WO2000072686A1

WO2000072686A1 - Regulation of systemic immune responses utilizing cytokines and antigens

Info

Publication number: WO2000072686A1
Application number: PCT/US2000/015190
Authority: WO
Inventors: Steve Hardy; Glenn Dranoff
Original assignee: Cell Genesys, Inc.
Priority date: 1999-06-02
Filing date: 2000-06-02
Publication date: 2000-12-07
Also published as: AU5458500A

Abstract

The present invention in all of its associated aspects provides a method for stimulating a systemic immune response in a mammal to a tumor that may be pre-existing, or that may arise following treatment. More particularly, the present invention is concerned with co-administering a tumor cell and a cytokine, the tumor cell expressing at least one cytokine. The invention also relates to a recombinant virus comprising nucleic acid encoding a cytokine, such as human granulocyte-macrophage colony stimulating factor, and tumor cell transduced by such a recombinant virus, wherein the tumor cell expresses human granulocyte-macrophage colony stimulating factor.

Description

REGULATION OF SYSTEMIC IMMUNE RESPONSES UTILIZING CYTOKINES AND ANTIGENS

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a a continuation-in part of U.S. Application No. 08/486,854, filed June 7, 1995, which is a continuation-in-part of U.S. Application Serial No. 08/265,554, filed June 24, 1994, issued as U.S. Patent No. 5,637,483 which is a continuation of U.S. Application Serial No. 07/956,621, filed October 5, 1992, which is a continuation-in-part of U.S. Application Serial No. 07/771,194, filed October 4, 1991, the entire disclosures of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a method of altering an individual's immune response to a target antigen or antigens. More particularly, the present invention is concerned with co-administering a target antigen and at least one cytokine in such a manner as to either increase or decrease the individual's immune response. Possible antigens include tumor cells from the same individual (i.e., autologous) or from a different individual (i.e., allogeneic), tumor cell antigens, infectious agents, foreign antigens, and non-foreign components.

BACKGROUND OF THE INVENTION

The immune system plays a critical role in the pathogenesis of a wide range of important diseases and conditions, including infection, autoimmunity, allograft rejection and neoplasia, particularly cancer. The shortcomings of the immune system in these disorders can be broadly considered as either the failure to develop a sufficiently potent response to a deleterious target or the inappropriate generation of a destructive response against a desirable target. Standard medical treatments for a destructive response against a desirable target. Standard medical treatments for these diseases, including chemotherapy, surgery, and radiation therapy, have clear limitations with regard to both efficiency and toxicity. Moreover, these standard treatments have had little success in preventing the disease or condition. Thus, new strategies based on specific manipulation of the immune response are greatly needed. The use of autologous cancer cells as vaccines to augment anti-tumor immunity has been explored throughout this century (Oettgen et al., "The History of Cancer Immunotherapy," In: Biologic Therapy of Cancer, Devita et al. (eds.), J. Lippincott Co., pp. 87-119 (1991)). However, the immunogenicity of cancer cells is generally too weak to elicit a pronounced immune reaction sufficient to overcome the disease. Patient responses to these "raw" vaccines have generally been only partial and relatively short-lived. Strategies to improve the efficacy of such vaccinations, including the use of non-specific immunostimulants such as BCG and Corynebacterium parvum, have resulted in little improvement. One approach for increasing the immunogenicity of tumor cells has been to treat the cells in different ways. For example, U.S. Patent No. 4,931,275 describes using as an alleged vaccine either cells treated with pressure or cholesteryl hemisuccinate, or plasma membranes or membrane proteins from these cells. These methods attempt to enhance the exposure of surface antigens to render the antigens more immunogenic.

Another approach focuses on the interaction of cytokines and the immune system. Cytokines and combinations of cytokines have been shown to play an important role in the stimulation of the immune system. For example, U.S. Patent No. 5,098,702 describes using combinations of TNF, IL-2, and IFN-β in synergistically effective amounts to allegedly combat existing tumors. U.S. Patent No. 5,078,996 describes the alleged activation of macrophage nonspecific tumoricidal activity by injecting recombinant granulocyte-macrophage colony stimulating factor (GM-CSF) to treat patients with tumors. A further expansion of this approach involves the use of genetically modified tumor cells to evaluate the effects of cytokines on tumorigenicity. Since the doses of cytokines necessary to effect tumor development are often systemically toxic, direct treatment of patients is frequently not feasible (see, for example, Asher et al., /. Immun. 146: 3227-3234 (1991) and Havell et al, /. Exp. Med. 167: 1067-1085 (1988)). Localized high concentrations of certain cytokines, delivered by genetically modified cells, have allegedly been found to lead to tumor regression (Abe et al., /. Cane. Res. Clin. Oncol. 121: 587-592 (1995); Gansbacher et al, Cancer Res. 50: 7820-7825 (1990); Forni et al., Cancer and Met. Reviews 7: 289-309 (1988). For example, Frost et al. teach that transfection of a gene encoding a cytokine, such as IL-2, into a tumor cell allegedly results in an immunopotentiating tumor cell that enhances the responsiveness of an animal's immune system to a tumor present in the animal (Frost et al, WO 92/05262). Such studies allegedly demonstrate that expression in murine tumor cells of various cytokine genes can lead to the rejection of these genetically modified cells by syngeneic hosts. For example, growth of malignant mouse neuroblastoma cells injected into mice was allegedly suppressed when the cells constitutively express γ-IFN at high levels (Gansbacher et al., Cancer Res. 50: 7820-7825 (1990); Watanabe et al, Proc. Natl. Acad. Sci. USA 86: 9456-9460 (1989)). Injection of mammary adenocarcinoma tumor cells expressing IL-4, mixed with a variety of nontransfected tumor cells, allegedly inhibited or prevented tumor formation (Tepper et al, Cell 57: 503-512 (1990)). Similar results were allegedly seen for IL-2 (Fearon et al., Cell 60: 397-403 (1990); Gansbacher et al., /. Exp. Med. 172: 1217-1224 (1990)), TNF-α (Asher et al, supra; Biankenstein et al., /. Exp. Med. 173: 1047-1052 (1991); Teng et al, Proc. Natl. Acad. Sci. USA 88: 3535-3539 (1991)), G-CSF (Colombo et al, /. Exp. Med. 173: 889-897 (1991)), JE (Rollins et al, Mol. Cell. Bio. 11: 3125-3131 (1991)), and IL-7 (Hock et al, /. Exp. Med. 174: 1291-1298 (1991)). Other studies describe the direct injection into an established tumor of a recombinant virus (such as adenovirus) encoding murine IL-12 (Chen et al, /. Immunol. 159: 351-359 (1997); Caruso et al., Proc. Natl. Acad. Sci USA 93: 11302-11306 (1996)), murine IL-12 plus B7-1 (Putzer et al., Gene 193: 129-140 (1997)), or murine GM-CSF (Chen et al, Cancer Res. 56: 3758-3763 (1996)). These studies report the alleged facilitation of tumor regression in mice following such injection.

Yet another approach involves the use of genetically modified tumor cells as vaccines to prevent the subsequent development of a tumor. Injection of tumor cells expressing IL-2 allegedly not only suppressed tumor formation initially but allegedly conferred systemic immunity as well, thus allowing the mice to reject a subsequent challenge of tumor cells (Fearon et al., supra). However, the systemic immunity generated in this manner was short-lived; mice were able to reject tumor cells injected two weeks after the vaccine but were not able to reject tumor cells injected four weeks after the vaccine was administered. Similar results were allegedly seen with TNF-α, where animals that experienced initial tumor rejection were challenged two weeks later and did not develop new tumors for at least 40 days (Asher et al., supra). A somewhat longer systemic immunity was allegedly seen with IFN-γ: mice vaccinated with IFN-γ -producing tumor cells were able to reject unmodified tumor cells injected six weeks later. The efficacy of these vaccines to stimulate the immune system to attack a previously existing tumor, which is the more desirable trait of a preventative vaccine, is not as clear. For example, injection of IL-4-, IFN-γ-, or IL-2-producing tumor cells allegedly did not affect the growth of non-modified tumor cells at a different site on the animal (Tepper et al., supra; Gansbacher et al., Cancer Res. 50: 7820-7825 (1990); Gansbacher et al., /. Exp. Med. 172: 1217-1224 (1990); and Fearon et al, supra, respectively). However, in one isolated case, tumor cells engineered to secrete IL-4 allegedly successfully mediated rejection of established renal cancer cells in animals

(Golumbek et al., Science 254: 713-716 (1991)).

An alternative approach taught by Sobol et al. involves the use of genetically modified, autologous, non-tumor cells as a alleged vaccine (Sobol et al., U.S. Patent No. 5,674,486). Autologous fibroblasts were genetically modified to secrete a cytokine, such as IL-2, and were administered with a tumor antigen (which the fibroblast may also express) at a site other than an active tumor site.

These prior studies suggest the potential use of gene transfer as a means of augmenting anti-tumor immunity. However, the previous methods lack the ability to successfully treat pre-existing cancers. Thus, the ability to systemically treat existing cancers is particularly appealing as a method to treat cancer patients once diagnosed.

In addition, the ability of these methods to prevent subsequent development of tumors is very important, particularly in precancerous patients predisposed to develop cancer (e.g., immunocompromised patients (e.g., AIDS patients), or patients diagnosed as having an aberrant gene known to be involved in cancer formation (e.g., mutation in BRCA-1 or BRCA-2, which is known to be involved in breast cancer). Accordingly, given the inadequacies of the known methods for augmenting anti-tumor immunity, there exists a need for new anti-tumor strategies that allow the treatment of tumors not only pre-existing in the animal prior to treatment, but also tumors arising after treatment of the animal.

SUMMARY OF THE INVENTION

The present invention provides an anti-tumor method that enables the treatment of tumors. The invention is based on the surprising discovery that tumor cells expressing certain cytokines and combinations of cytokines can confer long term specific systemic immunity to individuals receiving injections of such cells. This discovery has been exploited to provide the present invention which is directed to the regulation, either in a stimulatory or suppressive way, of an individual's immune response to a useful antigen, such as a tumor antigen. The method of the present invention is useful for preventative purposes as well as therapeutic applications; i.e., it is useful to protect an individual against development and/or progression of a tumor, or the progression of a bacterial or viral infection such as AIDS, rejection of transplanted tissue, or autoimmune condition. The present invention also finds utility in the reversal or suppression of an existing tumor, condition or disease, such as established tumors, bacterial or viral infections such as AIDS, transplanted tissue rejection, or autoimmune responses. In addition, the present invention finds utility in the treatment of chronic and life threatening infections, such as the secondary infections associated with AIDS, as well as other bacterial, fungal, viral, parasitic and protozoal infections. A particular advantage of the present invention is that the cytokines may be selected to optimize effects in the individual and thus maximize the desired result. The invention also provides cells, recombinant viruses, and viral vectors that are useful in vitro for studies of the biology of cancer and to identify potential therapeutic drugs. Particularly useful recombinant viruses include retroviruses, as well as other recombinant viruses, such as adenoviruses, which are capable of infecting non-replicating mammalian cells. As one aspect of the present invention, there is disclosed a method for regulating the immune response of an individual to a target antigen. The regulation is achieved by co-administering to the individual the target antigen and at least one cytokine, adhesion or accessory molecules or combinations thereof, in such a manner as to elicit a systemic immune response. The antigen and the cytokine, adhesion or accessory molecules or combinations thereof, are co-administered in a therapeutically effective amount, which results in the systemic immune response. Another aspect of the present invention utilizes cells which express the target antigen and at least two cytokines, either naturally or as a result of genetic engineering, which are administered to an individual whose immune response to the target antigen is to be regulated. For example, a tumor cell of the type against which an enhanced immune response is desired can be engineered to express the cytokines to be administered. The resulting genetically engineered tumor cell is used as a vaccine to protect against future tumor development or as a delivery vehicle to result in the reversal or control of previously existing tumors. Specific embodiments of the present invention utilize tumor cells expressing the two cytokines, GM-CSF and IL-2, and utilize melanoma cells as the tumor cells to be modified. Another aspect of the present invention relates to the use of genetically modified, cytokine-expressing tumor cells which are irradiated or rendered proliferation incompetent prior to administration to an individual. The irradiated, modified cells are administered in therapeutically effective amounts to a human. Administration of these cells results in the regulation, in a stimulatory manner, of the individual's systemic immune response. Of particular utility are human tumor cells expressing GM-CSF, and specifically melanoma, prostate cancer, and non-small cell lung carcinoma cells expressing GM-CSF, IL-2, or both. Also of use in the present invention are irradiated tumor cells expressing GM-CSF, IL-4, IL-6, CD2, IL-12, and/or ICAM. In other aspects, the invention relates to the use of modified tumor cells expressing cytokines for the reversal or suppression of pre-existing tumors. In some embodiments, the tumor cells express two cytokines, for example IL-2 or IL-12 and GM-CSF. In other embodiments, the tumor cells express three cytokines, for example IL-2 (or IL-12), GM-CSF, and TNF-α, IL-4, CD2, or ICAM. In preferred embodiments, the tumor cells are irradiated or rendered proliferation-incompetent by other means prior to administration.

In another aspect, the present invention relates to the use of recombinant retroviruses to genetically engineer the cytokine-expressing tumor cells. In one embodiment, the invention utilizes a single rransduction of a tumor cell by a recombinant retrovirus encoding at least one cytokine (which may be the same or different cytokine). In other embodiments, the invention utilizes multiple transductions by multiple recombinant retroviruses encoding different cytokines.

In various embodiments, a variety of retroviral vectors are used to generate the recombinant retroviruses. The MFG, α-SGC, pLJ, and pEm vectors (and derivatives thereof, e.g., MFG-S) are more fully disclosed in U.S. Serial No. 07/786,015, filed October 31, 1991 (PCT/US91/08121, filed October 31, 1991), incorporated herein by reference, and will find particular utility in the present invention.

In other embodiments, alternative vectors, including other retroviral vectors or other types of vectors (e.g., vaccinia virus vectors) are used in the method of the invention, provided that they can express sufficient quantities of the cytokine(s) and the adhesion molecule(s) of interest for a sufficient length of time. Thus, various aspects of the invention include the use of a variety of adenovirus (AV) vectors, adeno-associated virus (AAV) vectors, lentivirus vectors, herpesvirus vectors, SV-40 vectors, and vaccinia virus vectors to generate recombinant viruses to transduce tumor cells and thus genetically engineer the tumor cells to express a cytokine. In one embodiment, the invention utilizes a single transduction of a tumor cell by one or more of these recombinant virus(es) encoding at least one cytokine, or it may utilize multiple transductions by such recombinant viruses encoding different cytokines. In one preferred embodiment, the vector used to generate the recombinant viruses is an adenovirus. In some embodiments, the resulting recombinant adenoviral vector contains an MD expression cassette. In one embodiment, the tumor cells are also transduced with an adenovirus vector lacking a cytokine gene to provide an adjuvant in activating the immune system of a mammal to which the transduced tumor cell is administered against tumor tissue. In one embodiment, the adenovirus lacking the cytokine gene is co-transduced into the tumor cell with the recombinant adenovirus containing at least one cytokine gene. In another aspect, the present invention provides a method of enhancing the immune response of a human to a tumor antigen. In this method, primary malignant tumor cells, such as melanoma or non-small cell lung carcinoma tumor cells, modified to express the tumor antigen and GM-CSF, are administered to the mammal. These tumor cells are transduced with a recombinant virus comprising a nucleic acid encoding GM-CSF. The recombinant virus is an adenovirus, an adeno- associated virus, a lentivirus, a herpes virus, an SV-40, or a vaccinia virus. The modified tumor cells are adrninistered in a therapeutically effective amount and in such a manner that regulation of the immune response of the mammal to the antigen is achieved. In some embodiments, the recombinant virus further comprises a nucleic acid encoding a cytokine other than GM-CSF, and the transduced cell expresses the additional cytokine as well as GM-CSF. In one preferred embodiment, the primary tumor cell is an autologous tumor cell (i.e., derived from the individual being treated). In another embodiment, the primary tumor cell is an allogeneic tumor cell (i.e., derived from an individual different than the individual being treated). In yet other preferred embodiments, the tumor cell is from a tumor cell line of the same type as the tumor or cancer being treated. In some embodiments, the tumor cell has been rendered proliferation-incompetent. In preferred embodiments, the cell has been irradiated such that it has been rendered proliferation-incompetent.

