肿瘤细胞治疗研究
英文原题：Combined treatment with anti-PSMA CAR NK-92 cell and anti-PD-L1 monoclonal antibody enhances the antitumour efficacy against castration-resistant prostate cancer.
CAR NK-92 与抗 PD-L1 单克隆抗体联合治疗提高了对 CRPC 的抗肿瘤疗效，这在 CRPC 的临床治疗中具有非凡的转化价值。
背景：靶向前列腺特异性膜抗原（PSMA）的嵌合抗原受体NK-92（CAR NK-92）细胞已在去势抵抗性前列腺癌（CRPC）中显示抗肿瘤作用。然而，CAR NK-92和CRPC细胞中程序性死亡配体1（PD-L1）的表达变化及其机制，以及抗PD-L1单克隆抗体（mAb）对CAR NK-92细胞表达PD-L1的作用仍不明确。方法：从健康供者血样中分离人树突状细胞和CD8+ T细胞，并与C4-2细胞共培养。采用流式细胞术检测PD-L1表达变化。通过RNA测序分析差异基因表达，采用蛋白质印迹研究PD-L1分子信号调控。使用Cell Counting Kit-8实验和绿色荧光蛋白标记C4-2细胞的生物发光强度（BLI）评估体外细胞毒性。体内则在雄性NOD/SCID小鼠皮下注射C4-2细胞并监测CRPC生长；小鼠静脉接受抗PD-L1/PD-1 mAb、CAR NK-92细胞或共培养CD8+ T细胞。结果：共培养的C4-2和CAR NK-92细胞中PD-L1表达均显著上调。此外，C4-2细胞中PD-L1表达上调依赖干扰素-γ；CAR NK-92细胞中则依赖经NK细胞组2成员D/磷脂酰肌醇3激酶/AKT通路介导的直接细胞间相互作用。抗PD-L1 mAb可直接作用于CAR NK-92细胞表达的PD-L1，并在体外增强CAR NK-92细胞对C4-2和一例患者来源CRPC细胞的细胞毒性。与单独CAR NK-92治疗相比，无论是否存在共培养CD8+ T细胞，CAR NK-92联合抗PD-L1 mAb在体内对CRPC细胞的抗肿瘤效果均显著增强；与CAR NK-92联合抗PD-1 mAb相比亦然。结论：CAR NK-92联合抗PD-L1 mAb可提高对CRPC的抗肿瘤疗效，在CRPC临床治疗中具有重要转化价值。
BACKGROUND: The chimeric antigen receptor NK-92 (CAR NK-92) cell targeting the prostate-specific membrane antigen (PSMA) has shown antitumour effects in castration-resistant prostate cancer (CRPC). However, the expression changes of programmed death ligand 1 (PD-L1) and its mechanisms on CAR NK-92 and CRPC cells and the effect of the anti-PD-L1 monoclonal antibody (mAb) on PD-L1 expressed on CAR NK-92 cells remain unknown. METHODS: Human dendritic cells and CD8 + T cells were acquired from blood samples of healthy donors and cocultured with C4-2 cells. Changes in PD-L1 expression were detected by flow cytometry. Differential gene expressions were investigated by RNA sequence analysis, while the regulation of PD-L1 molecular signaling was explored using western blotting. In vitro cytotoxicity was evaluated using the Cell Counting Kit-8 assay and the bioluminescent intensity (BLI) of green fluorescent protein-labelled C4-2 cells. CRPC growth in vivo was monitored using callipers and BLI in male NOD/SCID mice subcutaneously injected with C4-2 cells and treated intravenously with anti-PD-L1/PD-1 mAb, CAR NK-92 or cocultured CD8 + T cells. RESULTS: Significantly upregulated expression of PD-L1k was observed in cocultured C4-2 and CAR NK-92 cells. In addition, upregulation of PD-L1 expression was dependent on interferon- in C4-2 cells, while it was dependent on direct cell-to-cell interaction via the NK group 2 member D/ phosphatidylinositol 3-kinase/AKT pathway in CAR NK-92 cells. The anti-PD-L1 mAb directly acted on PD-L1 expressed on CAR NK-92 cells and augmented the cytotoxicity of CAR NK-92 cells against C4-2 and CRPC cells from one patient in vitro. Anti-PD-L1 mAb significantly enhanced the antitumour effect of CAR NK-92 cells against CRPC cells in vivo when compared to treatment with CAR NK-92 cells or combined with anti-PD-1 mAb in the absence or presence of cocultured CD8 + T cells. CONCLUSION: Combined treatment with CAR NK-92 and anti-PD-L1 mAb improved the antitumour efficacy against CRPC, which is of extraordinary translational value in the clinical treatment of CRPC.
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