肿瘤细胞治疗研究
英文原题：Ablation of Cbl-b in ROBO1 CAR-NK92 Cells Enhances Their Antitumor Efficacy.
体外实验中，在效靶比（E:T）为0.1:1时，ROBO1 CAR-NK92-Cbl-b-KO细胞（50.55%）与ROBO1阳性T47D靶细胞共培养3小时后，其细胞杀伤活性显著高于ROBO1 CAR-NK92（34.10%）、NK92-Cbl-b-KO（22.22%）和亲本NK-92细胞（3.28%）。
新兴证据表明，CAR-NK细胞疗法在癌症治疗中具有很大潜力。ROBO1在多种癌症中高表达，包括胶质母细胞瘤、肝细胞癌、肺癌、乳腺癌和子宫癌。我们及其他实验室的研究显示，ROBO1 CAR-NK细胞具有良好的肿瘤治疗效果，但仍存在一定局限。已有报道指出，E3泛素连接酶Cbl-b可负向调节NK细胞活化、稳态和抗肿瘤免疫。因此，我们尝试采用CRISPR/Cas9基因编辑技术敲除Cbl-b，进一步增强ROBO1 CAR-NK92细胞的抗肿瘤活性。本研究将Cbl-b sgRNA与Cas9蛋白结合形成核糖核蛋白复合物，通过电穿孔导入ROBO1 CAR-NK92细胞和作为对照的NK-92细胞。经荧光活化细胞分选、有限稀释和测序，获得Cbl-b敲除（KO）单克隆细胞系。通过体外细胞毒性试验和体内肿瘤异种移植实验，检验Cbl-b敲除是否增强ROBO1 CAR-NK92细胞的靶细胞杀伤和肿瘤抑制能力。研究成功建立ROBO1 CAR-NK92-Cbl-b-KO和NK92-Cbl-b-KO单克隆细胞系。体外实验中，在效应细胞与靶细胞比为0.1:1时，共培养3小时后，ROBO1 CAR-NK92-Cbl-b-KO细胞对ROBO1阳性T47D靶细胞的杀伤活性为50.55%，显著高于ROBO1 CAR-NK92（34.10%）、NK92-Cbl-b-KO（22.22%）和亲本NK-92细胞（3.28%）。体内实验中，肿瘤体积和重量测量显示，接受ROBO1 CAR-NK92-Cbl-b-KO细胞治疗的小鼠肿瘤显著小于所有对照组，肿瘤生长抑制率为32.45%，表明Cbl-b敲除增强了抗肿瘤疗效。体内外数据证实，敲除Cbl-b可增强ROBO1 CAR-NK92细胞的抗肿瘤效能。总体细胞毒能力排序为：ROBO1 CAR-NK92-Cbl-b-KO > ROBO1 CAR-NK92 > NK92-Cbl-b-KO > NK-92。
Emerging evidence suggests CAR-NK cell therapy shows great promise in cancer treatment. ROBO1 is highly expressed in various cancer types, including glioblastoma, hepatocellular carcinoma, lung cancer, breast cancer, and uterine cancer. Our and other laboratories' studies have shown that ROBO1 CAR-NK cells exhibit promising tumor therapeutic effects. However, the results still have some limitations. Cbl-b, an E3 ubiquitin ligase, has been reported to negatively regulate NK cell activation, homeostasis, and antitumor immunity. 1 Therefore, we attempted to further enhance the antitumor activity of ROBO1 CAR-NK92 cells by knocking out Cbl-b using CRISPR/Cas9 gene-editing technology. In this study, we conjugated Cbl-b sgRNA with Cas9 protein to form ribonucleoprotein complexes, which were then delivered into ROBO1 CAR-NK92 and NK-92 cells (control cells) via electroporation. Through fluorescence-activated cell sorting, limiting dilution, and sequencing, we obtained monoclonal Cbl-b-knock-out (KO) cell lines. Both in vitro cytotoxicity assays and in vivo tumor xenograft experiments were conducted to examine whether Cbl-b knockout enhances the target cell killing and tumor suppression capacities of ROBO1 CAR-NK92 cells. In this study, monoclonal cell lines of ROBO1 CAR-NK92-Cbl-b-KO and NK92-Cbl-b-KO were successfully established. In vitro , at an effector-to-target (E:T) ratio of 0.1:1, ROBO1 CAR-NK92-Cbl-b-KO (50.55%) cells exhibited significantly higher cytolytic activity against ROBO1-positive T47D target cells after 3 h of coculture than ROBO1 CAR-NK92 (34.10%), NK92-Cbl-b-KO (22.22%), and parental NK-92 cells (3.28%). In vivo , tumor volume and weight measurements demonstrated that mice treated with ROBO1 CAR-NK92-Cbl-b-KO cells developed significantly smaller tumors than all control groups, achieving a tumor growth inhibition (TGI) rate of 32.45%, indicating enhanced antitumor efficacy conferred by Cbl-b knockout. In vitro and in vivo data confirmed that Cbl-b knockout potentiates the antitumor efficacy of ROBO1 CAR-NK92 cells. The overall cytotoxic capability ranked as follows: ROBO1 CAR-NK92-Cbl-b-KO > ROBO1 CAR-NK92 > NK92-Cbl-b-KO > NK-92.
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