ENHANCEMENT OF CELLULAR-IMMUNITY IN MELANOMA PATIENTS IMMUNIZED WITH A PEPTIDE FROM MART-1 MELAN-A

PURPOSE In this study, we tested the effectiveness of a melanoma-assoc iated antigen-derived peptide, MART-1(27-35), in eliciting cellular im mune responses in vivo in the context of a phase I active immunization protocol. This peptide (AAGIGILTV) corresponds to residues 27-35 from the nonmutated melanoma-associated antigen MART-1/Melan A and is reco gnized by most melanoma-specific, HLA-A0201-restricted, tumor-infiltr ating lymphocytes. To test the in vivo induction of cytotoxic T lympho cyte (CTL) sensitization, we compared CTL reactivity in vitro from per ipheral blood mononuclear cell (PBMC) pools obtained before and after vaccination. PATIENTS AND METHODS MART-1(27-35) was administered to HL A-A0201 melanoma patients subcutaneously in an emulsification with in complete Freund's adjuvant. A vaccination course included four inocula tions of peptide at 3-week intervals. PBMC collected by leukapheresis and separated by Ficoll-Hypaque gradient before and after vaccination were analyzed in 18 patients by in vitro sensitization with MART-1(27- 35). To induce MART-1(27-35)-specific CTL, PBMC were incubated with I mu M peptide (on day 0) and interleukin-2 (IL-2) (300 IU/mL, on days 1 and 4 after each stimulation). At weekly intervals, cells were harves ted and an aliquot was cryopreserved for later analysis. The remaining cells were replated and restimulated using irradiated autologous PBMC pulsed with 1 mu M of relevant peptide After three restimulations, al l samples from one patient were tested simultaneously for HLA-A0201-r estricted anti-MART-1(27-35) reactivity by microcytotoxicity and cytok ine (IFN-gamma) release assays. RESULTS Toxicities were minimal and co nsisted oflocal irritation at the site of vaccine administration. None of the patients sustained a clinical response. The first eight patien ts were monitored by inducing CTL reactivity from PBMC obtained preimm unization and after two and four vaccinations. Only two prevaccination cultures were reactive to MART-I, compared with five and seven cultur es from PBMC obtained after two and four vaccinations, respectively. T hus, an enhancement in cytotoxic activity could be detected in postvac cination CTL cultures, and serial vaccine administrations appeared to boost the detectability of cytotoxicity in vitro. For completeness, th e analysis compared prevaccination with postvaccination PBMC cultures. Specific anti-MART1(27-35) cytotoxicity (greater than or equal to 10 lyric units) could be detected in two prevaccination and 12 postvaccin ation cultures after two in vitro stimulations. In 15 postvaccination CTL cultures, a more than threefold increase in specific release of IF N-gamma was noted, compared with prevaccination. DISCUSSION In vivo ad ministration of a melanoma-associated antigen peptide, emulsified in i ncomplete Freund's adjuvant, could safely augment CTL reactivity again st epitopes commonly expressed by melanoma cells. Although the enhance ment of CTL reactivity did not achieve tumor regression, it is possibl e that the use of recombinant immunogens with increased immunomodulato ry capabilities in future clinical trials could reach the threshold of CTL activation necessary for tumor regression.