Supplementary file 1_Dual-functioning Targeted ADAM17 Blocker CD16 (TAB16) mediates selective ADAM17 inhibition in NK cells and engages overexpressed ADAM17 in tumor cells to induce cytotoxicity.zip

IntroductionNatural killer (NK) cells are innate lymphocytes that kill tumor cells by natural cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC). Human NK cells mediate the latter process exclusively by the IgG Fc receptor CD16 (FcγRIIIA). Cell surface levels of this activating receptor are tightly regulated by the metalloprotease ADAM17, which cleaves CD16 upon NK cell activation or cellular stress. We have reported that Medi-1, a fully human IgG1 mAb, blocks ADAM17, and its Fc region is simultaneously engaged by CD16, inducing and prolonging its signaling, which synergizes with cytokine stimulation, such as IL-15. To exploit these distinctive features of Medi-1 while also addressing limitations of the mAb, such as the varied affinity by which CD16 binds to it due to receptor polymorphisms and the risk of broadly blocking ADAM17 activity, we engineered Targeted ADAM17 Blocker CD16 (TAB16).
MethodsTAB16 was generated with a camelid heavy-chain variable domain specific to CD16 linked to a single-chain variable fragment derived from Medi-1. TAB16 was further modified by the linkage of an IL-15 moiety to generate TAB16/15. Primary human NK cells were treated with TAB16 or TAB16/15 and evaluated for proliferation by cell dilution dye, ADAM17 blocking, activation marker expression, and cytotoxicity against ovarian cancer cell lines in real-time by IncuCyte assays.
ResultsThe TAB16 bispecific engager targeted NK cells and blocked ADAM17. A novel feature of TAB16 is its dual functionality, as it synergizes with IL-15 to enhance NK cell activation and proliferation and targets ADAM17 overexpressed on cancer cells to induce ADCC. TAB16 is a modifiable backbone to which additional functional components can be added, such as IL-15 (TAB16/15) for consolidated and multifaceted activity.
DiscussionOur ADAM17-engaging platform offers a unique approach for targeted ADAM17 inhibition to augment the anti-tumor function of endogenous and therapeutic NK cells.

引言
自然杀伤（NK）细胞是一类先天淋巴细胞，可通过自然细胞毒性以及抗体依赖性细胞介导的细胞毒性（ADCC）杀伤肿瘤细胞。人NK细胞仅能通过IgG Fc受体CD16（FcγRIIIA）介导后一杀伤过程。该激活受体的细胞表面表达水平受到金属蛋白酶ADAM17的严格调控，ADAM17可在NK细胞激活或细胞应激状态下切割CD16。本团队此前报道，全人源IgG1单克隆抗体（mAb）Medi-1可阻断ADAM17，且其Fc区域可同时与CD16结合，诱导并延长CD16的信号传导，该效应可与白细胞介素15（IL-15）等细胞因子的刺激产生协同作用。为利用Medi-1的这些独特特性，同时克服该单克隆抗体的局限性——例如因受体多态性导致CD16与Medi-1的结合亲和力存在差异，以及广泛阻断ADAM17活性的风险——我们构建了靶向ADAM17阻断剂CD16（TAB16）。

方法
TAB16由靶向CD16的骆驼科重链可变域与源自Medi-1的单链可变片段连接构建而成。我们进一步将IL-15组分与TAB16连接，得到修饰后的TAB16/15。将原代人NK细胞用TAB16或TAB16/15处理后，通过细胞增殖染料稀释法评估其增殖能力，同时检测ADAM17阻断效果、激活标志物表达水平，并采用IncuCyte检测技术实时评估NK细胞对卵巢癌细胞系的细胞毒性。

结果
双特异性接合剂TAB16可靶向结合NK细胞并阻断ADAM17活性。TAB16的一项全新特性是其双重功能：它可与IL-15协同增强NK细胞的激活与增殖，同时靶向癌细胞上过表达的ADAM17以诱导ADCC。此外，TAB16属于可修饰骨架，可添加额外功能组分，例如用于实现整合型多维度活性的IL-15（即TAB16/15）。

讨论
本ADAM17靶向结合平台为实现靶向ADAM17抑制提供了一种独特策略，可增强内源性及治疗性NK细胞的抗肿瘤功能。