for tumor regression. To specifically identify the T cell subset which is responsible for the cytotoxic antitumor response observed, we repeated three-day active treatment study while selectively depleting either CD4+ or CD8+ cells with anti-CD4 and anti-CD8 antibodies. In mice treated with the anti-CD4+ antibody FACS analysis revealed ~ 99.8~
depletion o~ CD4 cells, whereas in mice treated with the anti-CD8+ antibody, >97~ depletion of CD8+ was seen (Figs.
21a-21f). These results were confirmed in three other WO96111279 ~ ~o~/5 9~ PCT~S9~/12638 FACS analysis completed l day before and seven days after treatment. In the groups ;mmlln;zed with HBSS, no tumor regression was seen (Fig. 21g). In groups ;mmlln;zed with v. MCB7-l/b-gal, both the no depletion group and the CD8+
depletion group showed a significant reduction in the number of plllmo~ry metastases (p c 0.005, and p c 0.054, respectively). The mean reduction in the no depletion group was approximately lO fold greater than that seen in the CD8+ depleted group. Notably, in mice depleted of CD4+ cells, there was no tumor regression seen. This suggests a critical role of CD4+ cells in mediating this cellular immune phenom~non. Similarly, in the v. MCMHA/b-gal ;mmlln;zed group, no reduction in the number of metastases was seen in the CD4+ depletion group, yet a small but significant reduction was seen in both the no depletion group, and the CD8+ depletion group (p < 0.02, and p c 0.06, respectively) (data not shown). Mice ;mmlln;zed with v. MCB7-l/NP showed no reduction in the number of plllmon~ry metastases in any group (data not shown). These results ~mo~trate that the reduction in pll1mon~ry metastases is T cell mediated, and-that both CD8+ and CD4+ lymphocytes are required to mediate this phenomenon. These results are contrary to most transfection studies in which a cytotoxic CT~ response was induced in the absence of CD4+ cells. When the genes for B7-l or B7-2 are transfected into tumor cells, it is the tumor cells themselves which act to present a TAA in the context of MHC and B7 to responding T cell. Following vaccination with a rVV coexpressing both B7 and the TAA, all infected cells expressing MHC class I molecules would theoretically function to activate T cells. In the transfection studies, CD+ 8 depletion abrogated the antitumor immune response, by removing the responding cytotoxic population. Following v. MCB7-l/b-gal vaccination however, although CD8+ depletion would 3S significantly reduce the precursor frequency of responding W 0 96/11279 ~ 2 0 1 ~ 9 2 PCTrUS9~112638 cytotoxic cells (3~ remained in this study), the increased number of cells capable of presenting antigen and costimulating may blunt the e~fect of the depletion.
Thus, the incompleteness of the CD8+ depletion may be a 5 simple explanation ~or the observed results. However, the importance of CD4+ cells in providing necessary help to proliferate a CTL response in this model system cannot be m;n;m; zed. CD4+ depletion completely abrogated any tumor regression following vaccination. Whether this 10 phen.~m~no~ 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.
Example 26 Materials and Methods ~or IL-2 Enhancement o~
Recombinant Poxvirus-Based Pulmonary Metasta~es Cell Lines CT26 is an N-nitroso-N-metylurethane-indiced BALB/c (H-2d) undi~ferentiated 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. Miller (Fred Hutchinson Cancer Research Center, Seattle, WA) cont~;n;ng the gene for ~B-gal and a neo-resistance marker were used to construct an ecotropic producer cell line secreting the LZSN retroviral construct. This retroviral supernatant was used to transduce the CT26.WT cell line.
Transductants were selected in a G418 media and then subcloned by limiting dilution analysis at 0.3 cells/well.
Subclones that expressed ,B-gal were screened by X-gal st~;n;ng and in 5ICr release assays with anti-~-gal effectors. The subclone CT26.CL25 was selected for use in all studies because of its stable expression of both ~B-gal and the class I molecule H-2 Ld. A clone of the mouse W O 96/11279 ~ ~ ~ /S-~ ~ PCTrUS95/12638 ~
f o thymoma EL4 (H-2b) stably transfected with ~-gal, termed B22 (provided by Y. Paterson, (Department of Microbiology, University of Penn~ylvania, Philadelphia) was used as a negative control in slCr release assays. BSC-1 cells (American Type Culture Collection (ATCC), Rockville, MD;
CCL 26) were used to expand and titer all W. 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 (National Institutes of Health Media Center, Bethesda, MD). CT26.CL25 and E22 were maintained in media cont~;n;ng 400 ~g/ml G418 (Life Technologies, Inc. Grand Island, NY).

