shRNA靶向抑制CD38的抗CD38 CAR-T细胞构建及其功能的初步鉴定

Construction of anti-CD38 CAR-T cells with targeted inhibition of CD38 by shRNA and preliminary identification of its functions

目的:探讨shRNA靶向抑制CD38的方法能否增强抗CD38 CAR-T细胞的抗癌功能。方法:构建shRNA靶向抑制CD38的抗CD38 CAR-T细胞的CAR分子,利用逆转录病毒载体包装成功后转导人原代T细胞,制备CAR-T细胞。实验分为shRNA1 CD38 CAR-T组、shRNA2 CD38 CAR-T组和对照组(shR-NC-CD38 CAR-T细胞)。采用qPCR法检测CAR-T细胞CD38 mRNA相对表达水平,计算CAR-T细胞培养0~14 d的增殖倍数,CFSE法检测CAR-T细胞与人Burkitt淋巴瘤细胞Raji-luc或人多发性骨髓瘤外周血B淋巴细胞RPMI-8226-luc共培养时的增殖情况,荧光素酶化学发光法检测CAR-T细胞在不同效靶比(1∶1、1∶2、1∶4、1∶8)时对Raji-luc和RPMI-8226-luc细胞的杀伤效率,ELISA法检测CAR-T细胞杀伤Raji-luc或RPMI-8226-luc细胞时上清液中IFN-γ水平,FCM检测CAR-T细胞表面耗竭T细胞生物标志物PD-1的表达水平。结果:shR-NC-CD38 CAR、shRNA1-CD38 CAR和shRNA2-CD38 CAR逆转录病毒载体的滴度均为1×107拷贝/mL,转导T细胞后,shR-NC-CD38 CAR-T、shRNA1-CD38 CAR-T和shRNA2-CD38 CAR-T细胞的转导效率(CAR的阳性率)分别为60.3%、67.0%和57.4%。与对照组比较,shRNA2-CD38 CAR-T组细胞中CD38 m RNA的表达水平显著降低(P<0.01),显示shRNA-CD38 CAR-T细胞构建成功。shRNA2-CD38 CAR-T组细胞在体外培养增殖能力更强(P<0.05),对2种CD38阳性的肿瘤细胞的杀伤效率更高(均P<0.05)、IFN-γ释放水平更高(均P<0.05)、细胞表面PD-1的表达水平更低(P<0.05)。结论:成功构建一种shRNA靶向抑制CD38的抗CD38 CAR-T细胞,其抗癌功能表现出明显的优势。

Objective:To investigate whether targeted inhibition of CD38 by shRNA can enhance the anticancer function of anti-CD38 CAR T cells. Methods: The anti-CD38 CAR molecules with targeted inhibition of CD38 by shRNA were constructed. After being successfully packaged with retroviral vector, the CAR molecules were transferred into human primary T cells to prepare CAR-T cells,which were then divided into shRNA1-CD38 CAR-T group, shRNA2-CD38 CAR-T group and control group(shR-NC-CD38 CAR-T cells). The relative expression of CD38 mRNA in CAR-T cells was detected by qPCR method, and the proliferation index of CAR-T cells cultured ex vivo for 0-14 days was calculated. The proliferation of CAR-T cells co-cultured with human Burkitt lymphoma Raji-luc cells or human multiple myeloma peripheral blood B lymphocytes RPMI-8226-luc was detected by CFSE method. The killing efficiency of CAR-T cells against Raji-luc and RPMI-8226-luc cells at different effector-target ratios(1∶1, 1∶2, 1∶4, 1∶8) was detected by luciferase chemiluminescence. The level of IFN-γ in the supernatant of CAR-T cells co-cultured with Raji-luc cells or RPMI-8226-luc cells was detected by ELISA. The expression of PD-1, one of the T cell exhaustion biomarkers, on the surface of CAR-T cells was detected by flow cytometry. Results: The titers of shR-NC-CD38 CAR, shRNA1-CD38 CAR and shRNA2-CD38 CAR retroviral vectors were all about 1×107 copies/mL. The transduction efficiency(CAR positive rate) in cells of shR-NC-CD38 CAR-T group, shRNA1-CD38 CAR-T group and shRNA2-CD38 CAR-T group was 60.3%, 67.0% and 57.4%, respectively. Compared with the control group, the expression level of CD38 mRNA in shRNA2-CD38 CAR-T group was significantly lower(P<0.01),indicating the successful construction of shRNA-CD38 CAR-T cells. For the cells in shRNA2-CD38 CAR-T group, their ex vivo proliferation ability was stronger, the killing efficiency against two CD38 positive tumor cells was higher, the release level of IFN-γ was higher, and the surface expression level of PD-1 was lower, as compared with the other two groups(P<0.05). Conclusion: A novel antiCD38 CAR-T cells with targeted inhibition of CD38 by shRNA was successfully constructed, which exhibited obvious advantages in antitumor function.