Study On The Killing Effect Of H3 Chimeric Antigen Receptor T Cells On MDA-MB-231 Cells

Objective Constructing H3(abbreviation of B7H3)chimeric antigen receptor T cells,then exploring its killing effects on MDA-MB-231 cells and the feasibility and effectiveness of chimeric antigen receptor T cells targeting human H3 tumor surface antigen in triple negative breast cancer treatment.Methods 1 Detection of H3 protein expression in TNBC and non-TNBC tissues.1)78 cases of TNBC and 54 cases of non-TNBC were collected in this study.2)The expression of H3 protein in TNBC and non-TNBC tumor tissue were detected by Immunohistochemistry.2 Construction of H3 CAR expression plasmid1)PLVX-CD19-CD137-CD3 plasmid and PCDH-EF1-H3scFV plasmid doubledigestion(XbaI/SgrAI)to obtain the linear vector(vector containing-CD137-CD3) and target fragment(H3 scFV).2)The target fragment(H3 scFV)and the linear vector(vector containing-CD137-CD3)were ligated by T4 ligase to obtain a H3 CAR expression plasmid.3 Packaging,concentration,titer determination of H3 lentiviral vector and detection of H3 protein in TNBC cells.1)The H3 CAR expression plasmid and two packaging plasmids(pSPAX2,pM-D2.G)were transfected into 293FT cells(human embryonic kidney cells)by the calcium phosphate method.2)Collecting the cell supernatant of 48h and 72h,centrifuge to remove the cell debris,concentrate the virus using an ultracentrifuge,dissolve the viruspellet in 1640 medium,dispense,and freeze with the liquid nitrogen and store in a refrigerator at-80℃.3)The 293FT cells were infected after diluting the virus.After 48 hours,the percentage of H3-positive cells was detected by flow cytometry,and the virus biological titer was calculated.4)The TNBC cells MDA-MB-231 and MDA-MB-231 H3KO were cultured,and2.5×10~5 cells with good growth status were taken respectively.The expression level of H3 protein on the surface of TNBC was detected by flow cytometry.4 Lentivirus infection of PBMCs 1)Peripheral blood mononuclear cells(PMBCs)from healthy humans were isolated,and using the CD3/CD28 magnetic beads to stimulate the activation and proliferation of T cells.2)After 48h of stimulation,H3 lentivirus(MOI=20)infected PBMCs.After 96h of infection,the expression rate of H3 CAR and the CAR T cells phenotype were detected by flow cytometry.5 the function of CAR T cells targeted killing MDA-MB-231 cells 1)H3 CAR T cells and uninfected T cells were co-cultured with MDA-MB-231cells and MDA-MB-231 H3KO cells,respectively,and compared the killing effect of on the MDA-MB-231 cells and MDA-MB-231 H3KO cells.2)H3-CAR T cells and uninfected T cells were co-cultured with MDA-MB-231cells and MDA-MB-231 H3KO cells,respectively,to compare the release levels of IL-2、TNF and INF-γ.3)H3-CAR T cells and uninfected T cells were co-cultured with MDA-MB-231cells and MDA-MB-231 H3KO cells,respectively,and the release levels of IL-2 and INF-γwere again verified by intracellular staining.Result 1 Detection of H3 protein expression in TNBC and non-TNBC tissues.The proportion of patients with positive H3 expression in TNBC and non-TNBC were respectively 67% and 30%.The expression of H3 protein was significantly different between TNBC and non-TNBC(p<0.001).2 Construction of H3 CAR expression plasmid and determination of H3 lentivirus titer.1)After the enzyme digestion and ligation,pick the monoclonal,pick up the plasmid and run the gel for identification,pick the cloned plasmid with the correct size for DNA sequencing,and the sequencing result is correct.2)The H3 CAR lentivirus titer is 1.5×10~8 TU/ml.3 H3 CAR T cell preparation and phenotypic detection 1)The H3 CAR expression rate was 63.4%after H3 CAR lentivirus infected T cells.2)CAR T cell phenotype detection:The results of single positive:CD3~+T cells accounted for 96.2%,CD4~+T cells accounted for 33.89%,and CD8~+T cells accounted for 56.6%,CAR T cells accounted for 64.6%.Double positive results:CAR~+CD3~+T cells accounted for 64.6%,CAR~+CD-4~+T cells accounted for 27%,and CAR~+CD8~+T cells accounted for 39.6%.The CAR T cells are mainly composed of CD3~+CD8~+T cells.4 Functional detection of CAR T cell targeted killing MDA-MB-231 cells1)In vitro killing assay showed that the killing effect of H3 CAR T cells on MDA-MB-231 cells was significantly different from that of control T cells(p<0.001)and the killing effect of H3 CAR T cells to MDA-MB-231 and MDA-MB-231 H3KO cells was significantly different(p<0.001).2)After the co-culture of H3 CAR T cells and control T cells with MDA-MB-231 and MDA-MB-231 H3KO,CAR T cells co-cultured with MDA-MB-231 cells secrete more IL-2 and IFN-γthan control T cells.Therefore there was a significant difference in the level of cytokines secreted by the both(p<0.001).CAR T cells co-cultured with MDA-MB-231 secrete more IL-2 and IFN-γthan CAR T cells co-cultured with MDA-MB-231 H3KO,so the level of cytokines secreted by the both is significant difference(p<0.001).Conclusion 1.H3 CAR T cells were successfully prepared,and the H3 CAR molecule expressed well on T cells.2.H3 CAR T cells can target recognize and specifically kill H3~+MDA-MB-231cells in vitro.3.The killing effect of tumor cells by H3-CAR T cells depends on the number of CAR T cells and the expression level of H3 on tumor cells.4.This study provides theoretical and experimental basis for the clinical treatment of TNBC with H3 CAR T cells.