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
邹婷 1马岩松 2陈泽建 3李长林 4王蓉蓉 5唐颖鑫 3周文婷 3陈德玉 3周芳 3向双林6
作者信息
- 1. 湖南师范大学生命科学学院,长沙 410081;湖南省药品监督管理局,长沙 410013
- 2. 国家药品监督管理局食品药品审核查验中心,北京 100044
- 3. 湖南远泰生物技术有限公司,长沙 410221
- 4. 湖南省药品监督管理局,长沙 410013
- 5. 湖南省药品检验检测研究院,长沙 410001;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
- 6. 湖南师范大学生命科学学院,长沙 410081;湖南远泰生物技术有限公司,长沙 410221
- 折叠
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.
Abstract
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Key words
memory CAR-T cell/IL-7/IL-15/TransAct引用本文复制引用
基金项目
湖南省科药联合基金(2022JJ80019)
出版年
2024
NETL
NSTL
万方数据