In one embodiment, the method further comprises administering an adjuvant comprising a proliferation-incompetent tumor cell which has not been transduced with genetic material encoding a cytokine, the adjuvant being administered concomitant with the administration of the cytokine-expressing tumor cell.

In another aspect, the present invention provides an isolated, genetically modified, primary malignant human tumor cell expressing at least a tumor antigen and a cytokine. In one embodiment, the tumor cell is a melanoma cell. In another embodiment, the tumor cell is a non-small lung cell carcinoma cell. The tumor cell is transduced with a recombinant virus comprising a nucleic acid encoding the cytokine. The recombinant virus is an adenovirus, an adeno-associated virus, a lentivirus, a herpesvirus, an SV-40, or a vaccinia virus. In some preferred embodiments, the cytokine is GM-CSF. In other embodiments, the cell also expresses a cytokine other than GM-CSF. In certain preferred embodiments, the tumor cell is an autologous primary tumor cell, an allogeneic primary tumor cell and/ or a cultured tumor cell of the same type. In preferred embodiments, the tumor cell is rendered proliferation-incompetent. In some embodiments, irradiation renders the tumor cell proliferation-incompetent. In a preferred embodiment, the antigenic characteristics of the tumor cell are preserved.

In yet another aspect, the invention provides a method of treating a tumor in a mammal. In this method, a therapeutically effective amount of primary tumor cells expressing GM-CSF is administered to the mammal, such as a human. The tumor cells then stimulate a systemic immune response to the tumor in the mammal. The tumor and the primary tumor cells are of the same type and may be of any forms of cancer, including, but not limited to, leukemia, malignant melanoma, non-small lung cell carcinoma, breast, ovarian, cervical, epidermal, or prostate cancer, pre-neoplastic lesions, or cancerous polyps. The primary tumor cells are transduced with a recombinant virus comprising genetic material encoding GM-CSF, the viral vector used to generate the recombinant virus being an adenovirus vector, an adeno- associated virus vector, a lentivirus vector, a herpesvirus vector, an SV-40 vector, or a vaccinia virus vector. In one embodiment, the tumor is malignant melanoma. In another embodiment, the tumor is non-small lung cell carcinoma.

Yet other aspects of the invention relate to kits for regulating the immune response of a mammal to a tumor antigen. These kits include a recombinant virus comprising a nucleic acid encoding GM-CSF, the recombinant virus being an adenovirus, an adeno-associated virus, a lentivirus, a herpesvirus, an SV-40 virus, a vaccinia virus, or a retrovirus. The kit further includes a container useful, e.g., for placing and containing a tumor cell or tumor fragment, and within which a tumor cell is transduced with the recombinant virus. In some embodiments, the kit further includes a digestive enzyme which converts the tumor tissue to a single cell suspension. In some embodiments, the enzyme is collagenase. In some embodiments, the kit further includes an adjuvant which comprises proliferation-incompetent tumor cells which have not been transduced with the recombinant virus comprising genetic material encoding a cytokine.

In another aspect, the invention provides a method for stimulating a systemic immune response in a mammal to a tumor, or to an antigen thereof, comprising administering a therapeutically effective amount of proliferation-incompetent tumor cells that have been genetically modified by transduction with a recombinant virus comprising a nucleic acid encoding a human cytokine so as to express at least one human cytokine, wherein the tumor cell and the tumor are of the same type and express the antigen. Preferably, the recombinant virus is an adenovirus, a lentivirus, an adeno-associated virus, an SV-40, a herpes virus, or a vaccinia virus. In certain embodiments, the systemic immune response to the tumor results in tumor regression or inhibits the growth of the tumor. In certain preferred embodiments, the recombinant virus is replication- deficient. In some embodiments, the human cytokine is human GM-CSF. In specific embodiments, the tumor cells are rendered proliferation-incompetent by irradiation. In one embodiment, the tumor is an established tumor that is present in the mammal prior to the adnriinistration step. In another embodiment, the tumor is one that arises in the mammal subsequent to the administration step. In a some embodiments, the tumor is a metastatic tumor.

In one embodiment, the tumor cell is derived from the mammal harboring a tumor. In a preferred embodiment, the tumor cell is derived from a human. In some embodiments, the tumor cells are cryopreserved prior to administration. In a preferred embodiment, the tumor cell and the tumor are derived from the same individual. In another preferred embodiment, the tumor cell and the tumor are derived from different individuals. In certain preferred embodiments, the tumor cell is from a tumor cell line of the same type as the tumor or cancer being treated. In certain preferred embodiments, the type of tumor cell and tumor being treated is selected from the group consisting of prostate tumor, melanoma, non-small cell lung carcinoma, breast cancer, epidermal cancer, pre-neoplastic lesion, cancerous polyp, ovarian cancer, cervical cancer, leukemia, and renal carcinoma. In a preferred embodiment, the tumor cell line is a prostate tumor cell line. In a certain preferred embodiment, the prostate tumor cell line is selected from the group consisting of DU- 145, PC-3, and LnCaP.

In yet another aspect, the invention provides a recombinant virus comprising a nucleic acid encoding human GM-CSF, wherein a cell transduced with the recombinant virus expresses human GM-CSF, and wherein the recombinant virus is selected from the group consisting of adenovirus, adeno-associated virus, lentivirus, SV-40, herpes virus, and vaccinia virus. In a preferred embodiment, the recombinant virus is replication-defective, the cell is a tumor cell, and the recombinant virus is generated in a packaging cell transfected with a viral vector genetically modified to encode human GM-CSF.

In yet another aspect, the invention provides a genetically modified, proliferation-incompetent tumor cell that expresses at least one cytokine, wherein the cytokine is human GM-CSF. In a preferred embodiment, the genetic modification is by transduction with a recombinant virus that encodes human GM-CSF, wherein a cell transduced with the recombinant virus expresses human GM-CSF, and wherein the recombinant virus is selected from the group consisting of adenovirus, adeno- associated virus, lentivirus, SV-40, herpes virus, and vaccinia virus. In a preferred embodiment, the recombinant virus is an adenovirus.

In one embodiment, the tumor cell is selected from the group consisting of an autologous tumor cell, an allogeneic tumor cell, or a tumor cell line. In certain embodiments, the tumor cell line is a prostate tumor cell line. In some particular embodiments, the prostate tumor cell line is selected from the group consisting of an LnCaP cell, a DU-145 cell, and a PC-3 cell.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects of the present invention, the various features thereof, as well as the invention itself may be more fully understood from the following description, when read together with the accompanying drawings in which:

FIG. 1A is a detailed schematic representation of the MFG vector, a recombinant retroviral vector useful in the present invention;

FIG. IB is a schematic representation of the pLJ vector, a recombinant retroviral vector useful in the present invention; FIG. 1C is a schematic representation of the pEm vector, a recombinant retroviral vector useful in the present invention;

FIG. ID is a schematic representation of the α-SGC vector, a recombinant retroviral vector useful in the present invention;

FIG. 2A is a graphic representation showing the ability of B16 melanoma cells expressing IL-2 to protect against subsequent challenge with wild-type B16 melanoma cells at various times after vaccination;

FIG. 2B is a graphic representation showing the ability of B16 melanoma cells expressing IL-2 and a second cytokine (as indicated) after vaccination to protect against challenge with wild-type B16 melanoma cells. The symbol O represents the animals that succumbed to tumor challenge and the symbol • represents the animals protected from tumor challenge;

FIG. 2C is a graphic representation showing the ability of B16 melanoma cells expressing IL-2 and GM-CSF and a third cytokine (as indicated) to protect against subsequent challenge with wild-type B16 melanoma cells; FIG. 3A is a graphic representation showing the ability of irradiated B16 melanoma cells expressing GM-CSF to protect against challenge with wild-type B16 melanoma cells. The symbol O represents the animals that succumbed to tumor challenge and the symbol • represents the animals protected from tumor challenge; FIG. 3B is a graphic representation showing the ability of irradiated B16 melanoma cells expressing GM-CSF to protect against challenge with wild-type B16 melanoma cells either at varying doses or when mixed with non-transduced B16 melanoma cells. The symbol O represents the animals that succumbed to tumor challenge and the symbol • represents the animals protected from tumor challenge;

FIG. 3C is a graphic representation showing the ability of irradiated B16 melanoma cells expressing GM-CSF to increase the immune response after inoculation with live non-transduced B16 melanoma cells (pre-established tumor) as indicated. The symbol O represents the animals that succumbed to tumor challenge and the symbol • represents the animals protected from tumor challenge;

FIG. 4 is a graphic representation showing the ability of irradiated B16 melanoma cells expressing a cytokine (as indicated) to protect against subsequent challenge with wild-type B16 melanoma cells. The symbol O represents the animals that succumbed to tumor challenge and the symbol • represents the animals protected from tumor challenge;

FIG. 5A is a photographic representation showing the histological analysis of the site of irradiated GM-CSF transduced B16 melanoma vaccination;

FIG. 5B is a photographic representation showing the histological analysis of the site of irradiated nontransduced tumor vaccination; FIG. 5C is a photographic representation showing the histological analysis of the draining lymph node from the irradiated GM-CSF transduced tumor vaccination;

FIG. 5D is a photographic representation showing the histological analysis of the draining lymph node from the irradiated non-transduced tumor vaccination;

FIG. 5E is a photographic representation showing the histological analysis of the challenge site of a mouse vaccinated with irradiated GM-CSF transduced tumor cells;

FIG. 5F is a photographic representation showing the histological analysis of the challenge site of a mouse vaccinated with irradiated non-transduced tumor cells; FIG. 5G is a photographic representation showing the histological analysis of the challenge site in a naive mouse;

FIG. 6A is a graphic representation showing the contribution of lymphocyte subsets to systemic immunity generated by irradiated GM-CSF transduced B16 melanoma cells. The symbol O represents the animals that succumbed to tumor challenge and the symbol • represents the animals protected from tumor challenge;

FIG. 6B is a graphic representation showing the CD8 blockable CTL activity, determined by a 4 hour ⁵¹Cr release assay on gamma-interferon treated B16 target cells at various effector:target ratios. Fourteen days after vaccination with either irradiated GM-CSF transduced or irradiated non-transduced B16 cells, splenocytes were harvested and stimulated in vitro for 5 days with gamma-interferon treated B16 cells;

FIG. 7 is a graphic representation showing the immunogenicity of irradiated, non-transduced murine tumor cell lines used in previous cytokine transfection studies. The symbol O represents the animals that succumbed to tumor challenge and the symbol • represents the animals protected from tumor challenge;

FIG. 8 is a graphic representation showing the ability of GM-CSF to enhance the systemic immunity of irradiated tumor cells of the types indicated relative to non-transduced irradiated tumor cells. The symbol O represents the animals that succumbed to tumor challenge and the symbol • represents the animals protected from tumor challenge;

FIG. 9 is a graphic representation showing the effect of B16 melanoma cells expressing IL-2, GM-CSF and a third cytokine, as indicated, on a pre-existing tumor. The symbol O represents the animals that succumbed to tumor challenge and the symbol • represents the animals protected from tumor challenge; FIG. 10 is a graphic representation showing a time course of the average percentage of eosinophils in the test subjects during the 64 day period in which the transduced melanoma cells were injected into the patients. The arrows indicate that the injections were made at days 1, 22, and 43. The circles represent the average data of the low dose patients and the triangles indicate the average data of the high dose patients;

FIG. 11A is a schematic representation of a GM-CSF-encoding adenovirus vector according to the invention which is used to generate a recombinant adenovirus encoding GM-CSF (AV-GM-CSF); FIG. 1 IB is a schematic representation of a recombinant adenovirus vector according to the invention which encodes two cytokines, GM-CSF and IL-2, substituted into the El and E3 regions, respectively, as two separate cassettes including separate promoters;

FIG. 11C is a schematic representation of a recombinant adenovirus vector according to the invention which encodes two cytokines, GM-CSF and IL-2, substituted into the El region as two separate cassettes, each including a promoter;

FIG. 1 ID is a schematic representation of a recombinant adenovirus vector according to the invention which encodes two cytokines, GM-CSF and IL-2, substituted as one cassette with a single promoter into the El region. Alternative attachment of the splice acceptor (SA) (in front of each cDNA) to the splice donor (DS) produces two different mRNA's encoding two different proteins;

FIG. 12 is a schematic representation of a recombinant adeno-associated viral (AAV) vector (SSV9/MD2-hGM) according to the invention;

FIG. 13A is a schematic representation of a recombinant lentivirus vector, according to the invention, which contains a GM-CSF expression cassette flanked by HIV LTRS;

FIG. 13B is a schematic representation of a recombinant, self-inactivating lentiviral vector according to the invention; FIG. 14A is a schematic representation of an HSV-based amplicon, according to the invention, which contains a GM-CSF expression cassette;

FIG. 14B is a schematic representation of an HSV-1-based vector, according to the invention, which contains a GM-CSF expression cassette in replacing the ICP22 HSV gene;

FIG. 15A is a schematic representation of an SV-40-based vector (pSV HD GM- CSFII), according to the invention, which includes a GM-CSF expression cassette, the SV-40 origin of replication, and viral late genes;

FIG. 15B is a schematic representation of an SV-40 vector (pSV MD GM-CSF) according to the invention, which includes a GM-CSF expression cassette and the SV- 40 origin of replication;

FIG. 16 is a schematic representation of a vaccinia virus vector expression cassette including a vaccinia virus promoter and termination sequence;

FIG. 17 is a graphic representation of the results of a B16 tumor protection assay in which B16 tissue culture cells were transduced with recombinant retrovirus- GM-CSF, AV-GM-CSF, AV, or no virus, injected into a mouse, and challenged one week later by injection with live B16 cells. The mice were monitored for survival;

FIG. 18 is a graphic representation of the results of a tumor protection assay (as described in FIG. 17) in which the effectiveness of vaccines prepared from B16 tissue culture cells (B16 Retro-GM-CSF or B16 AV-GM-CSF) was compared with cells prepared from primary B16 tumor tissue obtained from mice (tumor-AV-GM-CSF or tumor- A V); and

FIG. 19 is a graphic representation of the results of a tumor protection assay as described in FIG. 18, in which dispersed primary B16 tumor tissue was used for the live cell tumor challenge. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The published patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. The publications cited herein, including, without limitation, issued U.S. patents, allowed applications, published foreign applications, GenBank Sequences, and references, are hereby incorporated by reference to the same extent as if each independent publication was specifically and individually indicated to be incorporated by reference.

The present invention discloses methods of regulating the immune response of a mammal to a tumor antigen. In this method, a tumor antigen and a cytokine are administered to the mammal. This tumor antigen may be a tumor cell that has been transduced with a recombinant virus comprising a nucleic acid encoding the cytokine. The modified tumor cells are administered to the mammal in a therapeutically effective amount and in such a manner that regulation of the immune response of the mammal to the antigen is achieved.

By the term "regulating the immune response" or grammatical equivalents, herein is meant any alteration in any cell type involved in the immune response. The definition is meant to include an increase or decrease in the number of cells involved in the immune response, an increase or decrease in the activity of the cells, or any other changes which can occur within the immune system. The cell type involved in the immune response may be, without limitation, a T lymphocyte, B lymphocyte, natural killer (NK) cell, macrophage, eosinophil, mast cell, dendritic cell, or neutrophil. The definition encompasses both a stimulation or enhancement of the immune system to develop a sufficiently potent response to a deleterious target, as well as a suppression of the immune system to avoid a destructive response to a desirable target. In the case of stimulation of the immune system, the definition includes future protection against subsequent tumor challenge. By the term "cytokine" or grammatical equivalents, herein is meant the general class of hormones of the cells of the immune system, including lymphokines, monokines, and others. The definition includes, without limitation, those hormones that act locally and do not circulate in the blood, and which, when used in accord with the present invention, will result in an alteration of an individual's immune response. Also included in the definition of cytokine are adhesion or accessory molecules which result in an alteration of an individual's immune response. Thus, a cytokine may be, without limitation, IL-1 (α or β), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, GM-CSF, M-CSF, G-CSF, LIF, LT, TGF-β, γ-IFN, α-IFN, β-IFN, TNF-α, BCGF, CD2, or ICAM. Descriptions of the aforementioned cytokines as well as other applicable immunomodulatory agents may be found in "Cytokines and Cytokine Receptors," A.S. Hamblin, D. Male (ed.), Oxford University Press, New York, NY (1993)), or the "Guidebook to Cytokines and Their Receptors," N.A. Nicola (ed.), Oxford University Press, New York, NY (1995)) herein incorporated by reference. Where therapeutic use in humans is contemplated, the cytokines will preferably be substantially similar to the human form of the protein or will have been derived from human sequences (i.e., of human origin).

