肿瘤细胞治疗研究
英文原题：NK-92MI Cells Engineered with Anti-claudin-6 Chimeric Antigen Receptors in Immunotherapy for Ovarian Cancer.
这些结果表明，CLDN6-CAR NK细胞具有强大的抗肿瘤活性，是卵巢癌一种有前景的免疫治疗方式。
背景：嵌合抗原受体（CAR）NK细胞在实体瘤中的应用受到缺乏肿瘤特异性靶点和CAR-NK细胞疗效低下的阻碍。据报道，Claudin-6（CLDN6）在卵巢癌中过表达，可能是CAR-NK细胞免疫治疗的一个有吸引力的靶点。然而，使用抗CLDN6 CAR-NK细胞治疗卵巢癌的可行性仍有待探索。 方法：通过免疫组织化学和western blot检测原代人卵巢癌、正常组织和细胞系中CLDN6的表达。通过慢病毒转染构建了两种靶向CLDN6的第三代CAR NK-92MI细胞，即含有自激活元件结构域（NKG2D、2B4）的CLDN6-CAR1 NK-92MI细胞和含有经典结构域（CD28、4-1BB）的CLDN6-CAR2 NK-92MI细胞，并通过流式细胞术分选，通过western blot和qPCR验证。转导了表达GFP和荧光素酶基因的OVCAR-3、SK-OV-3、A2780、Hey和PC-3细胞。通过NSG小鼠建立皮下和腹腔肿瘤模型。通过活细胞成像和生物发光成像在体外和体内评估CLDN6-CAR NK细胞杀伤CLDN6阳性卵巢癌细胞的能力。 结果：在体外，CLDN6-CAR1和CLDN6-CAR2 NK-92MI细胞均可特异性杀伤CLDN6阳性卵巢癌细胞（OVCAR-3、SK-OV-3、A2780和Hey），而非CLDN6阴性细胞（PC-3）。含有自激活元件结构域（NKG2D、2B4）的CLDN6-CAR1 NK-92MI细胞表现出比含有经典结构域（CD28、4-1BB）的CLDN6-CAR2 NK-92MI细胞更强的细胞毒性。此外，CLDN6-CAR1 NK细胞可有效清除皮下和腹腔肿瘤模型中的卵巢癌细胞。更重要的是，CAR-NK细胞与免疫检查点抑制剂anti-PD-L1联合使用，可协同增强CLDN6靶向CAR-NK细胞的抗肿瘤疗效。结论：这些结果表明，CLDN6-CAR NK细胞具有较强的抗肿瘤活性，是卵巢癌一种有前景的免疫治疗方式。
Background : The application of chimeric antigen receptor (CAR) NK cells in solid tumors is hindered by lack of tumor-specific targets and inefficient CAR-NK cell efficacy. Claudin-6 (CLDN6) has been reported to be overexpressed in ovarian cancer and may be an attractive target for CAR-NK cells immunotherapy. However, the feasibility of using anti-CLDN6 CAR-NK cells to treat ovarian cancer remains to be explored. Methods : CLDN6 expression in primary human ovarian cancer, normal tissues and cell lines were detected by immunohistochemistry and western blot. Two types of third-generation CAR NK-92MI cells targeting CLDN6, CLDN6-CAR1 NK-92MI cells with domains containing self-activated elements (NKG2D, 2B4) and CLDN6-CAR2 NK-92MI cells with classical domains (CD28, 4-1BB) were constructed by lentivirus transfection, sorted by flow cytometry and verified by western blot and qPCR. OVCAR-3, SK-OV-3, A2780, Hey and PC-3 cells expressing the GFP and luciferase genes were transduced. Subcutaneous and intraperitoneal tumor models were established via NSG mice. The ability of CLDN6-CAR NK cells to kill CLDN6-positive ovarian cancer cells were evaluated in vitro and in vivo by live cell imaging and bioluminescence imaging. Results : Both CLDN6-CAR1 and CLDN6-CAR2 NK-92MI cells could specifically killed CLDN6-positive ovarian cancer cells (OVCAR-3, SK-OV-3, A2780 and Hey), rather than CLDN6 negative cell (PC-3), in vitro. CLDN6-CAR1 NK-92MI cells with domains containing self-activated elements (NKG2D, 2B4) exhibited stronger cytotoxicity than CLDN6-CAR2 NK-92MI cells with classical domains (CD28, 4-1BB). Furthermore, CLDN6-CAR1 NK cells could effectively eliminate ovarian cancer cells in subcutaneous and intraperitoneal tumor models. More importantly, CAR-NK cells combined with immune checkpoint inhibitors, anti-PD-L1, could synergistically enhance the antitumor efficacy of CLDN6-targeted CAR-NK cells. Conclusions : These results indicate that CLDN6-CAR NK cells possess strong antitumor activity and represent a promising immunotherapeutic modality for ovarian cancer.
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