rW and rFPV
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. The rV W used in a single experiment were titered together to m~;m;ze the accuracy of the relative titers. Preparation of rW expressing the influenze A/PR/8/34 nucteoprotein (NP) was previously described (Smith, G.L. et al Viroloqy 160:336, 1987). In the HPV16-E6 Vac, Escherichia coli 30 lacZ was under the control of the early promoter element of the W p75 promoter from plasmid pSC65 (S. Chakrabarti, J. Sisler, and B. Moss, NIAID, NIH, Bethesda, MD); this construct was named VJS6 for simplicity. Control CR19 W
(wild-type vaccinia) was kindly provided by J. Yewdell and J. B~nn;nk (NIAID, NIH, Bethesda, MD). Murine IL-2 cDNA

W096/11279 22 0 1 5 9 2 PCT~S95112638 was amplified by PCR from pBMGNomIL-2 and ligated into the - SmaI-Ba~HI site of a vaccinia expression vector, pMW601 (a gift from B. Moss), which contains the ~-gal gene under the control of the p7 5 early vaccinia promoter (Whitman, E.D. et al, Surqery 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.
The POXVAX-TC strain of FPV was used in the~e 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 VV 4Ok promoter, placed in the BamHI J region of the FPV genome.
Peptides The synthetic peptide, TPHPARIGL, spanning a-m--ino acids 876-884 o~ ~-gal, the naturally proce~sed H-2 Ld-restricted epitope (Gavin, M.A. et al, ~. Tmmllnol.
151:3971, 1993), was synthesized by Peptide Technologies (Washington, D.C.) to a purity of greater than 99~ as determined by HPLC and amino acid analysis.
Evaluation of primary response Primary lymphocyte populations were obt~;ne~ by injecting 8- to 12-wk-old fe-m~ e BA~B/c mice (~n;m~l Production Colonies, Frederick Cancer Research Facility, National Institutes of Health, Frederick, MD) i.v. with varying PFUs of recombinant viruses. The spleens were taken on day 6 after ;mmlln;zation, separated into a single cell suspension and tested for their ability to lyse ~-gal-positive targets in a 6 h5~Cr release assay.
Splenocytes were resuspended in complete me~;~ including RPMI 1640 with 10~ FCS tBiofluids, Rockville, MD), 0.1 mM
nonessential amino acids, 1 mM sodium pyruvate (Biofluids) and 5 x 10-5 ~M2-ME (Life Technologies, Inc., Rockville, WO96/11279 PCT~S95112638 MD), and used in 6-hslCR release assays as described elsewhere (Restifo, N.P. et al J. Immunol. 147:1453, 1991). To summarize, 2 x lo6 target cells were treated with 200 mCi Na5~CrO4(51Cr) for 90 min. Peptide-pulsed CT26.WT were incubated with l ~g/ml of peptide during labeling. Target and effector cells were m;xer7~ at appropriate ratios for 6 h. The amount of 5~Cr released was determined by gamma-counting and the percent specific lysis was calculated from triplicate samples according to the following formula:
[(experimental cpm - spontaneous cpm)/
(maximal cpm - spontaneous cpm] x l00.

Detection of cytokine production Cytokine production was determined by ELISA. The ELISA kits for detection of murine GM-CSF, IFN-~, TNF-~, and IL-2 from Endogen (Endogen, Boston, MA) were used according to the manufacturer's instructions. IL-2 concentrations were sometimes given in Cetus units, l Cetus unit/ml corresponding to 600 pg/ml of IL-2.
