嵌合抗原受体T细胞靶向LMP1抗原治疗EB病毒阳性淋巴瘤的功能研究  

Functional investigation of chimeric antigen receptor T cells targeting LMP1 antigen

作　　者：何慧珍 邢妍妍 张瑜 徐颖茜 田征 邢海燕 唐克晶 饶青 王建祥 王敏 He Huizhen;Xing Yanyan;Zhang Yu;Xu Yingxi;Tian Zheng;Xing Haiyan;Tang Kejing;Rao Qing;Wang Jianxiang;Wang Min(State Key Laboratory of Experimental Hematology,National Clinical Research Center for Blood Diseases,Institute of Hematology&Blood Diseases Hospital,Peking Union Medical University,CAMS&PUMC,Tianjin Key Laboratory of Cell Therapy for Blood Diseases,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,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3期229-234,共6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-I2M-1-041)。

摘　　要：目的制备一种靶向LMP1抗原的嵌合抗原受体T细胞(CAR-T细胞),研究其对EB病毒(EBV)阳性淋巴瘤的免疫治疗作用。方法应用分子克隆技术构建二代LMP1 CAR表达质粒,通过慢病毒包装体系包装病毒后感染人T细胞,获得LMP1 CAR-T细胞,体外实验验证LMP1 CAR-T细胞对感染EBV后的LMP1阳性淋巴瘤细胞的特异性细胞毒性作用。结果①LMP1蛋白表达于EBV阳性的淋巴瘤细胞表面;②成功构建了二代LMP1 CAR慢病毒载体,感染人T细胞,获取LMP1 CAR-T细胞,感染效率大于80%;③LMP1 CAR-T细胞可特异性杀伤LMP1阳性淋巴瘤细胞,当效靶比按4∶1共培养48 h后,LMP1 CAR-T细胞对Raji细胞的杀伤作用增强,对Ramos细胞无明显杀伤作用;④与LMP1阳性淋巴瘤细胞按1∶1共培养5 h后,LMP1 CAR-T细胞处理组CD107a^(+)T细胞比例显著高于Vector-T细胞组[(13.25±2.94)%对(1.55±0.05)%,t=3.972,P=0.017],脱颗粒效果增强;⑤与LMP1阳性淋巴瘤细胞共培养后,LMP1 CAR-T细胞组CD69+、CD25+T细胞比例显高于Vector-T细胞组[(7.40±0.41)%对(3.48±0.47)%,t=6.268,P=0.003;(73.00±4.73)%对(57.67±2.60)%,t=2.842,P=0.047],活化效应增强。⑥与LMP1阳性淋巴瘤细胞共培养后,LMP1 CAR-T细胞组细胞因子分泌增强,高于Vector-T细胞组[IFN-γ:(703±73)ng/L对(422±87)ng/L,t=2.478,P=0.068;TNF-α:(215±35)ng/L对(125±2)ng/L,t=2.536,P=0.064]。结论该研究证实EBV阳性淋巴瘤细胞表面可特异表达LMP1蛋白,成功构建了LMP1 CAR慢病毒载体并感染人T细胞,成功获得LMP1 CAR-T细胞。体外实验证实:与LMP1阳性淋巴瘤细胞共培养后,LMP1 CAR-T细胞脱颗粒效果增强,活化效应增强,高效分泌细胞因子,可特异杀伤LMP1阳性淋巴瘤细胞,具有潜在的临床应用前景。Objective This study aimed to create a type of CAR-T cells that targets LMP1 antigen and study its immunotherapeutic effect on LMP1-positive hematological malignancies.Methods To generate LMP1 CAR-T cells,a plasmid expressing LMP1 CAR was created using molecular cloning technology,and T cells were infected with LMP1 CAR lentivirus.The effects of LMP1 CAR-T cells on specific cytotoxicity against LMP1-positive tumor cell lines infected with the EB virus had been confirmed.Results①LMP1 protein expressing on EB virus-positive lymphoma cells surface was verified.②The LMP1 CAR-expressing plasmid was created,and LMP1 CAR-T cells were obtained by infecting T cells with a lentivirus packaging system,with an infection efficiency of more than 80%.③LMP1 CAR-T cells have a 4∶1 effect-to-target ratio in killing LMP1-positive lymphoma cells.The killing effect of LMP1 CAR-T cells on Raji cells was enhanced after 48 h of coculture,but there was no significant killing effect on Ramos,which are LMP1-negative lymphoma cells.④After coculture with LMP1-positive lymphoma cells at a ratio of 1∶1 for 5 h,the degranulation effect was enhanced.The proportion of CD107a^(+)T cells in the LMP1 CAR-T cell treatment group was significantly higher than that in the vector-T cell group[(13.25±2.94)%vs(1.55±0.05)%,t=3.972,P=0.017].⑤After coculture with LMP1-positive lymphoma cells,the proportion of CD69+and CD25+T cells in the LMP1 CAR-T cell group was significantly higher than that in vector-T cell group[(7.40±0.41)%vs(3.48±0.47)%,t=6.268,P=0.003;(73.00±4.73)%vs(57.67±2.60)%,t=2.842,P=0.047].⑥After coculture with LMP1-positive lymphoma cells,cytokine secretion in the LMP1 CAR-T cell group was higher than that in the vector-T cell group[interferon-gamma:(703±73)ng/L vs(422±87)ng/L,t=2.478,P=0.068;tumor necrosis factor-alpha:(215±35)ng/L vs(125±2)ng/L,t=2.536,P=0.064].Conclusion In this study,we found that the LMP1 protein is only found on the surface of the EBV-positive tumor cell.Simultaneously,we created an LMP1 CAR-expr

关 键 词：EB病毒 LMP1抗原 嵌合抗原受体T细胞 

分 类 号：R733.1[医药卫生—肿瘤]