Point Mutation Screening Of Tumour Neoantigens And Induced Specific CTLs Using The Cancer Genome Atlas Database

Objective: Based on TCGA database of mutations sequencing data to predict tumor antigen,the new design of bioinformatics antigen epitope peptide,immunogenicity experiment screened by the ideal of antigen epitope peptide,can efficiently activate cellular immune response of specific CTL clone,and analyze the TCR gene expression status,analysis the new specific tumor antigen as the possibility of anti-tumor therapeutic targets.Methods: 1.The high frequency mutation sites of the top 10 singlesubstituted bases of colorectal cancer,lung cancer and liver cancer genes were screened out from the TCGA database,and three groups of antigen epitope peptides with high affinity to MHC were selected and synthesized by using the bioinformatics computer prediction algorithm.Based on TCGA database,the survival data of patients with KRAS G12 V,TP53 R158 L,CTNNB1 K335 I mutant and wild-type cancer were searched,and their survival curves were analyzed.To investigate the incidence and coverage of KRAS G12 V,TP53 R158 L and CTNNB1 K335 I mutation hotspots in various cancers.2.The affinity between three groups of antigen epitope peptides and MHC molecules was preliminarily detected by flow cytometry,and the specific cytotoxic T lymphocytes were induced in vitro by mutants and wild peptides in each group.The killing function of peptide-induced specific CTL on peptide-t2 cell model was preliminarily verified by the secretion of IFN-? and the detection of Calcein-AM.3.Recombinant eukaryotic expression plasmids expressing the three point mutant genes were constructed and transfected into HCT116 colon cancer cells,NCI-H292 lung cancer cells and HepG2 liver cancer cell lines,respectively.The transfection efficiency and expression of q PCR detection point mutant genes in tumor cell lines were detected by flow cytometry.The cytotoxic effect of peptide-induced specific CTLs on wild-type and mutated tumor cells was verified by Calcein-AM and CFSE-PI staining.Flow detection was performed to detect the expression of peptide-specific CTL killing Gram enzyme B in wild-type and mutated tumor cells,as well as the secretion of IFN-? and perforin by ELISA.4.RNA samples of pre-induction specific T cells induced by original PBMC cells and CTNNB1 mutant peptide(IMRTYTYEI)were extracted and transcribed,and the gene expression of CDR3 region in specific T cells induced by CTNNB1 mutant peptide(IMRTYTYEI)was detected by multiple PCR amplification and capillary electrophoresis.Results: 1.In combination with TCGA database,3 groups of highfrequency gene mutation sites of KRAS G12 V,TP53 R158 L and CTNNB1 K335 I were found,and 3 groups of wild peptides and mutated peptides were screened by the peptide prediction algorithm,including KRAS G12 V wild peptides and mutated peptides,TP53 R158 L wild peptides and mutated peptides,and CTNNB1 K335 I wild peptides and mutated peptides.Survival analysis showed that the overall survival rate(OS)of colon cancer patients with KRAS G12 V point mutation was higher than that of other colon cancer patients with KRAS G12 V point mutation.The three predicted neoantigens have a certain coverage rate in tumor patients and have good clinical application value.2.By flow detection,the affinity between the mutated peptides in CTNNB1 group and HLA-A2 molecule was better(P < 0.001).The antigenspecific CTL cells induced by DC-loaded peptide were mainly CD8+T cytotoxic Tc cells.Elisa preliminary detection showed that the peptideinduced specific CTL in the CTNNB1 group had the highest amount of IFN-? secretion on the loaded peptid-t2 cells(P < 0.05),followed by the TP53 group and the KRAS group.The killing rate of mutant peptide-specific CTL in CTNNB1 group on the model of loaded peptide-T2 cells was higher than that in the other two groups.3.The transfection efficiency of the three groups of spot mutant genes in the tumor cell line was high and they were overexpressed.By the detection of Calcein-AM and CFSE-PI,it was found that the killing rate of CTL induced by mutated peptide was higher than that of CTL induced by wild peptide(P < 0.001).However,compared with the TP53 group and the KRAS group,the specific CTL induced by the mutant peptide in the CTNNB1 group had a stronger cytotoxic effect on mutant and wild-type tumor cells(P < 0.01).Flow detection of Gram enzyme B and ELISA detection of the secretion of perforin and IFN-?,it was found that the secretion concentrations of Gram enzyme B,perforin and IFN-? in the CTNNB1 mutant peptide group were much higher than those of the TP53 group and KRAS group(P < 0.01).4.By analysis of capillary electrophoresis,CTL cells induced by the CTNNB1 mutant peptide(IMRTYTYEI)were found to have clonal proliferation of part of the TCR V serpentine CDR3 subfamily in the V?14,V?7,V?9 subfamily and TCR V?1,V?15,V?18 serpentine subfamily.Conclusions: In this study,it was found that all the three groups of selected point mutant antigen epitopes could improve the cytotoxic effect of specific T cells,among which the CTNNB1 mutant peptide(IMRTYTYEI)had the strongest induction effect,and could effectively activate the immune response of T cells and mediate the specific cytotoxic effect on tumor cells.This study suggests that CTNNB1 K335 I point mutation epitope peptide may have the potential to be used in personalized cellular immunotherapy.