Specific Cytotoxicity Of EGFRvⅢ Oriented Chimeric Antigen Receptor-engineered T Cells On SMMC-7721

BackgroundLiver cancer is a common tumor of the digestive system,and current treatment methods cannot improve the quality of patients’life and their prognosis.In recent years,CAR-T immunotherapy has shown great potential in the field of tumor therapy.The application of CAR-T cell immunotherapy in the treatment of hepatocellular carcinoma has attracted much attention.The ability of targeted CAR-T cells to kill HCC cells remains to be demonstrated.Studies have found that liver cancer cell SMMC-7721 express EGFRvⅢ,SMMC-7721 was selected in this study as the target cell,and the specific killing activity of the constructed CAR-T cells was observed to provide experimental basis for CAR-T treatment of liver cancer,and for cellular immunotherapy of EGFRvⅢ positive tumors.ObjectiveTo observe the specific killing effect of anti-EGFRvⅢ chimeric antigen receptor modified T cells(EGFRvⅢ/2CAR-T)on liver cancer cell SMMC-7721.Methods1.Expression cassette of EGFRvⅢ/2CAR(1374bp)was inserted into XbaI and BamHI sites of Piggybac transposon vector PB513B-1(PB-EGFRvⅢ/2CAR).2.The latter together with transposase plasmid was then transducted into human peripheral blood T cells by electroporation(1ⅹ20ms,840V).The expression of EGFRvⅢ/2CAR on T cells was detected by flow cytometry.3.The expression of EGFRvⅢ in SMMC-7721 was detected by Western blot.4.EGFRvⅢ/2CAR-T cells were co-cultured with SMMC-7721 for 24h,then the killing effect was detected by LDH release test and cytokine IFN-γsecretion.Results1.The recombinant vector PB-EGFRvⅢ/2CAR was confirmed by XbaI and BamHI enzyme digestion,electrophoresis and DNA sequencing analysis.2.The expression of EGFRvⅢ/2CAR on T cells was detected by Flow cytometry(24h,55.5%).3.Western blot analysis showed that a protein band of approximate 145 000 molecular weight was observed in SMMC-7721 cells,which corresponds to the calculated molecular weight of EGFRvⅢ.4.EGFRvⅢ/2CAR-T cells were co-cultured with SMMC-7721 for 24h,LDH release test showed that tumor specific killing was positively correlated with E:T ratio(E:T=1:1,5:1,10:1,15:1,20:1);and the mortality rate of liver cancer cell SMMC-7721 was significantly higher than that of the control group at the E:T ratio of 5:1 to 20:1;ELISA showed that cytokine IFN-γsecretion was(1060.1±89)pg·ml-1,which was significantly higher than control group(P<0.001).ConclusionAnti-EGFRvⅢ CAR-T cells can kill specifically EGFRvⅢ~+liver cancer cell SMMC-7721,providing research basis for immunotherapy of hepatic cancer.