Construction of Chimeric Antigen Receptor T Cells Targeting Human BCMA and its Antitumor Activity in Vitro
Objective:To construct and screen the new type of chimeric antigen receptor CAR-T cell targeting human-derived B cell maturation antigen(BCMA),By modifying the ScFv single-chain variable region of the CAR structure,a novel chimeric antigen receptor(CAR)-T cell targeting human B-cell maturation antigen(BCMA)with a stronger ability to kill tumor cells was constructed and screened.Methods:We constructed a CAR molecule targeting BCMA human ScFv,and after successful packaging with retroviral vector,transducing T cells from healthy volunteers to prepare Anti-BCMA-CAR-T cells.Anti-BCMA-CAR-T cells were used as the observation group and normal T cells were used as the control group,they were co-cultured with RPMI-8226 cells,and the proliferation ability of the two groups in vitro was observed by the T cell proliferation experiment stained with CFSE.The killing efficiency of two groups of cells on RPMI-8226 cells at different effect-to-target ratios(1∶8,1∶4,1∶2,1∶1,2∶1,4∶1)was detected by luciferase chemiluminescence assay.Flow cytometry was used to detect the killing efficiency of the two groups of cells on RPMI-8226 cells at different effect-to-target ratios(1∶4,1∶2,1∶1,2∶1,4∶1).Results:CFSE detection results showed that compared with the control group,the FITC signal in the observation group shifted significantly to the left,indicating that the T cell proliferation ability was stronger.The results of flow cytometry showed that at the same effect target ratio,the killing efficiency of the observation group on RPMI-8226 cells was higher than that of the control group(both P＜0.05);the results of luciferase chemiluminescence experiment showed that the same effect target ratio,the killing efficiency of RPMI-8226 cells in the observation group was higher than that in the control group(P＜0.05).When the effect-to-target ratio was 4∶1,the killing efficiencies of CAR 170-T(unmodified conventional ScFv)cells and CAR174-T(modified ScFv)cells reached 88.5±0.3％and 98.5±0.7％,respectively.Conclusion:We successfully constructed a novel type of BCMA-targeting CAR-T cells by modifying the ScFv single-chain variable region of the CAR structure,which can maintain strong proliferative activity and have a stronger ability to kill tumor cells.
B cell maturation antigenChimeric antigen receptorScFv single-chain variable regionT cell
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