Strategy Study Of Neo-epitopes Presented By MHC-? By Combining Mass Spectrometry And Next-Generation Sequencing

Cancer is caused by the accumulation of somatic mutations in the genome.Neo-epitopes are mutated peptides caused by mutations and presented by MHC-? molecules through a series of biochemical reactions in the cell.They are only expressed by cancer cells and not subject to immunologic tolerance,which makes them become optimal targets for cancer immunotherapy.In the thesis,combining with mass spectrometry and next generation sequencing,we tried to use two strategies to identify neo-epitopes from both cancer cells and solid tumor tissues.We identified 758 MHC-? loaded peptides from one NSCLC cell line(NCI-H460)by immunoprecipitation,one of which is cancer testis epitope(EVSEPSMLQK).Synthesis of four peptides including EVSEPSMLQK further proved our result.However,no neo-epitopes have been identified from either human cancer cell lines or solid tumor tissues.Methonal/chloroform extraction followed with a trypsin digest was found to be an optimal method to identify neo-antigens and Triton X-114 could expand the amount of identification by a comparison of three protein extraction avenues.Feasibility of two methods was proved by application in different solid tumor tissues.We synthesized one of mutant peptides to obtain further prove.We used this proved neo-antigen for in slico prediction and got one neo-epitope with binding affinity 63.1 nM.Thus,identified neo-antigens can be used for the prediction of neo-epitopes KAYHEQLSV and have priority in immunogenicity testing.A new method for identification of neo-epitopes from solid tumor should be developed in the future.