Prediction And Identification Of HLA-A0201 Restricted Neoantigen In Breast Cancer

BackgroundBreast cancer is one of malignant tumors that seriously threaten the health of women around the world.Regardless of its morbidity or mortality,it ranks among the top malignant tumors in Chinese women.In recent years,the age of onset of breast cancer has become more and more young.Based on the development of multi-disciplinary treatments such as radiotherapy,chemotherapy,endocrine therapy and molecular targeted drug therapy,although the therapeutic effect of early breast cancer has been improved,its malignancy progress has not yet been fully and effectively controlled.Immunotherapy is an emerging field in cancer treatment that is expected to make up for the insufficient of traditional treatment methods.The immune mechanism is a key step which affects the development of tumors.Cytotic T Lymphocytes?CTL?are the main force that kill tumor cells,the key is that CTL can recognize specific neoantigens on the tumor surface due to gene mutations,while tumor-specific neoantigens can be identified by CTL and the process requires the function of antigen presentation from human leukocyte antigen?HLA?.HLA-A0201 is one of the HLA-A subtypes,which accounts for a high proportion of approximately 13%in the Chinese population.Currently,the main difficulty in academic research is the recognition and identification of tumor-specific neoantigens.Therefore,the urgent event to be solved in tumor immunocytotherapy research is to use a rapid and effective method to find neoantigens that can induce specific CTL to kill tumors,which will be a new hope for treating malignant tumors including breast cancer.ObjectiveThe purpose of this study was to quickly and efficiently identify neoantigen capable of inducing specific CTL responses in breast cancer by bioinformatics prediction technology combined with the experiment method of immunological,to provide an effective new target for tumor immunotherapy of breast cancer and lay a foundation for research.Methods1.We applied single nucleotide variation?SNVs?data in breast cancer from The Cancer Genome Atlas?TCGA?,the International Cancer Genome Consortium?ICGC?and the literature from Nature,using bioinformatics combined with affinity prediction software to predict human leukocyte antigen-A0201?HLA-A0201?restricted neoantigen-epitopes in breast cancer and chemical synthesis.The peptide binding assay was carried out by T2 cells to detect the binding affinity and stability of the neoantigen peptide,and the high affinity neoantigen peptide was optimized.2.CD8⁺T lymphocytes and dendritic cells?DC?were isolated from healthy donors'peripheral blood mononuclear cells?PBMC?.Cytokines of GM-CSF,IL-4,IFN-?and CD40L were used to induce differentiation and maturation of DC cells,and then DCs were loaded with high affinity neoantigen peptides and co-cultured with CD8⁺T lymphocytes to stimulate and induce specific CTLs.Finally,we collected specific CTLs.3.Immunogenicity assay of Enzyme-linked immunospot?ELISPOT?was used to detect the potency of CD8⁺T cells producing specific CTLs,CD8⁺T cells were stimulated by DCs loaded with neoantigen peptides;lactate dehydrogenase?LDH?cytotoxicity assay kit was used to determine the cytotoxicity of specific CTLs induced by DC cells loaded with neoantigen peptides.4.The neoantigen peptide encoded by TWISTNB_130-138 were screened to determine whether their mutant-type and wild-type peptides produced immunologic cross-reactivity by ELISPOT assay.The ELISPOT assay was performed to detect whether the surface of HCT116 cancer cells stably transfected with TWISTNB_130-13830-138 can present TWISTNB_130-13830-138 neoantigen peptide and were recognized by specific CTLs induced by TWISTNB_130-138.5.We expanded the sample size of healthy donators and tested whether DC cells sensitized by TWISTNB_130-138 neoantigen peptide can induce specific CTLs by ELISPOT assay.Results1.HLA-A0201 restricted 9mer peptides were successfully predicted