Additionally, cytokines of other mammals with substantial structural homology and/or amino acid sequence identity to the human forms of IL-2, GM-CSF, TNF-α, and others, will be useful in the invention when demonstrated to exhibit similar activity on the immune system. Similarly, proteins that are substantially analogous to any particular cytokine, but have conservative changes of protein sequence, will also find use in the present invention. Thus, conservative substitutions in protein sequence may be possible without disturbing the functional abilities of the protein molecule, and thus proteins can be made that function as cytokines in the present invention but have amino acid sequences that differ slightly from currently known sequences. Such conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

Finally, the use of either the singular or plural form of the word "cytokine" in this application is not determinative and should not limit interpretation of the present invention and claims. In addition to the cytokines, adhesion or accessory molecules or combinations thereof, may be employed alone or in combination with the cytokines.

By the term "antigen from a tumor cell" or grammatical equivalents thereof, herein is meant any protein, carbohydrate or other component from a tumor cell capable of eliciting an immune response. The definition is meant to include, but is not limited to, using the whole tumor cell with all of its associated antigens as an antigen, as well as any component separated from the body of the cell, such as plasma membrane, cytoplasmic proteins, transmembrane proteins, proteins purified from the cell surface or membrane, or unique carbohydrate moieties associated with the cell surface. The definition also includes those antigens from the surface of the cell which require special treatment of the cells to access.

By the term "systemic immune response or grammatical equivalents herein is meant an immune response which is not localized, but affects the individual as a whole, thus allowing specific subsequent responses to the same stimulus. By the term "co-administering" or grammatical equivalents herein, is meant a process whereby the target antigen and the selected cytokine or cytokines are encountered by the individual's immune system at essentially the same time. The components need not be administered by means of the same vehicle. If they are administered in two separate vehicles, they must be administered sufficiently closely, both in time and by route of administration, that they are encountered essentially simultaneously by the individual's immune system to achieve the desired specificity. By the term "tumor cell" is meant a cell that shows aberrant cell growth, such as increased cell growth. A tumor cell may be a hyperplastic cell, a cell that shows a lack of contact inhibition of growth in vitro, a cell that is incapable of metastasis in vivo, or a cell that is capable of metastasis in vivo.

By the term "reversal of an established tumor" or grammatical equivalents herein is meant the suppression, regression, or partial or complete disappearance of a pre-existing tumor. The definition is meant to include any diminution in the size, potency, growth rate, appearance or feel of a pre-existing tumor.

By the term "retarding the growth of a tumor" is meant the slowing of the growth rate of a tumor, the inhibition of an increase in tumor size or tumor cell number, or the reduction in tumor cell number, tumor size, or numbers of tumors. By the term "transfection" is meant the introduction of exogenous nucleic acid into a cell by physical means. As used herein, by "transduction" is meant the introduction of exogenous nucleic acid into a cell using a viral particle, preferably a genetically engineered (i.e., recombinant) viral particle. For various techniques for manipulating mammalian cells, see Keown et al., Methods of Enzymology 185: 527-537 (1990).

One of ordinary skill will appreciate that, from a medical practitioner's or patient's perspective, virtually any alleviation or prevention of an undesirable symptom (e.g., symptoms related to disease, sensitivity to environmental factors, normal aging, and the like) would be desirable. Thus, for the purposes of this Application, the terms "treatment," "therapeutic use," or "medicinal use," as used herein, shall refer to any and all uses of the claimed compositions which remedy a disease state or symptoms, or otherwise prevent, hinder, retard, or reverse the progression of disease or other undesirable symptoms in any way whatsoever.

By the term "therapeutically effective amount" or grammatical equivalents herein refers to an amount of the preparation that is sufficient to regulate, either by stimulation or suppression, the systemic immune response of an individual. This amount may be different for different individuals, different tumor types, and different preparations. An appropriate dosage of transduced cells, or derivatives thereof, may be determined by any of several well established methodologies. For instance, animal studies are commonly used to determine the maximal tolerable dose, or "MTD," of bioactive agent per kilogram weight. In general, at least one of the animal species tested is a mammal. Those skilled in the art regularly extrapolate doses for efficacy and avoiding toxicity to other species, including human. Before human studies of efficacy are undertaken, Phase I clinical studies in normal subjects help establish safe doses. Alternatively, initial toxicity studies may involve individuals that are at the terminal stages of the disease progression.

By the term "rejection" or grammatical equivalents herein is meant a systemic immune response that does not allow the establishment of new tumor growth. By the term "challenge" or grammatical equivalents herein is meant a subsequent introduction of tumor cells to an individual. Thus a "challenge dose 5 days post vaccination" means that on the fifth day after vaccination with tumor cells expressing cytokines or irradiated tumor cells, or both, a dose of unmodified tumor cells was administered. "Challenge tumor" means the tumor resulting from such challenge.

By the term "days to sacrifice" or grammatical equivalents herein, is meant that period of time before mice were sacrificed. Generally, mice were sacrificed when challenge tumors reached 2 - 3 centimeters in longest diameter, or if severe ulceration or bleeding developed.

By the terms "inactivated cells" and "proliferation-incompetent cells" or grammatical equivalents herein are meant cells inactivated by treatment rendering them proliferation-incompetent. This treatment results in cells which are unable to undergo mitosis, but still retain the capability to express proteins such as cytokines. An "irradiated cell" is one example of such an inactivated cell. Such irradiated cells have been exposed to sufficient irradiation to render them proliferation-incompetent. Typically a minimum dose of about 3500 Rads is sufficient to inactivate a cell and render it proliferation-incompetent, although doses up to about 30,000 Rads are acceptable. Preferably, a dose of about 10,000 Rads is used to inactivate a cell and render it proliferation-incompetent. It is understood that irradiation is but one way to render cells proliferation-incompetent, and that other methods of inactivation which result in cells incapable of cell division but that retain the ability to express cytokines are included in the present invention (e.g., treatment with mitomycin C, cycloheximide, and conceptually analogous agents, or incorporation of a suicide gene by the cell).

By the term "individual" or grammatical equivalents herein is meant any one individual mammal. The present invention relates to a method for regulating the immune response of an individual to target antigens by administering the target antigen or antigens with a cytokine, or cytokines, in such a manner that the immune system of the individual is either stimulated or suppressed. In either case, the present invention provides a means of controlling the activity of the cytokines with the result that they provide an unusual protective or therapeutic effect.

In one embodiment of the present invention, the target antigen (or antigens) and one or more cytokines are administered to an individual in a composition which provides for transfer or release of the cytokine(s) in direct proximity or in combination with the target antigen(s). The antigen and cytokine need not be administered in the same vehicle. If they are administered in two separate vehicles, they must be administered sufficiently closely, both in time and by route of administration, that they are encountered essentially simultaneously by the individual's immune system. That is, the cytokine(s) may be co-administered with the target antigen in any manner which provides transfer or delivery of the cytokine in the context of the target antigen in relation to which the immune response is to be regulated. For example, this can be accomplished by using slow or sustained release delivery systems, or direct injection. As a result of this co-administration, the cytokine has the specific effect of amplifying or altering the specific immune response to the target antigen. The emphasis is on local interaction of the cytokine and the target antigen to mimic the physiological occurrence of simultaneous presentation of cytokine and antigen, to maximize efficacy and minimize toxicity.

In a preferred embodiment, cells which express the target antigen(s), such as tumor cells, are themselves genetically engineered to express the cytokine(s) to be administered. The resulting modified tumor cells present to the host immune system not only the antigen against which an immune response is sought, but also the cytokine(s) whose expression determines the type and extent of the resulting immune response (i.e., whose expression stimulates or suppresses the resulting immune response to the antigen). Alternatively, cells that naturally express the target antigen(s) and the cytokine(s) can be used. The resulting cells, which express both the target antigen(s) and the cytokine(s) can be used as a vaccine to protect against future tumor development and/or to result in the reversal of already existing tumors. Conversely, a target antigen against which a decreased immune response is sought can be used in conjunction with the appropriate cytokine mixture.

In one embodiment, the individual's systemic immune response is increased or enhanced beyond that which would occur in the absence of the co-administered molecules. The systemic response is sufficient for the individual to mount an immune response against the target antigen, such as an antigen from a tumor cell, and may cause the tumor cell to be rejected by the individual.

In another embodiment, the individual's systemic immune response is reduced or suppressed, either partially or completely, to such an extent that the target antigen, such as antigens on transplanted cells or tissue, or cells of the individual incorrectly recognized as foreign (e.g., β -islet cells in individuals suffering from Diabetes mellitus), is not rejected by the individual, or is rejected to a lesser extent than would occur if the present method were not used.

In one embodiment of the present invention, the target antigen can be a tumor cell, a tumor cell antigen, an infectious agent or other foreign agent against which an W

enhanced immune response is desired. For example, any antigen implicated in the progression of a chronic and life threatening infection, such as the AIDS virus or a secondary infection associated with AIDS, or other bacterial, fungal, viral, parasitic and protozoal antigens, may be utilized in the present invention. In one embodiment, the target antigen is a tumor cell. Preferably, the invention utilizes melanoma cells. In a certain preferred embodiment, the invention utilizes carcinoma cells, such as (but not limited to) non-small cell lung carcinoma. Yet another embodiment utilizes prostate cancer cells. Other tumor cells such as (but not limited to) leukemic cells, breast cancer cells, cervical cancer cells, ovarian cancer cells, skin cancer cells, cancerous polyps or preneoplastic lesions, or cells engineered to express an oncogene (e.g., ras, p53) may also be used. Furthermore, the tumor cells used as the target antigen and expressing cytokines can be either an unselected population of cells or specific clones of modified cells. These cells may be primary tumor cells excised from a tumor within the individual to be treated, or may be cultured tumor cells.

In one preferred embodiment, tumor cells are modified to express the cytokines IL-2 and GM-CSF. This combination is particularly desirable since it results in the long term systemic immune protection against subsequent challenge with wild type tumor cells. In one embodiment of the present invention, tumor cells expressing the target antigen and the cytokines are made proliferation-incompetent before administration to an individual. The resulting nonviable tumor cells may be used to regulate the immune response of the individual. In a preferred embodiment, the cells are made proliferation-incompetent by irradiation. The administration of such irradiated cytokine producing tumor cells has been shown to be useful in inhibiting the establishment of tumors of the same type, presumably through a vaccine-like mechanism. This procedure has particular importance since the introduction of live tumor-producing cancer cells to an individual is undesirable. This has additional significance since primary tumor explants likely contain non-neoplastic elements as well, irradiation of the tumor samples prior to vaccination will also prevent the possibility of autonomous growth or proliferation of non-neoplastic cells induced by autocrine synthesis of their own growth factors. In a preferred embodiment, the irradiated tumor cells used to regulate the immune response of an individual are capable of expressing GM-CSF, IL-4, IL-6, CD2, ICAM, IL-12, or a combination thereof. In the most preferred embodiment, the irradiated tumor cells used to regulate the immune response of an individual express GM-CSF. In one embodiment, the administration of the combination of target antigen and cytokine(s) can be used to reverse established tumors. This may be accomplished by taking tumor cells from an individual with a pre-existing tumor and modifying these tumor cells to express a cytokine(s). Upon subsequent re-introduction of the modified cells into the individual, a systemic immune response capable of reversing the pre-existing tumor is produced. Alternatively, tumor cells of the type of the pre-existing cancer may be obtained from other sources, such as another individual or a culture of tumor cells of the same type of cancer. The identification of the cytokines to be expressed will depend on the type of tumor, and other factors. However, a GM-CSF vaccine is effective in more than one type of cancer, as illustrated by its effectiveness in rat prostate (Sanda et al., /. Virol. 155: 622- 623 ((1994)) as well as murine melanoma models (Dranoff et al, Proc. Natl. Acad. Sci. USA 90: 3539-3543 (1993)).

In one embodiment of the present invention, the rumor cells express IL-2, GM-CSF, and a third cytokine selected from the group consisting of TNF-α, IL-4, CD2, and ICAM. For example, the tumor cells may express IL-2, GM-CSF, and/or TNF-α. In a certain preferred embodiment, the modified tumor cells used for treatment are irradiated prior to administration to the individual. In a preferred embodiment, the irradiated tumor cells used to reverse an established tumor express GM-CSF. In one embodiment of the present invention, a vector such as the retroviral MFG vector herein described and schematically represented in FIG. 1A is used generate recombinant retrovirus to transduce and thus genetically alter the vaccinating cells. The MFG vector has particular utility since its use makes it feasible, for the first time, to rapidly screen a large number of potential immunomodulators for their effects on the generation of systemic immunity and to assess the activity of complex combinations of molecules. The ability of the MFG vector to generate recombinant viruses having a combination of high titer and high gene expression obviates the need for selection of transduced cells among the bulk target population. This minimizes the time required for culturing primary tumor cells prior to vaccination, and maximizing the antigenic heterogeneity represented in the vaccinating inoculum.

Other retroviral vectors find use in the present invention. For example, pLJ, pEm, and α-SGC may be used. pLJ, previously described in (Korman et al., Proc. Natl. Acad. Sci. USA 84: 2150-2154 (1987)), is capable of expressing two types of genes: the gene of interest and a dominant selectable marker gene, such as the neo gene. The structure of pLJ is represented in FIG. IB. pEm is a simple vector where the gene of interest replaces the gag, pol, and env coding sequences of the wild-type virus. The gene of interest is the only gene expressed. The structure of pEm is represented in FIG. 1C. The vector α-SGC utilizes transcriptional promoter sequences from the α-globin gene to regulate expression of the gene of interest. The structure of α-SGC is represented in FIG. ID.

On October 3, 1991, Applicants deposited with the American Type Culture Collection (ATCC), Rockville, Md. USA the plasmid MFG with the factor VIE insertion (ATCC accession no. 68726), plasmid MFG, described herein, with the tPA W

insertion (ATCC accession no. 68727), the plasmid α-SGC, described herein, with the factor VUI insertion (ATCC accession no. 68728), and plasmid α-SGC, described herein, with the tPA insertion (ATCC accession no. 68729). On October 9, 1991, Applicants deposited with the ATCC the plasmid MFG (ATCC accession no. 68754), and plasmid α SGC (ATCC accession no. 68755). These deposits were made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the purposes of patent procedure and the Regulations thereunder (Budapest Treaty). This assures maintenance of a viable culture for 30 years from the date of deposit. The organisms will be made available by the ATCC under the terms of the Budapest Treaty, and subject to an agreement between

Applicants and ATCC which assures unrestricted availability upon issuance of the pertinent U.S. patent. Availability of the deposited strains is not be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws. Alternatively, other recombinant viruses may be used to transduce the tumor cell. For example, particularly useful recombinant viruses are those capable of infecting mammalian cells which are not replicating, allowing for efficient transduction of cells without the necessity of cell culture.

One such recombinant virus is constructed from the adenovirus. Adenoviruses present a very favorable profile, as they are endemic in the general population and are associated with mild, self-limited illnesses usually requiring no specific therapy to cure. Adenoviruses are nonenveloped particles composed of an icosahedral protein shell and a single linear 36 kb, double-stranded DNA genome. Infection begins with binding of the viral fiber protein to a cellular receptor followed by receptor mediated endocytosis. The adenovirus escapes the endosome in a pH dependent manner and then moves to the nucleus where viral transcription begins. Initiation of the early phase of adenoviral transcription results in cell cycle progression, production of adenoviral gene products necessary for adenoviral DNA replication, and protection of the infected cell from immune surveillance. These events are critically dependent upon expression of the El genes. After DNA replication is underway, late stage transcripts are turned on resulting in the production of adenoviral structural proteins and the formation of adenoviral particles. FIGS. 11A-D show representative vector plasmids constructed from the adenovirus genome. These vectors may encode one (FIG. 11 A) or more than one cytokine. If the vector encodes two or more cytokines, the cytokine genes may be substituted into the genome as multiple cassettes, each with their own promoters (FIGS. 11B and 11C) or as a single cassette with one promoter driving both genes (FIG. 11D). These cassettes may be substituted anywhere in the viral genome which will not interfere with the ability of the virus to infect and express its genome. For example, the cytokine-encoding cassette(s) may be substituted into the El and/or E3 genes of the adenovirus genome. One such cassette is the MD cassette.

