靶向人CD70的嵌合抗原受体巨噬细胞制备及其对肾癌的杀伤作用的研究

Construction of Chimeric Antigen Receptor Macrophages Targeting Hu-man CD70 and Their Killing Effect on Renal Carcinoma

目的:本研究旨在探讨稳定感染靶向人CD70蛋白的嵌合抗原受体巨噬细胞(chimeric antigen receptor ma-cophages,CAR-M)对人肾癌细胞系(786-O、OS-RC-2和ACHN)的体外杀伤作用,开发靶向人CD70的肾癌细胞的新型细胞疗法。方法:将含人CD70抗体scFv片段的CAR序列插入含有GFP序列的慢病毒质粒中,制备CD70-scFV-CAR-GFP的慢病毒。通过慢病毒感染小鼠巨噬细胞RAW264.7制备CD70-CAR-M,通过流式细胞仪分选出含有绿色荧光标记的CD70-CAR-M细胞并在体外筛选稳定表达细胞株。将CD70-CAR-M细胞与不同类型的肾癌类器官按照1∶1的比例分别共培养后,利用流式细胞术检测CD70-CAR-M对肾癌细胞的杀伤作用。qRT-PCR检测CD70-CAR-M在和肾癌细胞系共培养后TNF-α和TGF-β的表达水平改变情况。结果:通过测序确认了CD70-scFV序列,并成功制备了相应的慢病毒。利用该慢病毒感染巨噬细胞RAW264.7,通过qRT-PCR技术证明CD70-CAR分子稳定表达在RAW264.7。通过和不同类型的肾癌细胞系共培养后,流式细胞术结果显示,与对照组相比,CD70-CAR-M共培养后的肿瘤细胞数量明显减少。同时发现CD70-CAR-M的TNF-α表达水平明显增加,而TGF-β的表达水平明显降低,从而对肿瘤产生较强的杀伤作用。结论:成功构建了靶向人CD70的CAR-M巨噬细胞,并且在体外证明其能对CD70阳性肾癌细胞产生杀伤作用。

Objective:The aim of this study was to generate stable chimeric antigen receptor macrophages(CAR-M)tar-geting human CD70 proteins,detect their killing effect on human renal cell carcinomas(786-O,OS-RC-2,ACHN)in vitro,and develop novel cell therapy targeting CD70-positive renal cell carcinomas(RCC).Methods:The CAR sequence contai-ning the scFv fragment of human CD70 antibody was inserted into lentivirus plasmids containing the GFP sequence to prepare CD70-scFV-CAR-GFP lentivirus.Mouse macrophage cell line RAW264.7 cells were infected with CD70-CAR-GFP lentivirus.Infected CD70-CAR-M marked by green fluorescent were sorted out by FACS and cultured in vitro.CD70-CAR-M was co-cultured with renal RCC with a 1∶1 ratio,and the killing effect of CD70-CAR-M on RCCs was detected by flow cy-tometry.Real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)was used to detect the expression levels of TNF-αand TGF-βafter CD70-CAR-M had been co-cultured with RCCs.Results:The sequence of human CD70-scFV was confirmed by sequencing,and the corresponding lentivirus was successfully generated.qRT-PCR results showed that CD70-CAR molecule was stably expressed in RAW264.7 after lentivirus infection.Flow cytometry showed that compared with the control group,CD70-CAR-M significantly reduced the number of cells.In addition,we found that CD70-CAR-M ex-pressed high level of TNF-αbut low level of TGF-βafter being co-cultured with cancer cells.Conclusion:We have success-fully generated the CD70-CAR-M targeting human CD70 proteins,and have demonstrated that they have a strong killing effect on CD70-positive RCCs in vitro.