双靶点CD38/CD138 CAR-T细胞的构建及其对多发性骨髓瘤细胞的体外杀伤效果  

Construction of CD38/CD138 dual-targeted CAR-T cell and it’s in vitro cytotoxicity against multiple myeloma cells

作　　者：潘璐 刘航宇 王景鸿 孙大玮 赵松柏 鞠吉雨 宋绚丽 PAN Lu;LIU Hangyu;WANG Jinghong;SUN Dawei;ZHAO Songbo;JU Jiyu;SONG Xuanli(Department of Immunology,School of Basic Medicine,Shandong Second Medical University,Weifang 261053,Shandong,China,Provincial Hospital Affiliated to Shandong First Medical University,Jinan 250021,Shandong,China;Department of Clinical Laboratory Medicine,Provincial Hospital Affiliated to Shandong First Medical University,Jinan 250021,Shandong,China;Department of Medical Imaging;Central Laboratory,Provincial Hospital Affiliated to Shandong First Medical University,Jinan 250021,Shandong,China;Clinical Laboratory,Junan People's Hospital of Linyi City,Linyi 276600,Shandong,China;Institute of Bacterial Disease Laboratory,Jinan Center for Disease Control and Prevention,Jinan 250021,Shandong,China)

机构地区：[1]山东第二医科大学基础医学院免疫学教研室,山东潍坊261053 [2]山东第一医科大学附属省立医院临床医学检验部,山东济南250021 [3]山东第一医科大学附属省立医院医学影像科,山东济南250021 [4]山东第一医科大学附属省立医院中心实验室,山东济南250021 [5]临沂市莒南县人民医院检验科,山东临沂276600 [6]济南市疾病预防控制中心细菌性疾病检验所,山东济南250021

出　　处：《中国肿瘤生物治疗杂志》2024年第12期1186-1193,共8页Chinese Journal of Cancer Biotherapy

基　　金：山东省自然科学基金(No.ZR2023QC179)。

摘　　要：目的:构建靶向CD38和CD138分子抗原的双靶点嵌合抗原受体基因修饰T淋巴细胞(CD38/CD138 CAR-T细胞),探讨其对多发性骨髓瘤(MM)细胞的体外杀伤作用。方法:利用CAR-T细胞技术,基于MM细胞高表达CD38和CD138抗原,分别构建靶向CD38、CD138的CD38 CAR-T与CD138 CAR-T细胞,以及同时靶向CD38与CD138的CD38/CD138 CAR-T细胞,实验分为未处理T、CD38 CAR-T、CD138 CAR-T和CD38/CD138 CAR-T细胞组。采用流式细胞术检测CAR-T细胞的表型,利用LDH释放法检测各种CAR-T细胞对MM细胞RPMI8226和U266的体外杀伤作用。结果:成功构建CD38 CAR-T、CD138 CAR-T和CD38/CD138 CAR-T细胞。CD38/CD138 CAR-T细胞倾向于向记忆表型分化,表达较高水平的增殖分子(CD25)、激活分子(CD27)和较低水平的耗竭分子(PD-1、CTLA-4、TIM-3)(均P<0.001),而且CD38/CD138 CAR-T细胞不易于耗竭和衰老,且表达较低水平的r-H2AX、p-p53、p21和p16蛋白(均P<0.01)。在不同效靶比条件下,CD38/CD138 CAR-T细胞较CD38 CAR-T、CD138 CAR-T细胞对RPMI8226和U266细胞具有更强的杀伤作用(均P<0.001)。结论:靶向CD38和CD138治疗MM的CD38/CD138 CAR-T细胞在体外具有较优表型及较强的抗肿瘤功能。Objective:To construct dual-targeting(CD38 and CD138)chimeric antigen receptor(CAR)gene-modified T cells(CD38/CD138 CAR-T cells)and explore their in vitro cytotoxicity against multiple myeloma(MM)cells.Methods:Based on the high expression of CD38 and CD138 antigens in MM cells,CD38 CAR-T cells and CD138 CAR-T cells targeting CD38 and CD138 respectively,and CD38/CD138 dual-targeted CAR-T cells targeting both CD38 and CD138 were constructed using CAR-T cell technology.The experimental groups included untreated T cells,CD38 CAR-T,CD138 CAR-T,and CD38/CD138 CAR-T cells.The phenotype of CAR-T cells was detected by flow cytometry.The cytotoxicity of various CAR-T cells against MM cells(RPMI8226 and U266)was assessed using the LDH release assay.Results:Three types of CAR-T cells,CD38 CAR-T,CD138 CAR-T,and CD38/CD138 CAR-T cells,were successfully constructed.The CD38/CD138 CAR-T cells tended to differentiate into a memory phenotype,expressing higher levels of proliferation marker(CD25),activation marker(CD27),and lower levels of exhaustion markers(PD-1,CTLA-4,TIM-3)(all P<0.001).Moreover,CD38/CD138 CAR-T cells were less prone to exhaustion and senescence,and expressed lower levels of r-H2AX,p-p53,p21,and p16 proteins(all P<0.01).Under different effector-to-target cell ratios,CD38/CD138 CAR-T cells exhibited stronger cytotoxic effects against RPMI8226 and U266 cells compared to CD38 CAR-T and CD138 CAR-T cells(all P<0.001).Conclusion:CD38/CD138 CAR-T cells targeting both CD38 and CD138 demonstrate an optimal phenotype and enhanced anti-tumor activity in vitro,offering promising potential for immunotherapy in multiple myeloma.

关 键 词：多发性骨髓瘤 嵌合抗原受体基因修饰T淋巴细胞 CD38/CD138 CAR-T细胞 免疫治疗 

分 类 号：R733.3[医药卫生—肿瘤] R730.51[医药卫生—临床医学] R392.12