靶向PSCA的缺氧诱导型CAR-T的构建及体外效能研究

Construction and In Vitro Potency Investigation of Hypoxia-Inducible CAR-T Targeting PSCA

该文针对肿瘤微环境中的缺氧环境,利用缺氧相关的启动子,设计和制备在缺氧环境中诱导表达靶向前列腺干细胞抗原(PSCA)的嵌合抗原受体修饰的T细胞(CAR-T),研究缺氧条件下其嵌合抗原受体(CAR)的表达及对肿瘤细胞的体外杀伤作用。研究者通过基因合成缺氧启动子(5H1P)和CAR的DNA片段,利用酶切方法将其插入到慢病毒载体中,然后感染T淋巴细胞。在体外用CoCl2构建缺氧模型,通过流式细胞术检测CoCl2未诱导组CAR有42%的弱阳性表达,诱导后阳性率增加到84.4%,且荧光强度显著增加。结果表明,诱导组比不诱导对照组抗肿瘤效果更好。综上所述,该实验成功构建了靶向PSCA的缺氧诱导型CAR-T,使其在缺氧的环境中效能增强,这为CAR-T在实体瘤的治疗方面提供了新的方案。

Considering anoxic tumor microenvironment,we designed a hypoxia-inducible chimeric antigen receptor-modified T cells(CAR-T)targeting prostate stem cell antigen(PSCA),and tested the cytotoxicity of these cells against tumor cells in vitro.5 H1 P and the sequences encoding the light and heavy chain variable regions of a monoclonal antibody targeting PSCA were synthesized and integrated into lentiviral vectors using restriction enzymes.Then lentivirus were infected to human T lymphocytes isolated from peripheral blood mononuclear cells to preparation CAR-T cells.Taking use of CoCl2,we emulated the hypoxia model in vitro.The positivity rate of PSCA-CAR expression in the T lymphocytes was detected by flow cytometry with or without CoCl2 treatment.There was 42%weak positive subpopulation in uninduced group and increased to 84.4%after CoCl2 induction,in which the fluorescence intensity increased significantly.Cytotoxic assay was performed to evaluate the specific cytotoxicity of the genetically modified T cells against the PSCA-positive HeLa cells,and ELISA was used to detect cytokine secretion by the cells.Results showed that induction group had better cytotoxicity than noninduced control group(P<0.05),and secreted significantly higher levels of cytokines(P<0.01).In conclusion,we successfully constructed the hypoxia-inducible CAR-T targeting PSCA with enhanced potency in anoxic environment.It will provide a new solution for CAR-T treatment in solid tumors.