CYTOTOXIC CD4(-LYMPHOCYTES, GENERATED BY MUTANT P21-RAS (12VAL) PEPTIDE VACCINATION OF A PATIENT, RECOGNIZE 12VAL-DEPENDENT NESTED EPITOPESPRESENT WITHIN THE VACCINE PEPTIDE AND KILL AUTOLOGOUS TUMOR-CELLS CARRYING THIS MUTATION() AND CD8(+) T)

Mutant p21-ras proteins contain sequences that distinguish them from n ormal ras, and represent unique epitopes for T-cell recognition of ant igen-bearing tumour cells, Here, we examined the capacity of CD4(+) an d CD8(+) T cells, generated simultaneously by mutant-ras-peptide vacci nation of a pancreatic-adenocarcinoma patient, to recognize and lyse a utologous tumour cells harbouring corresponding activated K-ras epitop es, The patient was vaccinated with a purified 17mer ras peptide (KLVV VGAVGVGKSALTI), 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. Transi ent pancreatic-adenocarcinoma cell lines(CPE) were established in cell culture from malignant ascites of the patient, and were shown to harb our the same K-ras mutation as found in the primary tumour, These cell s were efficiently killed by the T-cell clones and CD8(+)-mediated cyt otoxicity was HLA-class-1-restricted, as demonstrated by inhibition of lysis by anti-class-1 monoclonal antibodies, By employing as targets different class-1-matched tumour cell lines expressing a 12Val mutatio n, we were able to demonstrate HLA-B35 as the restriction molecule, an d further use of peptide-sensitized EBV-B cells as target cells identi fied VVVGAVGVG as the nonamer peptide responsible for CD8(+)-T-cell re cognition. These data demonstrate that peptide vaccination with a sing le mutant p21-ras derived peptide induces CD4(+) and CD8(+) CTL specif ic for nested epitopes, including the Gly --> Val substitution at codo n 12, and that both these T-cell sub-sets specifically recognize tumou r cells harbouring the corresponding K-ras mutation. (C) 1997 Wiley-Li ss, Inc.