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降低亲和力提高HER2-CAR-T细胞治疗的安全性

章浩,  叶真龙,  钱其军

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文章导航 > 药学实践与服务  > 2016 >  34(3): 261-266
章浩, 叶真龙, 钱其军. 降低亲和力提高HER2-CAR-T细胞治疗的安全性[J]. 药学实践与服务, 2016, 34(3): 261-266. doi: 10.3969/j.issn.1006-0111.2016.03.018
引用本文: 章浩, 叶真龙, 钱其军. 降低亲和力提高HER2-CAR-T细胞治疗的安全性[J]. 药学实践与服务, 2016, 34(3): 261-266. doi: 10.3969/j.issn.1006-0111.2016.03.018
shu
ZHANG Hao, YE Zhenlong, QIAN Qijun. Decreasing affinity of CAR-T cells targeting HER2 to increase therapeutic outcome against tumors[J]. Journal of Pharmaceutical Practice and Service, 2016, 34(3): 261-266. doi: 10.3969/j.issn.1006-0111.2016.03.018
Citation: ZHANG Hao, YE Zhenlong, QIAN Qijun. Decreasing affinity of CAR-T cells targeting HER2 to increase therapeutic outcome against tumors[J]. Journal of Pharmaceutical Practice and Service, 2016, 34(3): 261-266. doi: 10.3969/j.issn.1006-0111.2016.03.018
shu

降低亲和力提高HER2-CAR-T细胞治疗的安全性

doi: 10.3969/j.issn.1006-0111.2016.03.018

  • 第二军医大学东方肝胆外科医院病毒基因治疗实验室, 上海 200438

Decreasing affinity of CAR-T cells targeting HER2 to increase therapeutic outcome against tumors

  • Laboratory of Viral and Gene Therapy, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai 200438, China

  • 摘要: 目的 探讨降低CAR-T细胞亲和力是否能有效提高其杀伤特异性,减少"脱靶效应"。 方法 构建靶向HER2的中等亲和力和高亲和力的La-G3HER2-CAR和Ha-G3HER2-CAR并电穿孔转染T细胞,采用Western Blot、FCM技术和xCELLigence RTCA DP进行CAR载体表达和杀伤功能检测。 结果 La-G3HER2-CAR-T细胞和Ha-G3HER2-CAR-T细胞分别表达43000和58000的外源CAR载体片段,转染效率分别为58.1%和69.0%。高亲和力的Ha-G3HER2-CAR-T细胞对高、中、低水平表达HER2的6种靶细胞均有效杀伤,而低亲和力的La-G3HER2-CAR-T细胞高效杀伤HER2高表达的SK-OV-3和BT474细胞,对HER2中表达的MDA-MB-231和HCC-202的杀伤作用较弱,而对低水平表达HER2的MCF-7和293细胞不杀伤。进一步的机制研究发现,HER2中水平表达的MDA-MB-231细胞共培养对La-G3HER2-CAR-T细胞和Ha-G3HER2-CAR-T细胞激活和细胞因子分泌诱导水平不同(CD107a:8.2% vs 71.6%; IFN-γ:66.3% vs 83.4%; TNF-α:73.4% vs 94.1%)。 结论 中等亲和力La-G3HER2-CAR-T细胞比高亲和力的Ha-G3HER2-CAR-T细胞的杀伤作用特异性更强,降低CAR-T细胞的亲和力可以提高治疗的安全性。
    Abstract: Objective To investigate whether decreasing affinity of CAR-T cells can increase their therapeutic outcome or not. Methods Moderate affinity La-G3HER2-CAR and high affinity Ha-G3HER2-CAR were constructed, and electroporated to modify T cells. Western blot assay, FCM assay and the RTCA DP cytotoxic equipment were applied to test the CAR expression and cytotoxic function of CAR-T cells. Results 43000 and 58000 exogenous CD3ζ fragments were expressed by both La-G3HER2-CAR-T cells and Ha-G3HER2-CAR-T cells with 58.1% and 69.0% transfection rate respectively. High affinity Ha-G3HER2-CAR-T cells effectively killed all target tumor cells by which HER2 was expressed at variable expression levels, while moderate affinity La-G3HER2-CAR-T cells specifically killed HER2 high-level expressing SK-OV-3 and BT474 cells, and showed weaker cytotoxicity on HER2 moderate-level expressing MDA-MB-231 and HCC-202 cells, and showed no cytotoxicity on HER2 low-level expressing MCF-7 and 293 cells. The underlying mechanic investigation found that La-G3HER2-CAR-T cells and Ha-G3HER2-CAR-T cells were differentially activated by co-culture with MDA-MB-231(CD107a:8.2% vs 71.6%, IFN-γ:66.3% vs 83.4%, TNF-α:73.4% vs 94.1%). Conclusion Moderate affinity La-G3HER2-CAR-T cells have enhanced specific cytotoxicity toward target tumor cells compared to high affinity Ha-G3HER2-CAR-T cells, decreasing affinity of CAR-T cell is a promising strategy to increase the therapeutic outcome of CAR-T cell based immunotherapies.
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  • 收稿日期:  2016-04-20
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降低亲和力提高HER2-CAR-T细胞治疗的安全性