Other viral vectors useful to generate recombinant viruses that may be used in the methods and kits of the invention include, but are not limited to, an adeno- associated viral (AAV) vector, lentivirus vectors, herpesvirus vectors, SV-40 virus vectors, and a pox virus vector such as, but not limited to, a vaccinia virus vector. The representative AAV vector depicted in FIG. 12 lies between the inverted terminal repeats (ITR) of AAV and contains an expression cassette for GM-CSF. It also contains the cytomegalovirus (CMV) immediate early gene promoter/enhancer (Genbank Accession No. X03922) from pBC12/CMV/IL-2 (Cullen, Cell 46: 973-982 (1986)) and a shortened second intervening sequence (IVS2). Recombinant AAV may be produced by cotransfecting this vector with an AAV helper plasmid into 293 cells and then infecting them with adenovirus dl312 as described in Samulski et al. (]. Virol 63:3822-3828 (1989)).

FIG. 13A illustrates a representative recombinant lentivirus vector containing a GM-CSF expression cassette flanked by HIV LTRS. Downstream of the 5' LTR, the vector contains the HIV leader sequence, the major 5' splice donor site (SD), the packaging sequence (Ψ), the first 43 bps of the H1N gag gene, the HIV Rev Response Element (RRE), and the splice accepter sites (SA) of the second exon of HIV tat and HTV rev.

Another recombinant lentiviral vector is shown in FIG. 13B. This self- inactivating vector contains a GM-CSF expression cassette flanked by a 5' HIV LTR having a substituted U3 region and a 3' HIV LTR, or a 5' HTV LTR and a deleted 3' HTV LTR. This vector further contains downstream from the 5' LTR and HIV leader sequence, the major 5' SD, Ψ, the first 43 bps of the gag gene, the RRE, and the SA of the second exon at tat and rev. FIG. 14A depicts an HSV-based construct or amplicon which contains a GM-

CSF expression cassette. The HSV "a" and ori permit replication and packaging in complementing cells infected with the dl20 helper virus. The complementing cells supply ICP4 function missing from the dl20 (see, e.g., Spete et al., Cell 30: 295-304 (1982)). Another useful herpesvirus vector is the HSV-1 based vector delineated in FIG.

14B. Many regions of the HSV genome not needed for growth in cultured cells can be removed and cytokine expression cassette(s) substituted in. In the example shown, the ICP22 gene has been substituted with a GM-CSF cassette. The resulting vector is defective for growth on normal cells as the ICP4 genes have also been removed. This vector may be propagated in a cell line such as 7B which provides the ICP4 and ICP27 proteins (see, e.g., Glorioso et al, Ann. Rev. Microbiol. 49:675-710 (1995)).

FIG. 15A illustrates one representative SV40-based GM-CSF vector which contains the GM-CSF expression cassette and the SV-40 ori plus the viral late genes (SV-40 nucleotides 5177-2770). The total size of the vector is preferably less than 5243 bases. Packaging of the vector can be accomplished, for example, in COS-7 cells as described in Fang et al. (Analyt. Biochem. 254:139-143 (1997)). This vector can be generated from a larger plasmid containing a plasmid origin and selectable marker. The sequences are removed by restriction digestion, and the ends of the vector fragment are joined to make a circle by T4 DNA ligase.

An alternative SV-40 based recombinant vector plasmid including a GM-CSF expression cassette and the SV-40 ori (SV-40 nucleotides 5177-290) is shown in FIG. 15B. The total size of the plasmid is preferably less than 5243 bases and packaging can be accomplished, for example, in COS-7 cells according to Fang et al. (supra), as described above.

The vaccinia virus expression cassette shown in FIG. 16 is also useful in the methods and kits of the invention. As vaccinia viruses do not rely on the host cell's expression machinery, the cytokine (e.g., GM-CSF) cDNA must be flanked with the appropriate vaccinia sequences. The promoter is preferably a vaccinia virus promoter such as H6 which is active in both early and late phases of the vaccinia life cycle. Additionally, after the gene a termination sequence of Ti l l TNT is required in the early phase. Using homologous recombination, this cassette can be inserted into any region of the vaccinia genome which is dispensable for growth in cells (see, e.g., Perkus et al., /. Virol. 63: 3829-3836 (1989)).

One of ordinary skill will realize that the above vectors may be subtly altered in a manner such that the operative features of the generated recombinant virus, vis-a-vis transduction efficacy, are substantially maintained. As such, the present invention also contemplates the use of any and all operative (transfection competent) derivatives of the above retroviral, adenoviral, adeno-associated viral, lentiviral, SV- 40, herpes viral and vaccinia viral vectors to deliver genes encoding immunomodulatory agents to packaging cells. For example, the retroviral vector MFG-S comprises three point mutations in the MFG vector which theoretically improve the safety of the vector (although there is no evidence that MFG is unsafe) while retaining substantially identical transfection efficiency. Specifically, MFG-S has an A to T change at nucleotide 1256, a C to T mutation at nucleotide 1478, and a T to an A at nucleotide 1273. Similarly, the adenovirus vector AV-GM-CSF may have various deletions within the El and E3 regions of its viral genome. Of course, other or additional deletions in the same or other regions of the virus may be possible while retaining relative transfection efficiency.

It follows from the results herein that a variety of hormones, antigens, and cytokines will find use in the present invention. For example, cytokines, antigens, or hormones of other mammals with substantial homology to the human forms of the cytokines, antigens, and hormones, will be useful in the invention when demonstrated to exhibit similar activity on the immune system. Thus, the method of the present invention can include treatment with antigens, hormones, and cytokines, adhesion or accessory molecules or combinations thereof, combined with other systemic therapy such as chemotherapy, radiation treatments and other biological response modifiers.

The method of the present invention is also used to regulate an individual's systemic immune response to a variety of antigens. In one embodiment, the present invention is used to treat chronic and life threatening conditions, such as infection with the AIDS virus, as well as the secondary infections associated with AIDS. Other embodiments embrace the treatment of other bacterial, fungal, viral, parasitic, and protozoal infections described in "Principles and Practice of Infectious Disease," Mandell et al. (eds.), Churchill Livingstone, Inc., New York, NY (1990), herein incorporated by reference.

In one embodiment, treatment and prevention of HTV-related conditions is achieved. For example, in the case of the human immunodeficiency virus (HIV), and/or Simian immunodeficiency virus (SIV) there are at least six known target antigens (Clerici et al., /. Immunol. 146: 2214-2219 (1991); Venet et al, /. Immunol. 148, 2899-2908 (1992); Hosmalin et al, /. Immunol. 146: 1667-1673 (1991)) against which an immune response can be enhanced using the present invention. An appropriate host cell expressing the HIV target antigen can be modified to express one or more cytokines and administered to an uninfected individual, to serve as a vaccine and elicit an enhanced immune response to confer the ability to resist subsequent infection by the AIDS virus.

Alternatively, the antigen and the cytokines may be administered without a host cell. Furthermore, co-administration of the target antigen and cytokines may be given to an HIV-positive individual, in order to limit the existing infection or reverse it.

In another aspect, the invention provides kits for regulating the immune response of a human or other mammal to a tumor antigen. These kits enable preparation required for such treatment to be accomplished easily, quickly, and within the proximity of the patient being treated. Such a kit includes a recombinant virus useful in the methods of the invention, or source thereof, such as a packaging cell transfected with a viral vector that generates such a recombinant virus. It also includes a container into which a tumor or portion thereof may be placed after excision or biopsy from the affected individual. This container may be a sterile, flexible, plastic container or bag with a standard closure and septa useful for the application of needles. The tumor sample in the container is treated to obtain a single cell dispersion or suspension. Such treatment may be mechanical disruption, chemical digestion, or a combination of mechanical disruption and chemical digestion. The kit may further include an enzyme such as a digestive enzyme useful for separating the individuals cells of the tumor tissue to be placed in the container. Examples of particularly useful enzymes include, but are not limited to, collagenase, trypsin, and chymotrypsin. After disruption of the tumor tissue, the cells may be suspended in a wash buffer within the same or another container to facilitate the removal of resulting cellular adhesion materials and debris. Centrifugation may also be used to facilitate this process. Thus, the kit may further include any useful known and/or commercially available wash buffer which will not lyse or kill the cells, as well as known and/or commercially available cell media in which to grow the washed cells once they are transduced with the cytokine-encoding recombinant virus in the same or other container. Before or after transduction, the cells may be rendered proliferation-incompetent, for example, by irradiation within the same container. Treated tumor cells may then be removed for administration to the individual to be treated. Having described the particular methods employed in the present invention for the regulation of systemic immune responses to target antigens, and detailing how these methods may be utilized, and showing the successful regulation of systemic immune responses, the present disclosure is sufficient to enable one skilled in the art to use this knowledge to produce the end results by equivalent means using generally available techniques.

The following examples serve to more fully describe the manner of making and using the above-described invention, as well as to set forth the best modes contemplated for carrying out various aspects of the invention. It is understood that these examples in no way serve to limit the true scope of this invention, but rather are presented for illustrative purposes.

EXAMPLE 1 Generation of Recombinant Retroviral Genomes Encoding Cytokines

Construction of the retroviral vectors employs standard ligation and restriction techniques which are well understood in the art. A variety of retroviral vectors containing a gene or genes encoding a cytokine of interest were used. The MFG vector is described in co-pending U.S.S.N. 07/607,252 entitled "Genetic Modification of Endothelial Cells," filed October 31, 1990, U.S.S.N. 07/786,015; "Retroviral Vectors Useful in Gene Therapy," filed October 31, 1991, and PCT/US91/08121, filed October 31, 1991, the teachings of which are incorporated herein by reference. They are also described below with particular reference to the incorporation and expression of genes encoding cytokines. Furthermore, several MFG vectors have been deposited with the ATCC as described above.

The MFG vector is similar to the pEm vector, described below and depicted in FIG. 1, but contains 1038 base pairs of the gag sequence for MoMuLV, to increase the encapsidation of recombinant genomes in the packaging cells lines, and 350 base pairs derived from MOV-9 which contains the splice acceptor sequence followed by the initiation codon. An 18 base pair oligonucleotide containing Nco I and BamHl sites directly follows the MOV-9 sequence and allows for the convenient insertion of genes with compatible sites. In each case, the coding region of the gene was introduced into the backbone of the MFG vector at the Nco I site and BamHl site. In each case, the ATG initiator methionine codon was subcloned in frame into the Nco I site and little, if any, sequence beyond the stop codon was included, in order to avoid destabilizing the product and introducing cryptic sites. As a result, the ATG of the insert was present in the vector at the site at which the wild-type virus ATG occurs. Thus the splice was essentially the same as occurs in Moloney Murine Leukemia virus and the virus worked very well. The MoMuLV LTR controls transcription and the resulting mRNA contains the authentic 5' untranslated region of the native gag transcript followed directly by the open reading frame of the inserted gene. In this vector, Moloney murine leukemia virus (Mo-MuLV) long terminal repeat sequences were used to generate both a full length viral RNA (for encapsidation into virus particles), and a subgenomic mRNA (analogous to the Mo-MuLV env mRNA) which is responsible for the expression of inserted sequences. The vector retained both sequences in the viral gag region shown to improve the encapsidation of viral RNA (Armentano et al., /. Virol. 61: 1647-1650 (1987)) and the normal 5' and 3' splice sites necessary for the generation of the env mRNA. All oligonucleotide junctions were sequenced using the dideoxy termination method (Sanger et al., Proc. Natl. Acad. Sci. USA 74: 5463-5467 (1977)) and T7 DNA polymerase (Sequenase 2). The structure of MFG is represented in FIG. 1A. As can be seen, the virus is marker-free in that it does not comprise a dominant selectable marker (although one may optionally be inserted), and, given the high levels of transduction efficiency and expression inherent in the structure of the vector, transduction with MFG derivatives generally does not involve or require a lengthy selection step. MFG vectors containing genes for the following proteins were constructed: murine IL-2, GM-CSF, IL-4, IL-5, γ-IFN, IL-6, ICAM, CD2, TNF-α, and IL-l-RA (interleukin-1-receptor antagonist). In addition, human sequences encoding TNF-α, GM-CSF and IL-2 were constructed (See Table I). It is also possible to make MFG vectors containing a gene encoding one or more of the following: VCAM, ELAM, macrophage inflammatory protein, heat shock proteins (e.g., hsp60), M-CSF, G-CSF, IL-1, IL-3, IL-7, IL-10, TGF-β, B7, MIP-2, MIP-1 α, and MIP-1 β.

Precise cDNA sequences subcloned into MFG were as follows: murine IL-2 (Yokota et al., Proc. Natl. Acad. Sci. USA 82: 68-72 (1985)) base pairs 49-564; murine IL-4 (Lee et al., Proc. Natl. Acad. Sci. USA 83: 2061-2065 (1986)) base paris 56-479; murine IL-5 (Campbell et al, Europ. ]. Biochem. 17.: 345-352 (1988)) base pairs 44-462; murine GM-CSF (Gough et al., EMBO J. 4: 645-653 (1985)) base pairs 70-561; murine ICAM-1 (Horley et al, EMBO f. 8: 2889-2896 (1989)) base pairs 30-1657; murine CD2 (Yagita et al., /. Immunol. 140: 1321-1326 (1988)) base pairs 48-1079; murine IL-1 receptor antagonist (Matsushimi et al., Blood 78: 616-623 (1991)) base pairs 16-563; human TNF-α (Wang et al., Science 228: 149-154 (1985)) base pairs 86-788.

EXAMPLE 2 Cytokine Assay

Cytokines secreted by the transduced, unselected B16 populations were assayed 48 hours after plating 1 x 10⁶ cells in 10 cm dishes containing 10 ml of medium. IL-l-RA secretion was measured from transduced, unselected 3T3 cells 24 hours after plating 5 x 10⁶ cells in a 10 cm dish containing 10 ml of medium. Cytokines were assayed as follows: murine IL-2 using CTLL cells (Gearing et al., 'Production and assay of interleukin 2.' In: Lymphokines and Interferons: A Practical Approach, eds. Clemens et al. (IRL Press, Oxford, 1987) pp. 296-299.) and ELISA (Collaborative Biomedical,); murine IL-4 using CT4R cells (Hu-Li et al., /. Immunol. 142: 800-807 (1989)) and ELISA (Endogen, Woburn, MA); murine IL-5 using an ELISA (Endogen); murine IL-6 using T1165 cells (Le et al., Lymphokine Res. 7: 99-106 (1988); Coligan et al., Current Protocols in Immunology (Greene Publishing Associates and Wiley-Interscience) 1991) and ELISA (Endogen); murine GM-CSF using FDCP-1 cells (Dexter et al., /. Exp. Med. 152: 1036-1047 (1980)) and ELISA (Endogen); murine γ-IFN using vesicular stomatitis viral inhibition (Dugre et al., Acta. Pathol. Microbiol.

Immunol. Scand. 80: 863-870 (1972)) and ELISA (Genzyme, Cambridge, MA); human TNF-α using L929 cells (Oliff et al., Cell 50: 555-563 (1987)) and ELISA (R&D Systems, Minneapolis, MN); murine IL-l-RA using ¹²⁵I-IL1B binding inhibition (Hannum et al., Nature 343: 336-340 (1990)). Expression of murine ICAM-1 and CD2 in B16 target cells was determined with standard procedures (Coligan et al., Current Protocols in Immunology (Greene Publishing Associates and Wiley-Interscience) 1991) on an EPICS-C FACS analyzer (Coulter, Miami, FL) using antibodies YN1/1.47 (Takei, et al., /. Immunol. 134: 1403-1407 (1985)) and RM2/1, respectively.

