Recognition of Multiple Epitopes in the Human Melanoma Antigen gplOO by Peripheral Blood Lymphocytes Stimulated in Vitro with Synthetic Peptides

gplOO is a melanocyte lineage-specific antigen recognized by tumor-infiltrating lymphocytes whose adoptive transfer has been associated with tumor regression in patients with metastatic melanoma. The peripheral blood mononuclear cells of five melanoma patients were sensitized in vitro with synthetic peptides to elicit antigen-specific cytotoxic T lymphocyte (CTL) lines against four gplOO epitopes. These epitope-specific CTL lines were generated following weekly in vitro stimulation with the synthetic decamer G10476 (V-L-Y-R-Y-G-S-F-S-V) or the nonamers G9280 (Y-L-E-P-G-P-V-T-A), G9154 (K-T-W-G-Q-Y-W-Q-V), or G9209 (I-T-D-Q-V-P-F-S-V) pulsed onto autologous irradiated peripheral blood mononuclear cells. These lines grew as long as 4 months in culture in low-dose interleukin 2 (30 IU/ml) and exhibited antigen-specific, MHC class I-restricted lysis of peptide-pulsed T2 cells and HLA-A2+, gplOO+ established melanoma cell lines. G10476- and G9280-specific CTLs demonstrated specific release of granulocyte-macrophage-colony-stimnlating factor and tumor necrosis factor a in response to T2 cells pulsed with relevant peptide, as well as to gplOO+ melanoma cell lines. These results demonstrate that several peptides derived from the gplOO protein are presented on the surface of melanoma cells and are sufficiently immunogenic to generate, in vitro, potent CTLs capable of cytolysis and the secretion of cytokines. Therefore, for HLA-A2+ melanoma patients, these and possibly other gplOO peptides could represent good candidates for antigen-specific immunotherapy either singly or in a multivalent regimen.