背景:自然杀伤(NK)细胞作为癌症的细胞疗法正在被广泛研究。这些细胞通过识别肿瘤细胞上的配体和抗原而被激活。细胞因子疗法,例如IL-15,也广泛用于刺激癌症患者的内源性和过继转移的NK细胞。这些刺激激活膜蛋白酶ADAM17,其作为负反馈回路裂解NK细胞上的各种细胞表面受体以限制其细胞溶解功能。ADAM17抑制可以在体外和体内增强IL-15介导的NK细胞增殖。在这项研究中,我们研究了这个过程的潜在机制。
方法:来自人外周血的外周血单核细胞(PBMC)或富集的NK细胞,未标记或用细胞增殖染料标记,在rhIL-15±ADAM17功能阻断抗体存在下培养长达7天。产生了不同的完全人类版本的抗体;Medi-1(IgG1),Medi-4(IgG4),Medi-PGLALA,Medi-F(ab')2和TAB16(抗ADAM17和抗CD16双特异性)调节CD16A结合。流式细胞术用于评估NK细胞增殖和表型标志物,免疫印迹检查CD16A信号,和基于IncuCyte的活细胞成像来测量NK细胞抗肿瘤活性。
结果:ADAM17功能阻断单克隆抗体(mAb)Medi-1显著增加了IL-15对早期NK细胞的活化。通过使用不同的工程化版本的抗体,我们展示了CD16A的参与,激活Fcγ受体和良好描述的ADAM17底物。因此,当结合到NK细胞上的ADAM17时,Medi-1被CD16A接合并阻断其脱落,诱导和延长其信号传导。此过程并未促进明显的NK细胞自相残杀或功能障碍。在存在PBMC辅助细胞或抗CD137激动性mAb的情况下,Medi-1和IL-15的协同信号传导增强了CD137在CD16ANK细胞上的上调,并增强了它们的增殖。
结论:我们的数据首次揭示CD16A和CD137支持Medi-1增强IL-15驱动的NK细胞活化和增殖,分别,后者需要PBMC辅助细胞。使用Medi-1代表了增强IL-15驱动的NK细胞增殖的新策略。通过增加癌症患者NK细胞的抗肿瘤活性可能具有治疗意义。
BACKGROUND: Natural killer (NK) cells are being extensively studied as a cell therapy for cancer. These cells are activated by recognition of ligands and antigens on tumor cells. Cytokine therapies, such as IL-15, are also broadly used to stimulate endogenous and adoptively transferred NK cells in patients with cancer. These stimuli activate the membrane protease ADAM17, which cleaves various cell-surface receptors on NK cells as a negative feedback loop to limit their cytolytic function. ADAM17 inhibition can enhance IL-15-mediated NK cell proliferation in vitro and in vivo. In this study, we investigated the underlying mechanism of this process.
METHODS: Peripheral blood mononuclear cells (PBMCs) or enriched NK cells from human peripheral blood, either unlabeled or labeled with a cell proliferation dye, were cultured for up to 7 days in the presence of rhIL-15±an ADAM17 function-blocking antibody. Different fully human versions of the antibody were generated; Medi-1 (IgG1), Medi-4 (IgG4), Medi-PGLALA, Medi-F(ab\')2, and TAB16 (anti-ADAM17 and anti-CD16 bispecific) to modulate CD16A binding. Flow cytometry was used to assess NK cell proliferation and phenotypic markers, immunoblotting to examine CD16A signaling, and IncuCyte-based live cell imaging to measure NK cell antitumor activity.
RESULTS: The ADAM17 function-blocking monoclonal antibody (mAb) Medi-1 markedly increased early NK cell activation by IL-15. By using different engineered versions of the antibody, we demonstrate involvement by CD16A, an activating Fcγ receptor and well-described ADAM17 substrate. Hence, Medi-1 when bound to ADAM17 on NK cells is engaged by CD16A and blocks its shedding, inducing and prolonging its signaling. This process did not promote evident NK cell fratricide or dysfunction. Synergistic signaling by Medi-1 and IL-15 enhanced the upregulation of CD137 on CD16A+ NK cells and augmented their proliferation in the presence of PBMC accessory cells or an anti-CD137 agonistic mAb.
CONCLUSIONS: Our data reveal for the first time that CD16A and CD137 underpin Medi-1 enhancement of IL-15-driven NK cell activation and proliferation, respectively, with the latter requiring PBMC accessory cells. The use of Medi-1 represents a novel strategy to enhance IL-15-driven NK cell proliferation, and it may be of therapeutic importance by increasing the antitumor activity of NK cells in patients with cancer.