In vivo treatment studies BALB/c mice were ;mmlln;zed with virus (5 x lo6 to 107 PFUs) 3 or 6 days after i.v. challenge with tumor cells (l05 to 5 x l05) to establish pulmonary metastases. All ~n;m;71s were rc7n~7.nm;zed before receiving virus. Treatment with IL-2 was initiated 12 h after ;mml~n;zation; 6 doses of high dose IL-2 (l00,000 Cetus units/injection) or l0 doses of low dose IL-2 (15,000 Cetus units/injection) were Aflm;n;stered to selected groups of mice. Mice were killed on day 12 and lung metastases were ~nl7m~rated in a blind fashion. Identically treated groups of mice were followed for survival.
Whole organ X-gal st;7;n;ng Lungs from mice treated with i.v. tumor challenge as described above were inflated with PBS (Biofluids) before X-gal st;7;n;ng. Lungs were fixed in a solution cont.7;n;ng WO~6111279 ' ~Z O 1 5 9 2 pcTluss~ll2638 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 37C. X-gal solution for whole organs was prepared by combining the ~ollowing: 0.02~
, (v/v) Nonidet P-40, 0.01~ (w/v) sodium deoxycholate, 1 mg/ml X-gal, 5 mM potassium ferricyanide, 5 ~I potassium ferrocyanide, 2 mM MgCl2 in PBS. After staining in X-gal solution, the lungs were rinsed briefly with 3~ (v/v) dimethyl sulfate, and then with PBS. The St~; n~l lungs were stored at 4C in 0.02~ (w/v) sodium azide in PBS
(Lin, W.C. et al, Cancer Res. 50:2808, 1990).
Statistical analysis The Wilcoxon-Mann-Whitney U test was used to ex~m;ne 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.
Example 27 }~rlm;n;~tration of Exogenous IL-2 with rW encoding the model TAA reduces the number of plllm~n~ry metastases and prolonqs survival When inoculated i.v. at a dose of 5 x 105 cells, both the parental line CT26.WT and the subclone CT26.CL25, transduced with a retrovirus encoding the model TAA ,B-gal, grew progressively and killed the ~n;m~ls in 11 to 15 days. At the time of death, these mice had greated than 500 plllmon~ry metastases. In the experiment presented in Table 8, control mice received no treatment and had plllmn~;lry metastases that were too numerous to count.
Neither the inoculation of moderate doses of rIL-2 for 5 days nor one i.v. injection of 5 x 106 PFU/mouse of a rVV-encoding ,~-gal (VJS6) induced a significant reduction of the number of plllmo~ry metastases counted 12 days after tumor inoculation. Tmmlln;zation with VJS6 3 days after tumor injection combined with rIL-2 ~lm;n;~tration induced a significant reduction in CT26.CL25 plllmon~ry metastases WO96111279 ~ ~ PCT~S95112638 -(p = 0.005) whereas no significant decrease in the number of pulmonary met~stases of the TAA-negative parental line was seen.
Table 8 5 ~ Treatment of Established plll mo~ry Metastases with rW and exogenous IL-2d Mice Inoculllted Wlth C126.WT Cl'26.CI25 rW Treatment AYer~geno. ' '`' Aver~geNo. met~ es `' /Mouse met-st-ses Noneb >SOO >500 x 5 >SOff >500 x 5 VJS6 >500 >500 x 5 405.2 >500 x 3, 267, 259 Exogenous rlL-2 > 500 > 500 x 5 > 500 > 500 x 5 FY~g,~ ^ RIL-2 + 406.4 >500 x 3, 298, 234 20.8 43, 6, 0, 52, 3 ' Five BALB/c mice per each treatment group were injected i.v. with 0.5 ml of HBSS containing 5 x 105 tumor cells of either CT26.WT or CT26.CL25. Three days later they received a single i.v. injection of 5 x lO PFU of ~-gal expressing rW, VJS6. Treatment with exogenous rI~-2 (15,000 Cetus U, twice a day, i.p.) was started 6 h after rW inoculation and continued ~or 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 ll and 12 (before lung harvest).
