Identification Of CD147 CTL Epitope And Induce Specific CTLs For Use In Anti-drug Resistant Tumors

Objective:By studying the role of CD147 in drug-resistant tumor cells,CD147 was selected as a target and CD147 was screened for adoptive immunotherapy against CTL epitopes,and the wild-type epitopes were modified and induced in vitro Specific CTLs have identified CTL epitopes that are resistant to resistant tumors.To lay the foundation for the development of new methods and new targets for the development of TCR gene therapy for the specific T cell adoptive tumor therapy of point-mutation-related antigens.Method:First,the construction of PX458-CD147 knockdown plasmid by CRISPR knockdown technique was used to detect the changes of drug resistance and IC50 before and after MCF-7/Adr knockdown of CD147 by MTT method to determine whether CD147 is involved in the drug resistance of drug-resistant tumor cells.Second,the use of classical CTL epitope predicting system BIMAS and SYFPEITHI on CD147 Protein sequence for predicting CTL epitopes,looking for a low-affinity wild-type HLA-A2-restricted 9 amino acid epitope CD147_126-134,point mutation design of the two anchor residues of the 9peptide,by comparing the mutation The online predictive score was screened before and after,and the mutated peptides with higher predictive score were screened.Then,the results of the peptide affinity test and the MHC peptide binding stability test were validated by T2 cells.Thirdly,with normal human PBMCs,monocytes were obtained to induce mature DCs stimulated in vitro,and the mature DCs were loaded with wild-type epitope peptide CD147_126-134 and mutant peptide CD147_126-134L2 in vitro to induce the co-culture with PBMC Out of CTL.The cytotoxicity of CTL against T2-targeted cells was assayed by IFN-?ELISpot and calcein method.The cytotoxicity of CTL on MCF-7/Adr?A2+,CD147high+?,MCF?A2+,CD147low+?,Hela?A2-,CD147+?and SKOV3?A2+,CD147-?.Finally,the HLA-A2 antibody blocking experiments were used to verify whether CTLs were killed in an HLA-A2-restricted manner Target cells.The RNA extracted from experimental group was used to detect the expression of V?and V?subfamilies of T cell antigen receptor?TCR?before and after stimulation with CD147_126-134L2 epitope peptide by multiplex PCR and mulLtiplex capillary electrophoresis.The effects of antigen peptide-induced CTL can identify mutant peptide CD147_126-134L2antigen-specific T cell clone proliferation.Result:After knocking out CD147 by CRISPR,the resistance of MCF-7/Adr-resistant cells to CD147 was significantly reduced compared to that before knocking out.The mutated peptide was obtained after mutation of the wild-type CD147_126-134 epitope peptide at position 2 CD147_126-134L2,and the affinity and stability of the mutant peptide CD147_126-134L2 and HLA-A2were significantly increased.IFN-?ELISpot experiments showed that the CTLs induced by the mutant peptide CD147_126-134L2 not only can effectively recognize the T2 Target cells and had good cross-recognition effect on T2target cells loaded with wild-type peptide.Calcein assay cytotoxicity of CTLs on T2 target cells As can be seen with the gradual increase of effective target ratio,CTLs induced by mutant peptide CD147_126-134L2 showed effective recognition and killing effect on peptide-loaded T2 target cells CTLs induced by mutant peptide CD147_126-134L2 specifically recognize CD147-expressing tumor cells,and this cytotoxic effect is the most significant for MCF-7/Adr with high expression of CD147 The results showed that the wild-type CD147_126-134 epitope peptide could not be activated to induce CTL but could still be presented to the surface of tumor cells by CTLs induced by mutant peptide CD147_126-134L2.The HLA-A2antibody blocking experiment showed that this specificity killing was performed in a HLA-A2-restricted manner.Multiplex PCR combined with capillary electrophoresis showed clonal proliferation of these T cells in the V?2 family.Conclusion:Compared with normal tumor cells,CD147 is highly expressed in drug-resistant tumors and is involved in tumor drug resistance.In the subsequent study,we successfully identified the CD147 mutant epitope peptide CD147_126-134L2 with high affinity to HLA-A2 molecule by using point mutation technique and induced the CTL against this epitope peptide by using DC-loaded peptide method.The CTL could kill CD105-resistant tumor cells,and the antigen-specific T cells that recognize the mutant epitope peptide CD147_126-134L2 in CTL are a group of V?2-based cells.