| Objective:Recently,it has been found that gene mutation in non-coding region,abnormal splicing and modification of post-translational proteins are the main sources of tumor newantigen,which leads to low accuracy of prediction of tumor newantigen based on full exon sequencing,and limits the clinical application of newantigen.In this study,we directly detected human tumor cells and their derived normal cells by immunomagnetic bead precipitation,liquid chromatography combined with mass spectrometry,combined with whole exome sequencing,and obtained eight to eleven peptides unique to cancer cells but not normal cells after removing the contaminating peptides.Further,the binding affinity and binding stability of each selected peptide with HLA-Ⅰ allele molecules from patients of origin were determined by bioinformatics analysis software.According to the binding affinity and binding stability of each peptide,some of them were found to be able to present neoantigen peptides through HLA-class I molecules.The peptides before and after the above screening are randomly selected,the changes of immunocompetence before and after the screening are determined by in vitro immunological experiments and the changes of TCR activated by neoantigen peptides are detected by T cell receptor immunobanking technology to identify the discovered immunocompetent neoantigen peptides so as to confirm the accuracy and effectiveness of the neoantigen screening pipeline we developed.Methods:1.Tumor cell unique eight-eleven amino acid residue neoantigen peptide information acquisition:Collect tumor cells from cancer patients and their normal cells from tissue to obtain pMHC complexes using immunomagnetic bead precipitation,combined with liquid chromatography combined with mass spectrometry for detection to obtain cancer cell unique neoantigen peptides.Neoantigen peptides with a length of eight-eleven amino acid residues were retained,and four tumor neoantigen peptides with a peptide length of eight-eleven amino acid residues were obtained by primary screening after removing contaminating peptides（including peptides with a residue base of less than 8 or more than 11,peptides with anchor points or residue sequences significantly inconsistent with neoantigen peptide characteristics,and peptides with peptide mutant residues that did not bind to CDR3）.2.Whole exome sequencing was used to obtain HLA-Ⅰ molecular alleles and neoantigen peptide prediction in patients:whole exome sequencing was performed on tumor cells detected by mass spectrometry,and biological information analysis was used to obtain HLA-Ⅰmolecular allele typing and neoantigen peptide predicted by whole exome sequencing in derived patients.3.Prediction of tumor neoantigen peptides effectively presented by HLA-Ⅰ molecules:The IC50 of tumor neoantigen peptides with HLA-Ⅰ molecular alleles derived from each peptide and the predicted half-life value of pMHC complex were seized by NetMHC-4.0 and NetMHCstabpan-1.0 software.IC50 values<500 nm were considered to indicate a high binding affinity of tumor neoantigen peptides to HLA-Ⅰ molecules,and the predicted half-life of pMHC complexes>1 hour was considered to indicate a stable binding of tumor neoantigen peptides to HLA-Ⅰ molecules.According to similar studies,IC50 values<500 nm,and the expected half-life of pMHC complexes>1 hour were set as tumor neoantigen peptides that could be effectively presented by HLA-Ⅰ molecules.The neoantigen peptides before and after the above screening were verified by in vitro immunocompetence experiments.4.Immunogenicity test of ELISPOT assay in vitro:PBMC were obtained from peripheral blood of patients derived from samples by Ficoll density gradient centrifugation for in vitro culture.Differentiation into mature DC cells was induced with an equal mass of neoantigen peptide,and the immunological effects in vitro were detected by enzyme-linked immunological spot counting after co-culture with fresh PBMCs.5.TCR immune bank sequencing:Some tumor neoantigen peptides predicted to be able or unable to be effectively presented by HLA-Ⅰ molecules were randomly selected,PBMCs were induced to differentiate into mature DC cells with equal mass tumor neoantigen peptides and co-cultured with fresh PBMCs,and T cell immune bank sequencing technology was used to detect TCR-β chain changes before and after stimulation with two groups of neoantigen peptides.6.Statistical analysis:The total number of clones obtained by T’obtained by ELISPOT assay and T cell immune panel sequencing,and Clonality values were analyzed by independent sample t-test using SPSS 26 software,and P<0.05 was set as statistically significant.ResultsPart Ⅰ:Neoantigen peptides of eight-eleven peptides that were not present in normal tissues but unique to tumor cells were successfully obtained:a total of 5338 peptides were detected in tumor cells and normal cells of their derived tissues from four patients with different tumors using immunomagnetic bead precipitation combined with liquid chromatography combined with mass spectrometry.There were 650 neoantigen peptides unique to cancer cells,including 165 contaminating peptides,accounting for 25.38%.After removal,a total of 485 tumor neoantigen peptides with a length of eight-eleven amino acid residues were obtained,of which the nine-peptide was the highest,with a total of 57.32%.Part Ⅱ:1.According to the bioinformatics analysis of whole exome sequencing in four patients,the HLA-Ⅰ molecular allele type of each patient was successfully obtained,and the results were showed in table 3.2.The IC50 values of tumor neoantigen peptides detected by mass spectrometry and HLA-Ⅰ molecular alleles of patients derived from each peptide were seized by NetMHC-4.0 