It has been reported previously that antitumor cytolytic T lymphocyte (CTL) clones can be isolated from blood lymphocytes of HLA-A2 melanoma patients, after stimulation in vitro with autologous tumor cells, and that some of these CTL clones lyse most HLA-A2 melanomas. A first antigen recognized by such CTL clones was previously shown to be encoded by the tyrosinase gene. We report here the identification of another gene that also directs the expression of an antigen recognized on most melanomas by CTL clones that are restricted by HLA-A2. The gene, designated Melan-A, is unrelated to any known gene. It is 18 kb long and comprises five exons. Like the tyrosinase gene, it is expressed in most melanoma tumor samples and, among normal cells, only in melanocytes.

Lymphocytes of melanoma patients can be restimulated in vitro with autologous tumor cells to generate antitumor cytolytic T lymphocytes (CTL). Previous reports have indicated that, when such CTL are obtained from HLA-A2 melanoma patients, they often display broad reactivity on A2 melanoma cell lines. Such antitumor CTL clones, which appeared to recognize the same antigen, were isolated from two patients. We report here the cloning of a cDNA that directs the expression of the antigen recognized by these CTL. This cDNA corresponds to the transcript of the tyrosinase gene. The gene was found to be active in all tested melanoma samples and in most melanoma cell lines. Among normal cells, only melanocytes appear to express the gene. The tyrosinase antigen presented by HLA-A2 may therefore constitute a useful target for specific immunotherapy of melanoma. But possible adverse effects of antityrosinase immunization, such as the destruction of normal melanocytes and its consequences, will have to be examined before clinical pilot studies can be undertaken.

Transplantation experiments have demonstrated that most mouse tumors express antigens that can constitute targets for rejection responses mediated by syngeneic T lymphocytes. For human tumors, autologous cultures mixing tumor cells and blood lymphocytes or tumor-infiltrating lymphocytes have produced CD8+ and CD4+ cytolytic T cell (CTL) clones that recognize tumor cells specifically. Attempts to identify the target antigens by biochemical fractionation of tumor cells up to now have failed, with the important exception of the identification of underglycosylated mucins present on breast and pancreatic carcinomas. Gene transfection approaches have proved more successful. A gene family named MAGE codes for antigens recognized by autologous CTL on a melanoma tumor. These genes are not expressed in normal tissues except for testis. They are expressed in many tumors of several histological types. Differentiation antigens coded by genes such as tyrosinase are also recognized on human melanoma by autologous CTL. The identification of human tumor rejection antigens opens new possibilities for systematic approaches to the specific immune therapy of cancer.

SummaryT lymphocytes recognize antigens consisting ofpeptides presented by class I and II major histocompatibility complex (MHC) molecules. The peptides identified so far have been predictable from the amino acid sequences of proteins. We have identified the natural peptide target of a CTL clone that recognizes the tyrosinase gene product on melanoma cells. The peptide results from posttranslational conversion ofasparagine to aspartic acid. This change is of central importance for peptide recognition by melanoma-specific T cells, but has no impact on peptide binding to the MHC molecule. This posttranslational modification has not been previously described for any MHC-associated peptide and represents the first demonstration of posttranslational modification of a naturally processed class I-associated peptide. This observation is relevant to the identification and prediction of potential peptide antigens. The most likely mechanism for production of this peptide leads to the suggestion that antigenic peptides can be derived from proteins that are translated into the endoplasmic reticulum.C lass I molecules of MHC bind to peptides derived from intracellular pathogens or from proteins expressed in tumor cells, and present them on the cell surface to the host immune system (1-3). Identification of the specific peptides that constitute T cell epitopes has been difficult without prior knowledge of the source protein. However, peptides recognized by human melanoma--specific T cells have recently been identified from five proteins using two alternative strategies. One approach has been to generate genomic or cDNA libraries from tumor cells followed by transfection of progressively smaller subsets of these molecular clones into cells that express the appropriate MHC molecule, but not the tumor-specific epitope (4--14). Molecular clones that encode T cell epitopes are identified by their ability to reconstitute tumor-specific T cell recognition of the transfected cells. The exact T cell epitope is then identified by a combination of molecular subcloningThe contributions of the first two authors were equivalent and their order should be considered arbitrary. and the use of synthetic peptides based on the predicted amino acid sequence. This approach led to the identification of antigens encoded by genes whose expression is specific for tumors, such as MAGE and of other antigens related to melanocyte differentiation such as tyrosinase (4, 13). In the second approach, naturally occurring peptides associated with MHC molecules on the tumor cells are directly extracted, fractionated by HPLC, and used to reconstitute recognition by melanoma-specific CTL of a nonmelanoma cell expressing appropriate MHC molecules (15). The peptide epitope within a reconstituting peptide fraction is identified and sequenced by tandem mass spectrometry (16,17). Using this approach, a peptide, YLEPG-PVTA, from the protein Pmel-17/gp100, was identified as an epitope for HLA-A2.1-restricted, melanoma-specific CTL from multiple individuals (18).

SummaryWe have reported the identification of human gene MAGE-1, which directs the expression of an antigen recognized on a melanoma by autologous cytolytic T lymphocytes (CTL). We show here that CTL directed against this antigen, which was named MZ2-E, recognize a nonapeptide encoded by the third exon of gene MAGE-1 . The CTL also recognize this peptide when it is presented by mouse cells transfected with an HLA-A1 gene, confirming the association of antigen MZ2-E with the HLAA1 molecule. Other members of the MAGE gene family do not code for the same peptide, suggesting that only MAGE-1 produces the antigen recognized by the anti-MZ2-E CTL . Our results open the possibility of immunizing HLAA1 patients whose tumor expresses MAGE-1 either with the antigenic peptide or with autologous antigen-presenting cells pulsed with the peptide .