Isolation of human melanoma antigens recognized bt T cells for development of immunotherapy and gene therapy

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A total of 123 tumor infiltrating T lymphocytes (TIL) established from patients with metastatic melanoma was screened for identification of T cells that might recognize novel melanoma antigens or epitopes in previously identified antigens. Using 5 TIL that possibly recognized new antigens in the context of HLA-A 1, -A2 or -A3, antigens were isolated by cDNA expression cloning. New epitopes of the previously identified antigens (1 HLA-A1 binding tyrosinase peptide, 2 HLA-A2 binding gp100 peptides, 1 HLA-A3 binding gp100 peptide) were identified. Replacement of either cysteine to β-amino butyric acid in the gp100 peptide, RLPRIFCSC, enhanced the T cell recognition, suggesting that unoxidized cysteines were presented on the tumor cell surface. Since oxidation of the cysteines may easily occur in the synthetic peptides in in vitro culture, the modification of cysteines may have important implications for the development of peptide based vaccines. Using another HLA-A 1 restricted T cells, 8B6 antigen was isolated. The cDNA for the ubiquitously expressed 8B6 encoded an uncharacterized protein with a phosphate binding loop (P-loop) motif. A mutation was found in the P-loop of the 8B6 cDNA obtained from the 1362mel melanoma cell line, and the mutated 8B6 lost GTP binding ability. A T cell epitope with a glutamic acid encoded by the mutated sequence was identified, and the wild type peptide was not recognized by TIL 1362, suggesting that this T cell response was autologous tumor specific. Since many of the mutated antigens previously isolated with CD8+ T cells, including β-catenin, CDK4 and caspase 8, appeared to be involved in tumorigenesis, the mutated 8B6 may also be involved in the generation of melanoma possibly through inability to bind GTP. These newly identified melanoma antigens may be useful for development of immunotherapy as well as for understanding the biology of melanoma.