自杀基因作为一种“安全性开关”控制CAR-T细胞毒性的临床前研究  

Preclinical study of suicide gene as a safety switch to control CAR-T cell cytotoxicity

作　　者：张慧慧 孔群芳 吕晓菲 李想 孙玉桃 谭毅 ZHANG Huihui;KONG Qunfang;LYU Xiaofei;LI Xiang;SUN Yutao;TAN Yi(Qilu Cell Therapy Technology Co.,Ltd.,Jinan 250000,Shandong,China;Shandong Yinfeng Life Science Research Institute,Jinan 250000,Shandong,China)

机构地区：[1]山东省齐鲁细胞治疗工程技术有限公司,山东济南250000 [2]山东银丰生命科学研究院,山东济南250000

出　　处：《中国肿瘤生物治疗杂志》2021年第3期225-231,共7页Chinese Journal of Cancer Biotherapy

基　　金：山东省重点研发计划资助项目(No.2016ZDJS07B05)。

摘　　要：目的:探讨小分子化学诱导药AP1903能否在体内外终止过表达iCasp9自杀基因的CD19靶向嵌合抗原受体修饰T(CD19CAR-T)细胞毒性功能。方法:构建过表达iCasp9的CD19CAR-T(iCasp9-CD19CAR-T)细胞并和AP1903共孵育,采用流式细胞术检测细胞表型及凋亡的方法,分别在K562和T细胞上验证iCasp9/CID自杀基因系统,在体内(观察荷Raji细胞移植瘤NCG小鼠的生存率)和体外(流式细胞术检测细胞的杀伤功能)检测AP1903给药情况下iCasp9-CD19CAR-T细胞的杀伤功能。结果:和CD19CAR-T细胞相比,iCasp9-CD19CAR-T细胞的增殖能力、表型及体内外杀伤功能均无显著差异(均P>0.05)。AP1903给药2 h后双表达i Casp9和CD19CAR的K562和T细胞分别有(33.8±0.9)%和(27.95±0.35)%的细胞出现凋亡,AP1903给药24 h后双表达iCasp9和CD19CAR的K562和T细胞均已经全部死亡。检测AP1903给药和未给药两种条件下的iCasp9-CD19CAR-T细胞体外杀伤效率,前者明显低于后者(P<0.01);iCasp9-CD19CAR-T细胞治疗荷Raji细胞移植瘤NCG小鼠,其60 d生存率同样是AP1903给药的明显低于未给药的(P<0.01)。结论:小分子化学诱导药物AP1903能在体内外有效终止iCasp9-CD19CAR-T细胞毒性功能。Objective: To investigate whether AP1903, a small-molecule chemical inducer, can terminate the cytotoxicity of CD19 CAR-T cells over-expressing iCasp9 suicide gene in vivo and in vitro. Methods: CD19 CAR-T cells over-expressing iCasp9(iCasp9-CD19 CAR-T) were constructed and co-incubated with AP1903. Then, the cell phenotype and apoptosis were detected by Flow cytometry, and the iCasp9/CID suicide gene system was verified on K562 and T cells, respectively. The cytotoxicity of iCasp9-CD19 CAR-T cells was detected in vivo(survival rate of NCG mice bearing Raji cell transplanted xenograft) and in vitro(cell killing function was detected by Flow cytometry) under the administration of AP1903. Results: Compared with CD19 CAR-T cells, iCasp9-CD19 CAR-T cells showed in significant difference in proliferation, phenotype and cytotoxicity both in vitro and in vivo(all P>0.05). At2 h after AP1903 administration, the apoptosis rates of K562 and T cells co-expressing iCasp9 and CD19 CAR were(33.8±0.9)% and(27.95±0.35)%, respectively;and at 24 h after AP1903 administration, the apoptosis rates reached 100% in both cell lines. The in vitro cytotoxicity of iCasp9-CD19 CAR-T cells induced by AP1903 was significantly lower than that without AP1903 treatment(P<0.01);the60-day survival rate of mice bearing Raji cell transplanted tumor treated with AP1903-induced i Casp9-CD19 CAR-T cells was also significantly lower than those treated with i Casp9-CD19 CAR-T cells alone(P<0.01). Conclusion: AP1903 can effectively terminate the cytotoxicity of CD19 CAR-T cells over-expressing iCasp9 suicide gene in vitro and in vivo.

关 键 词：嵌合抗原受体修饰T细胞 CD19 安全性开关 诱导性自杀基因caspase9 细胞因子释放综合征 脱靶效应 K562细胞 

分 类 号：R739.12[医药卫生—肿瘤] R730.51[医药卫生—临床医学]