Antitumor Effect Of Modified TP53 Shared Neoantigen On Liver Cancer

Objective In recent years,tumor neoantigen have attracted much attention as targets of immunotherapy,to become the main force of personalized immunotherapy.Shared neoantigens are common among different patients and have wider applicability than individual neoantigen.In this study,the shared neoantigen produced by TP53 gene mutation were modified by amino acid substitution.The aim is to explore whether the modified shared neoantigen can enhance its immunogenicity,so as to better stimulate and activate T cells,and further enhance the anti-tumor effect of TP53 shared neoantigen.It lays the foundation for providing better neoantigen vaccine for patients with liver cancer and other tumors with the same mutant epitope.Methods 1.Hotspot mutations of liver cancer TP53 gene were found through literature.The shared neoantigen was obtained by antigen peptide prediction algorithm,and its affinity was predicted by bioinformatics software.TP53-Y220 C shared neoantigen with low affinity was selected for amino acid residue modification.The unmodified TP53-Y220 C neoantigen and the modified TP53-Y220C(L2)epitope were synthesized,and the affinity and stability of the neoantigen were detected with T2 cells.2.The synthesized neoantigens were loaded into Dendritic cells(DC),and cytotoxic T lymphocytes(CTL)were induced.Enzyme linked immunosorbent assay(ELISA)was used to detect the secretion of interferon-γ(IFN-γ)and the proliferation of CTL cells was detected by flow cytometry with CFSE staining.Lactic acid dehydrogenase(LDH)release method was used to detect the killing effect of CTL stimulated by different neoantigens on T2 cell loaded with TP53-Y220 C neoantigen.3.CTL stimulated by modified and unmodified TP53-Y220 C neoantigen were used as effector cells.Liver cancer cells with or without TP53-Y220 C neoantigen were used as target cells.In vitro,LDH release assay was used to detect the cytotoxicity of CTL stimulated by different neoantigens on tumor cells.In vivo,liver cancer cells were inoculated into the embryo of zebrafish,and then injected with different neoantigen stimulated CTL.The inhibitory effect of CTL on the growth of Liver cancer cells was observed 24 h later.4.LDH test was used to detect the killing effect of CTL stimulated by different neoantigens on gastric cancer cells,pancreatic cancer cells and esophageal cancer cells with or without TP53-Y220 C neoantigen.The release of IFN-γ and tumor necrosis factor-α(TNF-α)were detected by ELISA and enzyme-linked immunospot assay(ELISPOT).Results 1.Prediction with bioinformatics software showed that the affinity of modified TP53-Y220C(L2)neoantigen was increased.The in vitro affinity assay showed that the unmodified TP53-Y220 C epitope had an affinity of 3.10±0.35,and the modified TP53-Y220C(L2)epitope had an affinity of 3.61±0.31.The stability of the TP53-Y220 C neoantigen was <18h,while the TP53-Y220C(L2)neoantigen was >18h.The dissociation rate at 12 h and 18 h were significantly different between the two groups(P<0.01).2.T cells proliferation assay and ELISA assay showed that the modified TP53-Y220C(L2)neoantigen could stimulate more T cells proliferation and secrete more IFN-γ than the modified TP53-Y220 C neoantigen.3.According to the result of cross recognition experiment,both TP53-Y220 C neoantigen and TP53-Y220C(L2)neoantigen stimulated CTL could recognize T2 cells with TP53-Y220 C neoantigen.Moreover,CTL stimulated by the modified neoantigen secreted more IFN-γ and TNF-α after co-culture with T2 cells.4.According to the in vitro killing experiment to liver cancer.CTL stimulated by the modified TP53-Y220C(L2)neoantigen could kill more Hu H7-HLA-A0201 cells and secrete more IFN-γ and TNF-α than unmodified neoantigen.Zebrafish experiment showed that CTL stimulated by the modified TP53-Y220C(L2)neoantigen could reduce the fluorescence area of tumor cells in zebrafish more than the unmodified neoantigen within 24 h.5.LDH release assay showed that both TP53-Y220 C neoantigen and TP53-Y220C(L2)neoantigen stimulated CTL could recognize and kill gastric cancer cells,pancreatic cancer cells and esophageal cancer cells all with TP53-Y220 C epitope,and CTL group stimulated by TP53-Y220C(L2)neoantigen secreted more IFN-γ and TNF-α.Conclusion 1.The modified TP53-Y220C(L2)neoantigen got increased affinity and significantly higher stability than the unmodified,and the modified neoantigen showed greater immunogenicity and activation of more T cell proliferation.2.The in vitro killing test to liver cancer cells and the in vivo zebrafish test proved that CTL activated by TP53-Y220C(L2)neoantigen had better cytotoxicity to liver cancer cells than CTL activated by TP53-Y220 C neoantigen.3.CTL stimulated by TP53-Y220C(L2)neoantigen showed better killing effect on gastric cancer cells,pancreatic cancer cells and esophageal cancer cells with TP53-Y220 C epitope than CTL activated by TP53-Y220 C neoantigen.So the TP53-Y220 C neoantigen accorded with the shared neoantigen.The TP53-Y220C(L2)neoantigen with enhanced affinity and stability is expected to be used in DC vaccine or as peptide vaccine for anti-tumor immunotherapy in a variety of tumors.