DEVELOPMENT OF A MURINE MUTANT RAS CD8(-I BINDING AND IMMUNOGENIC PROPERTIES() CTL PEPTIDE EPITOPE VARIANT THAT POSSESSES ENHANCED MHC CLASS)

We recently identified a murine mutant Ras p21 CD8(+) CTL epitope refl ecting residues 4 to 12, containing the mutation of Gly to Val at codo n 12, that bound weakly to H-2K(d) in vitro and generated a weak prima ry CTL response in immunized BALB/c mice, Here, we explored the hypoth esis that specific modifications to the Ras(4-12) peptide sequence can improve MHC binding, leading to enhanced immunogenicity without alter ing immune specificity, We synthesized Ras(4-12) peptides in which Val at residue 12 was replaced with the more dominant H-2K(d) C-terminus anchor residue Leu or ne. In functional H-2K(d) binding assays, Ras(4- 12)(L12 or I12) peptide variants competed more effectively than the Ra s(4-12)(V12) peptide, Ras(4-12)(L12 or I12) peptide variants enhanced both in vitro cytotoxicity and proliferation responses of anti-Ras(4-1 2) CTL compared with the mutant Ras(4-12)(V12) peptide, Additionally, the Ras(4-12)(L12) peptide variant induced a quantitatively greater T cell response in vivo compared with that produced by Ras(4-12)(V12) as determined by IFN-gamma production, Mice immunized with Ras(4-12)(L12 ) peptide elicited CD8(+) CTL activity specific for target cells prese nting the Ras(4-12)(V12) epitope exogenously and endogenously, Moreove r, both anti-Ras(4-12)(V12)-derived and anti-Ras(4-12)(L12)-derived CT L lines were similar insofar as their TCR usage and amino acid contact residues in the Ras(4-12)(V12) peptide, These experiments demonstrate that modifications can be introduced in tumor-specific peptide epitop es to enhance both in vitro and in vivo immunogenicity. The design of oncogene-specific peptide epitope variants as immunogens may accelerat e the generation of anti-tumor T cell responses for cancer immunothera py.