Identification of tumor-associated MHC class I ligands by a novel T cell-independent approach

Specific immunotherapy of cancer utilizes tumor-directed cytotoxic T lympho cytes (CTL) that lyse tumor cells presenting MHC class I-associated peptide s derived from tumor-associated proteins. Many tumor-associated gene produc ts are known, but corresponding T cell epitopes are only known for relative ly few of these. The most commonly used approaches to identify such antigen s require pre-existing CTL lines or clones. By using a CTL-independent high performance liquid chromatography mass spectrometry (HPLC MS)-based approa ch we identified HLA-A2-presented peptides from carcinoembryonic antigen an d wild-type p53 with a copy number as low as eight molecules per cell. Pote ntial epitopes were predicted from the sequences of known tumor antigens an d the corresponding synthetic peptides were analyzed by nanocapillary HPLC MS. In parallel, peptides were extracted from fresh, solid tumor tissue or tumor cell lines and analyzed in the same way. Upon co-elution of a natural peptide with a predicted peptide of the same mass, the peptide sequence wa s confirmed by on-line tandem MS. This approach allows rapid screening of l arge numbers of tumor-associated gene products for naturally processed pept ides presented by different MHC class I molecules as a prerequisite for eff icient epitope identification and rapid transfer to therapeutic vaccine tri als.