Research On The Discovery Of Effective Antigenic Peptides For Anticancer Immunity Based On The Relevant Features Of Tumor Exon Mutations Such As Human Colon Cancer

Objective:Tumor neoantigen are weak immunogenicity.The neoantigen peptides did not elicit an effective immune response.The higher the killing activity of activated T cells,the stronger the immunogenicity of the antigenic peptide.This study through the second generation sequencing specimens of patients with colon cancer and other tumors of all exons sequencing,using bioinformatics analysis to determine the exons mutations,predict HLA class I molecules-presented neoantigen peptide synthesis,and determine the source of genetic mutation,the binding affinity of HLA-I allele and its products and the synthesis of neoantigen peptides.The immunogenicity of tumor neoantigen peptides was detected by immunological experiments,so as to clarify the relationship between the mutation of neoantigen peptides and the clinical immune effect of neoantigen peptides,so as to provide the basis for the rapid discovery of effective neoantigen peptides and the construction of highly effective tumor vaccines.Methods:Second-generation sequencing technology:whole-exome sequencing was performed on tumor samples from 11 patients with colon cancer and other tumors.Complete exon genomic data of each tumor specimen were obtained.The exon mutant genes were identified by bioinformatics analysis and software such as NetMHCPAN4.0,and the information characteristics of the neoantigen peptides were screened and synthesized artificially.From these tumor patients,fresh peripheral blood(PBMC)was extracted,and peripheral blood mononuclear cells were isolated.Immature dendritic cells(DCs)were induced to generate neoantigen peptide-activated mixed DCs and co-culture with PBMC to obtain activated T lymphocytes.The killing activity of T-effector lymphocytes activated by neoantigen peptide was detected by enzyme linked dot technique(ELISPOT test).The orthogonal design experiment was conducted according to 3 factors and 4 levels,and Table L16(34)was used.Results:Part1:The characteristics of neoantigen peptide mutation1.645 neoantigen peptides were predicted and screened,and various characteristic information of neoantigen peptides were identified.2.The HLA allele products presenting class ? antigens in each patient were generally type 3-6.The frequency and percentage of each HLA type in 11 patients were statistically shown in Figure 2,and we found that HLA-A*02:01 was the main type.3.A total of 645 HLA class I neoantigen peptides were measured in 11 patients,and we found that the length of neoantigen peptides was mainly 9 peptides.4.The binding affinity between each of the neoantigen peptides and HLA molecules could be obtained by whole exon sequencing(Figure 4).In this study,it was found that the affinity of 645 neoantigen peptides was mostly between 1 and 2.5.In the neoantigen polypeptides,the mutation of single amino acid was dominant,accounting for about 94.3%,and the mutation of single amino acid was mainly from polar amino acid to polar amino acid.Part 2:T effector lymphocyte activity induced by neoantigen peptidesOrthogonal experiment L16(34),using Elispot to detect the number of T cell activation of each peptide and quantify the immunogenicity of each peptide.The results of statistical analysis showed that Influences on the immunogenicity of neoantigen peptides in order:Influences on the immunogenicity of neoantigen peptides>Changes in HLA molecular affinity>The length of the neoantigen peptide,and the change degree of affinity between 1-2 mutated to the non-polar neoantigen nine peptides:ELISPOT had the highest value and CD8+T had the highest number.Conclusion:1.Among the mutations of amino acid residues of the neoantigen,nine peptides were the main alleles presenting the neoantigen peptides,and HLA-A*02:01 was the main alleles presenting the neoantigen peptides,with more affinity changes of 1-2.Single amino acid substitution mutation of polarity was the most common.2.Polarity mutation in the affinities between 1-2 was non-polar neoantigen nineptide:ELISPOT had the highest value and CD8+T cell had the highest number.3.Among the three factors,the influence of mutation mode is the most important.The mutation type from polar amino acid residues to non-polar amino acids enhances the immunogenicity.4.Using the second-generation sequencing technology to discover the characteristics of neoantigen.Based on the length of amino acid residues,amino acid mutation and HLA affinity variation of the neoantigen polypeptide,a new method can be developed to screen the neoantigen with strong immune activity quickly and easily.