Stabilizing the CH2 Domain of an Antibody by Engineering in an Enhanced Aromatic Sequon,ACS Chemical Biology

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Stabilizing the CH2 Domain of an Antibody by Engineering in an Enhanced Aromatic Sequon
ACS Chemical Biology ( IF 3.5 ) Pub Date : 2016-04-29 00:00:00 , DOI: 10.1021/acschembio.5b01035
Wentao Chen _{1,

2} , Leopold Kong ₃ , Stephen Connelly ₃ , Julia M. Dendle _{1,

2} , Yu Liu _{1,

2} , Ian A. Wilson _{3,

4} , Evan T. Powers ₂ , Jeffery W. Kelly _{1,

2,

4}

Affiliation

Monoclonal antibodies (mAbs) exhibiting highly selective binding to a protein target constitute a large and growing proportion of the therapeutics market. Aggregation of mAbs results in the loss of their therapeutic efficacy and can result in deleterious immune responses. The C_H2 domain comprising part of the Fc portion of Immunoglobulin G (IgG) is typically the least stable domain in IgG-type antibodies and therefore influences their aggregation propensity. We stabilized the C_H2 domain by engineering an enhanced aromatic sequon (EAS) into the N-glycosylated C′E loop and observed a 4.8 °C increase in the melting temperature of the purified IgG1 Fc fragment. This EAS-stabilized C_H2 domain also conferred enhanced stability against thermal and low pH induced aggregation in the context of a full-length monoclonal IgG1 antibody. The crystal structure of the EAS-stabilized (Q295F/Y296A) IgG1 Fc fragment confirms the design principle, i.e., the importance of the GlcNAc1•F295 interaction, and surprisingly reveals that the core fucose attached to GlcNAc1 also engages in an interaction with F295. Inhibition of core fucosylation confirms the contribution of the fucose–Phe interaction to the stabilization. The Q295F/Y296A mutations also modulate the binding affinity of the full-length antibody to Fc receptors by decreasing the binding to low affinity Fc gamma receptors (FcγRIIa, FcγRIIIa, and FcγRIIIb), while maintaining wild-type binding affinity to FcRn and FcγRI. Our results demonstrate that engineering an EAS into the N-glycosylated reverse turn on the C′E loop leads to stabilizing N-glycan–protein interactions in antibodies and that this modification modulates antibody–Fc receptor binding.

中文翻译：

通过在增强的芳香族序列中工程化来稳定抗体的C _H 2结构域

表现出与蛋白质靶标的高选择性结合的单克隆抗体（mAb）在治疗市场中占很大的比例，并且在不断增长。mAb的聚集导致其治疗功效的丧失，并可能导致有害的免疫反应。包含免疫球蛋白G（IgG）Fc部分的C _H 2结构域通常是IgG型抗体中最不稳定的结构域，因此会影响其聚集倾向。我们通过将增强的芳香族序列（EAS）工程化到N-糖基化的C'E环中来稳定C _H 2域，并观察到纯化的IgG1 Fc片段的解链温度增加了4.8°C。此EAS稳定的C _H在全长单克隆IgG1抗体的情况下，2结构域还赋予增强的针对热和低pH诱导的聚集的稳定性。EAS稳定的（Q295F / Y296A）IgG1 Fc片段的晶体结构证实了设计原理，即GlcNAc1•F295相互作用的重要性，并且令人惊讶地揭示，连接至GlcNAc1的核心岩藻糖也参与了与F295的相互作用。核心岩藻糖基化的抑制作用证实了岩藻糖-Phe相互作用对稳定作用的贡献。Q295F / Y296A突变还通过降低与低亲和力Fcγ受体（FcγRIIa，FcγRIIIa和FcγRIIIb）的结合来调节全长抗体与Fc受体的结合亲和力，同时保持对FcRn和FcγRI的野生型结合亲和力。

更新日期：2016-04-29

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