THE IMMUNOTHERAPY OF SOLID CANCERS BASED ON CLONING THE GENES ENCODING TUMOR-REJECTION ANTIGENS 1

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1 481 Annu. Rev. Med : THE IMMUNOTHERAPY OF SOLID CANCERS BASED ON CLONING THE GENES ENCODING TUMOR-REJECTION ANTIGENS 1 Steven A. Rosenberg, M.D., Ph.D. Department of Health and Human Services, National Cancer Institute, National Institutes of Health, Bethesda, Maryland KEY WORDS: interleukin-2, gp100, MART-1, melanoma, lymphocytes ABSTRACT Cellular immune reactions play a major role in the host reaction to growing cancers. Tumor infiltrating lymphocytes (TIL) can be isolated from melanomas and can specifically recognize unique tumor antigens. The adoptive transfer of TIL plus interleukin-2 can mediate tumor regression in patients with metastatic melanoma. TIL capable of mediating tumor regression have been used to clone and sequence a variety of the genes that encode the tumor-regression antigens recognized by these TIL. This information has opened new opportunities for the development of cancer immunotherapies. These gene products can be used to generate lymphocytes, in vitro, with improved antitumor activity for use in adoptive transfer. Active immunization can be performed using either the immunodominant peptides present in these proteins or by incorporating the tumor antigen genes into recombinant viruses. Cancer vaccine trials using many of these approaches have recently begun. Attempts to apply a similar strategy to epithelial tumors such as breast and ovarian cancer are underway. INTRODUCTION The rejection of tissue allografts and experimental murine tumors is mediated by cellular immune reactions. Thus, studies of cancer immunotherapy have 1 The US Government has the right to retain a nonexclusive, royalty-free license in and to any copyright covering this paper.

2 482 IMMUNOTHERAPY OF SOLID CANCERS traditionally concentrated on stimulating cellular immune reactions using either nonspecific or specific immune stimuli. Nonspecific immune stimulants such as bacillus Calmette Guerin (BCG), Corynebacterium parvum, muraramyl dipeptide, and other bacterial and synthetic substances have been administered to cancer patients with the expectation that the nonspecific stimulation resulting from these agents would lead to a concomitant increase in putative immune reactions against growing cancers. Attempts to more specifically target immune stimulation to growing cancer cells have involved injection either with intact irradiated cancer cells, or subfractions of these cells, or with chemically or genetically modified cancer cells. In the vast majority of experimental tumor models, these manipulations had little impact, and thus far they have found little application in the treatment of cancer in humans. More recently, the application of recombinant DNA techniques has allowed the production of large amounts of biologic molecules involved in immune regulation. These reagents provide a more potent means of stimulating endogenous immune reactions. In clinical trials assessing the administration of recombinant interleukin-2 (IL-2), approximately 20% of patients with metastatic melanoma or metastatic renal cell cancer showed objective clinical responses to treatment. IL-2 has been approved by the Food and Drug Administration as a treatment for patients with metastatic renal cell cancer (1). The administration of alpha interferon can also cause objective responses in selected patients and is approved by the Food and Drug Administration for the treatment of selected hematologic malignancies (2). These approaches, however, still often lack the specificity required to stimulate immune reactions against antigens that may be present on some cancers. The recent cloning of genes encoding cancer antigens in patients with melanoma has resulted in the identification of specific proteins and their immunodominant epitopes that appear to be the major targets of the reaction of the immune system against growing cancers (3, 4). This identification of the molecular nature of specific tumor antigens has opened new possibilities for the development of cancer immunotherapies. IL-2 AND TUMOR INFILTRATING LYMPHOCYTES: CLINICAL ASPECTS IL-2 is a cytokine that plays a major role in immune regulation. IL-2 has no direct impact on cancer cells and in preclinical models was shown to mediate all of its antitumor effects by stimulating host immune reactions to the growing cancer. These preclinical studies led to clinical evaluation of IL-2 as an anticancer agent. Patients with metastatic melanoma and metastatic renal cell cancer exhibited significant clinical responses to IL-2 administration in early clinical studies and this led to extensive evaluation of IL-2 treatment in pa-

