靶向CD52嵌合抗原受体T细胞的制备及其抗白血病作用研究  

Preparation of CD52-targeted chimeric antigen receptor-modified T cells and their anti-leukemia effects

作　　者：刘琰 刘钰 唐克晶 陈兆琪 牟峻黎 徐颖茜 邢海燕 田征 饶青 王敏 王建祥 Liu Yan;Liu Yu;Tang Kejing;Chen Zhaoqi;Mou Junli;Xu Yingxi;Xing Haiyan;Tian Zheng;Rao Qing;Wang Min;Wang Jianxiang(State Key Laboratory of Experimental Hematology,National Clinical Research Center for Blood Diseases,Haihe Laboratory of Cell Ecosystem,Institute of Hematology&Blood Diseases Hospital,Chinese Academy of Medical Sciences&Peking Union Medical College,Tianjin 300020,China)

机构地区：[1]中国医学科学院北京协和医学院血液病医院(中国医学科学院血液学研究所),实验血液学国家重点实验室,国家血液系统疾病临床医学研究中心,细胞生态海河实验室,天津市血液病细胞治疗研究重点实验室,天津300020

出　　处：《中华血液学杂志》2022年第4期279-286,共8页Chinese Journal of Hematology

基　　金：国家重点研发计划(2019YFA0110200);国家自然科学基金(81830005);中国医学科学院创新工程项目(2021-1-I2M-041)。

摘　　要：目的构建一种靶向CD52的嵌合抗原受体T细胞(CD52 CAR-T),探索其对CD52+白血病的治疗效果。方法应用分子克隆技术构建以CD52为抗原结合区的CD52 scFv、4-1BB为共刺激分子的二代CAR-T细胞。慢病毒载体包装后感染T细胞,制备CD52 CAR-T细胞,通过流式细胞术检测CD52 CAR-T细胞CD4、CD8细胞亚群及其分化状态。通过体外杀伤实验、脱颗粒实验及细胞因子的释放等功能实验,观察CD52 CAR-T细胞对CD52+白血病细胞的特异性细胞毒作用。结果①成功构建了pCDH-CD52 scFv-CD8α-4-1BB-CD3ζ-GFP表达载体,感染人T细胞,获得靶向CD52的CART细胞。②感染CD52 CAR表达载体的第6天,T细胞中表达CD52的细胞可被清除[CD52 CAR-T组CD52+T细胞为(4.48±4.99)%,Vector-T组为(56.58±19.8)%,P=0.011]。③CD52 CAR-T的自我杀伤不会影响T细胞的增殖,培养后期CD52 CAR-T组的增殖速度高于Vector-T组。④T细胞表型检测发现CD52 CAR可以促进CAR-T细胞向中心记忆T细胞及效应记忆T细胞分化,减少CAR-T细胞向终末效应细胞分化。⑤CD52 CAR-T细胞与HuT78-19t及MOLT4-19t靶细胞以效靶比1∶1共培养24 h,HuT78-19t细胞可被清除,而对MOLT4-19t细胞无明显杀伤作用[(2.66±1.60)%对(56.66%±5.74)%,P<0.001]。⑥脱颗粒实验显示,HuT78-19t细胞对CD52 CAR-T细胞的激活作用显著高于MOLT4-19t细胞[(57.34±11.25)%对(13.06±4.23)%,P<0.001]。⑦CD52 CAR-T与HuT78-19t细胞共培养48 h上清中细胞因子分泌水平[IFN-γ(3706±226)pg/ml、TNF-α(1732±560)pg/ml]远高于MOLT4-19t细胞组[IFN-γ(1577±846)pg/ml、TNF-α(74±12)pg/ml](P值均<0.01)。结论该研究成功制备了具有抗白血病作用的CD52 CAR-T细胞,为进一步的靶向CD52的免疫治疗奠定了基础。Objective To construct chimeric antigen receptor(CAR)T cells targeting CD52(CD52 CAR-T)and validate the effect of CD52 CAR-T cells on CD52-positive leukemia.Methods A second-generation CD52-targeting CAR bearing 4-1BB costimulatory domain was ligated into a lentiviral vector through molecular cloning.Lentivirus was prepared and packaged by 293 T cells with a four-plasmid system.Fluorescein was used to label cell surface antigens to evaluate the phenotype of CD52 CAR-T cells after infection.Flow cytometry and ELISA were used to evaluate the specific cytotoxicity of CD52 CAR-T cells to CD52-positive cell lines in vitro.Results①A pCDH-CD52scFv-CD8α-4-1BB-CD3ζ-GFP expressing plasmid was successfully constructed and used to transduce T cells expressing a novel CD52-targeting CAR.②On day 6,CD52-positive T cells were almost killed by CD52-targeted CAR-T post lentivirus transduction[CD52 CAR-T(4.48±4.99)%,vs Vector-T(56.58±19.8)%,P=0.011].③T cells transduced with the CAR targeting CD52 showed low levels of apoptosis and could be expanded long-term ex vivo.④The CD52 CAR could promote T cell differentiation into central and effector memory T cells,whereas the proportion of T cells with a CD45RA+effector memory phenotype were reduced.⑤CD52 CAR-T cells could specifically kill CD52-positive HuT78-19t cells but had no killing effect on CD52-negative MOLT4-19t cells.For CD52 CAR-T cells,the percentage of residual of HuT78-19t cells was(2.66±1.60)%at an the E:T ratio of 1∶1 for 24 h,while(56.66±5.74)%of MOLT4-19t cells survived(P<0.001).⑥The results of a degranulation experiment confirmed that HuT78-19t cells significantly activated CD52 CAR-T cells but not MOLT4-19t cells[(57.34±11.25)%vs(13.06±4.23)%,P<0.001].⑦CD52 CAR-T cells released more cytokines when co-cultured with HuT78-19t cells than that of vector-T cells[IFN-γ:(3706±226)pg/ml,P<0.001;TNF-α:(1732±560)pg/ml,P<0.01].Conclusions We successfully prepared CD52 CAR-T cells with anti-leukemia effects,which might provide the foundation for further

关 键 词：CD52 嵌合抗原受体 免疫治疗 

分 类 号：R733.7[医药卫生—肿瘤]