EXAMPLE 3 Production of B16 Melanoma Cells Containing Cytokine-Encoding Sequences

The above vector constructs were introduced by standard methods into the packaging cell lines known as Psi CRIP and Psi CRE (Danos et al., Proc. Natl. Acad. Sci. USA 85: 6460 (1988)). These cell lines have been shown to be useful to isolate clones that stably produce high titers of recombinant retroviruses with amphotropic and ecotropic host ranges, respectively. Psi CRIP packaging lines with amphotropic host range were generated by both transfection and electroporation with only small differences in efficiency. Calcium phosphate DNA coprecipitations were performed (Parker et al., /. Virol. 31: 360-369 (1979)) using 20 μg of vector and 1 μg of pSV2NEO (Southern and Berg, /. Mol. Appl. Genet. 1: 327-341 (1982)). Electroporations were performed after linearizing 40 μg of vector and 8 μg of pSV2NEO using the Gene Pulser electroporator (Bio-Rad). Conditions were 190 V and 960 μF. Producers were plated into selection in G418 (GIBCO) media at 1 mg/ml 36 hours after introduction of the DNA. Both clones and populations of producers were generated.

Viral titers were determined by Southern blot analysis (Southern, E.M., /. Mol. Biol. 98: 503-517 (1975)) foUowing transduction (Cone et al., Mol. Cell. Biol. 7: 887-897 (1987)) of B16 or 3T3 cells in medium containing 8 μg polybrene per ml. Ten μg of transduced target cell DNA as well as control DNA spiked with appropriate copy number standards were digested with Nhe I (an LTR cutter), resolved by electrophoresis in 1% agarose gels, and analyzed by the method of Southern using standard procedures (Sambrook et al, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press (1989)). Blots were probed with the appropriate sequences which had been labeled to high specific activity with p²P]dCTP by the random primer method (Feinberg et al, Anal. Biochem. 132: 6-13 (1983)). Probes used were all full-length cDNAs except for murine IL-2 which was the Sacl/Rsal fragment (base pairs 221-564). A titre of one copy per cell is approximately equivalent to 1 x 10⁶ retroviral particles per cell.

During the course of generating these products, we observed that cross-infection of packaging lines (CRE to CRIP or CRIP to CRE) with MFG vectors sometimes led to the mobilization of packaging function as determined by a sensitive his D mobilization assay (Danos et al., supra). Such cross-infection should thus be avoided. No mobilization of packaging function with producers generated by direct transfection or electroporation has been observed.

Viral titres and expression levels are shown in Table I below. Table I MFG Vector Constructs

The B16 melanoma tumor model (Fidler et al., Cancer Res. 35: 218-234 (1975)) was chosen for initial studies. Human melanoma has been shown to be sensitive to a variety of immunotherapies (Berd et al., /. Clin. Oncol. 8: 1858-1867 (1990); Rosenberg et al, New Engl }. Med. 319: 1676-1680 (1988)). To examine whether any of the gene products listed in Table I influenced the growth of B16 cells in vitro or in vivo, cells were exposed to viral supernatants and the transduced cells were characterized for their efficiency of transduction and secretion of gene product. Table I shows the approximate efficiency of transduction with each virus for the B16 melanoma cell line (a titer of one copy per cell corresponds to a titer of approximately 10⁶ infectious particles) and the corresponding level of secreted gene product. With the exception of the MFG γ-IFN construct, which transmitted at approximately 0.1 copies /cell, most of the viruses were capable of transducing a majority of tumor cells. γ-IFN secreting cells grew more slowly than non- transduced cells and adopted a flattened morphology relative to their wild type counterparts. In contrast, none of the other transduced cells displayed any altered in vitro growth characteristics. Both populations and specific clones of virus producing cells were used in these studies.

EXAMPLE 4 Vaccinations

Tumor cells were trypsinized, washed once in medium containing serum, and then twice in Hanks Balanced Saline Solution (GIBCO) prior to injection. Trypan blue resistant cells were suspended to the appropriate concentrations and injected in a volume of 0.5 cc HBSS. Vaccinations were administered subcutaneously in the abdomen and tumor challenges were injected in the dorsal midline of the back after anesthetizing the mice with Metaphane (Pitman-Moore). Mice were examined at 2-3 day intervals and the time to development of palpable tumor recorded. Animals were sacrificed when tumors reached 2-3 cm in diameter or if severe ulceration or bleeding developed. For antibody depletion experiments, mice were vaccinated in the right hind leg (volume 0.1 cc) and challenged in the left hind leg (volume 0.1 cc). For establishment of pulmonary metastases, cells were injected in the tail vein in a volume of 0.2 cc. Animals used were 6-12 week C57B1/6 females (Jackson Labs) for B16, Lewis Lung, and WP-4 experiments, and 6-12 week Balb/c females (Jackson Labs) for CT-26, RENCA, and CMS-5 studies.

When irradiated vaccines were employed, tumor cells (after suspension in HBSS) received 3500 Rads from a Cesium 137 source discharging 124 Rads/min. EXAMPLE 5 Histology

Tissues for histologic examination were fixed in 10% neutral buffered formalin, processed to paraffin embedment, and stained with hematoxylin and eosin. Tissues for immunoperoxidase staining were snap frozen in liquid N₂, frozen sections were prepared, fixed in acetone for 10 minutes, and stored at -20°C. Frozen sections were stained with primary antibodies, followed by anti-IgG antibodies (species specific), and then the antigen-antibody complexes visualized with avidin-biotin-peroxidase complexes (Vectastain, Vector Labs) and diaminobenzidine. Primary antibodies used were 500 A2 (hamster anti-CD3), GK 1.5 (rat anti-CD4), and GK2.43 (rat anti-CD8).

EXAMPLE 6 Vaccination Studies with Live, Transduced Tumor Cells

To directly assess the effect of the cytokine upon the tumorigenicity of B16 cells, either unmodified (wild type) B16 melanoma cells or the transduced modified cells were inoculated subcutaneously into C57B1/6 mice, their syngeneic host, and the mice were examined every few days for tumor formation. Typically, a vaccinating dose was 5 x 10⁵ transduced B16 cells.

Surprisingly, only tumor cells secreting IL-2 did not form tumors. All of the other nine cytokines tested resulted in tumor formation. Modest delays in tumor formation were associated with expression of IL-4, IL-6, γ-INF, and TNF-α. Several cytokines produced distinctive systemic syndromes, presumably as a consequence of the progressively increasing number of cells. GM-CSF-transduced cells induced a fatal toxicity manifested by profound leukocytosis (polymorphonuclear leukocytes, monocytes, and eosinophils), hepatosplenomegaly, and pulmonary hemorrhage, IL-5 expressing cells showed a striking peripheral eosinophilia and splenomegaly. IL-6 expressing cells caused hepatosplenomegaly and death. TNF-α expressing cells induced wasting, shivering, and death.

EXAMPLE 7 Systemic Antitumor Immunity Generated by Cytokine-Transduced Tumor Cells

The rejection of IL-2 transduced cells made it possible to examine their potential to generate systemic immunity. Mice were first inoculated with IL-2 expressing B16 melanoma cells, and subsequently challenged over the course of one month, with unmodified B16 cells. Typically, both the initial and challenge dose were 5 x 10⁵ live cells, cells which had been harvested from culture dishes and then extensively washed. Cells were injected subcutaneously in a volume of about 0.5 cc (in Hanks Buffered Saline (HBSS)). Groups of 5 mice were then challenged at a site different from that at which the vaccine was administered. A time course analysis was carried out by challenging groups of 5 animals every 3-4 days, beginning 3 days after the vaccine was administered and continuing to challenge the animals until a month after the vaccination.

The results demonstrated that IL-2 possessed a minimal ability to induce systemic protection, as measured by the ability of the unmodified tumor challenge to be rejected. In a typical experiment, most mice succumbed to the challenge tumor by 40 days after its inoculation. This is in direct contrast to the results of Fearon et al. (supra), since all animals succumbed to this challenge, regardless of the time of challenge, with only an occasional delay in tumor formation, FIG. 2A. Assessment of the effects of the transduced B16 cells was done through ascertaining when natural death or humane killing of the mice occurred. EXAMPLE 8 Systemic Antitumor Immunity Generated by Multiple Cytokine-Transduced Cells

As a result of the demonstration that tumor cells transduced with IL-2 alone may be effectively rejected, combinations of cytokines were tested. For this analysis, populations of B16 cells expressing both IL-2 and a second gene product were generated through the super-transduction of IL-2 transduced cells. Animals were vaccinated with the doubly transduced cells and then challenged with non-transduced cells after 7 to 14 days. Typically, both the initial and challenge doses were 5 x 10⁵ live cells, cells which had been harvested from culture dishes and then extensively washed. Cells were injected subcutaneously in a volume of roughly 0.5 cc of HBSS.

B16 melanoma cells were modified to express IL-2 plus one of the following: GM-CSF, IL-4, γ-IFN, ICAM, CD2, IL-l-RA, IL-6, or TNF-α. The results shown in FIG. 2B, indicate that cells expressing both IL-2 and

GM-CSF generate potent systemic immunity, with a majority of the mice surviving tumor challenge long term. For example, mice often survived as long as 120 days, at which time the mice were sacrificed. In total, 50 or 60 mice have been vaccinated with this combination, and the result show that mice were protected from challenge doses ranging from 10⁵ unmodified cells (essentially 70% of the animals were protected) to 5 x 10⁶ unmodified cells (which resulted in lesser, but still significant protection). This result is particularly surprising in light of the fact that tumor cells transduced with GM-CSF alone grew progressively in the host, and, in fact, the host succumbed to the toxicity of GM-CSF secreted systemically. A small degree of protection was observed with several other combinations. TNF-α and IL-4 had a very slight effect in combination with IL-2. IL-6 also showed slight activity when used in combination with IL-2. Under the conditions used, γ-IFN, ICAM, CD2 and IL-1 receptor antagonists were inactive. Assessment of the effects of the transduced B16 cells was done through ascertaining when natural death or humane killing of the mice occurred.

In a further set of experiments, the ability of a third cytokine (in addition to IL-2 and GM-CSF) to affect the ability of that combination to protect against wild type challenge was also assessed. Thus, B16 melanoma cells expressing IL-2, GM-CSF plus either γ-IFN, IL-4, or both, were constructed.

Preliminary results suggest that the addition of IL-4 to IL-2 with GM-CSF improves the efficacy of this approach. See FIG. 2C. Perhaps more intriguing is that the addition of γ-IFN to IL-2 with GM-CSF compromises the ability of the latter combination to function as an effective vaccine.

These data suggest that the various combinations of cytokines can both enhance as well as attenuate the magnitude of the immune response.

EXAMPLE 9 Vaccination Studies With Irradiated Cells

A. Immunogenicity of Non-Transduced, Irradiated Cells

The fact that cells expressing both IL-2 and GM-CSF, but not IL-2 alone, conferred systemic protection upon vaccinated hosts, and that cells secreting GM-CSF alone actually grew progressively, suggested the possibility that the IL-2 might be functioning primarily to mediate rejection of the vaccinating cells.

Thus, an important control was to assess the vaccination potential of non-transduced irradiated cells, since many previous studies have shown that irradiated tumor cells can possess significant vaccination activity (Foley, Cancer Res. 13: 835-837 (1953); Klein et al, Cancer Res. 20: 1561-1572 (1960); Prehn et al, /. Natl.

Cancer Inst. 18: 769-778 (1957); Revesz, Cancer Res. 20: 443-451 (I960)). Irradiation and vaccination were done as outlined above. The data in FIG. 3C indicates that non-transduced irradiated B16 cells elicited only minimal effects upon the growth of challenge cells at the vaccine and challenge doses tested.

However, a number of mouse tumor cell lines previously used to identify the anti-tumor activity of specific cytokines are inherently immunogenic, as revealed by studies involving vaccination with irradiated, non-transduced cells. Furthermore, irradiated cells alone are able to confer systemic immunity at levels comparable to those induced by live transduced cells. For these experiments, several tumor models which had been used previously by other investigators to identify cytokines possessing activity in tumor rejection or tumor challenge assays were used. These tumors included: (i) CT26, a colon carcinoma derived cell line (Brattain et al., Cancer Res. 40: 2142-2146 (1980)), used in studies which identified the activity of IL-2 (Fearon et al, supra); (ii) CMS-5, a fibrosarcoma derived cell line (Deleo et al, /. Med. Exp. 146: 720-734 (1977)), used to identify the activity of IL-2 (Gansbacher et al, /. Exp. Med. 172: 1217-1224 (1990)) and γ-IFN (Gansbacher et al., Cancer Res.50: 7820-7825 (1990)); (iii) RENCA, a renal cell carcinoma derived cell line (Murphy et al., /. Natl. Cancer Inst. 50: 1013-1025 (1973)), used to identify the activity of IL-4 (Golumbek et al., supra; and (iv) WP-4, a fibrosarcoma derived cell line (Asher et al., supra), used to identify the activity of TNF-α (Asher et al., supra). For these initial studies, the vaccine and challenge doses were comparable to those used previously in the cytokine gene transfer studies. The results of tumor challenge assays in which mice vaccinated with irradiated cells were challenged with tumor cells 7-14 days later are shown in FIG. 7.

Surprisingly, in each case, the irradiated cells possessed potent vaccination activity, comparable to that reported previously with live cells expressing the various cytokines tested above. In contrast, as shown earlier (FIG. 3C), irradiated B16 cells induced little if any systemic immunity. This has particular significance since it suggests that previous work may be misleading. Many of the tumor cells used in previous studies could have been inherently immunogenic, but the lethality of the live tumor cells masks such characteristics.

The expression of a cytokine may merely mediate the death of the live tumor cells, thus allowing the natural immunogenicity to be expressed. In contrast, the systemic immune responses of the present invention are due to a real interaction of the immune system and the simultaneous presentation of cytokine and antigen.

B. Vaccination With Transduced Irradiated B16 Cells

Irradiation of B16 cells expressing both IL-2 and GM-CSF did not abrogate either their secretion of cytokines in vitro, as shown above, or their vaccination activity in vivo (data not shown). Based on this result, irradiated B16 cells expressing GM-CSF alone were used to vaccinate mice, and subsequently challenged 7 to 14 days later. Cells were irradiated using 3500 Rads. Typically the irradiated cells were dosed at 5 x 10⁵, with subsequent challenge with the same dose of live cells. As shown in FIG. 3A, such a vaccination led to potent anti-tumor immunity, with most of the mice surviving their tumor challenge. When non-irradiated cells were used as a vaccine, 1 out of 19 animals was ultimately protected; when irradiated cells were used, 16 out of 20 mice were protected. No mice demonstrated the toxicity observed in mice injected with non-irradiated GM-CSF expressing cells (see Example 6) and circulating GM-CSF in the sera of vaccinated mice were unable to be detected using an ELISA sensitive to 10 pg/ml. The systemic immunity was also shown to be long lasting, in that most of the mice vaccinated with irradiated cells which expressed GM-CSF and subsequently challenged with non-transduced cells two months after vaccination remained tumor free. The systemic immunity was also specific, in that GM-CSF expressing B16 cells did not protect mice from a challenge by Lewis Lung carcinoma cells (Bertram et al., Cancer Letters 11: 63-73 (1980)), another tumor of C57B1/6 origin, and GM-CSF expressing Lewis Lung carcinoma cells did not protect mice from a challenge of non-transduced B16 cells.

The efficiency of the irradiated transduced cells as a vaccine was apparent over a wide range of GM-CSF concentrations in that transduced cells could be admixed with a one hundred fold excess of non-transduced cells, with little compromise in systemic immunity (FIG. 3B). This result may reflect the relatively high levels of GM-CSF expression afforded by the MFG vector. In contrast, protection was highly sensitive to the total inoculum of vaccinating cells, as activity was severely compromised with the use of ten fold fewer vaccinating cells (FIG. 3B). In addition to conferring potent protection against challenge with non- transduced cells, irradiated B16 cells expressing GM-CSF were also more capable of mediating the rejection of pre-established tumors than were irradiated cells alone (FIG. 3C). Similar results were also obtained in studies in which established metastases were generated through the intravenous injection of non-transduced cells. Surprisingly, the localized destruction of vaccinating cells does not always lead to systemic immunity, as evidenced by the B16 results. While live IL-2 expressing B16 cells were rejected by the syngeneic host, this vaccination did not generate protection against subsequent challenge of non-transduced B16 cells. Similarly, vaccination with non-transduced irradiated B16 cells also failed to induce systemic protection. The generation of systemic immunity in non- or poorly immunogenic tumor models may thus require qualitatively different mechanisms than those responsible for inducing this immunity in more immunogenic tumor models.

