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 recognized by the majority of HLA-A2-restricted tumor-specific tumor-infiltrating lymphocytes (TIL) from melanoma patients. In the present study we have analyzed 10 potential 9-mer epitopes containing the HLA-A2.1 binding 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 induced, 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 well as from 2 of 4 PBL from HLA-A2⁺ healthy donors by in vitro stimulation with autologous PBMC pulsed with the synthetic MART-1_27-35 peptide. These CTL lines specifically lysed and release cytokines (TNF-α, IFN-γ, 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 generated with MART-1_27-35 also lysed uncultured HLA-A2⁺ melanoma cells derived from tumor biopsies, indicating that this MART-1 epitope is likely to be expressed in association with HLA-A2 on the surface of tumor cells in vivo. CTL lines generated with MART-1_27-35 mediated 25- to 100-fold higher lytic activity than MART-1 -reactive CTL grown from TIL in the presence of high dose IL-2. These results demonstrate that MART-1_27-35 peptide may represent an ideal candidate for Ag-specific immunotherapy in melanoma patients.