Active immunotherapy with the combination of exogenous rIL-2 and rW also prolonged the survival of mice bearing 3-day-old plllmo~ry metastases (Figs. 22a and 22b). Two ~m; n; stration regimes for IL-2 were chosen:
lO0,000 U rIL-2 for 3 days (high dose) and 15,000 U for 5 days (low dose) were administeredd i.p. following a single i.v. injection of VJS6. As an additional negative control, a group of mice was treated with the highest dose of exogenous rIL-2 together with a TK-disrupted rW
expressing a protein different from ~-gal, the influenze virus NP (V69; Figs. 22a and 22b). 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 WO96/11279 2 2 0 1 5 9 2 PCT~S95112638 survival was obtained when exogenous rIL-2 was A~m;n;sted with the ~-gal-encoding rVv 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 ~-gal but not with the wild-type virus (Figs. 23a and 23b). The effect of a single i.v. injection of 107 PFU of the recombinant FPV.bg40k, expressing ~-gal under the control of the 40k vaccinia promoter, on the survival of mice bearing the ~-gal-positive tumor was limited and consistent with a partial reduction in the number o~
plllmonAry metastases. However, daily inoculation of high IL-2 doses significantly increased the therapeutic effect, resulting in 40~ of mice still surviving 2 mo a~ter treatment.
To ~Am; n~ a more advanced disease model, mice were injected with 105 tumor cells and treated on day 6.
Following the i.v. inoculation of 105 CT26.CL25 tumor cells, all the untreated mice died by day 22 (Fig. 24).
Lungs examined after 6 days of tumor growth revealed the 25 presence of more than l00 macroscopically visible nodules (not shown). At this time point, a single inoculation o~
V~S6 was able to slightly increase survival, but all the mice died within 24 days. Addition of high rIL-2 dose treatment resulted in a significant survival benefit (p =
30 0 005) also in this advanced disease model, with two of five mice surviving until day 35. Once again, the response was specific and limited to the combination of ~-gal-positive tumor and rW; the survival of CT26.WT tumor-bearing mice was not affected (data not shown).

wos6/11279 ~ ~ ~ 9 PCT~S95/12638 -o Example 28 Therapeutic efficacy of IL-2 produced from drVV also expressing the model TAA
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, 1982j. 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 ~-gal and IL-2 was previously described (Flexner, C.
et al, Vaccine 8:17, 1990). On the basis of the above results with passively ~m; n~ stered IL-2, a new set of dr W in which cytokine production was driven by a powerful synthetic promoter (Davison, A.J., Nucleic Acids Res.
18:4285, 1990) was constructed. The murine cytokines included in this study were the following: IL-2, GM-CSF, IFN-~, and TNF-~. Because the same plasmid was used to construct the various TK~drW, a similar level of ~-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 I~-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 ~-gal.

WO961ll279 2 2 0 1 5 9 2 PCT~S95112638 o Table 9 - Detection of cytokine production following in vitro infection of BSC-1 cells with 1:1 moi of different rW' Cytokine production after 12 h rW IL-2 GM-CSF IFN-~ TNF-~
VJS6 c34 c15 c47 c30 I~-2 2 x 106 c15 c47 c30 GM-CSF c34 5.1 x 105 ~47 ~30 IFN-~ c34 ~15 5.6 x 104 ~30 TNF-~ c34 ~15 ~47 2.05 x 103 Cytokine production after 36 h VJS6 c34 c15 ~47 ~30 IL-2 3.9 x 106 c15 c47 ~30 GM-CSF 54 4.2 x 106 ~47 c30 IFN-~ 94 c15 6.7 x 104 c30 TNF-~ ~34 2.2 x 1o3b ~47 1.35 x 105 - Duplicate wells of 105 BSC-1 cells were infected with di~ferent rW at a multiplicity of infection (moi) of 1 PFU/cell. At different intervals, supernatants were removed, centrifuged to eliminate cellular debris, serially diluted, and used to estim~te cytokine production by using an ELISA assay specific for the murine cytokines indicated in the table. Values are expressed as pg/ml of supernatant.
b- Only one of the two wells was positive. No positivity was detected in subsequent det~rm;n~tions.
When a single i.v. injection of 5 x lo6 PFU/mouse was used to treat mice bearing 3-day-old pl71mon~ry metastases, only the IL-2 drW was able to significantly reduce the number of pnlmon~ry nodules in mice inoculated with the ~-gal-positive tumor cell line (p = 0.005) (Fig. 25b). A
partial decrease was obtained with the other cytokine-encoding viruses and with VJS6. No appreciable change was observed in the number of metastases of the parental tumor cell line (Fig. 25a). The effect on plllmon~ry metastases also correlated with an increased survival (see below).
