N-糖基化是一种翻译后修饰，对于单克隆抗体 (mAb) 的开发非常重要，因为它调节单克隆抗体的生物活性，特别是在免疫效应子功能方面。虽然通常在 Fc 水平添加，但大约 15-25% 的循环抗体也在 Fab 结构域中表现出糖基化。据我们所知，西妥昔单抗（爱必妥®）是全球范围内唯一获得批准的针对表皮生长因子受体的具有 Fab 糖基化的治疗性抗体，用于治疗转移性结直肠癌和头颈癌。此外，它还可以触发抗体依赖性细胞毒性 (ADCC)，这种反应通常受到 Fc 水平 N-糖基化的影响。然而，Fab 糖基化在西妥昔单抗中的作用仍知之甚少。因此，本研究旨在研究 Fab 糖基化对西妥昔单抗构象行为的结构作用。该研究是通过加速分子动力学模拟在计算机上进行的。将商业西妥昔单抗与其不带 Fab 糖基化的形式进行比较，并评估结构描述符以确定构象差异。结果清楚地显示 Fab 糖基化和结构描述符之间的相关性，可能调节抗体的构象自由度，可能影响 Fc 效应子功能，并表明 Fab 糖基化对与 FcγRIIIa 的相互作用具有负面作用。西妥昔单抗的 Fab 糖基化是生物仿制药开发中最关键的挑战，但这项工作中强调的关于其非糖基化形式的差异可以提高知识并代表一个巨大的机会开发新的生物治疗策略。版权所有 © 2024 Saporiti、Bianchi、Ben Mariem、Rossi、Guerrini、Eberini 和 Centola。

N-glycosylation is a post-translational modification that is highly important for the development of monoclonal antibodies (mAbs), as it regulates their biological activity, particularly in terms of immune effector functions. While typically added at the Fc level, approximately 15-25% of circulating antibodies exhibit glycosylation in the Fab domains as well. To the best of our knowledge, cetuximab (Erbitux®) is the only therapeutic antibody presenting Fab glycosylation approved world-wide targeting the epidermal growth factor receptor for the treatment of metastatic-colorectal and head and neck cancers. Additionally, it can trigger antibody-dependent cell cytotoxicity (ADCC), a response that typically is influenced by N-glycosylation at Fc level. However, the role of Fab glycosylation in cetuximab remains poorly understood. Hence, this study aims to investigate the structural role of Fab glycosylation on the conformational behavior of cetuximab.The study was performed in silico via accelerated molecular dynamics simulations. The commercial cetuximab was compared to its form without Fab glycosylation and structural descriptors were evaluated to establish conformational differences.The results clearly show a correlation between the Fab glycosylation and structural descriptors that may modulate the conformational freedom of the antibody, potentially affecting Fc effector functions, and suggesting a negative role of Fab glycosylation on the interaction with FcγRIIIa.Fab glycosylation of cetuximab is the most critical challenge for biosimilar development, but the differences highlighted in this work with respect to its aglycosylated form can improve the knowledge and represent also a great opportunity to develop novel strategies of biotherapeutics.Copyright © 2024 Saporiti, Bianchi, Ben Mariem, Rossi, Guerrini, Eberini and Centola.