靶向CD138嵌合抗原受体修饰T细胞的构建及其抗多发性骨髓瘤作用研究  

Construction of CD138-targeted chimeric antigen receptor-modified T cells and their effect in multiple myeloma therapy

作　　者：国承彩 卢杨 唐克晶 邢海燕 田征 饶青 王敏 熊冬生 王建祥 Guo Chengcai;Lu Yang;Tang Kejing;Xing Haiyan;Tian Zheng;Rao Qing;Wang Min;Xiong Dongsheng;Wang Jianxiang(State Key Laboratory of Experimental Hematology,National Clinical Research Center for Blood Diseases,Tianjin Key Laboratory of Cell Therapy 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;Tianjin Institutes of Health Science,Tianjin 301600,China)

机构地区：[1]中国医学科学院血液病医院(中国医学科学院血液学研究所)北京协和医学院,血液与健康全国重点实验室,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,细胞生态海河实验室,天津300020 [2]天津医学健康研究院,天津301600

出　　处：《中华血液学杂志》2024年第5期436-444,共9页Chinese Journal of Hematology

基　　金：国家自然科学基金(82341213);中国医学科学院医学与健康科技创新工程项目(2021-I2M-1-041)。

摘　　要：目的构建一种新型靶向CD138的嵌合抗原受体T(CAR-T)细胞,探索其抗恶性浆细胞肿瘤作用。方法通过单克隆抗体制备及筛选技术,获得能稳定分泌CD138抗体的杂交瘤细胞株;将杂交瘤细胞接种至小鼠腹腔,收集腹水并纯化得到抗CD138抗体纯品,进一步检测抗体特异性及亲和力;RT-PCR扩增其可变区序列,以此作为抗原识别域构建CD138 CAR,并表达于T细胞表面,制备CD138 CAR-T;流式细胞术检测CAR-T细胞表型特征;体外杀伤及脱颗粒实验检测其抗肿瘤作用。结果①成功制备抗人CD138抗体杂交瘤细胞株,并筛选获得稳定分泌抗人CD138抗体的杂交瘤细胞株5G2。②CD138(5G2)抗体可以特异性识别CD138+细胞,与CD138蛋白亲和常数(KD)为6.011×10^(-9)mol/L,与CD138-细胞无明显交叉反应。③应用分子克隆技术扩增得到CD138(5G2)抗体可变区序列,成功构建CD138(5G2)CAR慢病毒载体,通过感染T细胞获得的CD138(5G2)CAR-T细胞,可以有效结合人CD138重组蛋白。④CD138(5G2)CAR-T可以有效大量扩增,表型检测发现CD138(5G2)CAR-T细胞更多的向中心记忆T及记忆干细胞方向分化,终末分化效应T细胞比例降低。⑤与靶细胞共培养48 h后,与Vector-T细胞相比,CD138(5G2)CAR-T细胞可以有效杀伤CD138+骨髓瘤细胞系H929[效靶比为1∶2,(12.92±8.02)%对(54.25±15.79)%,P<0.001]。但对CD138-K562细胞系无明显杀伤作用。⑥脱颗粒实验显示,H929细胞可以显著激活CD138(5G2)CAR-T细胞,但对Vector-T细胞无明显激活作用[(25.78±3.35)%对(6.13±1.30)%,P<0.001]。⑦与CD138+细胞共培养后CD138(5G2)CAR-T分泌细胞因子水平较Vector-T组明显升高[IL-2:(1697.52±599.05)pg/ml对(5.07±1.17)pg/ml,P<0.001;IFN-γ:(3312.20±486.38)pg/ml对(9.28±1.46)pg/ml,P<0.001;TNF-α:(1837.43±640.49)pg/ml对(8.75±1.65)pg/ml,P<0.001],但与CD138-细胞共培养后两组间细胞因子分泌水平无明显差异。结论本研究成功制备了抗CD138单克隆抗体,以其抗原识别域构建�Objective To construct a novel chimeric antigen receptor T(CAR-T)cell targeting CD138 and to investigate its cytotoxicity against myeloma cells.Methods The hybridoma strain that can stably secrete the CD138 monoclonal antibody(mAb)was prepared and obtained through monoclonal antibody screening technology.The hybridoma strain cells were intraperitoneally injected into mice to produce ascites containing monoclonal antibodies,which were then collected and purified to obtain pure CD138 mAb.Further examinations were performed to assess the biological characteristics of CD138 mAb.The variable region sequence of this antibody was amplified through reverse transcription polymerase chain reaction and was used as the antigen recognition domain of CD138 CAR,which was subsequently expressed on the surface of T cells by lentiviral infection.Flow cytometry was employed to assess the phenotype of CD138 CAR-T cells.In vitro cytotoxicity and degranulation assays were performed to evaluate their antitumor effects.Results①We successfully prepared anti-human CD138 antibody hybridoma cell lines and screened a hybridoma cell strain,5G2,which could persistently and stably secrete the anti-CD138 antibody.②The purified CD138(5G2)mAb can especially recognize CD138+cells with a binding affinity constant(KD)of 6.011×10^(-9) mol/L and showed no significant binding activity with CD138-cells.③The variable region sequence of the CD138(5G2)antibody was obtained using molecular cloning technology,and CD138(5G2)CAR was successfully constructed and expressed on T cells through lentivirus infection and,concurrently,demonstrated effective binding to recombinant human CD138 protein.④The proliferation of T cells transduced with the CD138(5G2)CAR was highly efficient.The phenotype analysis revealed that CD138(5G2)CAR-T cells exhibited a greater tendency to differentiate into central memory T cells and memory stem T cells,with a reduced proportion of terminally differentiated effector memory subsets.⑤CD138(5G2)CAR-T cells demonstrated specific

关 键 词：嵌合抗原受体 抗原识别区 多发性骨髓瘤 CD138 免疫治疗 

分 类 号：R733.3[医药卫生—肿瘤]