Efficient identification of novel HLA-A*0201-presented cytotoxic T lymphocyte epitopes in the widely expressed tumor antigen PRAME by proteasome-mediated digestion analysis

We report the efficient identification of four human histocompatibility leu kocyte antigen (HLA)-A*0201-presented cytotoxic T lymphocyte (CTL) epitopes in the tumor-associated antigen PRAME using an Improved "reverse immunolog y" strategy. Next to motif-based HLA-A*0201 binding prediction and actual b inding and stability assays, analysis of in vitro proteasome-mediated diges tions of polypeptides encompassing candidate epitopes was incorporated in t he epitope prediction procedure. Proteasome cleavage pattern analysis, in p articular determination of correct COOH-terminal cleavage of the putative e pitope. allows a far more accurate and selective prediction of CTL epitopes . Only 4 of 19 high affinity HLA-A*0201 binding peptides (21%) were found t o be efficiently generated by the proteasome in vitro. This approach avoids laborious CTL response inductions against high affinity binding peptides t hat are not processed and limits the number of peptides to be assayed for b inding. CTL clones induced against the four identified epitopes (VLDGLDVLL, PRA(100-108); SLYSFPEPEA, PRA(142-151); ALYVDSLFFL, PRA(300-309); and SLLQ HLIGL, PRA(425-433)) lysed melanoma, renal cell carcinoma, lung carcinoma, and mammary carcinoma cell lines expressing PRAME and HLA-A*0201. This indi cates that these epitopes are expressed on cancer cells of diverse histolog ic origin, making them attractive targets for immunotherapy of cancer.