3 tients with these diagnoses (5 7). In a series of 283 consecutive patients with metastatic melanoma or renal cell cancer treated with high-dose, bolus IL-2 (8), objective clinical responses were seen in 17 and 20% of patients, respectively; 7 and 9% of patients, respectively, experienced complete cancer regression (Table 1). Of 24 patients who underwent complete regression of their metastatic cancer, 19 patients remain in complete response at 28 to 103 months after initiation of therapy (Table 2). The presence of significant clinical responses in patients to the administration of IL-2 combined with the known effects of IL-2 on stimulating T-cell immune responses suggested that in cancer patients T lymphocytes existed that were capable of recognizing tumor-associated antigens. These observations stimulated attempts to identify the molecular nature of the antigens recognized by the cellular immune system. Studies of tumor infiltrating lymphocytes (TIL) have provided a means for identifying cancer-related antigens. TIL are lymphoid cells that infiltrate into solid tumors and can be grown from single cell suspensions of these tumors by incubation in IL-2. In approximately 50% of patients with melanoma, TIL exhibit specific immune recognition of tumor antigens present on fresh and cultured autologous melanoma cells (9 11). Several characteristics of the recognition of tumor cells by TIL were relevant to the clinical use of these cells. TIL appeared to recognize tumor cells in a major histocompatibility complex (MHC)-restricted fashion. Thus, TIL obtained from patients who were HLA-A2 were capable of recognizing autologous tumor cells but also often recognized allogeneic melanomas that shared HLA-A2 (12, 13). Thus, it appeared that a common melanoma tumor antigen was present on the surface of melanoma cells. The presence of widely shared melanoma antigens was verified by studies in which genes encoding HLA-A2 antigens were transfected into non-hla-a2 melanoma cells. Transfection conferred susceptibility to recognition by HLA-A2-specific TIL (14, 15). The presence of shared antigens restricted by other HLA antigens such as HLA- A24 and HLA-A31 was also demonstrated (12, 16). TIL did not recognize other tumor types or normal cells or non-mhc-matched melanomas. A notable exception, however, was the recognition by melanoma-reactive TIL of normal cultured melanocytes in an HLA-restricted fashion, which indicated Table 1 Results of immunotherapy with high-dose bolus IL-2 in patients with advanced cancer a ROSENBERG 483 Diagnosis Total CR PR CR + PR Melanoma (7%) 13 (10%) 23 (17%) Renal cell cancer (9%) 16 (11%) 30 (20%) Total (9%) 29 (10%) 53 (19%) a Shows number of patients; percentage of patients in parenthesis. CR, complete regression; PR, partial regression.

4 484 IMMUNOTHERAPY OF SOLID CANCERS Table 2 Duration of response of patients with advanced cancer treated with high-dose bolus IL-2 a Diagnosis CR PR Melanoma 103+, 57+, 56+, 47+, 45+, 36+, 36+, 35+, 16, 12 35, 31, 19 17, 10, 8, 8, 7, 5, 5, 4, 4, 2 Renal cell cancer 99+, 91+, 88+, 87+, 59+, 48+, 44+, 44+, 35+, 35+, 52, 30, 30, 24, 22, 21, 16, 14,13,11,8,8,7,6,4,4 26+, 28+, 19, 19 a Shows length of regression in months. Of 24 patients with complete regression (CR), 19 remain in CR at 28 to 103 months. PR, Partial regression. that TIL were recognizing melanocyte differentiation antigens expressed on melanomas (17, 18). In clinical trials, TIL were administered to cancer patients along with their requisite growth factor, IL-2 (19, 20). The results of the adoptive transfer of TIL plus IL-2 to 86 patients with metastatic melanoma is shown in Table 3. Of the 86 patients, 34% experienced objective cancer regressions. Responses were seen in patients who had previously failed IL-2 alone. Thus, it appeared that TIL were capable of recognizing tumor-associated antigens modulating in vivo cancer regression. Attempts were then made to identify the genes in melanoma cells that encoded the antigens recognized by these clinically effective TIL. Other methods for generating lymphocytes reactive with tumor-associated antigens on melanomas have been described. The repeated in vitro sensitization of peripheral blood lymphocytes from melanoma patients by using autologous or HLA-matched melanoma cells can give rise to lymphocyte populations with reactivity to melanoma. Boon and colleagues utilized the peripheral blood lymphocytes of a patient who received multiple immunizations with mutagenized cancer cells to raise in vitro lymphocyte lines reactive with tumor-associated antigens. They have subsequently used these cell lines to identify a series of cancer-related antigens (3, 21). A major advantage of using TIL to identify tumor-associated antigens is that it is possible to use TIL capable not only of recognizing cancer cells in Table 3 Treatment of patients with metastatic melanoma using TIL plus IL-2 a Patients Total Objective response Received prior IL (32%) No prior IL (34%) Total b (34%) a Shows number of patients. Percentage of patients in parenthesis. b Some patients (57) also received a single dose of cyclophosphamide; response rates were similar with (35%) and without (31%) cyclophosphamide.