Consistent with this hypothesis is that a screen of many gene products for anti- tumor activity in the B16 model, including all of the molecules identified in other systems as able to augment such immunity, showed that a previously unidentified cytokine, GM-CSF, is quite potent. Demonstration of the advantage of GM-CSF transduction even in tumor models that are somewhat immunogenic further suggests the relative potency of GM-CSF expression in comparison to other means of revealing tumor immunogenicity. Finally, to determine whether irradiated cells expressing other gene products might also confer systemic immunity upon vaccinated hosts, a survey of each of the cell populations expressing different gene products (after irradiation) for vaccination activity was done. B16 cells expressing IL-l-RA, IL-2, IL-4, IL-5, IL-6, GM-CSF, γ-IFN, TNF, ICAM, and CD2 were constructed as described previously. Vaccination and challenge doses and methods were done as described previously.

The results in FIG. 4 show that B16 cells expressing GM-CSF prior to irradiation appeared to be the most potent, with IL-4 and IL-6 modified cells showing slightly reduced activity.

C. Vaccination with Irradiated, GM-CSF-Expressing Cells in Other Murine Tumor Models Because the significant vaccination activity of the irradiated cells of the other murine tumor models alone precluded examination of the activity of GM-CSF- expressing cells, either the challenge or vaccine dose of a number of the tumors examined above were manipulated, in the hopes of establishing conditions where the relative efficiency of non-transduced irradiated cells and irradiated cells expressing GM-CSF could be evaluated. These conditions were the same as the ones employed relating to FIG. 3B. These conditions made possible the comparison of non-transduced irradiated cells and GM-CSF-expressing irradiated cells shown in FIG. 8. While the relative efficacy of GM-CSF-expressing cells was somewhat variable from cell line to cell line, GM-CSF-expressing cells in all cases were more efficacious than irradiated cells alone in eliciting systemic immunity.

As outlined above, included in this analysis was a study of the Lewis Lung carcinoma cell line (Bertram et al., Cancer Letters 11: 63-73 (1980)), a tumor not previously employed in cytokine gene transfer studies. While a representative experiment illustrating the efficacy of GM-CSF transduction is shown, the precise dose which demonstrates this effect has been somewhat variable. However, the results show that Lewis Lung carcinoma cells which express GM-CSF prior to irradiation show a specific systemic immunity, in that mice were protected from a subsequent challenge with unmodified Lewis Lung carcinoma cells but not from challenge with unmodified B16 melanoma cells (data not shown). The WP-4 cell line was not included in this experiment, since even large challenge doses were eventually rejected after vaccination with non-transduced cells (data not shown).

EXAMPLE 10 Reversal of Pre-Existing Tumors Using Cytokine-Transduced Tumor Cells

The ability of B16 melanoma cells expressing mixtures of cytokines to affect growth of a pre-existing tumor was tested. All experiments used mice subcutaneously injected with unmodified B16 cells on day 1. By day 7, tumors were microscopically established. On day 7, B16 melanoma cells expressing various cytokines were injected at a different subcutaneous site. Typically, both the initial and challenge doses were 5 x 10⁵ live cells, cells which had been harvested from culture dishes and then washed extensively. Cells were injected subcutaneously in a volume of roughly 0.5 cc of HBSS.

B16 melanoma cells were modified to express the following combinations of cytokines: IL-2 and GM-CSF; IL-2, GM-CSF and TNF-α; IL-2, GM-CSF and IL-4; IL-2, GM-CSF and IL-6; IL-2, GM-CSF and ICAM; IL-2, GM-CSF and CD2; and IL-2, GM-CSF and IL-l-RA. The results of these studies are shown in FIG. 9.

The experiments showed that if mice with pre-existing tumors were treated with modified B16 cells containing IL-2 and GM-CSF, there was a slight prolongation of the animal's life relative to controls. No mice were cured. Conversely, treatment with the IL-2, GM-CSF, TNF-α combination resulted in the apparent cure of 3 out of 5 mice, as evidenced by survival in excess of 120 days. Two of the three mice survived a subsequent challenge of wild-type B16 cells. The addition of IL^l, ICAM or CD2, individually, to IL-2 and GM-CSF expressing cells resulted in an apparent cure of 1 out of 5 mice (the mice survived up to the end of the study, and displayed no overt signs of tumor-associated illness). If treatment with the modified IL-2, GM-CSF, and TNF-α cells began 3 days after the establishment of the tumor, more animals were apparently tumor free, and, in some groups, 10 out of 10 mice were apparently cured. Reversal of established tumors was also shown using irradiated cells, as outlined in Example 9. As shown in FIG. 3C, irradiated B16 cells expressing GM-CSF were capable of mediating the rejection of pre-established tumors, while irradiated non-transduced cells were not, at least at the doses tested. FIG. 3C shows that the extent of protection was dependent upon both the dose of challenge cells and the time at which the therapy was initiated. Similar results were also obtained in studies in which established metastases were generated through the intravenous injection of non-transduced cells (data not shown); doses were 5 x 10⁵ live cells, cells which had been harvested from culture dishes and then washed extensively. Cells were injected subcutaneously in a volume of roughly 0.5 cc of HBSS.

EXAMPLE 11 Characteristics of the Immune Response Elicited by GM-CSF Expressing Tumor Cells

The immune response elicited by vaccination with irradiated B16 melanoma cells expressing GM-CSF was characterized in a number of ways. Histological examination of the site of injection of irradiated GM-CSF expressing cells five to seven days after injection revealed an extensive local influx of immature, dividing monocytes, granulocytes (predominantly eosinophils), and activated lymphocytes (FIG. 5, panel A). .Within one week after injection, the draining lymph node had dramatically enlarged, showing paracortical hyperplasia and some germinal center formation (FIG. 5, panel C). In contrast, in mice vaccinated with non-transduced irradiated cells, only a mild influx of inflammatory cells was seen (FIG. 5, panel B), which consisted primarily of lymphocytes, and a comparatively smaller enlargement of the draining lymph node was observed (FIG. 5, panel D). At the challenge site of mice vaccinated with irradiated GM-CSF expressing cells (FIG. 5, panel E), a large number of eosinophils, monocytes, and lymphocytes were evident after five days, while only patches of lymphocytes were seen at the challenge site in mice vaccinated with irradiated cells (FIG. 5, panel F). Virtually no responding cells were observed in naive animals challenged with live B16 cells (FIG. 5, panel G).

To determine which cells were critical for systemic immunity, a series of mice were depleted of CD4+, CD8+, or natural killer cells by the administration of antibodies in vivo and subsequently vaccinated with irradiated GM-CSF expressing cells. Specifically, beginning one week prior to vaccination, mice were depleted of either CD4+ cells with mAB GK1.5, CD8+ cells with mAB 2.43, or NK cells with PK 136. The antibodies were partially purified by ammonium sulfate precipitation. Delipidated ascites was mixed 1:1 with saturated ammonium sulfate and the precipitate was collected by centrifugation, dried, and resuspended in a volume of dH_zO equivalent to the original volume of ascites. The antibody was dialyzed extensively against PBS and passed through a 0.45 μm filter. Antibody titre was tested by staining 1 x 10⁶ splenocytes with serially diluted antibody and determining saturation by FACSCAN analysis. All preparations were titered beyond 1:2000. Antibodies were injected at a dose of 0.15 ml i.p. and 0.1 ml i.p. on day 1 of depletion, and 0.1 ml i.p. every week thereafter. Depletion of lymphocyte subsets was assessed on the day of vaccination (early depletions), on the day of wild type tumor challenge (early and late depletions), and weekly thereafter. Flow cytometric analysis of lymph node cells and splenocytes stained with 2.43 or GK1.5 followed by fluorescein isothiocyanate labeled goat antibody to rat IgG or with Pkl 36 followed by fluorescein isothiocyanate-labeled goat antibody to mouse IgG revealed that the depleted subset represented <0.5% of the total lymphocytes, with normal levels of the other subsets present. Both CD4+ and CD8+ T cells were required for effective vaccination, since depletion of either T cell subset prior to vaccination abrogated the development of systemic immunity, whereas depletion of NK cells had little or no effect (FIG. 6, panel A).

In addition, when various T cell subsets were depleted subsequent to immunization but prior to challenge, both CD4+ and CD8+ T cells were again found to be important (data not shown), thus indicating a role for both subsets at the effector as well as the priming phase of the response.

Finally, the generation of tumor specific CD8+ cytotoxic T cells in mice vaccinated with either non-transduced irradiated cells or irradiated cells expressing

GM-CSF was examined. In this series of experiments, 14 days after vaccination with either irradiated GM-CSF transduced or irradiated, non-transduced B16 cells, splenocytes were harvested and stimulated in vitro for 5 days with gamma-interferon treated B16 cells. CD8 blockable CTL activity was determined in a 4 hour ⁵¹Cr release assay on gamma-interferon treated B16 targets at various effector: target ratios.

Splenocytes from naive C57B1/6 and Balb/c mice served as controls. The data are shown in FIG. 6B, where the hollow square represents GM-CSF expressing, the solid circle represents Allo, the hollow diamond represents naive and the solid triangle represents B16 irradiated.

While mice vaccinated with irradiated non-transduced cells possessed little detectable CD8+ blockable killing, the level of CD8 blockable killing was significantly enhanced in a sample of cells isolated from mice vaccinated with irradiated GM-CSF expressing cells.

Previous studies had suggested that cytokine expressing tumor cells might enhance the generation of systemic anti-tumor immunity through the ability of cytokine expression to bypass T cell help (Fearon et al., supra). This model was based on the finding that IL-2 expressing tumor cells elicited systemic immunity in both the CT-26 colon carcinoma and B16 melanoma model, and that the tumor immunity depended only upon CD8+ cells. However, the inability to generate systemic immunity with IL-2 expressing B16 cells and the dependence of the GM-CSF effect upon CD4+ cells suggests that GM-CSF expressing cells may elicit systemic immunity in a fundamentally different manner.

Several characteristics of the immune response induced by vaccination with irradiated cells expressing GM-CSF suggest that the underlying mechanism may involve enhanced presentation of tumor-specific antigens by host antigen presenting cells. While the extent of systemic immunity induced by GM-CSF expressing cells was not appreciably affected by the level of GM-CSF secretion examined (FIG. 3B), the absolute number of vaccinating tumor cells was critical. This finding suggests that the absolute amount of tumor specific antigen available for vaccination may normally be limiting. Also, the injection of GM-CSF expressing cells led to a dramatic influx of monocytic cells, known to be potent antigen presenting cells (Morrissey et al., /. Immunol. 139: 1113-1119 (1987)), with lesser numbers of other cell types. Furthermore, both CD4+ and CD8+ cells were necessary for the immune response. Since the B16 cells used in this invention do not express detectable amounts of class II MHC molecules, even after γ-IFN treatment (unpublished results), it is likely that host antigen presenting cells, rather than the tumor cells themselves, were responsible for the priming of CD4+ T cells. These data are consistent with the hypothesis that local production of GM-CSF modifies the presentation of tumor antigens such that collaboration between CD4+ helper and CD8+ cytotoxic killer T cells is established or improved in vivo. EXAMPLE 12 Use of Other Retroviral Vectors

A. pLJ

The characteristics of this vector have been described in (Korman et al., supra), and in U.S.S.N. 07/786,015, filed October 31, 1991, and in PCT/US91/08121, filed October 31, 1991. This vector is capable of expressing two types of genes: the gene of interest and a dominant selectable marker gene, such as the neo gene. pLJ vectors containing murine IL-2, GM-CSF, IL-4, IL-5, γ-IFN, IL-6, ICAM, CD2 TNF-α, and IL- l-RA (interleukin-1-receptor antagonist) are made. In addition, human sequences encoding TNF-α, GM-CSF and IL-2 are constructed. The gene of interest is cloned in direct orientation into a BamHI/Smal/Sall cloning site just distal to the 5' LTR. The neo gene is placed distal to an internal promoter (from SV-40) which is farther 3' than the cloning site (i.e., it is located 3' of the cloning site). Transcription from pLJ is initiated at two sites: the 5' LTR, which is responsible for the gene of interest, and the internal SV-40 promoter, which is responsible for expression of the selectable marker gene. The structure pf pLJ is represented in FIG. IB.

B. pEm

In this simple vector, described in U.S.S.N. 07/786,015, filed October 31, 1991, and in PCT/US91/08121, filed October 31, 1991, the entire coding sequence for gag, pol, and env of the wild type virus is replaced with the gene of interest, which is the only gene expressed. The components of the pEm vector are described below. The 5' flanking sequence, 5' LTR and 400 base pairs of contiguous sequence (up to the

BamHl site) are from pZIP. The 3' flanking sequence and LTR are also from pZIP; however, the Clal site 150 base pairs upstream from the 3' LTR has been ligated with synthetic BamHl linkers and forms the other half of the BamHl cloning site present in the vector. The Hindlll/EcoRI fragment of pBR322 forms the plasmid backbone. This vector is derived from sequences cloned from a strain of Moloney Murine Leukemia virus (MMLV). pEm vectors containing murine IL-2, GM-CSF, IL-4, IL-5, γ-IFN, IL-6, ICAM, CD2, TNF-α, and IL-l-RA (interleukin-1-receptor antagonist) are made. In addition, human sequences encoding TNF-α, GM-CSF and IL-2 are constructed.

C. a-SGC

The α-SGC vector, described in U.S.S.N. 07/786,015, filed October 31, 1991, and in PCT/US91/08121, filed October 31, 1991, utilizes transcriptional promoter sequences from the α-globin gene to regulate expression of the cytokine-encoding gene. The 600 base pair fragment containing the promoter element additionally contains the sequences for the transcriptional initiation and 5' untranslated region of the authentic α-globin mRNA. A 360 base pair fragment which includes the transcriptional enhancer from cytomegalovirus precedes the α-globin promoter and is used to enhance transcription from this element. Additionally, the MMLV enhancer is deleted from the 3' LTR. This deletion is transferred to the 5' LTR upon infection and essentially inactivates the transcriptional activating activity of the element. The structure of α-SGC is represented in FIG. ID. α-SGC vectors containing murine IL-2, GM-CSF, IL-4, IL-5, γ-IFN, IL-6, ICAM, CD2, TNF-α, and IL-l-RA (interleukin-1 -receptor antagonist) are made. In addition, human sequences encoding TNF-α, GM-CSF, and IL-2 are constructed.

EXAMPLE 13 Co-administration of Viral Antigens and Cytokines

An appropriate vaccinating cell (which may include but is not restricted to, fibroblasts, keratinocytes, endothelial cells, monocytes, dendritic cells, B-cells, or T-cells) is transduced with a retrovirus expressing an individual or combination of HIV antigens (including, but not limited to gag, env, pol, rev, nef, tat, and vif) and a single or combination of cytokines (such as GM-CSF), is irradiated or otherwise rendered proliferation incompetent, and is administered to a patient.

EXAMPLE 14

Suppression of Immune Responses

An appropriate vaccinating cell (see Example 13) as well as particular cells of an allograft (i.e., renal tubular epithelial cells in the kidney) would be transduced with a virus expressing a single or combination of cytokines (such as gamma-interferon), irradiated or otherwise rendered proliferation incompetent, and administered to a patient to reduce the immune response against the allograft.

Additionally, the present invention can be used to suppress autoimmune diseases. The autoimmune response against a host target antigen could also be modified in a similar way. For example, the present invention could be used to treat multiple sclerosis. An appropriate cell (see Example 13) would be transduced with a retrovirus expressing myelin basic protein as well as a cytokine (such as gamma-interferon) to reduce the immune response directed against myelin basic protein in the patient.

