高亲和力人源化CD19 CAR-T细胞对Raji细胞株体外、体内杀伤活性研究  

In vitro and in vivo cytotoxic activity of humanized CD19 CAR-T cells against Raji cell line

作　　者：孟娟霞[1] 牟男 杨振兴 王嘉[1] 李新[1] 江嫣雨 袁婷 邓琦[1] Meng Juanxia;Mou Nan;Yang Zhenxing;Wang Jia;Li Xin;Jiang Yanyu;Yuan Ting(Department of Hematology, Tianjin First Central Hospital, Tianjin 300192, China;Shanghai Genbase Biotechnology Co., Ltd., Shanghai 201203, China)

机构地区：[1]天津市第一中心医院血液科,300192 [2]上海吉倍生物技术有限公司,201203

出　　处：《中华微生物学和免疫学杂志》2019年第9期662-667,共6页Chinese Journal of Microbiology and Immunology

摘　　要：目的通过体内、外试验比较人源化及鼠源化CD19 CAR-T细胞对Raji细胞株的不同杀伤活性。方法收集8例拟行CD19 CAR-T治疗的淋巴瘤患者外周血,作为外周血单个核细胞(PBMC)、鼠源与人源化CD19 CAR-T细胞培养的T细胞来源。CCK-8法检测细胞增殖、LDH法检测细胞杀伤活性;BALB/c雌性裸鼠Raji荧光细胞株成瘤,活体成像技术观察裸鼠体内瘤体变化;流式细胞术检测细胞转染率及裸鼠体内CAR-T细胞比例。结果两组CAR-T细胞转染率差异无统计学意义,其增殖活性于培养24 h人源化组细胞高于鼠源组,差异无统计学意义(P=0.104),48 h人源化CAR-T细胞增殖活性明显高于鼠源组(P=0.009)。杀伤活性检测,设定不同效靶比(1∶1, 4∶1),作用24 h两种CAR-T对靶细胞的杀伤活性差异无统计学意义(P=0.102和P=0.095);48 h时不同效靶比人源化CD19 CAR-T细胞的杀伤活性高于鼠源组,差异有统计学意义(P=0.005和P=0.008),其杀伤活性均高于PBMC对照组。尾静脉注射细胞治疗Raji荷瘤裸鼠后14 d,鼠源和人源化CAR-T组裸鼠体内瘤体均缩小,对照组裸鼠瘤体增大,治疗21 d,鼠源组开始出现瘤体增大,而人源化组无明显变化。鼠源组裸鼠于25 d全部死亡,而人源化组裸鼠于31 d开始死亡。裸鼠体内CD19 CAR-T细胞检测示两组裸鼠均于输注后7 d达峰值,然人源化组细胞高于鼠源组(P4 d=0.001,P7 d=0.000,P14 d=0.003),鼠源组CAR-T细胞于21 d消失,人源化CAR-T细胞至35 d消失;生存期分析,对照组于治疗后20 d全部死亡,鼠源组裸鼠于治疗后25 d全部死亡,人源化组裸鼠于治疗后53 d全部死亡。结论人源化与鼠源CD19 CAR-T细胞相比,其增殖活性强、杀伤活性高、体内存留时间长,提示人源化CD19 CAR-T在B细胞淋巴瘤治疗的临床疗效将优于鼠源CD19 CAR-T。Objective To investigate the different functions of humanized and murinized CD19 chimeric antigen receptor (CAR)-T cells against Raji cell line in vitro and in vivo. Methods Peripheral blood samples were collected from eight patients with lymphoma who were going to receive CD19 CAR-T cell therapy and used for the preparation of peripheral blood mononuclear cells (PBMC) as well as humanized and murinized CAR-T cells. Cell proliferation and cytotoxicity were detected with CCK-8 and LDH assays, respectively. A tumor-bearing mouse model was established by injecting BALB/c female nude mice with fluorescent Raji cells. Changes in tumor volume in these mice were observed by in vivo imaging technology. The transfection efficiency and amount of CAR-T cells in the mice were detected with flow cytometry. Results No statistical difference in transfection efficiency was found between humanized and murinized CAR-T cells, nor in cell proliferation at 24 h of culture in vitro(P=0.104). The proliferation of humanized CAR-T cells showed a significant increase compared with that of murinized CAR-T cells at 48 h of culture (P=0.009). Similarly, the cytotoxicity of the two types of CAR-T cells against Raji cells showed no significant difference at 24 h at any effector/target (E/T) ratio (1∶1 or 4∶1), and that of humanized CAR-T cells was higher than that of murinized CAR-T cells at both E/T ratios at 48 h (E/T ratio=1∶1, P=0.005;E/T ratio=4∶1, P=0.008). Moreover, the cytotoxicity of CAR-T cells was higher than that of PBMC in any case. Tumor volumes in mice were reduced 14 d after humanized or murinized CAR-T cell therapy, while the mice in the PBMC control group suffered tumor progression. Tumor volume began to increase in mice 21 d after murinized CAR-T cell therapy, while no significant change was observed in the mice treated with humanized CAR-T cells. All of the mice died 25 d after murinized CAR-T cell therapy, while the deaths among those under humanized CAR-T cell therapy occurred on 31 d. The proportion of CAR-T cell

关 键 词：鼠源 人源 CD19 CAR-T细胞 RAJI细胞株 杀伤活性 

分 类 号：R392[医药卫生—免疫学]