doi: 10.3969/j.issn.1006-0111.2016.03.018
  • 第二军医大学东方肝胆外科医院病毒基因治疗实验室, 上海 200438
  • 收稿日期: 2016-04-20
  • 修回日期: 2016-05-04

摘要: 目的 探讨降低CAR-T细胞亲和力是否能有效提高其杀伤特异性,减少"脱靶效应"。 方法 构建靶向HER2的中等亲和力和高亲和力的La-G3HER2-CAR和Ha-G3HER2-CAR并电穿孔转染T细胞,采用Western Blot、FCM技术和xCELLigence RTCA DP进行CAR载体表达和杀伤功能检测。 结果 La-G3HER2-CAR-T细胞和Ha-G3HER2-CAR-T细胞分别表达43000和58000的外源CAR载体片段,转染效率分别为58.1%和69.0%。高亲和力的Ha-G3HER2-CAR-T细胞对高、中、低水平表达HER2的6种靶细胞均有效杀伤,而低亲和力的La-G3HER2-CAR-T细胞高效杀伤HER2高表达的SK-OV-3和BT474细胞,对HER2中表达的MDA-MB-231和HCC-202的杀伤作用较弱,而对低水平表达HER2的MCF-7和293细胞不杀伤。进一步的机制研究发现,HER2中水平表达的MDA-MB-231细胞共培养对La-G3HER2-CAR-T细胞和Ha-G3HER2-CAR-T细胞激活和细胞因子分泌诱导水平不同(CD107a:8.2% vs 71.6%; IFN-γ:66.3% vs 83.4%; TNF-α:73.4% vs 94.1%)。 结论 中等亲和力La-G3HER2-CAR-T细胞比高亲和力的Ha-G3HER2-CAR-T细胞的杀伤作用特异性更强,降低CAR-T细胞的亲和力可以提高治疗的安全性。

English Abstract

章浩, 叶真龙, 钱其军. 降低亲和力提高HER2-CAR-T细胞治疗的安全性[J]. 药学实践与服务, 2016, 34(3): 261-266. doi: 10.3969/j.issn.1006-0111.2016.03.018
引用本文: 章浩, 叶真龙, 钱其军. 降低亲和力提高HER2-CAR-T细胞治疗的安全性[J]. 药学实践与服务, 2016, 34(3): 261-266. doi: 10.3969/j.issn.1006-0111.2016.03.018
shu
ZHANG Hao, YE Zhenlong, QIAN Qijun. Decreasing affinity of CAR-T cells targeting HER2 to increase therapeutic outcome against tumors[J]. Journal of Pharmaceutical Practice and Service, 2016, 34(3): 261-266. doi: 10.3969/j.issn.1006-0111.2016.03.018
Citation: ZHANG Hao, YE Zhenlong, QIAN Qijun. Decreasing affinity of CAR-T cells targeting HER2 to increase therapeutic outcome against tumors[J]. Journal of Pharmaceutical Practice and Service, 2016, 34(3): 261-266. doi: 10.3969/j.issn.1006-0111.2016.03.018
shu

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