Cytotoxic CD4+ and CD8+ T lymphocytes, generated by mutant p21-ras (12Val) peptide vaccination of a patient, recognize 12Val-dependent nested epitopes present within the vaccine peptide and kill autologous tumour cells carrying this mutation.

Mutant p21-ras proteins contain sequences that distinguish them from normal ras, and represent unique epitopes for T-cell recognition of antigen-bearing tumour cells. Here, we examined the capacity of CD4+ and CD8+ T cells, generated simultaneously by mutant-ras-peptide vaccination of a pancreatic-adenocarcinoma patient, to recognize and lyse autologous tumour cells harbouring corresponding activated K-ras epitopes. The patient was vaccinated with a purified 17mer ras peptide (KLVVVGAVGVGKSALTI), containing the Gly12 --> Val substitution. Responding T cells were cloned following peptide stimulation, and CD4+ and CD8+ peptide-specific cytotoxic T lymphocytes(CTL) were obtained. Transient pancreatic-adenocarcinoma cell lines(CPE) were established in cell culture from malignant ascites of the patient, and were shown to harbour the same K-ras mutation as found in the primary tumour. These cells were efficiently killed by the T-cell clones and CD8+-mediated cytotoxicity was HLA-class-I-restricted, as demonstrated by inhibition of lysis by anti-class-I monoclonal antibodies. By employing as targets different class-I-matched tumour cell lines expressing a 12Val mutation, we were able to demonstrate HLA-B35 as the restriction molecule, and further use of peptide-sensitized EBV-B cells as target cells identified VVVGAVGVG as the nonamer peptide responsible for CD8+-T-cell recognition. These data demonstrate that peptide vaccination with a single mutant p21-ras-derived peptide induces CD4+ and CD8+ CTL specific for nested epitopes, including the Gly --> Val substitution at codon 12, and that both these T-cell sub-sets specifically recognize tumour cells harbouring the corresponding K-ras mutation.