IMMUNIZATION AGAINST EPITOPES IN THE HUMAN-MELANOMA ANTIGEN GP100 FOLLOWING PATIENT IMMUNIZATION WITH SYNTHETIC PEPTIDES

gp100 is a melanocytic lineage-specific antigen recognized by tumor-in filtrating lymphocytes, the adoptive transfer of which is associated w ith tumor regression in melanoma patients. In this study, peripheral b lood mononuclear cells (PBMCs) were harvested from HLA-A2(+) melanoma patients before and after immunization with G9-209 (ITDQVPFSY), G9-280 (YLEPGPVTA), or G9-154 (KTWGQYWQV) peptides in Incomplete Freund's Ad juvant and were tested for the ability to be sensitized in vitro using PBMCs pulsed with the native peptides. In addition, PBMCs from patien ts receiving the G9-209 or G9-280 peptide were stimulated in vitro wit h peptides modified at anchor residues to enhance binding to HLA-AZ: G 9-209/2M (IMDQVPFSY) or G9-280-9V (YLEPGPVTV). In patients immunized w ith G9-209, a single in vitro restimulation with G9-209/2M resulted in the generation of specific antipeptide lymphocytes from seven of seve n postimmune PBMCs and only three of seven preimmune PBMCs. In patient s immunized with G9-280, a single in vitro restimulation with G9-280/9 V resulted in the generation of specific antipeptide lymphocytes from five of six postimmune PBMCs and four of six preimmune PBMCs. In almos t all cases, CTLs raised against modified epitopes were capable of rec ognizing targets displaying the native nonamers. Several anti-G9-209 a nd anti-G9-209/2M CTLs also demonstrated specific lysis of, and specif ic IFN-gamma release in response to, gp100(+)-established cell lines. Thus, using peptides modified to enhance immunogenicity for in vitro s timulation improved the sensitivity of immune monitoring of patients i mmunized with synthetic peptides. These results demonstrate that immun ization with a peptide derived from a tumor-associated protein such as gp100 can provoke a measurable antitumor immune response in cancer pa tients.