IDENTIFICATION OF OVERLAPPING EPITOPES IN MUTANT RAS ONCOGENE PEPTIDES THAT ACTIVATE CD4(+) AND CD8(+)T CELL RESPONSES

Mutant ras p21 proteins contain sequences which distinguish them from normal endogenous rats and. thus, may represent unique epitopes for T cell recognition of antigen bearing tumor cells. Here. we examined the capacity of a mutant K-ras 9-mer peptide to induce in vivo CDS cytoto xic T lymphocytes (CTL). The peptide chosen reflected positions 4-12 o f the point-mutated sequence of the K-ras oncogene encoding the Gly to Val substitution at codon 12. The overall rationale for selecting thi s particular 9-mer sequence was threefold: the mutant peptide containe d a putative major histocompatibility complex (MHC) class I consensus anchor motif for murine H-2K(1): specific binding to MHC class I may t hen create an immunogenic complex for the induction of anti-ras CD8(+) CTL: and finally. the mutant sequence overlapped with a newly charact erized anti-ras CD4(+) T helper type I epitope. which may have implica tions for the coordination and activation of both anti-ras immune mech anisms against the same target cell antigenic determinant. A functiona l interaction with H-2K(d) was demonstrated with the mutant ras4-12(V1 2) peptide. but not the normal ras4-12(G12) peptide. which specificall y inhibited an H-2K(d)-restricted. anti-nucleoprotein NP147-155 CTL re sponse in a dose-dependent fashion. An anti-ras CD8(+) T cell line was then established from immune splenocytes of BALB/c (H-2(d)) mice inje cted with ras4-12(V12) in adjuvant. which mediated peptide-specific ly sis of syngeneic P815 tumor targets. Cytotoxicity was restricted by H- 2K(d) and strongly specific for the mutant ras peptide. Importantly, t hese anti-ras CTL specifically lysed a syngeneic tumor line (i.e. A20 lymphoma) transduced with the corresponding point-mutated ras oncogene . suggesting T cell receptor recognition of endogenously derived antig en. Overall, these data demonstrated that mutant ras p21 at codon 12 ( Gly --> Val) contained a peptide sequence which exhibited specific fun ctional binding to a murine MHC class I molecule: the ability of the m utant. but not the normal sequence to bind selectively to murine MHC c lass I likely reflected the generation of a C-terminal anchor residue: and the ras 4-12(V12) peptide was immunogenic for the production of a ntigen-specific CD8(+) CTL. which lysed in vitro a syngeneic tumor cel l line harboring the mutant K-ras oncogene.