Skipper, John Charles Alexander; (1993) Cytotoxic T lymphocyte responses to Ras protein. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The possibility of inducing cytotoxic T lymphocytes (CTL) to mutant or normal Ras has been investigated using recombinant vaccinia viruses expressing these proteins. Mutant Ras or over expression of Ras is frequently associated with human malignancies, being implicated in 40% of colorectal cancers and 95% of pancreatic cancers. Oncogenic activation occurs by single point mutation of amino acid 12, 13 or 61. The structurally mutated or over expressed Ras oncogene has the potential of producing novel peptide antigens, which if presented on the cell surface by class I MHC molecules may be recognized by cytotoxic T cells. These may serve as targets for immunotherapy of certain cancers. Recombinant vaccinia viruses expressing normal (Vac-Ras) or mutant (Vac-Ras6l) Ras were constructed. Vac-Ras61 immunized C57Bl/10 mice (H-2b) developed Ras61-specific CTL which recognized normal Ras inefficiently. CTL isolated from Vac-Ras immune mice showed the opposite specificity. Endogenous levels of Ras expression were insufficient for lysis. Synthetic Ras peptides with an H-2Kb motif were identified. Peptide binding of MHC class I was demonstrated using the H-2b derived RMA-S mutant thymoma cell line in which peptide induced class I stabilisation was detected by immunofluorescence. One CTL epitope mapped to amino acids 60-67 and residue 61 was critical for T cell recognition. Anti-Ras61 CTL recognised peptide 60-67 containing mutant residue 61, while anti-Ras CTL recognised wild type 60-67. A second epitope mapped to residues 152-159 of Ras and was recognised equally well by CTL raised to normal and mutant Ras. These peptides could stimulate in vitro proliferation of specific CTL which recognised target cells pulsed with the appropriate peptides. The murine data suggest the possibility of exploiting Ras-specific CTL for targeted immunotherapy of certain human cancers.

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