a Department of Chemical Engineering , Polytechnique Montréal , Montréal , Québec , Canada.
b Human Health Therapeutics Research Center , National Research Council Canada , Montréal , Québec , Canada.
MAbs. 2019 Apr;11(3):435-452. doi: 10.1080/19420862.2019.1581017. Epub 2019 Mar 5.
The N-glycosylation profile of immunoglobulin G (IgG) is considered a critical quality attribute due to its impact on IgG-Fc gamma receptor (FcγR) interactions, which subsequently affect antibody-dependent cell-based immune responses. In this study, we investigated the impact of the FcγR capture method, as well as FcγR N-glycosylation, on the kinetics of interaction with various glycoforms of trastuzumab (TZM) in a surface plasmon resonance (SPR) biosensor assay. More specifically, we developed a novel strategy based on coiled-coil interactions for the stable and oriented capture of coil-tagged FcγRs at the biosensor surface. Coil-tagged FcγR capture outperformed all other capture strategies applied to the SPR study of IgG-FcγR interactions, as the robustness and reproducibility of the assay and the shelf life of the biosensor chip were excellent (> 1,000 IgG injections with the same biosensor surface). Coil-tagged FcγRs displaying different N-glycosylation profiles were generated either by different expression systems, in vitro glycoengineering or by size-exclusion chromatography, and roughly characterized by lectin blotting. Of salient interest, the overlay of their kinetics of interaction with several TZM glycoforms revealed key differences on both association and dissociation kinetics, confirming a complex influence of the FcγR N-glycosylation and its inherent heterogeneity upon receptor interaction with mAbs. This work is thus an important step towards better understanding of the impact of glycosylation upon binding of IgGs, either natural or engineered, to their receptors.
免疫球蛋白 G（IgG）的 N-糖基化谱被认为是一个关键的质量属性，因为它会影响 IgG-Fcγ 受体（FcγR）的相互作用，进而影响抗体依赖的细胞免疫反应。在这项研究中，我们研究了 FcγR 捕获方法以及 FcγR N-糖基化对表面等离子体共振（SPR）生物传感器分析中各种曲妥珠单抗（TZM）糖型与 IgG-FcγR 相互作用动力学的影响。更具体地说，我们开发了一种基于卷曲螺旋相互作用的新策略，用于在生物传感器表面稳定且定向地捕获带有卷曲螺旋标签的 FcγR。与应用于 IgG-FcγR 相互作用的 SPR 研究的所有其他捕获策略相比，带有卷曲螺旋标签的 FcγR 捕获具有更好的稳健性和重现性，并且该测定法的保质期和生物传感器芯片的保质期都非常长（>1000 次 IgG 注射，使用相同的生物传感器表面）。通过不同的表达系统、体外糖基工程或分子筛层析法生成了具有不同 N-糖基化谱的带有卷曲螺旋标签的 FcγR，并通过凝集素印迹进行了大致表征。特别有趣的是，将它们与几种 TZM 糖型相互作用的动力学叠加，揭示了在结合和解离动力学方面的关键差异，证实了 FcγR N-糖基化及其固有异质性对受体与 mAb 相互作用的复杂影响。因此，这项工作是朝着更好地理解糖基化对天然或工程 IgG 与其受体结合的影响迈出的重要一步。
