Mucin-16 (MUC16) is a target for antibody-mediated immunotherapy in pancreatic ductal adenocarcinoma (PDAC) amongst other malignancies. The MUC16 specific monoclonal antibody AR9.6 has shown promise for PDAC immunotherapy and imaging. Here, we report the structural and biological characterization of the humanized AR9.6 antibody (huAR9.6). The structure of huAR9.6 was determined in complex with a MUC16 SEA (Sea urchin sperm, Enterokinase, Agrin) domain. Binding of huAR9.6 to recombinant, shed, and cell-surface MUC16 was characterized, and anti-PDAC activity was evaluated in vitro and in vivo. huAR9.6 bound a discontinuous, SEA domain epitope with an affinity of ~90 nM. Binding affinity depended on the specific SEA domain(s) present, and glycosylation enhanced affinity by 3-7-fold driven by favorable entropy and enthalpy and via distinct transition state thermodynamic pathways. Treatment with huAR9.6 reduced the in vitro growth, migration, invasion, and clonogenicity of MUC16-positive PDAC cells and patient-derived organoids (PDOs). HuAR9.6 blocked MUC16-mediated ErbB and AKT activation in PDAC cells, PDOs and patient-derived xenografts and induced antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. More importantly, huAR9.6 treatment caused substantial PDAC regression in subcutaneous and orthotopic tumor models. The mechanism of action of huAR9.6 may depend on dense avid binding to homologous SEA domains on MUC16. The results of this study validate the translational therapeutic potential of huAR9.6 against MUC16-positive PDACs.

中文翻译：

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人源化抗体 AR9.6 多价 MUC16 识别和强大抗胰腺癌活性的结构基础

Mucin-16 (MUC16) 是胰腺导管腺癌 (PDAC) 及其他恶性肿瘤中抗体介导的免疫治疗的靶点。MUC16 特异性单克隆抗体 AR9.6 在 PDAC 免疫治疗和成像方面显示出前景。在这里，我们报告了人源化 AR9.6 抗体 (huAR9.6) 的结构和生物学特征。huAR9.6 的结构是在与 MUC16 SEA（海胆精子、肠激酶、Agrin）结构域的复合物中确定的。对 huAR9.6 与重组、脱落和细胞表面 MUC16 的结合进行了表征，并在体外和体内评估了抗 PDAC 活性。huAR9.6 结合不连续的 SEA 结构域表位，亲和力约为 90 nM。结合亲和力取决于存在的特定 SEA 结构域，并且糖基化在有利的熵和焓的驱动下并通过不同的过渡态热力学途径将亲和力增强了 3-7 倍。huAR9.6 治疗降低了 MUC16 阳性 PDAC 细胞和患者来源的类器官 (PDO) 的体外生长、迁移、侵袭和克隆形成。HuAR9.6 阻断 PDAC 细胞、PDO 和患者来源的异种移植物中 MUC16 介导的 ErbB 和 AKT 激活，并诱导抗体依赖性细胞毒性和补体依赖性细胞毒性。更重要的是，huAR9.6治疗导致皮下和原位肿瘤模型中PDAC显着消退。huAR9.6 的作用机制可能取决于与 MUC16 上同源 SEA 结构域的密集结合。本研究的结果验证了 huAR9.6 针对 MUC16 阳性 PDAC 的转化治疗潜力。