EXAMPLE 15 Enhancement of Immune Response

It is also possible to use the present invention to enhance the immune response to a variety of infectious diseases. For example, the present invention could be utilized in the treatment of malaria. An appropriate vaccinating cell (see Example 13) would be transduced with a retrovirus expressing the gene for circumsporouzoite surface protein and a cytokine (such as GM-CSF), irradiated or otherwise rendered proliferation incompetent, and then administered to a patient to induce protection of a therapeutic response against malaria

EXAMPLE 16 Human Clinical Studies Using Retroviral Transduced Cells

A. Melanoma Trial

Melanoma patients in which the disease had progressed to a stage where the surgery did not remain as an effective option were studied. The study also required that the patients showed no evidence of autoimmune disease, serious allergy, or brain metastases (as determined by magnetic resonance imaging, MRI), and retained adequate immune, hepatic, renal, and bone marrow function.

The human GM-CSF gene was subcloned into the insertion site of the retroviral vector MFG-S (a derivative of MFG) in a proper context and orientation for expression of the gene. The recombinant vector was subsequently transfected into the amphotropic packaging cell line psi CRIP and high producer transfectants were isolated. High titer stocks of helper free retroviral particles (i.e., recombinant retrovirus) which harbor the human GM-CSF gene were then harvested from the culture medium of the transfected producer cell lines essentially as described above. Tumor cells were resected from cutaneous and subcutaneous lesions, lymph nodes, and lung, liver, and soft tissue metastases. After resection, the specimens were aseptically transferred to a sterile container and transported to the laboratory. All subsequent procedures were conducted under aseptic conditions. The tumor tissues were disaggregated by either collagenase treatment or by mechanical dissociation and subsequently grown in culture and/or frozen in DMSO with 50 percent FCS (collagenase treated) or human albumin (mechanical dissociation). The numbers of the primary and secondary autologous tumor cells were expanded in culture, and a portion of the cells were subsequently transduced to produce huGM-CSF using a recombinant retrovirus generated by transfection with a MFG-S construct.

After verification that the transduced cells were sterile, viable and producing cytokine, (see Table II), a total of either 5 x 10⁶ (low dose) or 5 x 10⁷ (high dose) of the transduced tumor cells were irradiated, suspended in normal saline, and injected (both intradermally and subdermally at up to five different sites in volumes of 0.25-1.0 ml) back into the original donors. Up to three injections at the given dosages were applied to the individuals at approximately 21 day intervals. Control individuals were separately injected with irradiated non-transduced cells or with levels of GM-CSF analogous to the amounts of GM-CSF produced by the transduced cells which were injected into the test subjects.

TABLE II GM-CSF Production (ng/10^ cells/24 hrs)

15

20

25

After injection, tests were performed to confirm that the transduced tumor cells elicited an immune response. These tests included assaying for delayed type hypersensitivity (DTH) reactions to intradermally injected (irradiated) tumor cells, and biopsies of the vaccination and injection sites. The data indicate that T-cells are present in the inflamed tissues, and that there is a reproducible increase in the number of monocytes. The control subjects (who were injected with either nontransduced tumor cells or huGM-CSF at levels analogous to those produced by the transduced autologous tumor cells) showed little to none of the differences seen in the test subjects. Immunohistochemistry indicated that a tendency for antigenic drift correlated with decreased expression of the Mel 4 and HMB45 markers.

Additional tests were conducted to determine whether or not an increase in the number of eosinophils correlated with the injections. As seen in FIG. 10, after each vaccination, a transient increase in the percentage of eosinophils in the bloodstream generally followed.

The above data indicate that immune stimulation had occurred, and that the observed immune stimulation could be associated with the fact that the autologous tumor cells were expressing human GM-CSF in vivo.

B. Renal Cell Carcinoma Trials

Eighteen patients with advanced renal cell carcinoma were vaccinated up to four times (at approximately one month intervals) with either 4 x 10⁶, 4 x 10⁷, or 4 x 10^s transduced and irradiated tumor cells. Tumor tissue was surgically resected (see above), and the population of primary and secondary tumor cells was expanded by in vitro culture. The cultured cells were subsequently transduced with the recombinant retrovirus generated by transfection of packaging cells with the human GM-CSF/MFG-S vector described above, and tested for GM-CSF production. Transduced autologous cells which produced acceptable quantities of cytokine were irradiated and injected into the donor individuals as described above.

At the lowest dose of injected cells, the levels of GM-CSF secreted ranged between about 17 and 189 ng/10⁶ cells /24 hrs. At the middle dose, the transduced cells constructed from the various individuals' tumors secreted between about 42 and 149 ng of GM-CSF/ 10⁶ cells/24 hrs.

Control individuals were injected with irradiated non-transduced tumor cells. At the lowest dose, three subjects received non-transduced cells, at the middle dose of cells, five subjects received non-transduced cells, and the two patients that were injected with the highest dosage of cells only received nontransduced cells. Patient responses to the vaccinations included marked erythema and induration at the injection site (Dose 1 showed the least response), which became more pronounced after multiple vaccinations (this was especially the case when transduced cells were injected). Moreover, test subjects who were multiply injected with transduced cells also displayed positive DTH reactions, as well as an increase in the amount of eosinophils (following vaccination). As in the melanoma study, apart from local symptoms (erythema, induration and itching), no significant adverse events were observed.

This data reveals that the injection of irradiated autologous tumor cells expressing GM-CSF into human test subjects stimulated an observable in vivo tumor-specific immune response against the parent tumor cells.

EXAMPLE 17 Generation of Recombinant Adenovirus Encoding a Cytokine

The recombinant adenovirus encoding human GM-CSF (AV-GM-CSF) was generated with an adenoviral vector containing a GM-CSF expression cassette substituted for the El genes of adenovirus type 5 with an additional deletion in the viral genome in the E3 region (see FIG. 11 A). According to the complete GenBank sequence for Ad5 (Accession no. M73260), the deletions are from 455 to 3327 in the El region (see Table III). Numbering begins with the first base of the left inverted terminal repeat. TABLE III

FEATURES OF AD-GM ADENOVIRAL VECTOR

5

Construction of the adenoviral vectors employs standard ligation and restriction techniques which are well understood in the art. The E3 deletion was introduced by overlap recombination between wild type 300 (from H. Ginsberg) (0 to 27330) and dl324 (Thimmappaya et al. (1982) Cell 31:543-551) (21561 to the right end). The GM-CSF expression cassette was added to the El region by cre/lox mediated recombination between pAdlox MC hGM and E3 deleted adenovirus in CRE8 cells (Hardy et al, f. Virol. 71:1842-1949 (1997)). pAdlox MD hGM was derived from pAdlox (Hardy et al, supra) and pMD.G (Naldini et al, Science 272:263-267 (1996)) and contains the following sequences: 0 to 455 from Ad5, the cytomegalovirus (CMV) immediate early gene promoter /enhancer (nucleotide positions -670 to +72, GenBank accession no. X03922) from pBC12/CMV/IL-2 (Cullen, Cell 46: 973-982 (1986)), a small region of human β-globin exon 2 and a shortened second intervening sequence (IVS2) (nucleotide position 62613-62720 plus 63088-63532, GenBank accession no. J00179)), exon 3 and the poly adenylation signal from human β- globin(nucleotide positions 63532-64297), the GM-CSF cDNA inserted into exon 3 (position 63530), a second poly adenylation site from SV-40 (position 2681-2534), GenBank accession no. J02400) and a loxP site followed by bacterial sequences from Bluescript. The GM-CSF cDNA was obtained from a plasmid (DNAX Research Institute of Molecular and Cellular Biology (Palo Alto, CA). The DNA sequence was isolated from cDNA libraries prepared from Concanavalin A-activated human T-cell clones by functional expression in mammalian cells. The isolation and characterization of the cDNA including the entire sequence of the gene have been reported in the scientific literature. (Lee, F. et al., Proc. Natl. Acad. Sci. USA 82:4360- 4364 (1985)). The identity of the clone was verified by restriction endonuclease digestion upon receipt. The MD expression cassette was modified by PCR to include restriction sites for Pmll, EcoRI and Bgl II for insertion of a transgene downstream of the IVS2. The GM-CSF cDNA was removed using Pmll and BamHl from pMFG S hGM (Dranoff et al, Human Gene Ther. 7:111-123 (1997)) and inserted into the Pmll and Bgl II sites in the MD expression cassette. The region of the pAdlox MD hGM plasmid that was incorporated into the Ad GM virus was sequenced on both strands. Correct structure of the initial viral construction was confirmed by restriction analysis and ELISA testing for GM-CSF production from infected HeLa cells. The recombinant virus was then subjected to two rounds of plaque purification. Recombinant virus from a plaque was restriction mapped and expanded two passages by growth in 293 cells (certified cells from Microbiological Associates) to produce a research virus stock. The sequence of the GM-CSF gene in the recombinant virus was determined by direct sequencing of viral DNA prepared from the research virus stock. Finally, recombinant virus from the research virus stock was tested for mycoplasma and sterility, and when found negative, used to infect cells for the master virus stock.

EXAMPLE 18 In Vitro Studies Using Recombinant Adenovirus Encoding GM-CSF to Transduce Human Tumors

In order to demonstrate the feasibility of transducing freshly explanted human tumor cells with adenovirus, pilot studies have been performed on six non-small cell lung carcinomas and three melanomas. In these experiments, single cell suspensions of tumor masses were prepared by mechanical dispersion and enzymatic (collagenase) digestion. The dispersed cells were transduced at high density for one hour with E1/E3 doubly-deleted adenoviral vectors expressing either beta- galactosidase or human GM-CSF (at a multiplicity of infection ranging from 1 to 500). The adenoviral vectors used for these experiments are replication-deficient and have less than one replication competent viral particle per 10⁹ pfu. After one hour, the cells were placed into petri dishes at 37°C overnight in 10 mis of DME plus 10% fetal calf serum and antibiotics. The following day the cells were washed extensively and then irradiated with 10,000 Rads (this dose eliminated clonogenicity of all melanoma and lung lines evaluated thus far). A portion of the material was placed into culture for 2 days and GM-CSF secretion measured by ELISA. Beta-galactosidase staining was detected using a simple histochemical assay. The average cell yield from 1 gram of tissue was approximately 1 x 10⁷ cells

(viability ranged from 40-70%). Transduction efficiency as assayed by beta-galactosidase staining exceeded 50% in all cases. GM-CSF secretion levels achieved ranged from 63-512 ng/10⁶ cells/ 24 hours. These levels are comparable to or increased relative to those obtained with the recombinant retroviruses used in current melanoma studies. No replication competent adenovirus was detected in the transduced tumors tested by plaque assay on A549 cells.

EXAMPLE 19 Preclinical Analysis of Adenovirus-Transduced Cells in a Murine Melanoma Model

The effectiveness of recombinant adenovirus as a GM-CSF transducing agent was compared with that of retrovirus in a B16 tumor protection assay. Briefly, 5 x 10⁵ B16 cells were transduced with recombinant adenovirus, cultured, irradiated, and then injected into a C57BL/6 mouse as a tumor vaccine. One week later the mice were challenged by injection of 5 x 10⁵ live B16 cells. Mice were monitored regularly and any mice surviving to 15 weeks after challenge were considered protected.

In the first experiment, B16 tissue culture cells were transduced with either recombinant adenovirus (AV-GM-CSF) or recombinant retrovirus (Retro-GM-CSF) (2,000 particles /cell) and were compared to sham transduced cells and cells transduced with a "vector only" control adenovirus, Psi5 (see Example 9B). As shown in FIG. 17, the best protection was seen from cells transduced with AV-GM-CSF providing 90% protection. Additionally, the control virus which lacked a transgene, produced a measurable protection. In the second experiment, the effectiveness of vaccines prepared from B16 tissue culture cells were compared with those prepared from primary B16 tumor tissue. The tumors were grown in mice, removed surgically, dispersed by combined collagenase digestion and mechanical disruption, and then washed. Briefly, mice were inoculated with 5 x 10⁵ live B16 cells that were suspended in 0.5 ml Hank's buffered saline solution. The tumors were removed for processing when they were 2-3 cm in diameter (see Example 20). The viability of the cells was measured. Cells were then transduced with AV-GM-CSF, followed by overnight incubation in petri dishes. The next day, the transduced cells were washed, irradiated, and injected into mice, the whole process taking about 24 hours. Once again, the AV-GM-CSF transduced cells produced significant protection and primary tumor tissue was comparable to the cell line at providing protection from tumor challenge (FIG. 18).

Finally, dispersed B16 tumor tissue was used for the live cell tumor challenge (see Example 20, below). Vaccines were prepared as in FIG. 18. Tumors were observed to occur more quickly (at one week for primary tumor challenge versus two weeks for cell line challenge). As shown in FIG. 19, the vaccine derived from primary tumor produced a 50% survival rate, while the vaccine derived from a similar number of tissue culture cells produced a 30% survival rate.

The results of these three experiments indicate that it is possible to prepare a tumor vaccine by transduction of cells modified to express GM-CSF after transduction with a recombinant adenovirus. The vaccination process took about 24 hours from the time the tumors were removed until the vaccine was injected into mice. Further, a vaccine prepared from dispersed tumor material appeared to serve as a very effective protective agent against challenge with primary tumor cells. This is important as it indicates that minimally processed tumor vaccines have a high likelihood of bearing a complete set of tumor antigens. Also, these experiments demonstrate that the adenovirus vector lacking a transgene provides an adjuvant in activating the immune system against tumor tissue. EXAMPLE 20

Clinical Trials Using Non-Small Cell Lung Carcinoma Cells Transduced With Recombinant Adenovirus Encoding GM-CSF

Phase I clinical trials are currently being conducted to determine the feasibility of preparing irradiated, autologous carcinoma cells engineered by recombinant adenovirus mediated gene transfer to secrete GM-CSF in patients with metastatic non-small cell lung carcinoma. In addition, these trials are being conducted to determine the safety and biologic activity of vaccination with autologous, irradiated carcinoma cells engineered by adenoviral mediated gene transfer to secrete GM-CSF. In this trial, 24 patients with metastatic non-small cell lung carcinoma (histologically documented) were treated. Cancer patients underwent a surgical procedure to obtain autologous tumor tissue for vaccine preparation. Vaccine cell preparation involved a 48 hour manufacturing process. Vaccines were manufactured from metastatic tumors procured from multiple clinical sites including lymph node, lung, pleural effusion, adrenal, thyroid subcutaneous nodule, and paraspinous mass. Briefly, a tumor specimen removed from each patient was washed with HBSS buffer and cut into portions weighing 3 grams or less. Each portion was placed into a separate Petri dish into which 5 ml collagenase/gram of tissue was added. The tumor tissue was minced into 1 to 3 mm³ sections and placed in a Stomacher bag. The volume was less than 20 ml. The bag was sealed and placed inside a mechanical homogenator or Stomacher Lab Blender and homogenized until the digestion was complete (usually 30 to 40 minutes, i.e., until the contents of the bag was a thick milky suspension with few, if any, undigested pieces of tissue). The homogenate was then diluted with 20 ml growth media, brought to a final volume of 40 ml, and centrifuged at 2,000 RPM, for 10 minutes at 4°C. The supernatant was removed and the cell pellet resuspended in 10 ml growth media on ice. These steps were repeated until the entire specimen was digested and washed free of enzymes (e.g., collagenase) and in suspension in growth media. The number and viability of cells in the cell suspension was then determined with Trypan blue.

The cell suspension was then concentrated to the appropriate density for transduction via centrifugation at 2,000 rpm for 10 min. at 4°C. The pellet was then resuspended to a final cell density of 10⁷ cells/ml. Some cells were then set aside for Delayed Type Hypersensitivity (DTH) testing, establishment of cell line, and cryopreservation. The remaining cells were transduced with the recombinant adenovirus encoding human GM-CSF.

