记忆型双特异性CAR-T细胞的工艺优化研究  

Optimization of the process of memory dual specificity CAR-T cells

作　　者：邹婷 马岩松 陈泽建 李长林 王蓉蓉 唐颖鑫 周文婷 陈德玉 周芳 向双林[1,4] ZOU Ting;MA Yan-song;CHEN Ze-jian;LI Chang-lin;WANG Rong-rong;TANG Ying-xin;ZHOU Wen-ting;CHEN De-yu;ZHOU Fang;XIANG Shuang-lin(College of Life Sciences,Hunan Normal University,Changsha 410081;Hunan Provincial Drug Administration,Changsha 410013;Center for Food and Drug Inspection of NMPA,Beijing 100044;Forevertek Biotechnology Co.,Ltd.,Changsha 410221;Hunan Research Institute of Drug Testing,Changsha 410001;Laboratory for Critical Quality Attributes of Cell Therapy Products,Changsha 410001)

机构地区：[1]湖南师范大学生命科学学院,长沙410081 [2]湖南省药品监督管理局,长沙410013 [3]国家药品监督管理局食品药品审核查验中心,北京100044 [4]湖南远泰生物技术有限公司,长沙410221 [5]湖南省药品检验检测研究院,长沙410001 [6]细胞治疗产品关键质量属性研究湖南省重点实验室,长沙410001

出　　处：《中南药学》2024年第5期1250-1255,共6页Central South Pharmacy

基　　金：湖南省科药联合基金项目(No.2022JJ80019)。

摘　　要：目的 研究能够显著降低CAR-T细胞的体外分化程度、维持记忆型CAR-T(CAR-Tm)细胞高浓度稳态的新工艺路径,提升和延长CAR-T细胞的肿瘤杀伤效果。方法 采用流式细胞分析(FACS)技术,对原工艺采用CD3/CD28磁珠分选CD3^(+)T细胞,在体外培养过程中不断补充IL-2细胞因子,和新工艺利用CD4/CD8磁珠分选出CD4^(+)和CD8^(+)T细胞并用耦联人源化抗体纳米粒(TransAct)活化,在体外培养过程中添加IL-7和IL-15培养所得T细胞分别进行CAR-Tm细胞亚群分析,并通过对比不同TransAct用量和培养时间获得最优制备工艺。结果 新工艺得到的低分化记忆型CAR-T(CAR-Tscm^(+)CAR-Tcm亚群)细胞占总T细胞和CAR-T细胞的比例显著高于原工艺(P<0.05),TransAct的最佳用量为20%,细胞体外最佳培养时间为12 d。结论 联用IL-7细胞因子、IL-15细胞因子、TransAct有利于维持靶向CD19/CD37双特异性低分化CAR-Tm细胞高位数量和维持时间,提高持久性,为其后续临床研究及产业化奠定了基础。Objective To develop a new process to reduce the differentiation degree of CAR-T cells in vitro,maintain the high concentration steady state of memory CAR-T(CAR-Tm),and enhance and prolong the tumor-killing effect of CAR-T cells.Methods We used flow cytometry(FACS)technology,to conduct memory CAR-T cell subpopulation analysis of T cells with the original process from CD3^(+)T cells which were sorted from CD3/CD28 magnetic beads,and were continuously supplemented by IL-2 cytokines in vitro,and by using the new process from CD4^(+)and CD8^(+)T cells were sorted from CD4/CD8 magnetic beads,and activated by TransAct in vitro,and were added by IL-7 and IL-15 cytokines.Then,by comparing different TransAct dosages and cultivation times,the best process was determined.Results The proportion of low-differentiated memory CAR-T cells(CAR-Tscm+CAR-Tcm)in total T cells and CAR-T cells by the new process method was significantly higher than that by the original process(P<0.05).The optimal amount of TransAct was 20%,and the optimal culture time was 12 days.Conclusion Combination of IL-7 cytokines,IL-15 cytokines and TransAct helps maintaing the high levels and numbers of tduration of low-differentiated targeted CD19/CD37 dual specificity memory CAR-T cells,laying a foundation for subsequent clinical research and industrialization.

关 键 词：CAR-Tm细胞 白细胞介素-7 白细胞介素-15 耦联人源化抗体纳米粒 

分 类 号：R730.51[医药卫生—肿瘤]