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. Int. J. Cancer 72:784–790, 1997. © 1997 Wiley‐Liss, Inc.