The number of plaque forming units (pfu) required to perform transduction was determined by multiplying the total number of cells (dead + viable, calculated after tumor digestion) by 10. The virus was then diluted in culture media to 10 times the final concentration. Then, 0.1 ml of the adenovirus-GM-CSF suspension per ml of a tumor suspension was added to cell suspension. The recombinant adenovirus used in this trial was a standard first generation El and E3 gene deleted replica tion- deficient virus described above in EXAMPLE 17. In the adenovirus vector used to generate this recombinant adenovirus, the human GM-CSF cDNA was introduced into the El deleted region. (Of course, the cytokine cDNA could have been introduced in other deleted regions of the viral genome.) The cells were incubated at room temperature (i.e., approximately 23-25°C) for 1 hour with gentle mixing every 10 minutes. The volume of tumor cell suspension was then doubled by adding the appropriate volume of growth media to this suspension. Five ml of the cell suspension was transferred to a Petri dish, and the cells incubated at 37°C

The tumor cells were transduced overnight at 37°C with replication-defective recombinant adenovirus transferring the human GM-CSF gene. After overnight transduction, the cells were washed extensively with HBSS and then irradiated within 24 hours with 10,000 Rads using a gamma irradiator. A small aliquot of cells (up to 10% of the total) was placed into culture for 48 hours in the presence of medium. GM-CSF secretion from transduced cells was determined by ELISA (for example, using a kit commercially available kit, for example, from R&D Laboratories, Minneapolis, MN) to document appropriate target levels of GM-CSF production. Preferably, the target level is at least 40 ng GM-CSF/ 10⁶ cells/24 hours. Even more preferably, the target level is at least 100 ng GM-CSF /10⁶ cells/ 24 hours. Most preferably, the target level is over 1000 ng GM-CSF/ 10⁶ cells/24 hours.

If the transduced cells did not reach at least 40 ng GM-CSF/10⁶ cells/24 hours, they were still used to vaccinate the patient in order to determine the minimum GM-CSF secretion rates necessary for biologic activity. The minimum target level of GM-CSF production was found to be about 20 ng/10⁶ cells/ 24 hours. In addition, routine cultures for bacteria and fungus were performed to insure sterility of the manipulated cells. Cells for vaccination and immunologic evaluation were then frozen in 10% DMSO/90% fetal calf serum and stored in liquid nitrogen. For total cell yields of 1 x 10⁸ or greater, the vaccination doses were about 1 x 10⁷ per dose (Dose Level 3); for cell yields ranging from 3 x 10⁷ to 1 x 10⁸, the vaccination doses were about 4 x 10⁶ per dose (Dose Level 2); and for cell yields up to 3 x 10⁷, the vaccination doses were about 1 x 10⁶ per dose (Dose Level 1). Of the patients treated, eight patients received Dose Level 1, ten received Dose Level 2, and four received Dose Level 3.

Vaccinations were administered on day 0, 7, 14, 21, and every two weeks thereafter until the supply of vaccine was either exhausted, or the patient was removed from the study. As indicated above, vaccine cell dosage is 1 x 10⁶, 4 x 10⁶, or 1 x 10⁷, depending on total cell yield. This variation in cell dosage was designed to maximize each patient's opportunity of receiving vaccinations and was not based on any expectation of significant differences in toxicity as a function of cell number. Patients received different numbers of vaccinations, depending on cell yield. Treated patients received a mean of 6.8 vaccinations (range 1-12).

A patient was considered evaluable for toxicity if at least six vaccinations were administered at a given dose level or if a patient had not received six vaccinations because of grade 3 or higher toxicity. There was no requirement for a particular number of patients at each cell dose level. A total of 24 evaluable patients formed the basis for this study. Up to five patients were initially enrolled at each dose level. If no grade 3 or 4 toxicities were observed in more than one patient by week 10 of treatment, then additional patients were treated at that dose level. No individual patient was treated with escalating doses.

The vaccination procedure used was as follows. Cells were thawed and washed twice with Hanks Buffered Salt (HBSS) solution. For administration, cells thawed not more one hour prior to administration were resuspended in a total volume of not more than 1 ml with sterile saline. A vaccination constituted a single injection in a volume of not more than 1 ml total given intradermally with a 23 or 25 gauge needle, into the upper arm or the thigh on an alternating basis. The total volume of each injection depended on the dose level administered. If sufficient cells were available, injections of non-transduced irradiated NSCLC cells (1 x 10⁶) were given on day 0 and given the fifth vaccination for evaluation of baseline and vaccine induced reactivity. These cells were resuspended in a volume of 0.5 ml and injected intradermally. In some cases, if the clinical status of the patient following completion of the first round of vaccination permitted, the option for a second round of tumor procurement, vaccine production, and vaccination were offered to certain patients. For patients with measurable disease at the time vaccination began, response was defined as follows. Complete response: disappearance of all measurable disease for 4 weeks; partial response (PR): when compared with the pre-treatment measurements, a reduction of greater than 50% in the sum of the products of the greatest perpendicular diameters of all measurable lesions for 4 weeks. If a lesion was measurable only in one dimension, then a reduction in the size of the lesion by 30% was required for classification as PR; stable disease: less than a 50% reduction or less than a 25% increase in the sum of the products of the greatest perpendicular diameters of all measurable lesions when compared with pre-treatment measurements, without appearance of new lesions for 4 weeks; and progression: greater than 25% increase in the sum of the products of the greatest perpendicular diameters of all measurable lesions when compared with pretreatment measurements, clear-cut increases in the size of valuable lesions, or the appearance of new lesions. Independent of the category assigned above, pathologic evaluation of resected lesions was performed in terms of assessing amount of inflammation, edema, fibrosis, and tumor cell death.

To characterize local reactions, punch biopsies were performed with the fifth vaccination. If cells were available, injections of 1 x 10⁶ non-transduced irradiated cells for Delayed Type Hypersensitivity reactions were given intradermally into the upper arm or thigh at both the time of beginning treatment as well as concurrent with the fifth vaccine. Histologic analyses for macrophages, dendritic cells, eosinophils, and lymphocytes were performed.

To characterize metastatic lesions, part of the tumor removed for the second round of vaccination was evaluated pathologically for lymphocyte, eosinophil, and plasma cell reactions. Attempts to establish cell lines and to harvest inflammatory cells from these sites were made. In the event that a patient did not undergo a second round of therapy, an attempt to perform a biopsy of residual disease was made to perform an assessment of immune response. To perform blood analyses, serum and circulating mononuclear cells were frozen prior to beginning vaccination and at monthly intervals thereafter. In some patients, cell lines were established from tumor removed for vaccine preparation. When this was possible, these cell lines were used to assay the development of anti-NSCLC humoral and cellular responses. For humoral responses, immunoblotting and FACS analysis were performed using pre- and post-sera against autologous tumor. For cellular responses, cytokine production and cytolytic activity against the autologous tumor pre- and post-immunization were measured. In addition, responses against adenoviral proteins expressed in the vector used for vaccine preparation were measured in a similar manner.

Endpoints included standard laboratory evaluations for clinical laboratory safety, delayed type hypersensitivity (DTH) reactivity, DTH and vaccine site biopsy, and evaluation of objective tumor response. DTH testing occurred at Day 0 and Day 28 and biopsies were performed 2-3 days after DTH testing. Vaccine site biopsies were performed 2-3 days after the fifth vaccination.

Evidence of anti-tumor immunity as measured by the development of delayed type hypersensitivity (DTH) reactions to injections of nontransduced tumor cells has been detected in patients receiving at least three vaccinations. One patient had a >50% reduction in lung and lymph node masses, but developed a new symptomatic bone metastasis. Biopsy of the responding lymph node and bone metastasis demonstrated inflammatory cell infiltration, tumor necrosis, and fibrosis, which is a pattern similar to that seen in earlier retroviral GMCSF transduced autologous melanoma vaccine trials. Two patients who had isolated metastatic sites resected for vaccine generation have remained without evidence of recurrent disease for nine and ten months and two additional patients have had stable disease for three months.

Thus, as expected, the administration to human patients of irradiated tumor cells expressing GM-CSF stimulated a detectable immune response against the parent cells, resulting in the development of delayed type hypersensitivity to non-transduced tumor cells, inflammatory cell infiltration into metastatic tumors, and, in some cases, alleviation of disease symptoms.

EXAMPLE 21

Clinical Trials Using Malignant Melanoma Cells Transduced with Recombinant Adenovirus Encoding GM-CSF

Phase I clinical trials were initiated to determine the feasibility of preparing autologous, lethally irradiated, melanoma cells engineered by adenoviral-mediated gene transfer to secrete GM-CSF in patients with metastatic melanoma. In addition, these trials are to determine the safety and biologic activity of vaccination with autologous, lethally irradiated melanoma cells engineered by adenoviral-mediated gene transfer to secrete GM-CSF.

Tumor specimens were removed from 25 patients with metastatic melanoma and processed, as described above in Example 20. The melanoma cells were transduced overnight at 37°C with supernatant containing recombinant replication- defective adenovirus transferring the human GM-CSF gene, as described in Example 17. If sufficient cells were available, a portion of untransduced cells was set aside for subsequent use in immunologic studies. After overnight transduction, the cells were washed extensively with HBSS and then irradiated within 24 hours with 10,000 Rads. A small aliquot of cells (up to 10% of the total) was placed into culture for 48 hours in the presence of medium. GM-CSF secretion from transduced cells was determined by ELISA to document appropriate target levels of GM-CSF production (>40 ng/10⁶ cells/24 hours). When this target level of GM-CSF production is not achieved, the patient is still vaccinated in order to determine the minimum GM-CSF secretion rates necessary for biologic activity. Routine cultures for bacteria and fungus were performed to insure sterility of the manipulated cells. Cells for vaccination and immunologic evaluation were frozen in 10% DMSO/90% fetal calf serum and stored in liquid nitrogen. For total cell yields of 1 x 10⁸ or greater, the vaccination doses are about 1 x 10⁷; for cell yields ranging from 3 x 10⁷ to 1 x 10⁸, the vaccination doses are about 4 x 10⁶; and for cell yields up to about 3 x 10⁷, the vaccination doses are about 1 x 10⁶.

Vaccinations were administered on days 0, 7, 14, 21, and every 14 days thereafter until the supply of vaccine is exhausted. Vaccine cell dosage was 1 x 10⁶, 4 x 10⁶, or 1 x 10⁷, depending on total cell yield. This variation in cell dosage is designed to maximize each patient's opportunity of receiving vaccinations and is not based on any expectation of significant differences in toxicity as a function of cell number. It is also recognized that patients will receive different numbers of vaccinations, depending on cell yield. A patient is considered evaluable for toxicity if at least six vaccinations have been administered at a given dose level or if someone has not received six vaccinations because of grade 3 or higher toxicity. A total of 25 valuable patients are the basis for this study. Up to five patients have been initially enrolled at each dose level. If no grade 3 or 4 toxicities are observed in more than one patient by week 10 of treatment, then additional patients may be treated at that dose level. No individual patient is being treated with escalating doses.

As expected, the administration of irradiated, autologous melanoma cells expressing GM-CSF to human subjects has stimulated an immune response against the parent melanoma cells in the form of the infiltration of immune cells seen in tumors, as well as delayed type hypersensitivity responses.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.

What is claimed is:

Claims

1. A method for stimulating a systemic immune response to a tumor in a mammal, or to an antigen thereof, comprising administering a therapeutically effective amount of a proliferation-incompetent tumor cell that has been genetically modified by transduction with a recombinant virus encoding human granulocyte- macrophage colony stimulating factor, wherein the transduced tumor cell expresses human granulocyte-macrophage colony stimulating factor, the tumor cell and the tumor are of the same type and express the antigen, and the recombinant virus is selected from the group consisting of adenovirus, lentivirus, adeno-associated virus, SV-40, herpes virus, and vaccinia virus.

2. The method of claim 1, wherein the recombinant virus is an adenovirus.

3. The method of claim 2, wherein the adenovirus comprises the MD expression cassette.

4. The method of claim 1, wherein the recombinant virus is a lentivirus.

5. The method of claim 1, wherein the recombinant virus is an adeno-associated virus.

6. The method of claim 1, wherein the recombinant virus is an SV-40 virus.

7. The method of claim 1, wherein the recombinant virus is a herpes virus.

8. The method of claim 1, wherein the recombinant virus is a vaccinia virus.

9. The method of claim 1, wherein the recombinant virus is replication-deficient.

10. The method of claim 1, wherein the tumor cells are rendered proliferation- incompetent by irradiation.

11. The method of claim 1, wherein the tumor is an established tumor that is present in the mammal prior to the administration step.

12. The method of claim 1, wherein the administration step inhibits the formation of the tumor in the mammal.

13. The method of claim 1, wherein the tumor cell is derived from the mammal.

14. The method of claim 1, wherein the mammal is a human.

15. The method of claim 1, wherein the type of the tumor cell and the tumor is selected from the group consisting of leukemia, prostate tumor, melanoma, non-small cell lung carcinoma, breast cancer, epidermal cancer, pre-neoplastic lesion, cancerous polyp, ovarian cancer, cervical cancer, and renal carcinoma.

16. The method of claim 15, wherein the type of the tumor cell and the tumor is a prostate tumor.

17. The method of claim 15, wherein the type of the tumor cell and the tumor is a non-small cell lung carcinoma.

18. The method of claim 15, wherein the type of the tumor cell and the tumor is a melanoma.

19. The method of claim 1, wherein the tumor is metastatic.

20. The method of claim 1, wherein the tumor cell and the tumor are derived from the same individual.

21. The method of claim 1, wherein the tumor cell and the tumor are derived from different individuals.

22. The method of claim 1, wherein the tumor cell is derived from a tumor cell line.

23. The method of claim 21, wherein the tumor cell line is selected from the group consisting of prostate tumor, melanoma, non-small cell lung carcinoma, breast cancer, epidermal cancer, pre-neoplastic lesion, cancerous polyp, ovarian cancer, cervical cancer, and renal carcinoma.

24. The method of claim 23, wherein the tumor cell line is a prostate tumor cell line.

25. The method of claim 24, wherein the prostate tumor cell line is selected from the group consisting of a LnCaP cell, a DU-145 cell, and a PC-3 cell.

26. The method of claim 1, wherein the systemic immune response to the tumor results in tumor regression.

27. The method of claim 1, wherein the systemic immune response to the tumor retards the growth of the tumor.

28. A recombinant virus comprising nucleic acid encoding human granulocyte- macrophage colony stimulating factor which is expressed in a tumor cell transduced with the recombinant virus, wherein the recombinant virus is selected from the group consisting of adenovirus, adeno-associated virus, lentivirus, SV-40, herpes virus, and vaccinia virus.

29. The recombinant virus of claim 28, wherein the recombinant virus is replication-defective.

30. The recombinant virus of claim 28, wherein the recombinant virus is an adenovirus.

31. The recombinant adenovirus of claim 30, comprising the MD expression cassette.

32. The recombinant virus of claim 28, wherein the recombinant virus is a lentivirus.

33. The recombinant virus of claim 28, wherein the recombinant virus is an adeno- associated virus.

34. The recombinant virus of claim 28, wherein the recombinant virus is an SV-40 virus.

35. The recombinant virus of claim 28, wherein the recombinant virus is a herpes virus.

36. The recombinant virus of claim 28, wherein the recombinant virus is a vaccinia virus.

37. The recombinant virus of claim 28, wherein the virus was generated in a packaging cell transfected with a viral vector genetically modified to encode human granulocyte-macrophage colony stimulating factor.

38. A genetically modified, proliferation-incompetent tumor cell that expresses at least one cytokine, wherein the cytokine is human granulocyte-macrophage colony stimulating factor, and wherein the genetic modification is by transduction with the recombinant virus of claim 28.

39. The tumor cell of claim 38, wherein the tumor cell is a human cell.

40. The tumor cell of claim 38, wherein the tumor cell is selected from the group consisting of an autologous tumor cell, an allogeneic primary tumor cell, and an allogeneic tumor cell line.

41. The tumor cell of claim 40, wherein the tumor cell is from a tumor cell line.

42. The tumor cell of claim 41, wherein the tumor cell line is a prostate tumor cell line.

43. The tumor cell of claim 42, wherein the prostate tumor cell line is selected from the group consisting of LnCaP cells, DU-145 cells, and PC-3 cells.

44. The tumor cell of claim 38, wherein the recombinant virus is an adenovirus.

45. The tumor cell of claim 44, wherein the recombinant adenovirus comprises the MD expression cassette.

46. A kit for stimulating a systemic immune response to a tumor or antigen thereof in a mammal, comprising the recombinant virus of claim 28 and a container for holding a tumor tissue or a portion thereof.

47. The kit of claim 46 further comprising an enzyme for separating the individual cells of the tumor or portion thereof.

48. The kit of claim 46, wherein the recombinant virus is provided in the form of a packaging cell transfected with a viral vector encoding human granulocyte- macrophage colony stimulating factor.