Screening And Identification Of MHC-Ⅰ Related Antigen Peptides Of Cervical Cancer Derived From Autologous Protein

Background:Cervical cancer,as the most common gynecological malignant tumor,seriously threatens women’s health and lives.The monoclonal antibody therapy that has been launched recently has not been effective in prolonging the survival of patients with advanced stages.For young patients who retain fertility,or patients with advanced cervical cancer,there is no more effective treatment.Tumor therapeutic peptide vaccines based on tumor-related/specific antigen peptides have multiple advantages such as individualization,stimulating the host’s active immunity,strong targeting,significantly enhancing the therapeutic effect of immune checkpoints,and low toxic and side effects.Ideal results have been obtained in clinical trials of small cell lung cancer.So far,the development of therapeutic vaccines for cervical cancer is still based on human papillomavirus(Human Papillomavirus,HPV)related antigens,and because of the low mutation rate of cervical cancer,the probability of finding new antigens derived from mutations is low and it is not universally applicable to the population.There are no research reports on cervical cancer-related/specific antigens derived from self-protein.The sum of peptides presented by Major Histocompatibility Complex(MHC)-Ⅰ molecules in all nucleated cells is called MHC Ⅰ-associated immunopeptidome(MIP),It is derived from proteins synthesized and degraded in cells.The composition of MIP is highly complex and plastic,and is tightly regulated by intracellular/extracellular factors.CD8+ T cells can play a major role in the anti-tumor immune response by monitoring tumor-related/specific antigen peptides that are naturally presented on the surface of tumor cells by MHC-Ⅰ molecules that are different from normal cells.In recent years,mass spectrometry has become an ideal strategy for high-throughput MIP analysis due to its powerful ability to analyze complex peptide mixtures,greatly advancing the discovery of tumor-related/specific antigen peptides.Purposes:Isolate and identify MIP directly from patients’ cervical cancer tissues and corresponding normal cervical tissues,identify cervical cancer-related/specific antigen peptides derived from autologous proteins naturally presented by MHC-Ⅰ molecules,and provide new opportunities for the development of therapeutic vaccines for cervical cancer Target.Establish methods and strategies for directly extracting,identifying and screening MHC-Ⅰ molecule-related antigen peptides from human carcinoma in situ tissues,providing new ideas and methods for the research of therapeutic peptide vaccines for other solid tumors.Method:Collect 21 cases of cancerous tissues and contralateral normal cervical tissues from patients diagnosed with cervical cancer.Immunoaffinity Chromatography(IAC)was used to separate and purify MIP from tissues,and Western blot was used to detect the concentration of enriched MHC-Ⅰ molecules.Liquid chromatography-tandem mass spectrometry(LC-MS/MS)was used to detect MIP in samples.The search software SEQUEST was used to load the conventional human protein database and the customized peptide database to match the mass spectrometry data.The HLA-A/B/C nucleic acid typing test(PCR-SBT)method was used to perform high-resolution identification of HLA types in the peripheral blood of patients from tissue samples.Net MHCpan 4.0 was used to predict the binding ability of candidate peptides to specific HLA-I molecular types.By comparing the difference between the MIP collection of cervical cancer tissues from all patients and the MIP collection of normal cervical tissues,combined with the analysis of the tumor-related protein database and the frequency of patient coverage,several antigen peptides and their source proteins that are unique and frequently occurring in cervical cancer are selected.Finally,enzyme-linked immunospot assay(ELISPOT)and flow cytometry(Flow Cytometry,FC)were used to verify the immunogenicity of the obtained antigen peptides.Research content and results:1.Separation of MIP from cervical cancer and normal cervical tissues and identification by mass spectrometry based on conventional protein databases.A total of 21 pairs of cervical cancer and normal cervical tissues that meet the inclusion criteria were obtained in the sample library,and MIP samples were successfully separated and purified by IAC.A total of 21 pairs of cervical cancer and normal cervical tissues that meet the inclusion criteria were obtained in the sample library.The MIP samples were successfully separated and purified by IAC.After LC-MS/MS detection and loading of conventional protein databases,the output peptides of each sample were used After net MHCpan4.0 predicts the affinity to the specific HLA type of the sample,it retains the MHC-class I molecule binding peptides with Rank% <2%,and different numbers of MIPs(3470-24392)were identified from each sample.2.MIP high-throughput mass spectrometry identification and characterization based on customized peptide database.According to the 8-12 mer length characteristics of MIP,use Seq Kit to cut the protein sequences in Universal Protein and Human isoform protein continuously,directionally and with limited length,construct 8mer to 12 mer custom peptide libraries respectively,and re-match 21 pairs of sample mass data.After analysis,it was found that the customized peptide library significantly improved the quantity and quality of MIP identification.The number of MIPs for each sample is significantly different,there are significant individual differences,and there is no difference in the distribution of the three HLA-I loci.The MIP of each sample conforms to the basic characteristics of HLA-I molecule binding peptides.Among them,9-mer-length peptides account for the highest proportion of MIPs,and most of the conservative sites in the MIP sequence are located at the second and last amino acids;and different types The amino acid types at the conservative sites of the binding peptides are different.3.Screening of cervical cancer related antigen peptidesCombine 21 pairs of MIPs identified in cervical cancer and normal cervical tissues and compare the differences in peptide source proteins,and screen out cervical cancer tissue-specific MIP source proteins.Combined with the tumor-related protein reference database,candidate tumor-related autologous proteins were further screened.According to the frequency of appearance in the sample MIP,these candidate autologous proteins are sorted,and the autologous proteins GPX1(Glutathione peroxidase),MYO9B(Myosin IXB),MCM4(Minichromosome maintenance proteins-4),PNKP(Polynucleotide kinase phosphatase)are finally selected.,CKAP5(Cytoskeleton associated protein 5),LAMB1(Laminin subunit beta 1)and EIF3D(Eukaryotic translation initiation factor 3 subunit D)12 MIPs as antigenic peptide candidates.4.Verification of the immunogenicity of cervical cancer related antigen peptidesSynthesize peptides based on the amino acid sequences of 12 candidate peptides and verify the consistency of mass spectrometry identification.PBMCs from healthy donors and cervical cancer patients corresponding to HLA types are used to stimulate PBMC to secrete IFN-γ through indirect and direct candidate peptide methods.ELISPOT results suggest that the candidate peptides significantly increase the secretion of IFN-γ in PBMC from healthy donors.The results of flow cytometry showed that the candidate peptides can stimulate a significant increase in the secretion of IFN-γ by CD8+ T cells from healthy donors and patients.Conclusion:1.Innovatively established a limited-length customized peptide database,and successfully identified MIP derived from cervical cancer and normal cervical tissue.2.Successfully established a strategy based on the analysis of MIP-derived proteins to screen the autologous protein-derived MHC-Ⅰ molecule-related cervical cancer antigen peptides,and screened a series of candidate cervical cancer-related autologous protein antigens.3.In vitro experiments verified the immunogenicity of the cervical cancer-related/specific antigen peptides derived from autologous proteins,and provided a new target for the development of therapeutic peptide vaccines for cervical cancer in the future.