Clinical Anti-cancer Immune Effect Of Human Colorectal Cancer Neoantigen Peptide Based On Mutation Of Amino Acid Residues

Objective: This study intends to sequence the entire exons of tumor cells from patients with colorectal cancer,synthesize neoantigen peptides in vitro based on biological information,and detect the immunogenicity of neoantigen peptides through immunological experiments and flow cytometry methods to clarify the relationship between the mutations of amino acid residues in neoantigen peptides.On this basic,we can explore a new method for the rapid discovery of effective neoantigen peptides and provide a new way to improve the efficacy of clinical immunotherapy of tumor.Methods: 1.Perform the entire exons sequencing on tumor samples of patients with colorectal cancer to obtain the entire exons genome data of the tumor;2.Randomly select neoantigen peptides from the neoantigen peptide library screened by the entire exons and entrust Wuhan Dangang Biotechnology Co.,Ltd.to synthesize neoantigen peptides according to the amino acid sequence of the neoantigen peptide;3.Extract fresh peripheral blood from patients to prepare PBMC and then induce differentiation into im DC;4.Prepare the DC vaccine loaded with neoantigen peptides;5.Complete co-culture of the DC vaccine and fresh PBMC to obtain a mixed DC lymphocyte vaccine;6.ELISPOT technology detects the activity of the T cell activated by the neoantigen peptide to represent the immunogenicity of neoantigen peptide;7.Flow cytometry is used to identify the PBMC before the new antigen peptide is loaded and the proportion of T,B,NK cells in the mixed DC lymphocyte vaccine.Results: 1.We randomly select a total of 95 neoantigen nonapeptides from the neoantigen nonapeptide library screened by whole exome sequencing from 6 tumor patients.Among them,87 neoantigen peptides are single amino acid residue mutations.There are 8 neoantigen peptides involved multiple amino acids residue mutations.There are 4 neoantigen peptides which mutated from a certain amino acid residue to any amino acid.And there are 18 neoantigen peptides amino acids residue mutations containing benzene ring.2.Divide the neoantigen peptides with single amino acid residue mutations into groups,respectively: group 1.polar →non-polar(polar amino acid residues are mutated to non-polar amino acid residues,the same below);group 2.non-polar→polar;group 3 polar→polar and non-polar→non-polar.After completing the normality test and the homogeneity of variance test,do a pairwise comparison of the one-way variance test.And then we can conclude that the difference between group 2 and group 1,group 2 and group 3 are statistically significant(P<0.05),and the mean value of group 2 is higher than that of group 1 and group 3.3.We group according to the polarity mutation type of amino acid residues,randomly select 1 mixed DC lymphocyte cells loaded with neoantigen peptides from each group of P5,and according to the results of flow cytometry,it is suggested that the group of the amino acid residue which is mutated from non-polar amino acid to polar amino acid has more activated T cells,and the proportion of CD8+ T cells is higher.4.This study analyzes the immunogenicity of neoantigen peptides formed by mutations of multiple amino acid residues.Group A is neoantigen peptides with mutations in multiple amino acid residues,and group B is neoantigen peptides with mutations in single amino acid residues.We have completed an independent sample t test,and the results show that the difference is statistically significant(P<0.05),and the mean value of group A is higher than that of group B.We complete the flow cytometry test,and the results show that the number of T lymphocytes increased after loading the neoantigen peptide,and the number of neoantigen peptide T lymphocytes containing multiple amino acid residue mutations increases more significantly.5.This study analyzes the neoantigen peptides containing mutations in the amino acid residues of the benzene ring,and completes the independent sample t test.The results show that the immunogenicity of the neoantigen peptides formed by the mutation of the amino acid residues of the benzene ring is not statistically significant.Conclusion: 1.Among the amino acid residue mutations of neoantigen peptides,the main ones are substitution mutations of single amino acid residues,and a few are mutations of multiple amino acid residues.2.Among the neoantigen peptides formed by mutations of different polar amino acid residues,the mutant neoantigen peptides in which non-polar amino acid residues are mutated to polar amino acids have the strongest immunogenicity.3.The immunogenicity of neoantigen peptides containing multiple amino acid residue mutations is stronger than that of single amino acid residue mutations.4.The mutation of amino acid residues containing benzene ring has nothing to do with the immunogenicity of the neoantigen peptide.5.The neoantigen peptide does have the effect of enhancing the specific cellular immune effect,and CD8+T cells are more activated after loading the neoantigen peptide.6.Based on the relationship between the characteristics of amino acid residues and immunogenicity of neoantigen peptides,a new method can be provided for rapid and effective screening of neoantigen peptides with strong immunogenicity.