靶向CLL-1嵌合抗原受体T细胞的构建及其功能验证

Development and functional verification of CAR-T cells targeting CLL-1

目的探索开发一种靶向CLL-1的嵌合抗原受体T细胞(CAR-T细胞)并验证其功能。方法通过流式细胞术检测急性髓系白血病(AML)细胞系和AML原代细胞CLL-1靶点的表达水平。构建CLL-1 CAR载体并制备出相应慢病毒,感染激活后T细胞生产出CAR-T细胞,并通过体外和体内实验验证CLL-1 CAR-T细胞的功能。结果AML细胞系和原代AML细胞中均表达CLL-1。制备的CLL-1 CAR-T细胞转导率为77.82%,在AML细胞系以及AML原代细胞中,CLL-1 CAR-T细胞能明显特异性杀伤表达CLL-1的靶细胞系和原代肿瘤细胞。相对于未转导的T细胞,CLL-1 CAR-T细胞在杀伤靶细胞和原代肿瘤细胞时分泌IL-6、IFN-γ等细胞因子水平更高(P值均<0.001)。在AML人源性异种移植小鼠模型中,相对于未转导的T细胞,CLL-1 CAR-T细胞表现出有效的抗白血病活性并延长小鼠存活时间[未达到对22(95%CI 19~24)d,P=0.002]。结论靶向CLL-1的CAR-T细胞成功开发并具有较好的肿瘤杀伤作用。

Objective To explore the development of a CAR-T cells targeting CLL-1 and verify its function.Methods The expression levels of CLL-1 targets in cell lines and primary cells were detected by flow cytometry.A CLL-1 CAR vector was constructed,and the corresponding lentivirus was prepared.After infection and activation of T cells,CAR-T cells targeting CLL-1 were produced and their function was verified in vitro and in vivo.Results CLL-1 was expressed in acute myeloid leukemia(AML)cell lines and primary AML cells.The transduction rate of the prepared CAR T cells was 77.82%.In AML cell lines and AML primary cells,CLL-1-targeting CAR-T cells significantly and specifically killed CLL-1-expressing cells.Compared to untransduced T cells,CAR-T cells killed target cells and secreted inflammatory cytokines,such as interleukin-6 and interferon-γ,at significantly higher levels(P<0.001).In an in vivo human xenograft mouse model of AML,CLL-1 CAR-T cells also exhibited potent antileukemic activity and induced prolonged mouse survival compared with untransduced T cells[not reached vs 22 days(95%CI 19-24 days),P=0.002].Conclusion CAR-T cells targeting CLL-1 have been successfully produced and have excellent functions.