Mass-spectrometric evaluation of HLA-A*0201-associated peptides identifiesdominant naturally processed forms of CTL epitopes from MART-1 and gp100

Melanoma-reactive human cytotoxic T lymphocytes (CTLs) mediate tumor regres sion in vivo through specific recognition of MHC-associated peptide epitope s, many of which are encoded by the melanocytic tissue differentiation prot eins gp100/Pme117 and MART-1/Melan-A. Vaccines using these peptides may ind uce 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 iden tified indirectly. Especially where these peptides may be used in human cli nical trials for the treatment or prevention of cancer, there is substantia l 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 I molecules of human melanoma ce lls and have determined that naturally processed epitopes for HLA-A*0201-re stricted, melanoma-reactive CTLs include the nonamers MART-1(27-35) (AAGIGI LTV), 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 gp100154-16 2 (KTWGQYWQV), The others are among the less abundant peptides, HLA-A*0201- restricted CTLs from one melanoma patient who has survived metastatic disea se recognized MART-1(27-35) (AAGIGILTV), gp100(280-288) (YLEPGPVTA) and gp1 00(154-162) (KTWGOYWQV) and were crossreactive on longer peptides that cont ained these nonamer sequences. These peptides, identified by both an indire ct genetic approach and by a direct peptide approach, can be used for tumor vaccine strategies with confidence that they are identical to the naturall y processed peptide epitopes presented at the surface of melanoma cells in association with HLA-A*0201 molecules, Int. J. Cancer 82:669-677, 1999, (C) 1999 Wiley-Liss, Inc.