Direct identification of clinically relevant neoepitopes presented on native human melanoma tissue by mass spectrometry

Although mutations may represent attractive targets for immunotherapy, direct identification of mutated peptide ligands isolated from human leukocyte antigens (HLA) on the surface of native tumor tissue has so far not been successful. Using advanced mass spectrometry (MS) analysis, we survey the melanoma-associated immunopeptidome to a depth of 95,500 patient-presented peptides. We thereby discover a large spectrum of attractive target antigen candidates including cancer testis antigens and phosphopeptides. Most importantly, we identify peptide ligands presented on native tumor tissue samples harboring somatic mutations. Four of eleven mutated ligands prove to be immunogenic by neoantigen-specific T-cell responses. Moreover, tumor-reactive T cells with specificity for selected neoantigens identified by MS are detected in the patient`s tumor and peripheral blood. We conclude that direct identification of mutated peptide ligands from primary tumor material by MS is possible and yields true neoepitopes with high relevance for immunotherapeutic strategies in cancer.