These resuls were particularly interesting because the drW expressed 20- to 50-fold less ~-gal enzyme activity than the VJS6 rW, as detected after infection of BSC-1 cells with an equivalent moi (data not shown) and WO96111279 PCT~S95/12638 ~
'9 ~

o - 90 also suggested that the production of IL-2 could overcome the lower production of the model TAA. When compared under the same experimental conditions, IL-2 drW together with exogenous IL-2 gave the best results in terms of prolongation of survival (p = 0.002), whereas the survival in the group treated with IL-2 rW or the combination VJS6 plus exogenous IL-2 was similar (Figs. 26a and 26b).
Example 29 IL-2 enhances the primary CTL response against W and heterologously expressed ~-qal Because the presence of CD8+ lymphocytes recognizing tumor determ;n~nts has been associated with an antitumor effect (Greenberg, P.D., Adv. Immunol. 49:281, l99l), the generation of effector CTL against ~-gal in the spleens of mice 6 days after ;mmlln;zation was studied. This time point represents the peak of the CD8+ CTL anti-vaccinia response (B~nn;nk, J.R., Curr. Top. Microbiol. Immunol.
163:153, 1986). BALB/c mice were ;mmlln;zed with different rW indicated in Figure 27, and the spleens of two mice, removed 6 days after ;mmlln;zation, were pooled and tested directly in a short term 51Cr release assay. The response of VJS6-;mmlln;zed splenocytes against the CT26.WT target cells infected with the crude l9 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-~) of the primary cytotoxic response to vaccinia Ags. This effect was even more pronounced when a comparison between the lytic units in each spleen was performed because the difference in spleen size among the different groups; in fact, the number of lytic units in GM-CSF and IL-2 groups were increase 8.3-and 6.4-fold, whereas TNF-~ and IFN-~ were reduced 5.l-and 2.9-fold, respectively, compared with the control ;: ~
~ WO 96tll279 PCT/US9~/12638 o - 91 2201592 cytokine-negative VJS6 (Fig. 27). In the same - experiments, the primary response against ~-gal-positivetarget cells was evaluated but no lysis was detected, with the exception of a small response (5 to 10~ specific s1Cr release) in mice ;mmlln;zed with IL-2 drW (see below).
After in vitro restimulation with the antigenic peptide from ~-gal (TPHPARIGL), splenocytes of BALB/c mice ;mmlln;zed 14 days before with 5 x 1o6 PFU of different drW generated effector CTL able to specifically recognize the ~-gal-positive clone CT26.CL25 or the parental cell line pulsed with the m;n~mAl deter~;n~nt antigenic peptide. Despite the clear effect of IL-2, either exogenously ~m; n; stered or endogenously produced by rW, in the treatment of tumor-bearing mice, no difference in the cytolytic activity against the specific target cells was observed in secondary cultures from ~n;m~l S ;mmllnl zed with different rW co-expressing the TAA with the different murine cytokines (data not shown).
Because analysis of primary and secondary responses did not show any clear difference in reactivity against ~-gal in normal mice inoculated with different rW, the possibility that the in vivo response could be influenced by the presence of growing tumor by comparing the primary cytotoxic activity in normal and in tumor-bearing mice was investigated. Groups of three mice were mock inoculated (HBSS) or injected with different doses (5 x 105, 105, 5 x 104) of either the parental CT26.WT or the ~-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 ;mmlln;zation~ were tested for primary CT~ activity. Figure 28 summarizes the results of one experiment representative of three others. As previously indicated, ;mm~ln;zation of normal BALB/c mice with IL-2 drW produced a small but consistent primary response against the ~-gal peptide-pulsed CT26.WT and the transduced CT26.C~25 target cells; this response was _ _ _ _ _ _ WO96/11279 ~ ~ ~9~ PCT~S95112638 -augmented in a dose-dependent fashion in mice bearing an increasing number of ~-gal-positive pl~1mon~ry metastases as a consequence of an increased inoculum of tumor cells.
Conversely, the specific anti-~-gal response in mice injected with the highest dose of CT26.WT tumor cells was not comparably increased (in Fig. 28 the 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 ~-gal-positive line expressing a different restriction element (H-2b). No response was observed in mice inoculated with 5 x 10~ CT26.CL25 cells and treated with VJS6 (Fig. 28) or drW expressing GM-CSF, IFN-~, or TNF-~ (now shown). The same enhancement of primary response was obt~;ne~ with exogenous IL-2 and VJS6 (data not shown).
