Recently, neoantigens have been paid great attention among cancer-specific antigens. Neoantigens are self-peptides that are displayed on major histocompatibility complexes (MHC) with cancer specific mutations that are never seen in normal tissues. We performed combined searching by the next generation sequencing (NGS) and mass spectrometry for MHC-associated neoantigens in the refractory cancers.
DNAs and RNAs are extracted and sequenced from cultured colon cancer cell lines and patient tissues (total 9 samples) from pancreatic and biliary cancers. Among the samples, 4 tissues are transplanted to mice to prepare xenograft mice. Proteins are extracted from the xenograft models, immunoprecipitated with anti-MHC antibody, and measured by a mass spectrometer. Peptide sequences are searched against database obtained from the exome and transcriptome from the identical tissue.
After a systematic examination of experimental conditions using the cultured cells, we have established a workflow that can detect above 1,000 MHC-associated peptides from 3 mg of protein lysates from xenograft mice. We have also identified several neoantigen candidates by DNA and RNA sequencing followed by peptide sequencing with mass spectrometric analysis.
In identifying neoantigens, we have found that peptidomics have been added the detection confidence of the identified neoantigens. We are further improving sensitivity to the level to detect directly from patient tissues without xenograft formation. The identified neoantigen candidates are tested for immunogenicity in vitro.