Combinatorial peptide libraries as an alternative approach to the identification of ligands for tumor-reactive cytolytic T lymphocytes

The recent identification of molecularly defined human tumor antigens recog nized by autologous CTLs has opened new opportunities for the development o f antigen-specific cancer vaccines. Despite extensive work, however, the nu mber of CTL-defined tumor antigens that are suitable targets for generic va ccination of cancer patients is still limited, mostly because of the painst aking and lengthy nature of the procedures currently used for their identif ication. A novel approach is based on the combined use of combinatorial pep tide libraries in positional scanning format (positional scanning synthetic combinatorial peptide libraries, PS-SCLs) and tumor-reactive CTL clones. T o validate this approach, we herein analyzed in detail the recognition of P S-SCLs by Melan-A-specific CTL clones. Our results indicate that, at least for some clones, most of the amino acids composing the native antigenic pep tide can be identified through the use of PS-SCLs, Interestingly, this anal ysis also allowed the identification of peptide analogues with increased an tigenic activity as well as agonist peptides containing multiple amino-acid substitutions. In addition, biometrical analysis of the data generated by PS-SCL screening allowed the identification of the native ligand in a publi c database. Overall, these data demonstrate the successful use of PS-SCLs f or the identification and optimization of tumor-associated CTL epitopes.