5 vitro, but also of mediating anticancer responses when adoptively transferred to melanoma patients. Thus, the antigens recognized by TIL are more likely to be those antigens involved in vivo in anti-cancer responses. IDENTIFICATION OF GENES ENCODING TUMOR ANTIGENS Lymphocytes with specific reactivity against melanoma were used to screen complementary (c) DNA or genomic libraries obtained from melanoma cells. Transfection of these genes into target cells expressing the appropriate MHC restriction element and subsequent testing for reactivity with tumor-specific lymphocytes has led to the identification of a variety of genes encoding tumor antigens (22 27). A summary of many of the genes isolated in the past several years is shown in Table 4. The cdna and amino acid sequence of these antigens have been defined. Table 4 Human melanoma antigens recognized by T cells Antigen Alternate name Presenting MHC Epitope TIL used for recognition Melanocyte lineage proteins gp100 HMB45 HLA-A2 KTWGQYWQV 1200 and 660 HMB-50 HLA-A2 ITDQVPFSV 620 NKI/beta HLA-A2 YLEPGPVTA 660 and 1143 HLA-A2 LLDGTATLRL 1200 HLA-A2 YRYGSFSV 660 MART-1 Melan-A HLA-A2 AAGIGILTV 1235 and others HLA-A2 ILTVILGVL TRP-1 gp75 HLA-A31 MSLQRQFLR 586 Tyrosinase HLA-A2 HLA-A2 MLLAVLYLL None a HLA-A2 YMNGTMSQV None a HLA-A24 AFLPWHRLF 1413 HLA-DR4 unknown 1088 Proteins expressed in cancers and testis MAGE-1 HLA-A1 EADPTGHSY None a HLA-Cw16 SAYGEPRKL None a MAGE-3 HLA-A1 EVDPIGHLY None a HLA-A2 FLWGPRALV None a BAGE HLA-Cw16 AARAVFLAL None a ROSENBERG 485 Tumor-specific antigens ß-Catenin HLA-A24 SYLDSGIHF 1290 p15 HLA-A24 AYGLDFYIL 1290 a These antigens were identified using peripheral lymphocytes repeatedly sensitized in vitro to tumors.

6 486 IMMUNOTHERAPY OF SOLID CANCERS The recognition of antigen by CD8 + T cells involves recognition of degraded peptides derived from intracellular proteins. CD8 + T cells recognize peptides of 8 10 amino acids in association with HLA class-i molecules, and CD4 + T cells recognize slightly larger peptides presented with surface HLA class-ii molecules. By pulsing individual peptides onto target cells that lack antigen expression but express the appropriate HLA molecule, it has been possible to identify the peptide fragments that represent the immunodominant epitopes in these tumor antigens (22, 28 30). These immunogenic peptides have been identified for most of the tumor antigens listed in Table 4. These tumor antigens fall into several general categories. Some melanoma tumor antigens are encoded by normal genes that are also expressed in melanocytes. These antigens, such as gp100, MART-1, TRP-1, and tyrosinase, represent differentiation antigens also present on normal melanocytes and retinal cells but not on other normal cells or on other tumor types (22, 28 30). A second class of antigens are those expressed on a variety of cancers and also on normal testis. The MAGE-1 antigen, for example, is present in about 30% of melanomas, sporadically in other tumors, and in normal testis (21). A third class of antigens represents those that are specifically recognized on tumors either because of mutations [e.g. ß-catenin (P Robbins, Y Kawakami, SA Rosenberg, submitted for publication)], or because of what appears to be selective expression on melanomas but not normal cells [e.g. p15 (26)]. The administration of TIL to patients provided an opportunity to identify those T cells capable of mediating tumor destruction in vivo. The identification of the antigens recognized by these clinically useful TIL maximized the chances that the antigens thus identified were clinically relevant. Of particular interest in this group is the gp100 antigen, which is recognized by a series of different TIL, each of which recognizes one or more epitopes in the gp100 protein (22). The ability to recognize the gp100 protein appears to be associated with clinical response in HLA-A2 + patients (P = 0.005). Of 10 HLA-A2- positive patients treated with tumor-specific TIL, four reacted with gp100 and all responded. None of the six patients treated with TIL nonreactive to gp100 exhibited objective responses. The expression of MART-1 and gp100 on melanocytes, as well as on melanomas, helps explain the presence of vitiligo in some melanoma patients treated with IL-2-based immunotherapies. In a prospective review of National Cancer Institute, Surgery Branch, patients treated with IL-2, none of 104 renal cell cancer patients followed for at least one year after receiving IL-2 developed vitiligo, compared with 12 cases of vitiligo in 69 patients with melanoma. Of further interest was the association of vitiligo with clinical response. None of the 27 nonresponding patients developed vitiligo, whereas 12 of 42 (29%) responding patients had evidence of vitiligo (P = 0.002).

