Selection,Identification And Application Of Bladder Cancer Neoantigen

Objective:Bladder cancer is one of the most common malignancies in the urinary system.Neoantigen-based immunotherapy is one of the hotspots in the field of immunotherapy against cancer.Benefit from mutation data from Broad Institute Genome Data Analysis,we intended to identify mutant peptides with neoantigen property in bladder cancer.Method:By using bioinformatics,HLA-A2 restricted mutant peptides and wild type counterparts were selected based on whole exosome sequencing data of bladder cancer from the database.The binding affinity of predicted peptide pairs were determined by using T2 cell-based assay.Peptides with high affinity to HLA-A2 molecules were subjected to the determination of immunoreactivity in bladder cancer patients by using Elispot assay upon in vitro stimulation.Clinical relevance to immune responses was analyzed.Result:920 paired peptides were predicted by bioinformatic screening based on the criteria of higher affinity of mutant-type peptides and lower affinity of wild-type peptides to HLA-A2 molecules.57 paired peptides were synthesized with the highest affinity and expression frequency and subjected to T2 cells based validation.Among them,12 MT peptides from 12 individual genes exhibited higher and stronger affinity to HLA-A2 molecules when compared to wild-type counterparts.Importantly,it was shown that bladder cancer patients exhibited higher responsiveness to mutant-type peptides than to wild-type peptides.The average number of peptide-specific IFNg spots from 40 HLA-A2 positive bladder cancer patients upon mutant-type peptide stimulation was dramatically higher than that upon wild-type stimulation(P=0.006).The clinical association analysis revealed that mutant-type peptide specific T cell responsiveness was negatively correlated to C-reactive protein(r=-0.425,P<0.01)and thrombocytocrit(r=-0.384,P<0.05)and platelet(r =-0.403,P<0.05).Conclusion:The results presented here illustrate that mutant-type peptides screened from the database can induce stronger immune reactivity than wild-type peptides in Chinese bladder cancer patients,making them neoantigen candidates for future immunotherapy against bladder cancer.The peptide-specific responses might be influenced by the inflammatory environment according to our study,providing potential strategy to enhance the efficacy of neoantigen-based vaccination against bladder cancer.