基础医学与临床2024，Vol.44Issue(6)：763-771,9.DOI:10.16352/j.issn.1001-6325.2024.06.0763
靶向BCMA的CAR-γδT细胞抗多发性骨髓瘤的疗效
Therapeutic effect of CAR-γδT cells targeting at BCMA in multiple myeloma
摘要
Abstract
Objective To construct chimeric antigen receptors modified γδT cells targeting at BCMA(BCMA CAR-γδT)and to evaluate its efficacy of anti-multiple myeloma in vitro.Methods Lentiviral vectors containing BCMA single-chain variable fragment were constructed and transiently transfected into 293T cells.The expression of foreign genes was verified by fluorescence microscopy and Western blot;the lentivirus was packaged and the virus titer was determined by flow cytometry.Human peripheral blood αβT cells were infected and γδT cells were examined for its infection efficiency;LDH release method was used to detect the cytotoxic activity of BCMA CAR-γδT cells against human multiple myeloma cell lines in vitro,and the difference of cytotoxic activity between CAR-γδT cells and CAR-αβT cells was compared by Incucyte S3 Live-Cell Analysis Instrument.Results Twenty-four hours after BCMA-CAR lentiviral vector was transferred into 293T cells,the expression of exogenous ZsGreen was microscopied by fluorescence microscope;CD3ζ was detected by Western blot,which showed that BCMA-CAR could be success-fully expressed.The lentivirus was packaged,collected and concentrated(virus titer of 2.23×108 Tu/mL).Infected αβT cells and γδT cells from human peripheral blood in MOI=5,and the results of flow cytometry showed that infection efficiency of αβT cells was 59.18%±2.56%,γδT cells was 48.15±9.86%.The cytotoxic activity of CAR-γδT cells against human myeloma cell lines MM1.S,H929 with high expression of BCMA and K562 cells with over-expression of BCMA was higher than that of empty vector control γδ T cells,which were signifi-cantly enhanced(P＜0.001),but there was no difference in cell lines negative for BCMA expression;Live-Cell Analysis Instrument results showed that the cytotoxic activity of BCMA CAR-γδT cells and BCMA CAR-αβT cells against H929 in vitro was significantly better than their vector control cells.There was no difference in the cytotoxic activities of BCMA CAR-γδT cells as compared with against BCMA negative cell lines,and so do BCMA CAR-T cells.Conclusions Cytotoxic activity of BCMA CAR-γδT targeting at BCMA in vitro was significantly enhanced,which is expected to serve as a novel allogeneic γδT cell product for cell a-doptive immunotherapy of multiple myeloma.关键词
B细胞成熟抗原/多发性骨髓瘤/嵌合抗原受体/γδT细胞Key words
B-cell maturation antigen/multiple myeloma/chimeric antigen receptors/γδT cells分类
医药卫生引用本文复制引用
李盈辉,许依,张建民,陈慧,何维..靶向BCMA的CAR-γδT细胞抗多发性骨髓瘤的疗效[J].基础医学与临床,2024,44(6):763-771,9.基金项目
中国医学科学院医学与健康科技创新工程项目(2021-I2M-1-035) （2021-I2M-1-035）
国家自然科学基金(32270915,U20A20374) （32270915,U20A20374）
