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首页> 外文期刊>Journal of Molecular Biology >Engineering Aglycosylated IgG Variants with Wild-Type or Improved Binding Affinity to Human Fc Gamma RIIA and Fc Gamma RIIIAs
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Engineering Aglycosylated IgG Variants with Wild-Type or Improved Binding Affinity to Human Fc Gamma RIIA and Fc Gamma RIIIAs

机译：用野生型或改善对人FCγRIIA和FCγRIIIAs的野生型或改善结合亲和力的工程糖糖化IgG变体

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The binding of human IgG1 to human Fc gamma receptors (hFc gamma Rs) is highly sensitive to the presence of a single N-linked glycosylation site at asparagine 297 of the Fc, with deglycosylation resulting in a complete loss of hFc gamma R binding. Previously, we demonstrated that aglycosylated human IgG1 Fc variants can engage the human Fc gamma RII class of the low-affinity hFc gamma Rs, demonstrating that N-linked glycosylation of the Fc is not a strict requirement for hFc gamma R engagement. In the present study, we demonstrate that aglycosylated IgG variants can be engineered to productively engage with Fc gamma RIIIA, as well as the human Fc gamma RII subset. We also assess the biophysical properties and serum half-life of the aglycosylated IgG variants to measure stability. Aglycosylated constructs N297D/S298T (DTT)-K326I/A327Y/L328G (IYG) and N297D/S298A IYG optimally drove tumor cell phagocytosis. A mathematical model of phagocytosis suggests that hFc gamma R) and hFc gamma RIIIA dimers were the main drivers of phagocytosis. In vivo tumor control of B16F10 lung metastases further confirmed the variant DTT IYG to be the best at restoring wild-type-like properties in prevention of lung metastases. While deuterium incorporation was similar across most of the protein, several peptides within the CH2 domain of DTT IYG showed differential deuterium uptake in the peptide region of the FG loop as compared to the aglycosylated N297Q. Thus, in this study, we have found an aglycosylated variant that may effectively substitute for wild-type Fc. These aglycosylated variants have the potential to allow therapeutic antibodies to be produced in virtually any expression system and still maintain effector function. (C) 2017 Elsevier Ltd. All rights reserved.
机译：人IgG1对人FCγ受体（HFCγRS）的结合对Fc的天冬酰胺297的单个N键合糖基化位点的存在非常敏感，具有脱糖基化，导致HFCγR结合的完全丧失。以前，我们证明糖糖化的人IgG1 Fc变体可以接合低亲和力HFCγrs的人FCγRII类，表明FC的N-连接糖基化不是HFCγR参与的严格要求。在本研究中，我们证明可以设计糖糖化的IgG变体以高效地与FcγRIIIA和人FCγRII子集进行高效地接合。我们还评估糖糖化IgG变体的生物物理和血清半衰期以测量稳定性。糖糖化构建体N297D / S298T（DTT）-K326I / A327Y / L328G（IYG）和N297D / S298A IYG最佳地推动肿瘤细胞吞噬作用。吞噬作用的数学模型表明，HFCγR）和HFCγRIIIA二聚体是吞噬作用的主要驱动因素。在B16F10的体内肿瘤控制中，进一步证实了变体DTT IyG在预防肺转移中恢复野生型性质。氘掺入在大多数蛋白质中相似，而DTT IYG的CH2结构域内的几种肽在FG环的肽区域中显示出差分氘摄取，与糖基化的N297Q相比。因此，在本研究中，我们发现了一种可有效替代野生型Fc的糖糖化变体。这些糖糖化变体具有允许在几乎任何表达系统中生产治疗性抗体并仍然保持效应功能。 （c）2017 Elsevier Ltd.保留所有权利。

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