肿瘤细胞治疗研究
英文原题：CRISPR knock-in of a chimeric antigen receptor into GAPDH 3'UTR locus generates potent B7H3-specific NK-92MI cells.
CRISPR knock-in of a chimeric antigen receptor into GAPDH 3'UTR locus generates potent B7H3-specific NK-92MI cells.
CAR-NK 疗法正成为一种有前景的实体瘤治疗方法。
CAR-NK 疗法正成为一种治疗实体瘤的有前景的方法。然而，常见制备方法导致的 CAR 基因随机插入和 CAR 表达不灵活显著影响其疗效和安全性。在此，我们通过将 CAR 序列整合到 NK-92MI 细胞的 GAPDH 3'UTR 位点，成功建立了一种新型 CAR-NK 细胞（CRISPR-CAR-NK），实现了 CAR 基因的位点特异性整合，并使内源性调控组分能够调控 CAR 表达。与慢病毒转导的对应细胞相比，CRISPR-CAR-NK 细胞具有相当的生长能力，但表现出更优的抗肿瘤活性。当与肿瘤细胞共培养时，它们更有效地活化和脱颗粒，这是由于激活性受体表达增加和抑制性分子表达减少。它们还增强了颗粒酶 B 和 IFN-γ 的产生，并更有效地触发 IFN-γ 通路。此外，CRISPR-CAR-NK 细胞在代谢特征和信号依赖性方面表现出与常规 CAR-NK 不同的特性。值得注意的是，CRISPR-CAR-NK 细胞表现出较低的代谢水平，但未损害抗肿瘤活性，并且其功能对 PI3K-AKT 通路的依赖较少，这提示 CRISPR-CAR-NK 细胞在增强与 AKT 抑制剂的协同作用以及适应肿瘤微环境中的营养应激方面具有显著潜力。这些发现为癌症免疫治疗提供了一种新的潜在策略，并为利用 CRISPR 技术优化 CAR-NK 细胞提供了实验基础和范式，凸显了 CRISPR 推动免疫治疗发展的潜力。
CAR-NK therapy is becoming a promising approach to treat solid tumors. However, the random insertion of the CAR gene and inflexible CAR expression caused by common preparation methods significantly impact its efficacy and safety. Here we successfully established a novel type of CAR-NK cells by integrating CAR sequences into the GAPDH 3'UTR locus of NK-92MI cells (CRISPR-CAR-NK), achieving site-specific integration of the CAR gene and allowing endogenous regulatory components to govern CAR expression. CRISPR-CAR-NK cells had comparable growth capacity but displayed superior anti-tumor activity compared with their lentiviral counterparts. They activated and degranulated more effectively when co-cultured with tumor cells, due to increased expression of activating receptors and decreased expression of inhibitory molecules. They also enhanced the production of Granzyme B and IFN- , and more effectively triggered the IFN- pathway. Moreover, CRISPR-CAR-NK cells demonstrated distinct properties from conventional CAR-NK concerning metabolic features and signal dependence. Notably, CRISPR-CAR-NK cells exhibited lower metabolic levels without compromising antitumor activity, and their function was less reliant on the PI3K-AKT pathway, implying that the CRISPR-CAR-NK cells have significant potential for enhanced synergy with AKT inhibitors and adaptation to nutrient stress within the tumor microenvironment. These findings provide a novel potential strategy for cancer immunotherapy and an experimental foundation and paradigm for optimizing CAR-NK cells utilizing CRISPR technology, highlighting the potential of CRISPR to advance immunotherapies.
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