We have continued our studies on the definition 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. Previously, 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) a processed form of the mutant ras protein. We plan to evaluate whether such peptide-based immunogens can mediate therapeutic antitumor effects in vivo. In human in vitro studies, we have developed methodology for derivation of Ag-specific CTL from naive precursor CD8+ lymphocytes. We have identified a 9-mer ras peptide, which incidently was identical in H-2d mice, as the mutant, but not the normal proto-ras form bound to HLA-A2. A CD8+ CTL line was produced, which displayed specific lysis against the HLA-A2+ SW480 colon carcinoma expressing the naturally-occurring Val12 mutation; albeit, maximal lysis occurred following IFN-gamma pretreatment of the target. A peptide-based phase I clinical trial was initiated, in which metastatic carcinoma patients were vaccinated with a mutated ras 13-mer peptide in DetoxTM adjuvant reflecting the codon 12 mutation found in their cancer (i.e., ras5-17 Gly12 to Val12, Asp12 or Cys12). Thus far, 3 of 8 evaluable patients have demonstrated Ag-specific cellular immune responses (CD4+ and/or CD8+), as determined by the production of T cell lines 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 future clinical implications for combining both active and passive immunotherapies, perhaps with exogenous cytokine(s). The identification of overlappping or """"""""nested"""""""" peptide 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 design ras peptide variants, which may: (1) increase binding to cell surface MHC class I/II molecules; (2) enhance peptide interaction with de novo-synthesized MHC class I molecules; and (3) act as T cell agonists.
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