7 ROSENBERG 487 The presence of TIL reactive with normal differentiation antigens in melanoma patients raises the interesting question of the mechanisms by which melanomas can break tolerance to these normal tissue antigens. The overexpression of genes encoding these differentiation antigens on tumors, or the unique inflammatory reactions that occur at tumor sites, might be involved in the breaking of self-tolerance. If specific differentiation proteins from melanocytes can serve as tumor-rejection antigens, then the possibility exists that other tissue-specific proteins in tumors derived from other nonessential organs can also serve as targets for immunotherapy. Possible examples include tumors arising in the thyroid, ovary, testis, breast, and prostate. The gene encoding ß-catenin presented on HLA-A24 represents a mutation of a normal protein. This cell adhesion protein serves as a tumor antigen and may also play a role in the malignant phenotype of these cells. This finding suggests that mutations in other genes involved in tumorigenesis, such as p53, ras, and others, might also serve as tumor antigens. OPPORTUNITIES FOR THE DEVELOPMENT OF NEW CANCER THERAPIES BASED ON THE IDENTIFICATION OF THE GENES ENCODING CANCER-REJECTION ANTIGENS Current cancer immunotherapies fall into passive and active categories. In passive immunotherapy, patients receive the administration of immune reagents (such as antibodies or immune cells) capable of recognizing tumor cells and that lead directly or indirectly to tumor destruction. With rare exceptions, passive immunotherapy with antibodies has not been successful. The adoptive transfer of immune lymphocytes has been shown to be capable of causing the rejection of an established growing tumor in mice and in human beings with melanoma (19, 20). Active immunotherapy relies on the immunization of the tumor-bearing host with materials that express tumor antigens. Most attempts to apply this approach have utilized either irradiated intact cancer cells or cancer cell fragments that contain a multitude of irrelevant proteins. More recently, attempts to genetically modify tumor cells, to increase their immunogenicity, have been widely applied. Only minor therapeutic activity has been seen with active immunotherapy in animals, and no successful consistent application of this approach to human beings has been reported. The recent identification of the genes encoding melanoma tumor antigens has opened a variety of new opportunities for the development of specific immunotherapies based on both passive and active approaches. A summary of some of these opportunities is presented in Table 5. A major challenge to the development of improved passive immunotherapies for the treatment of cancer has been the need to develop antitumor lym-

