INDUCTION OF TUMOR-REACTIVE CTL FROM PERIPHERAL-BLOOD AND TUMOR-INFILTRATING LYMPHOCYTES OF MELANOMA PATIENTS BY IN-VITRO STIMULATION WITH AN IMMUNODOMINANT PEPTIDE OF THE HUMAN-MELANOMA ANTIGEN MART-1

MART-1 is an Ag expressed on melanomas and melanocytes, and is recogni zed by the majority of HLA-A2-restricted tumor-specific tumor-infiltra ting lymphocytes (TIL) from melanoma patients. In the present study we have analyzed 10 potential 9-mer epitopes containing the HLA-A2.1 bin ding motifs for their ability to induce melanoma-specific T cell lines . Antimelanoma CTL could be generated only with MART-1(27-35) peptide, which has been previously shown to be recognized by a majority of HLA -A2-restricted TIL. Anti-MART-1(35-43)-specific CTL could also be indu ced, but these T cells did not recognize melanoma cells. MART-1(27-35) -specific CTL could be effectively generated from a total of 11 of 12 PBL and from 3 of 3 TIL derived from HLA-A2(+) melanoma patients, as w ell as from 2 of 4 PBL from HLA-A2(+) healthy donors by in vitro stimu lation with autologous PBMC pulsed with the synthetic MART-1(27-35) pe ptide. These CTL lines specifically lysed and release cytokines (TNF-a lpha, IFN-gamma, and GM-CSF) in response to T2 cells pulsed with MART- 1(27-35), as well as to HLA-A2(+) MART-1(+) melanoma cells. CTL genera ted with MART-1(27-35) also lysed uncultured HLA-A2(+) melanoma cells derived from tumor biopsies, indicating that this MART-1 epitope is li kely to be expressed in association with HLA-A2 on the surface of tumo r cells in vivo. CTL lines generated with MART-1(27-35) mediated 25- t o 100-fold higher lytic activity than MART-1-reactive CTL grown from T IL in the presence of high dose IL-2. These results demonstrate that M ART-1(27-35) peptide may represent an ideal candidate for Ag-specific immunotherapy in melanoma patients.