The cultured T-cell line TIL1200, established from the tumor-infiltrating lymphocytes (TILs) of a patient with advanced metastatic melanoma, recognized an antigen on most HLA-A2+ melanomas and on all HLA-A2+ cultured neonatal melanocytes in an HLA-A2 restricted manner but not on other types of tissues or cell lines tested. A cDNA encoding an antigen recognized by TIL1200 was isolated by screening an HLA-A2+ breast cancer cell line transfected with an expression cDNA library prepared from an HLA-A2+ melanoma cell line. The nucleotide and amino acid sequences of this cDNA were almost identical to the genes encoding glycoprotein gp100 or Pmel17 previously registered in the GenBank. Expression of this gene was restricted to melanoma and melanocyte cell lines and retina but was not expressed on other fresh or cultured normal tissues or other types of tumor tested. The cell line transfected with this cDNA also expressed antigen recognized by the melanoma-specific antibody HMB45 that bound to gp100. A synthetic 10-amino acid peptide derived from gp100 was recognized by TIL1200 in the context of HLA-A2.1. Since the administration of TIL1200 plus interleukin 2 resulted in regression of metastatic cancer in the autologous patient, gp100 is a possible tumor rejection antigen and may be useful for the development of immunotherapies for patients with melanoma.
The existence of lymphocytes within melanoma deposits that, when isolated, are capable ofrecognizing specific tumor antigens on autologous and allogeneic melanomas in a major histocompatibility complex (MHC) restricted fashion provides strong evidence that an immune response to cancer exists in humans (1-8). The ability of these tumor-infiltrating lymphocytes (TILs) to mediate the regression ofcancer when adoptively transferred into patients with metastatic melanoma attests to the clinical importance of the antigens recognized (9, 10). Characterization of these antigens may thus be important for development of strategies for cancer immunotherapy. ) have characterized two genes coding for melanoma antigens, MAGE-1 and tyrosinase, by using T-cell clones established from the peripheral blood of patients who were repetitively immunized in vivo with mutagenized tumor cells or whose peripheral blood lymphocytes were sensitized by repetitive in vitro stimulation with tumor. The strategy used in the present study attempted to identify the genes coding for tumor antigens that were recognized by TILs from tumor-bearing patients in the absence of immunization to enhance the possibility that genes would be identified that coded for antigens involved in the natural immune response against the growing cancer. Anti-melanoma T cells appear to be enriched in TILs probably as a consequence of clonal expansion and accumulation at the tumor site in vivo (14). We have now cloned and sequenced a gene coding for a shared, commonly expressed melanoma antigen, restricted by HLA-A2.1. § MATERIALS AND METHODS Generation of Cytotoxic T Lymphocytes (CTLs) and Culture of Cell Lines. CTLs were generated from excised tumor specimens by culturing a suspension of cells with interleukin 2 (IL-2) (6000 units/ml) (Chiron) for
SummaryFour melanoma proteins, MART-l, gpl00, tyrosinase, and tyrosinase-related protein-1 (gp75) were evaluated for recognition by HLA-A2-restricted melanoma-specific cytotoxic T lymphocytes (CTLs) derived from the tumor-infiltrating lymphocytes (TIL) of 10 different patients. 9 of 10 TIL recognized MART-l, 4 recognized gpl00 (including 3 that also recognized MART-l), but none of the TIL recognized tyrosinase or gp75. Based on the known HLA-A2.1 peptide binding motifs, 23 peptides from MART-1 were synthesized in an attempt to identify the epitopes recognized by TIL. Three peptides were recognized by TIL when pulsed on T2 target cells. One of the 9-met peptides, AAGIGILTV, was most effective in sensitizing the T2 cells for TIL lysis. This peptide was recognized by 9 of 10 HLA-A2-restricted melanoma-specific CTLs. Therefore, this peptide appears to be a very common immunogenic epitope for HLA-A2-restricted melanomaspecific TIL and may be useful for the development of immunotherapeutic strategies.
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