Differential immunopeptidome analysis revealed cancer specific amino acid usage of HLA class-I antigens and novel neoantigens of colorectal cancer

Knowing the nature of human leukocyte antigen (HLA) peptides, also called as immunopeptides, is indispensable to realize the cancer precision medicine such as cancer vaccination and the better prediction of efficacy for immunocheckpoint inhibitor (ICI) treatment. Direct interrogation of immunopeptides by mass spectrometry (MS) is of great use for that purpose but the reality in analyses of scarce tissue samples still confronts technical challenges. To elucidate characteristics of HLA class-I immunopeptides specifically presented on colorectal cancer (CRC), the optimized immunopeptide isolation method and differential ion mobility mass spectrometry (DIM-MS) with whole exome sequencing (WES)-based personalized database were established and subjected to differential analysis of tumor or normal tissues of CRC patients. From pilot experiments using 108 cells of colon cancer cell line HCT116, total 9,249 unique immunopeptides, including total 11 neoantigens, were identified. Next, approximately 40 mg of tumor or normal regions of CRC tissues were collected from 17 patients and analyzed by our personalized immunopeptidomic technology. As the result, 44,785 unique immunopeptides were profiled, in which 2 neoantigens carrying the mutation KRAS-G12V or CPPED1-R228Q were identified. Interestingly, specific amino acid usage of C-terminus trimming of immunopeptides was found in tumor-exclusive immunopeptides. Thus, our personalized immunopeptidome analysis significantly expands presented antigen knowledgebase and allows direct determination of neoantigens from scarce tissue specimens. This advanced immunopeptidomics holds promise to new outlook of research in immunopeptides both from basic and clinical aspects.