[CANCERRESEARcHst,5265—5268, October15,1994) Advances in Brief Identification of MART-i-specific T-Cell Receptors: T Cells Utilizing Distinct TCell Receptor Variable and Joining Regions Recognize the Same Tumor Epitope David J. Cole, Daniel P. Well, Peter Shamamlan, Licla Rivoltini, Yutaka Kawakami, Suzanne Topalian, Christopher Jennings, Slona Ellyahu, Steven A. Rosenberg, and Michael I. Nishlmura1 Surgery Branch, NO.tiOnOJ Cancer Institute, NIH, Bethesda, Mwyland 20892 Abstract Tumor-specific cytotoxic T lymphocytes (CTLs) can mediate tumor regression in patients with metastatic melanoma and play a central role in the Immune response to cancer. The recent Identification of shared mel anoma antigens has raised the possibifity of a limited melsnoina-speclflc T-cell receptor (TCR) repertoire, but subsequent studies have been con troversial and difficult to interpret without knowing which tumor-assod ateti antigens(TAAs)are beingrecognizedby specificTCRS.However,the recent cloning ofseveral melanoma TAAs now allows for the Identification ofthe specifically recognized TAA and Its epitope. We evaluated the TCR of two clonal CD8' CTL lines, A42 and 1E2, from two HLA-A2@pedents with metastatic melanoma. Both CTL lines were MART-i spedflc, and both demonstrate reactivity to the same epitope when presented in an HLA-A2.1 onntext The TCR genes of the two clones were sequenced. AU of the productively rearranged A42 TCR fi chain genes were Vfi7/D@32.1/ JfJ2.7IC@32; theTCRa chaingeneswereVa2lIJa42/CaThe1E2TCR @3 chain genes were V@33/DfI1.1/JfJ1.1/Cfi1, and TCR a chains were Va25/ JaM/Ca. This study Is the first report of TCR sequences specific for a melanoma epitope. These TCR clones may be usefUl for the development of more effective Immunotheraples and in studies of the mechanism of T-cell recognition oftumor antigen. They also provide direct evidence that the immunesystemcan providemorethanone TCRcapableof recogniz ing a TAA epitope. Introduction Melanoma-specific CFLs2 derived from ilLs are capable of mcdi ating tumor regressions in select patients with metastatic disease (1—2). In vitro, these T lymphocytes specifically lyse autologous and allogeneic melanoma cells sharing matched MHC restriction dc ments, suggesting the recognition of commonly expressed TAM (3—5).Recently, several different melanoma TAM have been de scribed based on their recognition by melanoma-specific CTL (6—11). These antigens are present in the majority of melanoma cell lines and are also expressed in normal melanocytes (tyrosinase, gplOO, and MART-i) or testes (MAGE-1, MAGE-3). Although the in vivo sig nificance of these antigens in the recognition of melanoma by T cells is yet to be defined, it is clear that CFL can play a centralrole in the immune response to melanoma. Antigen specific T-lymphocyte reactivity is provided by the TCR a and @3 chain heterodimer.Each chain is composed of a variable (V'), joining (J) and constant (C) region, as well as a diversity (D) region in the 13chain. The V-J and V-D-J junctions of the a and @3 chains code for the third complementarity-determining region, which plays Received 7/27/94; accepted 8/31)94. Thecostsof publication ofthisarticleweredefrayed inpartbythepayment of page charges. Thisarticlemusttherefore beherebymarkedadvertisement in accordance with 18 USC. Section 1734 solely to indicatethis fact. 1 To whom requests for reprints should be addressed, at Surgery Branch, National Cancer Institute, NIH@Building 10,Room 2B04, Bethesda, MD 20892. 2 The abbreviations used are: Cli, cytotoxic T lymphocyte; i'lL, tumor-infiltrating lymphocyte; MHC, major histocompatibiity complex; TAA, tumor-associated antigen; cDNA, complementary DNI¼ IFN-'y, -@‘ interferon; PCR, polymerase chain reaction; TCR, T-cell receptor. an importantrole in antigenspecificity (12, 13). Antigen recognition occurs via binding of the TCR to a complex composed of peptide, derived from an antigenic protein, presented in the context of an MHC molecule (14, 15). Until recently the actual identification of the structures of the TCR and peptideelements involved in T-cell recognitionof tumorantigens has been problematic. The presence of shared TAA in melanoma raised the possibility of a limited tumor antigen-specific TCR reper toire, and led to several conflicting studies of T-cell V gene usage in bulk TIL cultures (16—19).Even the subsequent use of clonal tumor reactive T-cell populations has demonstrated limited TCR usage in some studies (20, 21) and diverse V gene usage in others(22, 23). The possible recognition of several different tumor antigens by different T-lymphocyte clones makes the interpretation of these studies diffi cult without knowing which antigens and specific epitopes are being recognized. Recent cloning of several melanoma antigens recognized by T lymphocytes has been reported(6-11). Among these antigens, the MART-i antigen appearsto be the most prevalentbecause it is specifically recognized by the large majority of ilL cultures tested (24). The subsequentidentification of the iinmunodominantpeptide presented in the context of the MHC class I molecule HLA-A2 now makes possible the isolation of MART-i epitope-specific T-cell clones and their T-cdll receptors(24, 25). In this study we have identified the T-cell receptorsof two T-cell clones which recognize the same MART-i epitope. Each clone uses different variable and joining regions. These TCR clones may be useful for the development of more effective adoptive immunothera pies and in studies of the mechanisms of T-cell recognition of tumor antigen.They also provide direct evidence thatthe redundancyof the immune system can provide more than one TCR capable of recog rnzing a specific epitope on a tumor-associated antigen. Materials and Methods Generationof TIL Lines and Clonee.ilL weregenerated fromtumor biopsies of 2 patients with metastatic melanoma as described previously (2). clone A42 was established by limiting dilution at 100 cells/well, with a proliferating frequency of 1:800, and clone 1E2 was established at 1 cell/well with a proliferating frequency of 1:43. The clones were cultured in round bottomed microtiter plates with interleukin 2 (120 international units/mI) in the presence of feeders (1 x 10@allogeneic peripheral blood lymphocytes/well irradiated to 5000 rads) with weekly stimulation using 1X 10' autologous irradiated tumor cells/well. Cell Lines. Melanoma cell lines C32 and MaIme3M; breast carcinoma cell line MDA231 (American Type Qilture Collection, Rockville, MD); Ewing's sarcoma cell line RD-ES (M. Tsokos, NIH); COS-7 cells (W. Leonard, NIH); melanomacelllines397mel,5Olmel,526mel,624mel,677mel,7OSmel, and 888me1 [established in the Surgery Branch/National Cancer Institute labora tory as descrthed (5)J; and T2 cells (24) were maintained in RPMI with 10% fetal calf serum. Peptide Synthesis. Peptides (kindly provided by Drs. K. Sakaguchi and E. Appella,NIH)weresynthesizedby a solidphasemethodusinga multiple peptidesynthesizer (ModelAMS422;GilsonCo.,Inc,Worthington, OH)and 5265 Research. on January 14, 2022. © 1994 American Association for Cancer cancerres.aacrjournals.org Downloaded from Research. on January 14, 2022. © 1994 American Association for Cancer cancerres.aacrjournals.org Downloaded from Research. on January 14, 2022. © 1994 American Association for Cancer cancerres.aacrjournals.org Downloaded from