HLA BINDING CHARACTERISTICS AND GENERATION OF CYTOTOXIC LYMPHOCYTES AGAINST PEPTIDES DERIVED FROM ONCOGENIC PROTEINS

Aims and background: Structurally altered proteins (derived from chrom osomal translocations or gene mutations) can be considered tumor speci fic antigens and represent an attractive target for a T-cell mediated response. T lymphocytes recognize antigens in the form of peptides bou nd to HLA-molecules. Materials and methods: Peptides derived from onco genic proteins were screened fro the presence of HLA binding motifs; a ctual binding were evaluated by HLA stabilization experiments using tr ansfectants and specific anti-HLA antibodies. Specific lymphocytes wer e induced by in vitro peptide sensitization and screened by thymidine uptake or cellular cytotoxic assays. Results: We Identified peptides d erived from EWS/FLI-1 fusion protein and from mutated K-RAS protein (e ncompassing respectively the fusion point and the mutation at position 12) that showed binding motif for HLA-Cw0702 and HLA-A3 respectively . The actual binding of these peptides was analysed in a stabilization assay. We detected binding for the EWS/FLI-1 peptide and for 5 RAS pe ptides (1 wild type and 4 mutated). The effect of temperature, beta 2- microglobulin (beta 2-m) and fetal calf serum (FCS) on the binding and the stability of the HLA/peptide complex was studied,A low temperatur e (26 degrees C) increased the binding both in HLA-AB and HLA-Cw0702, while FCS reduced it. beta 2-m increased the binding to the HLA-A3 mo lecule but did not influence the binding to the HLA-Cw0702, The stabi lity of already formed complexed was somewhat different in the HLA-AB and HLA-Cw0702 system: both were more stable at 26 degrees C than at 37 degrees C but while the beta 2-m and FCS did not influence the stab ility of the HLA-A3/peptide complex, they seemed to cause opposite eff ects in the HLA-Cw0702 system (beta 2-m stabilized and FCS destabiliz ed the complex). Finally, we were able to generate a specific CD8(+) C TL line against a K-RAS mutated peptide, Conclusions: Although binding motifs and actual HLA binding can be detected in several cases, the g eneration of a cellular response is infrequent, confirming that HLA bi nding is necessary but not sufficient to obtain an in vitro response, Further optimization of culture conditions, type of Antigen Presenting Cells (APC), peptides, use of stabilizers like beta 2-m are still nee ded.