Example 30 plllmnn~ry metasases in mice dead after a prolonged survival do not present detectable levels of the model TAA
Tumor cells devoid of the ~-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). Lungs from untreated mice, dead after 12 to 16 days from the i.v. injection of 5 x 10~ CT26.CL25 tumor cells, showed an intense, dark-blue staining. Lungs removed from mice treated with the combination high dose exogenous rIL-2 and rW and found dead after 50 days of survival in the experiment described in Figs. 22a and 22b were completely negative for X-gal st~;n;ng as were the mice inoculated with the ~-gal-negative parental cell line (data not shown). The only slight positivity detected in one sample was associated with the mouse liver, strongly adherent to the lung through a ~-gal-negative invasive metastasis. This WO96/11279 2 2 0 1 5 ~ 2 PCT~S95/l2638 r positivit~ is probalby related to the ~-gal-like activity present in lysosomal compartment (Sanes, J.R. et al, EMB0 J. 5:3133, 1986)). A similar negativity to X-gal st~;n~ng was observed in plllmo~ry metastases from mice treated with the combination IL-2-rVV and exogenous rIL-2 in the survival study presented in Figs. 26a and 26b.
Example 31 The ef~ects of recombinant IL-10 on the therapeutic effectiveness of replicating and non-replicating poxviruses BA~B/c mice (5-10/group) were injected intravenously with 5 x 105 CT26-~-gal transfectants (CT-26.C.25) then randomized. Three days later, mice were treated with a recombinant vaccinia virus (designated VJS6) that encodes the model tumor antigen (full-length ~-galactosidase) at lo6 and 107 PFU either alone or in co-m-bination with O.l~g of IL-10 I.P. per day for 7 days starting 12 hrs after the virus was given. The source of IL-10 was human recombinant I~-10 obtained from Biological Response Modifiers Program (Frederick, MD). As designated, other groups of mice were treated with a non-replicating recombinant fowlpox virus that also encodes the model tumor antigen (full-length ~-galactosidase) at 106 and 107 PFU either alone or in combination with O.l~g of I~-10 I.P. per day for 7 days starting 12 hrs after the virus was given. plllmon~ry metastases were ~nllmerated on day 12 in a coded, blinded fashion. The data is provided in Table 10.

Wo 96111279 ,~ ' 9 ,~ PCTIUS9~/12638 Table 10 Adjuvant Therapy in a r~plicating and non-replicating setting Group Avg # Breakdown P2 Mets S No Tx 250 tntc*5 ---VJS6 lOe6 198 tntc*3,135,95,208 .05 (vs. no tx) VJS6 lOe6 + IL10 92 45,70,85,74,112,125,130,90 .0036 (vs. VJS6) VJS6 lOe7 49 44,40,41,84,41,40,55 .0028 (vs. no tx) VJS6 lOe7 + IL10 28 52,35,3,20,31 .005 (v. VJS6) IL-10 alone 250 tntc*5 ---FPV lOe6 83 84,89,40,94,84,65,49,61,125 .002 (vs. no tx) FPV lOe6 + IL10 20 15,4,6,8,0,12,40,82 .024 (v. FPV) ~PV lOe7 10 0*2,3,3,5,2,15,12,49 .002 (vs. no tx) FP lOe7 + IL10 24 0,0,2,4,29,30,55,38,45,46 >.05 In another experiment BA~B/c mice (5-lO~group) were injected intravenously with 5 x 105 CT26-~-gal transfectants (CT-26.C.25) then r~n~om;zed. 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-~ull-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. pnlmon~ry metastases were enumerated on day 12 in a coded, blinded fashion. ~n;m~l S receiving no treatment or IL-10 alone had an average of 250 plllmo~ry metastases (Figure 29).
