The major objective of this proposal is to develop a more effective form of active specific immunization with a vaccine made from synthetic peptides loaded on to dendritic cells in the human melanoma model. We have shown that a peptide and antigen presenting cell-based vaccine can induce a peptide specific and autologous melanoma reactive cytolytic T lymphocyte (CTL) response in melanoma patients. We have observed that in vitro stimulation of PBL from the immunized patients led to the rapid generation of peptide specific CTL, the CTL activities, however, often precipitously declined after repeated stimulation with the development of a Th2 type response. There was preliminary evidence of apoptosis in the process. These suggest that in vivo expansion of antigen specific CTL might also be regulated by a Th2 type response or through activation induced cell death (AICD). Hence, the proposed study will examine the mechanism of the decline of the CTL response with particular reference to the role of the Th2 cells and the role of AICD. Further, we shall test the hypothesis that a more effective peptide/APC-based immunization can be achieved with a number of innovations to the approach (such as by using dendritic cells, higher peptide loading, a different format of immunization consisting of priming and boosters, pretreating with cyclophosphamide to block the Th2 response, and using the type 1 cytokine IL-12 to induce a Th1 response). The study will be carried out with a clinical trial that will include these new approaches coordinated with extensive laboratory evaluations of the biologic response to test whether or not a Th1 type response can be induced, CTL response can be amplified, and memory response can be generated. The trial will be carried out with a vaccine made of DC (cultured in GM-CSF and IL-4) loaded with the HLA-A1 or - A2 determined peptides encoded by the following genes: MAGE-1, MAGE- 3, MART-1/Melan A. Thus, melanoma patients, who are HLA-A1 or HLA-A2 positive and whose melanomas express the above genes, will be primed with the vaccine and then given several boosters with a single low but biologically active dose of Interleukin-12 and a single injection of cyclophosphamide 3 days before immunization. A skin test with the naked peptide will be done after each vaccination and extensive analyses of the host immune responses in the draining lymph nodes, in tumors, and in circulation will be performed to determine whether or not the new approach could induce a Th1 type response and amplify specific CTL response and activate memory response.
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