8 488 IMMUNOTHERAPY OF SOLID CANCERS Table 5 Opportunities for the development of cancer immunotherapies based on cloning the genes encoding cancer antigens Passive Immunotherapy: Generation of antitumor lymphocytes 1. In vitro sensitization using repeated exposure of peripheral blood lymphocytes or TIL to: a. Immunodominant tumor peptides pulsed on antigen-presenting cells b. Antigen presenting cells transduced with genes encoding tumor antigens 2. Genetic modification of lymphocytes a. Genes encoding cytokines such as tumor necrosis factor or GM-colony stimulating factor. b. Genes encoding T-cell receptors capable of recognizing tumor antigens 3. Genetic modification of bone marrow-derived hematopoietic stem cells with the genes encoding T cell receptors capable of recognizing tumor antigens Active Immunotherapy 1. Purified tumor antigens obtained by expression of genes in E. coli, yeast, or baculovirus 2. Immunodominant peptides synthesized in vitro 3. Combination of tumor antigens or peptides with adjuvants, linked to lipids, pulsed on antigen presenting cells, etc 4. Naked DNA encoding tumor antigens 5. Antigen presenting cells expressing tumor antigens 6. Viruses (or other live vectors such as BCG) encoding tumor antigens a. Administered alone or with cytokines to enhance immunization b. Viruses encoding tumor antigens plus genes encoding cytokines, costimulatory, molecules, adhesion molecules, etc. phocytes with increased ability to recognize cancer-related antigens. Rivoltini et al demonstrated that repeated exposure of peripheral blood lymphocytes from melanoma patients to antigen-presenting cells pulsed with the immunodominant peptides of melanoma antigens such as MART-1 can lead to immune lymphocytes with times the per-cell potency of conventional TIL (31). Clinical trials utilizing the adoptive transfer of these in vitro sensitized lymphocytes are about to begin. The identification of the genes encoding cancer-rejection antigens has facilitated the identification of the T-cell receptors present on lymphocytes capable of reacting with these antigens. The genes encoding the alpha and beta chains of the T-cell receptor capable of recognizing the MART-1 immunodominant epitope have been cloned and sequenced (32 34). These genes have been inserted into alternative effector cells, which then acquired the ability to react with the MART-1 antigen (35). The genetic modification of lymphocytes or hematopoietic stem cells with these T-cell receptor genes is another approach to generating immune cells capable of reacting with tumor. New opportunities are being explored for active immunization against tumor-associated antigens that utilize the genes encoding cancer-rejection antigens or the purified protein products derived from these genes. By insertion of the genes encoding cancer-rejection antigens into high-efficiency expression systems such as Escherichia coli, yeast, or baculovirus, large amounts of

9 ROSENBERG 489 purified tumor antigens can be isolated and used for in vivo immunization. Similarly, the immunodominant peptides present in these proteins can be synthesized in large amounts and used for immunization either alone or in conjunction with a variety of adjuvants capable of improving the cellular immune response to peptides or proteins. Clinical protocols that utilize immunization with the MART-1 immunodominant peptide or with three of the immunodominant gp100 peptides emulsified in incomplete Freund s adjuvant have begun at the National Cancer Institute in the past year. Purified DNA encoding cancer-rejection antigens can be directly injected into tissue where the genes are transcribed and translated. This approach is capable of generating immune lymphocytes directed against the products of recombinant genes in experimental murine systems (36). This naked DNA can be injected intramuscularly or can be directly microinjected into cells using ballistic gene gun techniques. The greatest flexibility for immunization using the genes encoding cancerrejection antigens includes insertion of genes into live immunizing vectors (4, 37, 38). Recombinant viruses such as vaccinia, adenovirus, or fowlpox encoding cancer-rejection antigens have been developed for use in human immunization protocols. Second-generation viruses simultaneously encoding cancerrejection antigens and cytokines, adhesion molecules, or costimulatory factors represent additional approaches to improve the ability to immunize with virus. Active immunization can also be combined with passive immunization by isolating and expanding in vitro lymphoid cells from patients following active immunization in an attempt to obtain cells with increased activity against the cancer-rejection antigens. CONCLUDING COMMENTS Significant progress has been made in the past several years in identifying the molecular components of the immune response to human cancer. The genes encoding several cancer-rejection antigens have been cloned, as have the genes encoding the receptors that recognize these antigens. Studies utilizing recombinant DNA technology combined with increasing knowledge of the regulation of the cellular immune response to tumor antigens has led to an increased ability to manipulate the immune system and has opened significant new opportunities for cancer immunotherapy. Many of these approaches are currently being explored in clinical protocols. Any Annual Review chapter, as well as any article cited in an Annual Review chapter, may be purchased from the Annual Reviews Preprints and Reprints service ; ; arpr@class.org

