特殊结构双靶点CAR-T细胞对肿瘤细胞的杀伤作用  

Effect of Special Structure Bi-Specific Chimeric Antigen Receptor T Cell on Tumor Cells

作　　者：刘灿[1] 彭浩 曾伟杰 李薇[1] 陈可可[1] 李琬婷 曾桂芳 梁晓 胡隽源 周明[1] LIU Can;PENG Hao;ZENG Wei-Jie;LI Wei;CHEN Ke-Ke;LI Wan-Ting;ZENG G ui-Fang;LIANG Xiao;HU Juan-Yuan;ZHOU Ming(Department of Hematology,Hunan Provincial People’s Hospital,Changsha 410005,Hunan Province,China;Beike Biotechnology Co.Ltd,Shenzhen 518057,Guangdong Province,China)

机构地区：[1]湖南省人民医院(湖南师范大学附属第一医院)血液内科,湖南长沙410005 [2]深圳市北科生物科技有限公司,广东深圳518057

出　　处：《中国实验血液学杂志》2022年第6期1730-1740,共11页Journal of Experimental Hematology

基　　金：湖南省社会发展重点研发项目(2019SK2091)。

摘　　要：目的:探索并设计一种新颖的双靶点嵌合抗原受体(chimeric antigen receptor,CAR)结构,获得相应CAR-T细胞并验证其对肿瘤细胞的体内外杀伤效果。方法:构建并制备5种包含人源化CD19 scFv和CD79b scFv、CD8 hinge&TM-4-1BB-CD3ζ和/或CD3ε链胞内区的双靶点CAR-T细胞,荧光素酶法和ELISA法检测CAR-T细胞对3M-CD19-CD79b-Luc靶细胞的杀伤能力和细胞因子的分泌,选取最优结构CAR-T细胞治疗Daudi-Luc细胞构建的白血病小鼠模型并评估其疗效,同时使用其他靶点代入优选结构,相同方法验证该结构的稳定性及有效性。结果:CAR-19-79b-T细胞培养7 d后,CAR-19表达率为21.6%-36.3%,CAR-79b表达率为21.7%-37.8%。CAR-19-79b-T细胞在10∶1效靶比时,对3M-CD19-CD79b-Luc细胞的杀伤率均显著高于T细胞对照组,其中CARⅢ、CARⅣ号结构细胞杀伤能力最强,均显著高于T细胞组(P<0.01);CARⅣ与CAR V结构IFN-γ和TNF-α的分泌量均最低,且两者间均无统计学意义(P>0.05)。杀伤显著且分泌细胞因子水平低的CARⅣ细胞对Daudi-Luc白血病小鼠治疗效果明显,延长小鼠生存周期至64 d,T细胞对照组小鼠于(41.0±2.4)d全部死亡。相同结构构建的CAR-19-BCMA-T和CAR-19-22-T均表现出了显著的杀伤能力和较低的细胞因子水平。结论:成功设计了一种新颖的抗双靶点CAR-T细胞,能够高效杀伤相应的肿瘤细胞,同时分泌较少的细胞因子(如TNF-α,IFN-γ),而且对Daudi淋巴瘤小鼠动物模型有显著的治疗效果,该双靶点CAR结构表现出良好的杀伤特异性和安全性。Objective:To explore and design a novel bi-specific chimeric antigen receptor(CAR)structure.To obtain the corresponding CAR-T cells and verify killing effects on tumor cells in vitro and in vivo.Methods:Five kinds of bi-specific CAR structures including humanized CD19 scFv and CD79 b scFv,CD8 hinge&TM-4-1 BB-CD3ζand/or CD3εchain intracellular regions were constructed and prepared.CAR-19-79 b cells were obtained.Five kinds of CAR-T cells were co-incubated with the 3 M-CD19-CD79 b-Luc target cells.Luciferase assay and ELISA were used to detecte the killing ability of these five groups of CAR-T cells and the secretion of cytokines and compared.The optimal structure of CAR-T cells was used to treat the leukemia mouse model constructed by Daudi-Luc cells.And the treatment efficacy was evaluated.At the same time,other targets were used in this structure.With the same methods,the stability and effectiveness of the structure were verified.Results:CAR-19-79 b-T cells were cultured for 7 days,the expression rates of CAR-19 and CAR-79b were 21.6%-36.3%and 21.7%-37.8%,respectively.The killing rates of 5 kinds of CAR-19-79 b-T cells prepared by T cells from 3 healthy donors on 3M-CD19-CD79b-Luc cells were significantly higher than those of the T cell control group at the effect-target ratio of 10∶1.Among them,the killing rates of CAR-19-79 b-T cells with No.Ⅲand No.Ⅳstructures were the strongest.After co-incubation with 3M-CD19-CD79 b-Luc target cells,the amount of IFN-γand TNF-αsecreted by CAR-T cells with CARⅣand CARV structures was the lowest.And there was no significance between the two groups(P>0.05).CAR IV cells with remarkable killing effect and lowsecretion factor had obvious therapeutic effect on Daudi-Luc leukemia mice,extending the survival period of mice to 64 days.And all mice in the T cell control group died at 41.0±2.4 days.The CAR-19-BCM A-T and CAR-19-22-T with the same structure showed significant killing ability and lowcytokine expression levels.Conclusion:A novel bi-specific CAR structures was

关 键 词：CD19 CD79b 双靶点嵌合抗原受体 淋巴瘤 

分 类 号：R733[医药卫生—肿瘤]