IL-7的诱导表达增强靶向GPC3 CAR-T细胞的增殖及体外抗肿瘤活性

Inducible IL-7 expression enhances proliferation and in vitro antitumor activity of GPC3-specific CAR-T cells

目的:探讨IL-7的诱导表达对靶向磷脂酰肌醇蛋白聚糖3(GPC3)嵌合抗原受体基因修饰T淋巴细胞(CAR-T细胞)的增殖和体外抗肿瘤活性的影响。方法:通过无缝克隆将GPC3 CAR序列片段插入GV400载体的Bam HⅠ/Eco RⅠ位置,构建第二代CAR慢病毒载体GPC3-BBZ及GPC3-BBZ-NFAT-IL-7,以293T细胞包装相应的慢病毒载体后,感染人T细胞制备CAR-T细胞。实验分为未转导T细胞(NT)组、GPC3-BBZ CAR-T细胞组、GPC3-BBZ-NFAT-IL-7 CAR-T细胞组。采用流式细胞术检测各组CAR-T细胞中CAR的表达水平,qPCR法检测经GPC3蛋白激活的CAR-T细胞中IL-7 m RNA的表达水平,细胞计数法检测CAR-T细胞在GPC3抗原刺激下的增殖能力,ELISA检测CAR-T细胞在受到肿瘤细胞刺激后IL-7、IFN-γ和TNF-α的分泌水平。应用实时细胞分析(RTCA)技术检测CAR-T细胞对人肝癌Huh-7细胞的杀伤作用。结果:成功构建慢病毒载体GPC3-BBZ和GPC3-BBZ-NFAT-IL-7,制备出靶向GPC3的CAR-T细胞。经GPC3抗原激活后,GPC3-BBZ-NFAT-IL-7 CAR-T细胞可有效表达IL-7 mRNA(P<0.01),其表现出更强的增殖能力(P<0.05)。与GPC3-BBZ CAR-T细胞相比,GPC3-BBZ-NFAT-IL-7 CAR-T细胞与GPC3阳性靶细胞Huh-7细胞共培养后,分泌更高水平的IL-7、IFN-γ和TNF-α(P<0.01或P<0.001)。RTCA结果显示,GPC3-BBZ-NFAT-IL-7 CAR-T细胞对GPC3阳性Huh-7细胞的杀伤活性显著高于GPC3-BBZ CAR-T细胞(P<0.05)。结论:成功制备可诱导表达IL-7的靶向GPC3的CAR-T细胞,IL-7的诱导表达增强靶向GPC3 CAR-T细胞的免疫活性,在体外展现出较强的肿瘤细胞杀伤能力。

Objective:To explore the effects of induced IL-7 expression on proliferation and in vitro antitumor activities of glypican-3(GPC3)-specific chimeric antigen receptor gene modified-T(CAR-T)cells.Methods:The GPC3 CAR sequence fragment was inserted into the BamHⅠ/EcoRⅠsite of the GV400 vector using seamless cloning,constructing second-generation CAR lentiviral vectors GPC3-BBZ and GPC3-BBZ-NFAT-IL-7.The lentiviruses were packaged with 293T cells and transfected into healthy human T cells to prepare CAR-T cells,which were divided into non-transduced T cell(NT)group,GPC3-BBZ CAR-T cell group and GPC3-BBZ-NFAT-IL-7 CAR-T cell group.The expression of CAR in CAR-T cells of each group was determined by flow cytometry.IL-7 mRNA expression level in CAR-T cells activated by GPC3 protein was determined by qPCR.The proliferation ability of CAR-T cells under GPC3 antigen stimulation was evaluated by cell counting.The secretion levels of IL-7,IFN-γ,and TNF-αby CAR-T cells after stimulation with tumor cells was determined using ELISA.The cytotoxicity of CAR-T cells against human hepatocellular carcinoma Huh-7 cells was tested by real-time cell analyzer(RTCA).Results:Lentiviral vectors GPC3-BBZ and GPC3-BBZ-NFAT-IL-7 were successfully constructed,and GPC3-specific CAR-T cells were prepared.After activation with GPC3 antigen,GPC3-BBZ-NFAT-IL-7 CAR-T cells effectively expressed IL-7 mRNA(P<0.01)and exhibited stronger proliferation capacity(P<0.05).Compared with GPC3-BBZ CAR-T cells,GPC3-BBZ-NFAT-IL-7 CAR-T cells co-cultured with GPC3-positive Huh-7 cells secreted higher levels of IL-7,IFN-γ,and TNF-α(P<0.01 or P<0.001).The RTCA results showed that the cytotoxicity of GPC3-BBZ-NFAT-IL-7 CAR-T cells against GPC3-positive Huh-7 cells was significantly higher than that of GPC3-BBZ CAR-T cells(P<0.05).Conclusion:GPC3-specific CAR-T cells with inducible IL-7 expression were successfully prepared,which exhibited immune activity and tumor cell killing capacity in vitro.