Therapeutic effect of CAR-γδT cells targeting at BCMA in multiple myeloma

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 successfully 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 significantly 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 adoptive immunotherapy of multiple myeloma.

Keywords

• b-cell maturation antigen|multiple myeloma|chimeric antigen receptors|γδt cells