software.Accordingly,the affinity magnitude of 485 neoantigen peptides to HLA-Ⅰ molecules was determined.3.The predicted half-life of the pMHC complex between the tumor neoantigen peptide detected by mass spectrometry and the HLA-Ⅰ molecular allele of the patient from which each peptide was derived was seized by NetMHCstabpan-1.0 software.Based on this,485 tumor neoantigen peptides binding stability data to HLA-Ⅰ molecules were determined.4.The IC50 value<500 nm,and the predicted half-life of pMHC complex>1 hour were set as tumor neoantigen peptides that could be effectively presented by HLA-Ⅰ molecules.A total of 135 peptides that were effectively presented were selected from 485 tumor neoantigen peptides.5.Whole exome sequencing predicted 275 neoantigen peptides,of which only 2 appeared in the above 485 tumor neoantigen peptides and only 1 appeared in the above 135 peptides.Part Ⅲ:1.The mixed DC vaccine was successfully prepared by Ficoll density gradient centrifugation.2.ELISPOT in vitro immunocompetence test results:1)Independent sample t-test was performed for the immunocompetence T ’values of the higher and lower HLA-Ⅰ molecule binding affinity groups,and the result was P<0.001,and the difference was statistically significant.The results showed that the tumor neoantigen peptide with high binding affinity with HLA-Ⅰ had strong immune activity;2)Independent sample t-test was performed for predicting the T’values of the tumor neoantigen peptide in the more stable HLA-Ⅰ molecule binding group,P<0.001,and the difference was statistically significant.The results showed that the tumor neoantigen peptide with stable binding to HLA-had strong immune activity;3)Independent sample t-test was performed for the T ’values of the tumor neoantigen peptide group that could or could not be effectively presented by HLA-Ⅰ molecules,P<0.001,and the difference was statistically significant.It shows that the tumor neoantigen peptides that can be effectively presented by HLA-Ⅰ molecules have immunocompetence.3.Sequencing results of TCR β chain immune panel:1)Independent sample t-test was performed for the percentage change in the total number of clones obtained after detection of T cell receptor β chain after stimulation with neoantigen peptides predicted to be able or unable to be effectively presented by HLA-Ⅰmolecules,P=0.018,and the difference was statistically significant.Independent sample t-test was performed for the change in Clonality（i.e.,Clonality’）before and after stimulation with antigenic peptides in the two groups,P=0.007,and the difference was statistically significant.Compared with tumor neoantigen peptides that could not be effectively presented by HLA-Ⅰ molecules,tumor neoantigen peptides that could be effectively presented by HLA-Ⅰmolecules stimulated TCR clonal proliferation,confirming that tumor neoantigen peptides effectively presented by HLA-Ⅰ molecules selected using HLA-Ⅰ molecule binding affinity and binding stability were immunoeffective neoantigen peptides.2)The V-J gene rearrangement results of the TCR-β chain after stimulation with tumor neoantigen peptides predicted to be or not efficiently presented by HLA-Ⅰ were obtained.3)The CDR3 length changes of the TCR β chain after stimulation with tumor neoantigen peptides were obtained,and it was found that the CDR3 length range of the TCR β chain of after stimulation with tumor neoantigen peptides accounted for the highest proportion in the range of 40-50 bp.4)The predicted TCR-β chain clonal snail map after stimulation with tumor neoantigen peptides that could or could not be effectively presented by HLA-Ⅰ molecules was obtained,more than three TCR clone sequences and the first 20%TCR clone sequences could be measured in the obtained TCR β chain sequencing data after stimulation with tumor neoantigen peptides that could be effectively presented by HLA-Ⅰ were increased compared with the blank control group,and more than three TCR clone sequences and the first 20%TCR clone frequencies that could not be measured after stimulation with tumor neoantigen peptides that could not be effectively presented by HLA-Ⅰ were decreased compared with their corresponding blank control groups.The results showed that the tumor neoantigen peptides predicted to be effectively presented by HLA-Ⅰ were immunoeffective neoantigen peptides.ConclusionFirst,immunomagnetic bead precipitation combined with liquid chromatography combined with mass spectrometry can preliminarily screen the neoantigen peptides bound by HLA-Ⅰ molecules unique to tumor cells,but the proportion of contaminating peptides is relatively high;increasing the depth of mass spectrometry detection can reduce the omission of neoantigen peptides,but may increase the proportion of contaminating peptides.Second,the binding affinity and stability of neoantigen peptides to HLA-Ⅰ molecules can be used to simply and effectively screen immunocompetent neoantigen peptides.Third,TCR immune repertoire sequencing can be used to assess the dynamic changes of TCR immune repertoire before and after neoantigen peptide stimulation,confirm the immunological effect and immune specificity of neoantigen peptides,and help predict the therapeutic effect of individualized tumor vaccines.Fourth,the prediction of tumor neoantigen peptides based on whole exome sequencing alone has very low accuracy and limited clinical application value.Fifth,the combination of immunomagnetic bead precipitation,liquid chromatography combined with mass spectrometry,whole exome sequencing,ELISPOT technology and TCR immunobank sequencing can form an accurate screening pipeline for truly existing immunoeffective neoantigen peptides. |
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