Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands.
MAbs. 2013 Mar-Apr;5(2):219-28. doi: 10.4161/mabs.23532. Epub 2013 Feb 13.
Antibody engineering is increasingly being used to influence the properties of monoclonal antibodies to improve their biotherapeutic potential. One important aspect of this is the modulation of glycosylation as a strategy to improve efficacy. Here, we describe mutations of Y407 in the CH3 domain of IgG1 and IgG4 that significantly increase sialylation, galactosylation, and branching of the N-linked glycans in the CH2 domain. These mutations also promote the formation of monomeric assemblies (one heavy-light chain pair). Hydrogen-deuterium exchange mass spectrometry was used to probe conformational changes in IgG1-Y407E, revealing, as expected, a more exposed CH3-CH3 dimerization interface. Additionally, allosteric structural effects in the CH2 domain and in the CH2-CH3 interface were identified, providing a possible explanation for the dramatic change in glycosylation. Thus, the mutation of Y407 in the CH3 domain remarkably affects both antibody conformation and glycosylation, which not only alters our understanding of antibody structure, but also reveals possibilities for obtaining recombinant IgG with glycosylation tailored for clinical applications.
抗体工程越来越多地被用于影响单克隆抗体的性质，以提高其治疗潜力。其中一个重要方面是通过调节糖基化作为提高疗效的策略。在这里，我们描述了 IgG1 和 IgG4 的 CH3 结构域中 Y407 突变，这些突变显著增加了 CH2 结构域中 N 连接糖基化的唾液酸化、半乳糖基化和分支化。这些突变还促进了单体组装（一对重链-轻链）的形成。氢氘交换质谱用于探测 IgG1-Y407E 的构象变化，正如预期的那样，暴露出更暴露的 CH3-CH3 二聚化界面。此外，还确定了 CH2 结构域和 CH2-CH3 界面的变构结构效应，为糖基化的巨大变化提供了可能的解释。因此，CH3 结构域中 Y407 的突变显著影响抗体的构象和糖基化，这不仅改变了我们对抗体结构的理解，而且还揭示了获得针对临床应用定制糖基化的重组 IgG 的可能性。
