一种基于突变型IgG4长铰链区和4-1BB共刺激域的靶向CD19 CAR-T细胞构建及抗白血病的功能研究  

Evaluating anti-leukemia efficacy of a new anti-CD19 chimeric antigen receptor T cells using mutated IgG4 long spacer and 4-1BB costimulatory domain

作　　者：张灿 安志静 白玥 王琛琛 钟晓松 ZHANG Can;AN Zhijing;BAI Yue;WANG Chenchen;ZHONG Xiaosong(The Clinical Center of Gene and Cell Engineering,Beijing Shijitan Hospital,Capital Medical University,Beijing 100038,China)

机构地区：[1]首都医科大学附属北京世纪坛医院临床基因与细胞工程中心,北京100038

出　　处：《中国免疫学杂志》2022年第16期1988-1992,共5页Chinese Journal of Immunology

基　　金：北京市科技计划项目(Z161100000216136)资助。

摘　　要：目的:构建一种基于突变型IgG4长铰链区和4-1BB共刺激域的靶向CD19 CAR-T细胞(CART19),并在体内和体外证明其抗肿瘤的能力。方法:构建CD19 CAR的逆转录病毒载体,逆转录病毒转导植物血球凝集素(PHA)刺激的健康志愿者T细胞,从而获得CART19细胞;采用流式细胞术和Western blot检测转导效率;采用流式细胞术检测细胞内CD107a和IFN-γ水平,以及CFSE染色后的细胞增殖情况;4 h荧光杀伤实验评估CART19细胞的体外抗肿瘤能力;ELISA法检测共培养上清液的IFN-γ释放水平;建立白血病小鼠模型评估CART19细胞的体内抗肿瘤作用。结果:成功构建了一种新的CART19细胞;CD19^(+)细胞可以特异性刺激CART19细胞表达CD107a和IFN-γ、促进CART19细胞增殖以及分泌IFN-γ;4 h共培养荧光杀伤实验显示CART19细胞对NALM6-GL具有良好的体外抗肿瘤活性;CART19细胞可以显著提高白血病小鼠的生存率。结论:成功构建了可以有效治疗CD19^(+)肿瘤的基于突变型IgG4长铰链区和4-1BB共刺激域CART19细胞,为下一步临床治疗奠定了基础。Objective:To construct a CD19-targeting CAR-T cell(CART19)using mutated IgG4 long long spacer and 4-1BB costimulatory domain,and demonstrate its antitumor capability in vivo and in vitro.Methods:T cells were obtained from healthy donors,and then stimulated by using PHA. The new anti-CD19 CAR was transduced into primary T cells using retroviruses to form the CART19 cells. Transduction efficiency was measured by flow cytometry and Western blot. Flow cytometry was used to examine the intracellular CD107a and IFN-γ levels,and the cell proliferation after CFSE staining. The in vitro anti-tumor ability of CART19 was examined by evaluating the luciferase activity after 4 h co-culture. The cytokines levels in the cell medium were detected by ELISA. The efficacy against leukemia of CART19 cells in vivo was evaluated in a leukemia mouse model.Results:A new CART19 cell was successfully constructed. CART19 cells were specifically activated by CD19^(+)tumor cells,which have a good expansion,increased CD107a and IFN-γ expression,and IFN-γ secretion. CART19 exhibited good anti-tumor activity in vitro via killing NALM6-GL cells after 4 hours co-culture. CART19 cells can significantly improve the survival rate of leukemia mice.Conclusion:CART19 cells with mutated IgG4 long spacer and 4-1BB costimulatory domain have been successfully constructed for the effectively treatment of CD19^(+)tumor,which provide a good choice for clinical treatment.

关 键 词：嵌合抗原受体T细胞 CD19 突变型IgG4长铰链区 4-1BB共刺激域 白血病 

分 类 号：R392[医药卫生—免疫学]