Synthetic Biology Engineering Lab of Henan Province, School of Life Sciences and Technology, Xinxiang Medical University, Xinxiang, Henan Province, PR China.
Synthetic Biology Engineering Lab of Henan Province, School of Life Sciences and Technology, Xinxiang Medical University, Xinxiang, Henan Province, PR China.
Mol Immunol. 2023 Mar;155:91-99. doi: 10.1016/j.molimm.2023.01.010. Epub 2023 Feb 1.
Adoptive chimeric antigen receptor (CAR)-modified T or NK cells (CAR-T/NK) have emerged as a novel form of disease treatment. Lentiviral vectors (LVs) are commonly employed to engineer NK cells for the efficient expression of CARs. This study reported the influence of single-promoter and dual-promoter LVs on the CAR expression and cytotoxicity of engineered NK cells. We constructed a third-generation NKG2D-based CAR that kills cancer cells by targeting up to eight stress-induced ligands (NKG2DLs). Our results demonstrated that the CAR exhibits both a higher expression level and a higher coexpression concordance with the GFP reporter in HEK-293T or NK92 cells by utilizing the optimized single-promoter pCDHsp rather than the original dual-promoter pCDHdp. After puromycin selection, the pCDHsp produces robust CAR expression and enhanced in vitro cytotoxicity of engineered NK cells. Therefore, infection with a single-promoter pCDHsp lentivector is recommended to prepare CAR-engineered NK cells. This research helps to optimize the production of CAR-NK cells and enhance their functional activity, to provide CAR-NK cell products with better and more uniform quality.
过继性嵌合抗原受体 (CAR)-修饰的 T 或 NK 细胞 (CAR-T/NK) 已成为一种新型疾病治疗方法。慢病毒载体 (LV) 常用于工程化 NK 细胞以实现 CAR 的高效表达。本研究报告了单启动子和双启动子 LV 对工程化 NK 细胞 CAR 表达和细胞毒性的影响。我们构建了第三代基于 NKG2D 的 CAR，通过靶向多达八个应激诱导配体 (NKG2DL) 来杀死癌细胞。我们的结果表明，与原始的双启动子 pCDHdp 相比，利用优化的单启动子 pCDHsp，CAR 在 HEK-293T 或 NK92 细胞中的表达水平更高，且与 GFP 报告基因的共表达一致性更高。在嘌呤霉素选择后，pCDHsp 产生强大的 CAR 表达，并增强了工程化 NK 细胞的体外细胞毒性。因此，建议使用单启动子 pCDHsp 慢病毒载体进行感染，以制备 CAR 修饰的 NK 细胞。本研究有助于优化 CAR-NK 细胞的生产并增强其功能活性，为 CAR-NK 细胞产品提供更好、更均匀的质量。
