BACKGROUND Although immune checkpoint inhibitors have revolutionized cancer treatment, clinical benefit with this class of agents has been limited to a subset of patients. Hence, more effective means to target tumor cells that express immune checkpoint molecules should be developed. For the first time, we report a novel natural killer (NK) cell line, programmed death-ligand 1 (PD-L1) targeting high-affinity natural killer (t-haNK), which was derived from NK-92 and was engineered to express high-affinity CD16, endoplasmic reticulum-retained interleukin (IL)-2, and a PD-L1-specific chimeric antigen receptor (CAR). We show that PD-L1 t-haNK cells also retained the expression of native NK receptors and carried a high content of granzyme and perforin granules. METHODS NanoString, flow cytometry, and immunofluorescence analyses were performed to characterize the phenotype of irradiated PD-L1 t-haNK cells. In vitro PD-L1 t-haNK cell activity against cancer cell lines and human peripheral blood mononuclear cells (PBMCs) was determined via flow-based and 111In-release killing assays. The antitumor effect of PD-L1 t-haNK cells in vivo was investigated using MDA-MB-231, H460, and HTB1 xenograft models in NOD-scid IL2Rgammanull (NSG) mice. Additionally, the antitumor effect of PD-L1 t-haNK cells, in combination with anti-PD-1 and N-803, an IL-15 superagonist, was evaluated using mouse oral cancer 1 syngeneic model in C57BL/6 mice. RESULTS We show that PD-L1 t-haNK cells expressed PD-L1-targeting CAR and CD16, retained the expression of native NK receptors, and carried a high content of granzyme and perforin granules. In vitro, we demonstrate the ability of irradiated PD-L1 t-haNK cells to lyse 20 of the 20 human cancer cell lines tested, including triple negative breast cancer (TNBC) and lung, urogenital, and gastric cancer cells. The cytotoxicity of PD-L1 t-haNK cells was correlated to the PD-L1 expression of the tumor targets and can be improved by pretreating the targets with interferon (IFN)-γ. In vivo, irradiated PD-L1 t-haNK cells inhibited the growth of engrafted TNBC and lung and bladder tumors in NSG mice. The combination of PD-L1 t-haNK cells with N-803 and anti-PD-1 antibody resulted in superior tumor growth control of engrafted oral cavity squamous carcinoma tumors in C57BL/6 mice. In addition, when cocultured with human PBMCs, PD-L1 t-haNK cells preferentially lysed the myeloid-derived suppressor cell population but not other immune cell types. CONCLUSION These studies demonstrate the antitumor efficacy of PD-L1 t-haNK cells and provide a rationale for the potential use of these cells in clinical studies.

中文翻译：

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靶向高亲和力NK（t-haNK）细胞的PD-L1诱导直接的抗肿瘤作用，并靶向抑制性MDSC群体。

背景技术尽管免疫检查点抑制剂已经彻底改变了癌症治疗方法，但是这类药物的临床益处仅限于部分患者。因此，应该开发更有效的方法来靶向表达免疫检查点分子的肿瘤细胞。首次，我们报道了一种新型的自然杀伤（NK）细胞系，其靶向高亲和力自然杀伤（t-haNK）的程序化死亡配体1（PD-L1），该细胞系衍生自NK-92，并被工程改造为表达高亲和力CD16，内质网保留的白介素（IL）-2和PD-L1特异性嵌合抗原受体（CAR）。我们显示，PD-L1 t-haNK细胞还保留天然NK受体的表达，并携带高含量的颗粒酶和穿孔素颗粒。方法NanoString，流式细胞仪，并进行了免疫荧光分析以表征受辐照的PD-L1 t-haNK细胞的表型。通过基于流的杀灭和111In-释放杀伤试验确定了体外PD-L1 t-haNK细胞对癌细胞系和人外周血单核细胞（PBMC）的活性。使用MDA-MB-231，H460和HTB1异种移植模型在NOD-scid IL2Rgammanull（NSG）小鼠中研究了PD-L1 t-haNK细胞在体内的抗肿瘤作用。另外，使用小鼠口腔癌1同基因模型在C57BL / 6小鼠中评估了PD-L1 t-haNK细胞与抗PD-1和IL-15超激动剂N-803的抗肿瘤作用。结果我们显示，PD-L1 t-haNK细胞表达靶向PD-L1的CAR和CD16，保留了天然NK受体的表达，并携带高含量的颗粒酶和穿孔素颗粒。体外，我们证明了辐照的PD-L1 t-haNK细胞能够溶解20种测试的人类癌细胞系中的20种，包括三阴性乳腺癌（TNBC）以及肺癌，泌尿生殖器和胃癌细胞。PD-L1 t-haNK细胞的细胞毒性与肿瘤靶标的PD-L1表达相关，可以通过用干扰素（IFN）-γ预处理靶标来改善。在体内，辐照的PD-L1 t-haNK细胞抑制了NSG小鼠中移植的TNBC以及肺和膀胱肿瘤的生长。PD-L1 t-haNK细胞与N-803和抗PD-1抗体的组合可导致对C57BL / 6小鼠移植的口腔鳞状细胞癌的优异肿瘤生长控制。此外，当与人PBMC共培养时，PD-L1 t-haNK细胞优先裂解髓样来源的抑制细胞群，但不裂解其他免疫细胞类型。