Melanoma-reactive human cytotoxic T lymphocytes (CTLs) mediate tumor regression in vivo through specific recognition of MHC-associated peptide epitopes, many of which are encoded by the melanocytic tissue differentiation proteins gp100/Pme117 and MART-1/Melan-A. Vaccines using these peptides may induce protective or therapeutic immunity against melanoma. Rational design of such approaches is aided by a clear understanding of the identity of these antigenic peptides; however, most CTL epitopes described to date were identified indirectly. Especially where these peptides may be used in human clinical trials for the treatment or prevention of cancer, there is substantial need for direct evaluation of HLA-A*0201-associated peptides from MART-1 and gp100 that are naturally processed and presented. To that end, we have isolated peptides directly from HLA-A*0201 molecules of human melanoma cells and have determined that naturally processed epitopes for HLA-A*0201-restricted, melanoma-reactive CTLs include the nonamers MART-1(27-35) (AAGIGILTV), gp100(154-162) (KTWGQYWQV), gp100(209-217) (ITDQVPFSV) and gp100(280-288) (YLEPGPVTA) and the decamer gp100(476-485) (VLYRYGSFSV). Among these, the one that appears to be most abundant at the cell surface is gp100(154-162) (KTWGQYWQV). The others are among the less abundant peptides. HLA-A*0201-restricted CTLs from one melanoma patient who has survived metastatic disease recognized MART-1(27-35) (AAGIGILTV), gp100(280-288) (YLEPGPVTA) and gp100(154-162) (KTWGQYWQV) and were cross-reactive on longer peptides that contained these nonamer sequences. These peptides, identified by both an indirect genetic approach and by a direct peptide approach, can be used for tumor vaccine strategies with confidence that they are identical to the naturally processed peptide epitopes presented at the surface of melanoma cells in association with HLA-A*0201 molecules.

To identify shared epitopes for melanoma-reactive CTL restricted by MHC molecules other than HLA-A*0201, six human melanoma patient CTL lines expressing HLA-A1 were screened for reactivity against the melanocyte differentiation proteins Pmel-17/gp100, MART-1/Melan-A, and tyrosinase, expressed via recombinant vaccinia virus vectors, CTL from five of the six patients recognized epitopes from tyrosinase, and recognition of HLA-A1(+) target cells was strongly correlated with tyrosinase expression, Restriction by HLA-A1 was further demonstrated for two of those tyrosinase-reactive CTL lines, Screening of 119 synthetic tyrosinase peptides with the HLA-A1 binding motif demonstrated that nonamer, decamer, and dodecamer peptides containing the sequence KCDICTDEY (residues 243-251) all reconstituted the CTL epitope in vitro, Epitope reconstitution in vitro required high concentrations of these peptides, which was hypothesized to be a result of spontaneous modification of cysteine residues, interfering with MHC binding, Substitution of serine or alanine for the more N-terminal cysteine prevented modification at that residue and permitted target cell sensitization at peptide concentrations 2 to 3 orders of magnitude lower than that required for the wild-type peptide, Because spontaneous modification of sulfhydryl groups may also occur in vivo, tumor vaccines using this or other cysteine-containing peptides may be improved by amino acid substitutions at cysteine residues.

The genetic origins of CD8+ T cell–recognized unique antigens to which mice respond when immunized with syngeneic tumor cells are unknown. The ultraviolet light-induced murine tumor 8101 expresses an H-2Kb-restricted immunodominant antigen, A, that induces cytolytic CD8+ T cells in vivo A+ 8101 cells are rejected by naive mice while A− 8101 tumor cells grow. To identify the antigen H-2Kb molecules were immunoprecipitated from A+ 8101 cells and peptides were eluted by acid. The sensitizing peptide was isolated by sequential reverse-phase HPLC and sequenced using microcapillary HPLC-triple quadruple mass spectrometry. The peptide, SNFVFAGI, matched the sequence of the DEAD box protein p68 RNA helicase except for a single amino acid substitution, caused by a single nucleotide change. This mutation was somatic since fibroblasts from the mouse of tumor origin expressed the wild-type sequence. The amino acid substitution created an anchor for binding of the mutant peptide to H-2Kb. Our results are consistent with mutant p68 being responsible for rejection of the tumor. Several functions of p68, which include nucleolar assembly and inhibition of DNA unwinding, may be mediated through its IQ domain, which was altered by the mutation. This is the first description of a somatic tumor–specific mutation in the coding region of a nucleic acid helicase.

Human CD8+ CTL recognize peptides bound to class I MHC molecules on the surface of melanoma cells. Several peptides derived from melanocyte lineage-specific proteins have been identified as epitopes for HLA-A2 restricted melanoma-reactive CTL. Because less than half of melanoma patients express HLA-A2, it is important to identify CTL epitopes restricted by other common MHC molecules including HLA-A1 and -A3. We have generated HLA-A3-restricted human CTL that recognize one or more shared melanoma Ags. All of the melanomas recognized by one of these CTL lines express Pmel-17/gp100, and those that fail to express this Ag are not lysed. This CTL line also specifically recognizes the lymphoblastoid line C1R-A3 following infection with a recombinant vaccinia encoding the melanocyte lineage-specific protein Pmel-17/gp100. Thus, at least one Pmel-17/ gp100 peptide is an epitope for this CTL line. We have identified ALLAVGATK (Pmel-17/gp100 residues 17-25) as an epitope for this CTL line and have shown that it is naturally processed and presented by HLA-A3 on melanoma cells. A second HLA-A3-restricted melanoma-reactive CTL line recognizes at least one additional shared epitope. These findings suggest that cellular immune responses directed against multiple shared melanoma epitopes exist in the 20 to 25% of melanoma patients who express HLA-A3. In addition, immunotherapy directed against Pmel-17/gp100 and other shared melanoma Ags may be useful in a large subset of these patients.

T lymphocytes recognize antigens consisting of peptides 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 of asparagine 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.