We have conducted studies on the identification of potential mouse and human T cell (CD4+ and CD8+) epitopes reflecting point mutations in the ras proto-oncogenes at codon 12, and the utility of these oncogene- derived peptides to function as tumor-specific immunogens in the induction of antitumor T cell responses. In a murine system (BALB/c, H- 2d), we have synthesized peptides reflecting the substitution of glycine (Gly) to valine (Val) as a model target cell determinant and analyzed their immunogenicity (in adjuvant) in vivo. We have identified both MHC class II-restricted, CD4+ Th1-type and MHC class I-restricted, CD8+ cytotoxic T lymphocyte (CTL) peptide epitopes reflecting the Val12 mutation. Importantly, these peptide-derived T cell lines recognized antigen (Ag)-bearing syngeneic tumor cells in vitro endogenously expressing (via retrovirus transduction) processed forms of the mutant ras protein. Furthermore, we have developed a murine mutant ras CD8+ CTL peptide epitope variant that displayed enhanced binding to MHC class I molecules and exhibited improved Ag-specific immunogenicity in vitro and in vivo. We plan to develop a ras oncogene tumor model in vivo to evaluate the biologic activity and efficacy such peptide-based immunotherapies. In human in vitro studies, we have developed methodology for derivation of Ag-specific CTL from naive precursor CD8+ lymphocytes. A HLA-A2-restricted, mutant ras peptide-specific CD8+ CTL line was produced, which lysed the HLA-A2+ SW480 colon carcinoma expressing the naturally-occurring Val12 mutation. Maximal lysis occurred following IFN-gamma pretreatment of the target, which correlated with enhanced HLA-A2, ICAM-1 and Fas (CD95) expression. To determine whether ras peptides reflecting mutations in the ras proto- oncogenes are immunogenic in humans, a phase I anticancer vaccine clinical trial was initiated in metastatic carcinoma patients whose primary tumors harbored K-ras mutations at codon 12. Selected patients were inoculated, at 3 separate intervals, with a mutated ras 13-mer peptide in Detox adjuvant reflecting the codon 12 mutation found in their cancer (i.e., ras5-17 Gly12 to Val12, Asp12 or Cys12). Thus far, 3 of 10 evaluable patients have demonstrated Ag-specific cellular immune responses, as determined by the production of T cell lines (CD4+ and/or HLA-A2-restricted CD8+) from post-vaccinated lymphocytes. No specific responses were detectable against the normal ras sequence and no T cell line was produced from pre-immune lymphocytes. Together with our in vitro human studies, we have identified for the first time human CD8+ CTL epitopes reflecting specific point mutations in the ras proto- oncogenes at codon 12 (i.e., Asp12 and Val12). The ability to propagate Ag-specific CD4+ and CD8+ T cell lines from vaccinated patients may have important clinical implications for combining both active and passive immunotherapies, perhaps with exogenous cytokine(s) such as IL-2. The identification of overlapping or nested peptide epitope sequences may lead to the development of a diverse set of second generation peptide configurations containing multiple T cell subset-specific epitopes for coordinated activation of both types of cellular immune reactions. We plan to continue our studies on the role, characterization and modulation of the cellular immune response in the appropriate experimental model systems. These studies include: (1) to enhance peptide immunogenicity and potency for the induction and amplification of the Ag-specific T cell response; (2) to characterize mechanisms of tumor cell killing by anti-ras immune effector cells; and (3) to study principles of adoptive immunotherapy using epitope-specific CD4+ and/or CD8+ T cell populations in preclinical animal models.
Showing the most recent 10 out of 16 publications
