IMPROVED INDUCTION OF MELANOMA-REACTIVE CTL WITH PEPTIDES FROM THE MELANOMA ANTIGEN GP100 MODIFIED AT HLA-A-ASTERISK-0201-BINDING RESIDUES

Recognition of the melanoma Ag gp100 by tumor-infiltrating lymphocytes (TIL) in vitro has been correlated with tumor regression in patients with metastatic melanoma treated with the adoptive transfer of TIL plu s IL-2. Three common gp100 epitopes have been identified that are reco gnized in the context of HLA-A2 by TIL from different patients: G9(154 ) (KTWCQYWQV), C9(209) (ITDQVPFSV), and C9(280) (YLEPGPVTA). Upon stim ulation with these peptides, melanoma-reactive CTL could be induced in vitro from PBL of some HLA-A2(+) melanoma patients, However, numerous restimulations were required, and specific reactivity could not be ge nerated in many patients, Therefore, to enhance the immunogenicity of gp100 peptides, amino acid substitutions were introduced into G9(154), G9(209), and G9(280) at HLA-A0201-binding anchor positions, but not at TCR contact residues, to increase peptide class I MHC-binding affin ity. Several modified gp100 peptides bound with greater affinity to HL A-A0201 than unmodified peptides and were recognized by TIL specific for the natural epitopes, These peptides were used to sensitize PBL fr om HLA-A2(+) melanoma patients in vitro using peptide-pulsed autologou s PBMC as stimulators, After five weekly restimulations with either th e native C9(209) or G9(280) peptide, melanoma-reactive CTL could only be induced from two of seven patients, However, amino acid substitutio ns in these peptides enabled the induction of melanoma-reactive CTL fr om all seven patients, These results suggest that modified gp100 pepti des may be more immunogenic than the native epitopes, and may be usefu l in immunotherapy protocols for patients with melanoma.