In yet another experiment, mice were injected I.V.
with 5 x 105 CT26-~-gal transfectants. Three days later mice were treated with 10l, 102, 103, 104, 105, 106, or 107 PFU of VJS6 either alone or in combination with O.l~g of WO96111279 PCT~Ss~/l2638 IL-lO per day for 7 days starting 12 hrs after the virus - was given. pl7l mon~ry metastases were enumerated on day 12 as above. ~n;m~l S receiving no treatment or IL-lO alone had an average of 250 plllmon~ry metastases (Figure 30).
5 ~ While the invention is described above in relation to certain specific em.bodiments, it will be understood that many variations are possible, and that alternative materials and reagents can be used without departing from the i~vention. In some cases such variations and substitutions may require some experimentation, but will only involve routine testing.
The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific lS embodiments without departing from the generic concept, and therefore such adaptations and modifications are intended to be comprehended within the m~n; ng and range o~ equivalents of the disclosed em.bodiments.
All references and patents referred to are incorporated herein by reference.

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Claims (33)

1. A recombinant virus comprising a recombinant virus which has incorporated into a viral genome or portion thereof one or more genes or portion thereof encoding an antigen of a disease causing agent and one or more genes or portions thereof encoding an immunostimulatory molecule.
2. The recombinant virus according to claim 1 where the virus is nonpathogenic.
3. The recombinant virus according to claim 1 wherein the virus is nonreplicating.
4. The recombinant virus according to claim 1 wherein the virus is selected from the group consisting of retrovirus, baculovirus, Ankara virus, fowlpox, adenovirus, and vaccinia virus.
5. The recombinant virus according to claim 1 wherein the disease causing agent is a cancer or pathogenic microorganism.
6. The recombinant virus according to claim 5 wherein the cancer is a non-Hodgkin Lymphoma, leukemia, Hodgkins lymphoma, lung cancer, liver cancer, metastases, melanoma, adenocarcinoma, thymoma, colon cancer, uterine cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, bladder cancer, kidney cancer, pancreatic cancer or sarcoma.
7. The recombinant virus according to claim 5 wherein the pathogenic microorganism is virus, bacterium, protozoan, or yeast.
8. The recombinant virus according to claim 7 wherein the pathogenic virus is HIV, hepatitis virus, human papillomavirus, equine encephalitis virus, herpes simplex virus or influenza virus.
9. The recombinant virus according to claim 6 wherein the antigen is a tumor associated antigen.
10. The recombinant virus according to claim 9 wherein the tumor associated antigen is selected from the group consisting of oncofetal antigens, MART-1, Mage-1, Mage-3, gp100, tyrosinase, CEA, PSA, CA-171A, CA-19-A, CA-125, erb-2, TRP-1, P-15 and .beta.-galactosidase.
11. The recombinant virus according to claim 1 wherein the immunostimulatory molecule is selected from the group consisting of IL-2, B7.1, B7.2, ICAM-1, LFA-3, CD72, GM-CSF, TNF.alpha., IFN.gamma., IL-10, IL-12, IL-1, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-13, IL-14, IL-15, ICAM-2, LFA-1, G-CSF, M-CSF, IFN.alpha., CTAP III, ENA-78, GRO, I-309, PF-4, IP-10, LD-78, MGSA, MIP-1.alpha., MIP-1B, RANTES
and combinations thereof.
12. The recombinant virus according to claim 11 comprising the immunostimulatory molecule, IL-2 and B7.1.
13. The recombinant virus according to claim 1 wherein the recombinant virus is vaccinia virus.
14. The recombinant virus according to claim 13 wherein the gene encoding the antigen is incorporated into a HIND F13L region or TK region of the vaccinia virus.
15. The recombinant virus according to claim 13 wherein the gene encoding the immunostimulatory molecule is incorporated into a HIND F13L region or HA region of the vaccinia virus.
16. A recombinant virus comprising a recombinant vaccinia virus which has incorporated into a genome a gene encoding a tumor associated antigen and a gene encoding an immunostimulatory molecule.
17. The recombinant virus according to claim 16 wherein the tumor associated antigen is an oncofetal antigen, MART-1, Mage-1, Mage-3, gp100, tyrosinase, CEA, PSA, CA-171A, CA-19-A, CA-125, erb-2, TRP-1, P-15 or .beta.-galactosidase.
