共表达IL-15和CCL19的EGFRvⅢCAR-T细胞的构建和功能探究  

Construction and functional analysis of EGFRvIII CAR-T cells co-expressing IL-15 and CCL19

作　　者：陈婉琼 咸娜 林少梅 廖婉婷 陈明珠 CHEN Wanqiong;XIAN Na;LIN Shaomei;LIAO Wanting;CHEN Mingzhu(School of Pharmacy,Quanzhou Medical College,Quanzhou 362011,Fujian,China;Institute of Immunotherapy,Fujian Medical University,Fuzhou 350122,Fujian,China)

机构地区：[1]泉州医学高等专科学校药学院,福建泉州362011 [2]福建医科大学免疫治疗研究院,福建福州350122

出　　处：《生物工程学报》2023年第9期3787-3799,共13页Chinese Journal of Biotechnology

基　　金：泉州市科技计划项目(2021N130S);泉州医学高等专科学校校级青年科技课题(XJK2015B)。

摘　　要：本研究分析了共表达白细胞介素-15(interleukin-15,IL-15)和趋化因子配体19(C-C chemokine ligand 19,CCL19)的EGFRvⅢCAR-T细胞的功能特性及其体外特异性杀伤效果,旨在优化CAR-T细胞多项功能,提高靶向EGFRvⅢ的CAR-T细胞对胶质母细胞瘤(glioblastoma,GBM)的治疗效果。通过基因工程技术获得重组慢病毒质粒,转染293T细胞获得慢病毒并感染T细胞获得靶向EGFRvⅢ的第四代CAR-T细胞(EGFRvⅢ-IL-15-CCL19 CAR-T)。利用流式细胞仪、细胞计数仪、趋化小室、凋亡试剂盒等检测了第四代和第二代CAR-T细胞(EGFRvⅢCAR-T)的CAR分子表达率、增殖、趋化能力、体外特异性杀伤能力及抗凋亡能力等。结果表明,与EGFRvⅢCAR-T细胞相比,EGFRvⅢ-IL-15-CCL19 CAR-T细胞能成功分泌IL-15和CCL19,具有更强的体外增殖能力、趋化能力以及抗凋亡能力(P值均<0.05),而体外特异性杀伤能力无显著差异。因此,靶向EGFRvⅢ且同时分泌IL-15和CCL19的CAR-T细胞有望提高胶质母细胞瘤的治疗效果,为临床试验提供一定的参考依据。The aim of this study was to investigate the functional characteristics and in vitro specific killing effect of EGFRvIII CAR-T cells co-expressing interleukin-15 and chemokine CCL19,in order to optimize the multiple functions of CAR-T cells and improve the therapeutic effect of CAR-T cells targeting EGFRvIII on glioblastoma(GBM).The recombinant lentivirus plasmid was obtained by genetic engineering,transfected into 293T cells to obtain lentivirus and infected T cells to obtain the fourth generation CAR-T cells targeting EGFRvIII(EGFRvIII-IL-15-CCL19 CAR-T).The expression rate of CAR molecules,proliferation,chemotactic ability,in vitro specific killing ability and anti-apoptotic ability of the fourth and second generation CAR-T cells(EGFRvIII CAR-T)were detected by flow cytometry,cell counter,chemotaxis chamber and apoptosis kit.The results showed that compared with EGFRvIII CAR-T cells,EGFRvIII-IL-15-CCL19 CAR-T cells successfully secreted IL-15 and CCL19,and had stronger proliferation,chemotactic ability and anti-apoptosis ability in vitro(all P<0.05),while there was no significant difference in killing ability in vitro.Therefore,CAR-T cells targeting EGFRvIII and secreting IL-15 and CCL19 are expected to improve the therapeutic effect of glioblastoma and provide an experimental basis for clinical trials.

关 键 词：嵌合抗原受体修饰的T细胞 表皮生长因子受体突变体Ⅲ 胶质母细胞瘤 白细胞介素-15 趋化因子配体19 

分 类 号：R392[医药卫生—免疫学] Q789[医药卫生—基础医学]