by bioinformatics technology combined with 6 affinity prediction software,and 30 breast cancer neoantigen polypeptide sequences were screened according to affinity score and mutation frequency and were synthesized successfully.2.Using peptide binding experiments,T2 cells loaded with neoantigen peptides were used as target cells to optimize neoantigen candidate peptides.Ten neoantigen peptide sequences were successfully optimized by screening criteria:RHO_261-269?FLMCWVPYA?,SLC13A3375-383?FLYDAVTGV?,PIK3CA_451-459?GLKDLLNPI?,CLN3120-128?YLLPYSPRV?,PIK3CA338-346?ILCATYVKV?,MRGPRX4225-233?FLLCGLPFV?,COL14A1_1035-1043?FMVDGFWSI?,TWISTNB_130-138?KLMGIVYKV?,CCNA2213-221?ILVDWLFEV?,CDC37L1_196-204?YLILWCFYL?.3.Using ELISPOT assay to detect the ability of 10 neoantigen peptides induce specific CTLs in vitro.Except for RHO_261-269,CLN3_120-128 and CCNA2_213-221,the number of IFN-?spots formed by the remaining 7 peptide-stimulated CTLs were significantly increased compared with the irrelevant peptide group.Among them,TWISTNB_130-138 stimulated CTLs formed IFN-?spots of 565±9.19,and about 5.65%?565/10000?of CTLs secreted IFN-?,which was 25.7 times higher than irrelevant peptides?p<0.001?.4.In the cytotoxicity assay,specific CTLs induced by five candidate peptides have cytotoxicity against T2 target cells loaded with TWISTNB_130-138,PIK3CA_338-346,COL14A1_1035-1043,SLC13A3375-383 and CDC37L1_196-204.When the ratio of effector cells to target cells increased from 1:1 to 10:1,the average percentage of specific killings increased by 34.7%,11.73%,12.63%,9.77%and 9.10%respectively.The cytotoxicity of TWISTNB_130-138 group was the most significant.5.The ELISPOT result showed that when specific CTLs induced by TWISTNB_130-13830-138 mutant-type peptide,were stimulated by mutant type peptide-sensitized T2 cells,the number of IFN-?spots was highter than IFN-?spots produced by T2 cells sensitized by wild-type peptide and irrelevant peptide?P<0.01?.However,when specific CTLs that induced by TWISTNB_130-138 wild-type peptide,were stimulated by mutant peptide-sensitized T2 cells,the number of IFN-?spots was lower than IFN-?spots produced by T2 cells sensitized by wild-type peptides and was similar to IFN-?spots produced by T2cells sensitized by irrelevant peptides.6.The result of ELISPOT showed that positive spots produced by TWISTNB_130-138mutant peptide-specific CTLs stimulated by HCT116 cells which stably expressing TWISTNB_130-138 mutant gene,were much higher than those stimulated by HCT116 cells?P<0.01?.7.The immunogenicity of TWISTNB_130-13830-138 mutant peptide was verified by ELISPOT assay in other 7 healthy donors.The result showed that the number of IFN-?spots formed by TWISTNB_130-138 mutant peptide-stimulated CTLs were significantly higher than irrelevant peptides group?P<0.05?.Conclusions1.After stimulation with DCs loaded with HLA-A0201 restricted peptides of TWISTNB_130-138,PIK3CA338-346,COL14A1_1035-1043,SLC13A3375-383 and CDC37L1_196-204,specific CTLs can be efficiently activated and amplified in vitro,and these CTLs produced cytotoxicity to T2 target cells loaded with corresponding peptides.2.TWISTNB_130-138,a neoantigen mutant peptide,was successfully identified and able to induce specific CTLs and may have immunologic cross-reactivity with TWISTNB_130-138 wild-type peptide.3.Mutant peptides derived from TWISTNB_130-138 were able to be presented on HLA-A0201 restricted HCT116 tumor cells and were recognized by specific CTLs induced by TWISTNB130-138.4.Mutant peptides derived from TWISTNB_130-138 can induce specific CTLs in vitro in different healthy donors.5.In summary,using the existing sequencing data in the database combined with bio-information neoantigen prediction technology and immune cell experiments of healthy donators in vitro,it is possible to quickly and effectively screen out breast cancer neoantigens and provide research basis for breast cancer tumor immunotherapy.It also provides an effective way to identify a variety of tumor neantigens.