Optimization of the process of memory dual specificity CAR-T cells
Objective To develop a new process to reduce the differentiation degree of CAR-T cells in vitro,maintain the high concentration steady state of memory CAR-T(CAR-Tm),and enhance and prolong the tumor-killing effect of CAR-T cells.Methods We used flow cytometry(FACS)technology,to conduct memory CAR-T cell subpopulation analysis of T cells with the original process from CD3+T cells which were sorted from CD3/CD28 magnetic beads,and were continuously supplemented by IL-2 cytokines in vitro,and by using the new process from CD4+and CD8+T cells were sorted from CD4/CD8 magnetic beads,and activated by TransAct in vitro,and were added by IL-7 and IL-15 cytokines.Then,by comparing different TransAct dosages and cultivation times,the best process was determined.Results The proportion of low-differentiated memory CAR-T cells(CAR-Tscm+CAR-Tcm)in total T cells and CAR-T cells by the new process method was significantly higher than that by the original process(P＜0.05).The optimal amount of TransAct was 20％,and the optimal culture time was 12 days.Conclusion Combination of IL-7 cytokines,IL-15 cytokines and TransAct helps maintaing the high levels and numbers of tduration of low-differentiated targeted CD19/CD37 dual specificity memory CAR-T cells,laying a foundation for subsequent clinical research and industrialization.
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