10 490 IMMUNOTHERAPY OF SOLID CANCERS Literature Cited 1. Marincola FM, Rosenberg SA Melanoma. In Biologic Therapy of Cancer, ed. VT DeVita, S Hellman, SA Rosenberg, p Philadelphia: Lippincott. 2nd ed. 2. Foon KA Hairy cell leukemia, chronic myelogenousleukemia, and myeloproliferative disorders. In Biologic Therapy of Cancer, ed. VT DeVita, S Hellman, SA Rosenberg, p Philadelphia: Lippincott. 2nd ed. 3. Boon T Tumor antigens reocgnized by cytolytic T lymphocytes present perspectives for specific immunotherapy. Int. J. Cancer 54: Rosenberg SA A new perspective: the development of new cancer therapies based on the molecular identification of cancer regression antigens. Cancer J. Sci. Am. 1: Rosenberg SA, Lotze MT, Muul LM, et al Observations on the systemic administration of autologous lymphokine-activated killer cells and recombinant interleukin-2 to patients with metastatic cancer. N. Engl. J. Med. 313: Lotze MT, Chang AE, Seipp CA, et al High dose recombinant interleukin-2 in the treatment of patients with disseminated cancer: responses, treatment related morbidity and histologic findings. J. Am. Med. Assoc. 256: Lotze MT, Rosenberg SA Interleukin-2 therapy for disseminated cancer. JAMA 257: Rosenberg SA, Yang JC, Topalian SL, et al Treatment of 283 consecutive patients with metastatic melanoma or renal cell cancer using high-dose bolus interleukin-2. JAMA 271: Itoh K, Tilden AB, Balch CM Interleukin-2 activation of cytotoxic T-lymphocytes infiltrating into human metastatic melanomas. Cancer Res. 46: Muul LM, Spiess PJ, Director EP, Rosenberg SA Identification of specific cytolytic immune responses against autologous tumor in humans bearing malignant melanoma. J. Immunol. 138: Topalian SL, Solomon D, Rosenberg SA Tumor-specific cytolysis by lymphocytes infiltrating human melanomas. J. Immunol. 142: Hom SS, Topalian SL, Simoni ST, et al Common expression of melanoma tumor-associated antigens recognized by human tumor-infiltrating lymphocytes: analysis by human lymphocyte antigen restriction. J. Immunother. 10: Darrow TL, Slingluff CL, Seigler HF The role of class I antigens in recognition of melanoma cells by tumor-specific cytotoxic T lymphocytes: evidence for shared tumor antigens. J. Immunol. 142: Kawakami Y, Zakut R, Topalian SL, et al Shared human melanoma antigens. Recognition by tumor infiltrating lymphocytes in HLA-A2.1 transfected melanomas. J. Immunol. 148: O Neil BH, Kawakami Y, Restifo NP, et al Detection of shared MHC-restricted human melanoma antigens after vaccinia virus-mediated transduction of genes coding for HLA. J. Immunol. 151: Topalian SL, Hom SS, Kawakami Y, et al Recognition of shared melanoma antigens by human tumor infiltrating lymphocytes. J. Immunother. 12: Kawakami Y, Nishimura I, Restifo NP, et al T-cell recognition of human melanoma antigens. J. Immunother. 14: Anichini A, Maccalli C, Mortarini R, et al Melanoma cells and normal melanocytes share antigens recognized by HLA- A2 restricted cytotoxic T cell clones from melanoma patients. J. Exp. Med. 177: Rosenberg SA, Packard BS, Aebersold PM, et al Use of tumor infiltrating lymphocytes and interleukin-2 in the immunotherapy of patients with metastatic melanoma. Preliminary report. N. Engl. J. Med. 319: Rosenberg SA, Yannelli JR, Yang JC, et al Treatment of patients with metastatic melanoma using autologous tumor-infiltrating lymphocytes and interleukin-2. J. Natl. Cancer Inst. 86: Van der Bruggen P, Traversari C, Chomez P, et al A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science 254: Kawakami Y, Eliyahu S, Delgado CH, et al Identification of a human melanoma antigen recognized by tumor infiltrating lymphocytes associated with in vivo tumor rejection. Proc. Natl. Acad. Sci. USA 91: Kawakami Y, Eliyahu S, Delgado CH, et al Cloning of the gene coding for a shared human melanoma antigen recognized by autologous T cells infiltrating into tumor. Proc. Natl. Acad.Sci. USA 91: Robbins PF, El-Gamil M, Kawakami Y, et al Recognition of tyrosinase by tumor infiltrating lymphocytes from a patient responding to immunotherapy. Cancer Res. 54: Topalian SL, Rivoltini L, Mancini M, et al Human CD4 + T cells specifically recognize a shared melanoma-associated

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