18. The recombinant virus according to claim 16 wherein the immunostimulatory molecule is ICAM-1, LFA-3, CD72, GM-CSF, TNF.alpha., IFN.gamma., IL-12, RANTES, IL-10, IL-2, B7.1, B7.2, IL-1, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-13, IL-14, IL-15, ICAM-2, LFA-1, G-CSF, M-CSF, IFN.alpha., CTAP III, ENA-78, GRO, I-309, PF-4, IP-10, LD-78, MGSA, MIP-1.alpha., MIP-1B or combinations thereof.
19. A method of preventing a disease in a mammal comprising administering to the mammal an effective amount of a recombinant virus according to claim 1, alone or in combination with an exogenous immunostimulatory molecule said amount is effective in preventing or ameliorating said disease.
20. The method according to claim 19, wherein the disease is caused by a cancer or pathogenic microorganism.
21. The method according to claim 19, wherein the cancer is non-Hodgkin lymphoma, Hodgkins lymphoma, leukemia, lung cancer, liver cancer, metastases, melanoma, adenocarcinoma, thymoma, colon cancer, uterine cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, bladder cancer, kidney cancer, pancreatic cancer or sarcoma.
22. The method according to claim 19 wherein the pathogenic microorganism is an intracellular virus, bacterium, protozoan, or yeast.
23. The method of claim 19 wherein the exogenous immunostimulatory molecule is IL-2, GM-CSF, TNF.alpha., TNF.gamma., IL-12, RANTES, B7-1, B7-2, ICAM-1, LFA-3, IL-10, CD72, IL-1, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-13, IL-14, IL-15, ICAM-2, LFA-1, G-CSF, M-CSF, IFN.alpha., CTAP III, ENA-78, GRO, I-309, PF-4, IP-10, LD-78, MGSA, MIP-1.alpha., MIP-1B or combinations thereof.
24. A pharmaceutical composition comprising the recombinant virus according to claim 1 alone or in combination with an exogenous immunostimulatory molecule, chemotherapy drug, antibiotic, antifungal drug, antiviral drug or combination thereof and a pharmaceutically acceptable carrier.
25. The pharmaceutical composition according to claim 24 wherein the exogenous immunostimulatory molecule is ICAM-1, LFA-3, CD72, GM-CSF, TNF.alpha., IFN.gamma., IL-10, IL-12, RANTES, IL-2, B7.1, B7.2, IL-1, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-13, IL-14, IL-15, ICAM-2, LFA-1, G-CSF, M-CSF, IFN.alpha., CTAP III, ENA-78, GRO, I-309, PF-4, IP-10, LD-78, MGSA, MIP-1.alpha., MIP-1B or combination thereof.
26. A method of making an immune-enhancing recombinant virus against a disease causing agent comprising inserting a nucleic acid sequence encoding one or more antigens or immunodominant epitopes thereof and a nucleic acid sequence encoding one or more immunostimulatory molecules into the genome or portion thereof of a recombinant virus.
27. An immune-enhancing recombinant virus made according to the method of claim 26.
28. A pharmaceutical composition comprising a recombinant virus, one or more exogenous immunostimulatory molecules and a pharmaceutically acceptable carrier, said virus having incorporated into a viral genome or portion thereof one or more genes or portion thereof encoding an antigen of a disease causing agent.
29. The pharmaceutical composition according to claim 28 wherein the disease causing agent is a cancer or a pathogenic microorganism.
30. The pharmaceutical composition according to claim 28 wherein the antigen is a tumor associated antigen.
31. The pharmaceutical composition according to claim 28 wherein the exogenous immunostimulatory molecule is IL-2, GM-CSF, TNF.alpha., IFN.gamma., IL-10, IL-12, RANTES, B7.1, B7.2, ICAM-1, LFA-3, IL-10, CD72, IL-1, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-13, IL-14, IL-15, ICAM-2, LFA-1, G-CSF, M-CSF, IFN.alpha., CTAP III, ENA-78, GRO, I-309, PF-4, IP-10, LD-78, MGSA, MIP-.alpha., MIP-1B or combination thereof.
32. The pharmaceutical composition according to claim 30 wherein the exogenous immunostimulatory molecule is IL-2, IL-10, or combination thereof.
33. A method of preventing a disease in a mammal comprising administering to the mammal an effective amount of the composition according to claim 28, said amount is effective in preventing or ameliorating said disease.
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