Department of Chemical Engineering, Polytechnique Montréal, Montreal, Canada; Human Health Therapeutics Research Centre, National Research Council of Canada, Montreal, Canada.
Department of Chemical Engineering, Polytechnique Montréal, Montreal, Canada.
Mol Immunol. 2020 May;121:144-158. doi: 10.1016/j.molimm.2020.03.010. Epub 2020 Mar 26.
Most of therapeutic monoclonal antibodies belong to the immunoglobulin G1 (IgG1) family; they interact with the Fcγ receptors (FcγRs) at the surface of immune cells to trigger effector functions. The IgG1-Fc N-glycans impact the interaction with FcγRs and are considered a critical quality attribute. Pioneer studies on FcγR N-glycans have unveiled an additional complexity in that the N-glycan linked on the Asn-162 of FcγRIIIa was shown to be directly involved in the strong affinity for afucosylated IgG1. The last few years have thus seen the emergence of many studies investigating the complex influence of FcγRIIIa N-glycans on the interaction with IgG1 through their glycosylation sites or their glycoprofiles. In this context, we performed site-directed mutagenesis along with glycoengineering on FcγRs (FcγRI, FcγRIIa/b and FcγRIIIa) in an effort to elucidate the impact of FcγRs N-glycans on the interaction with IgG1. Furthermore, we assessed their binding to various trastuzumab glycoforms with an enhanced surface plasmon resonance assay. The FcγRIIIa N-glycans had the highest impact on the interaction with IgG1. More specifically, the N162 glycan positively influenced the affinity (15-fold) for afucosylated IgG1 while the N45 glycan presented a negative impact (2-fold) regardless of the IgG1 glycoforms. Interestingly, only the FcγRIIIa glycoprofile had an impact on the interaction with IgG1 with a 1.5-fold affinity increase when FcγRIIIa displays high-mannose glycans. These results provide invaluable insights into the complex and strong influence of N-glycosylation upon FcγRs/IgG1 binding and are instrumental to further understand the impact of FcγRs N-glycosylation in their natural forms.
大多数治疗性单克隆抗体属于免疫球蛋白 G1（IgG1）家族；它们与免疫细胞表面的 Fcγ 受体（FcγRs）相互作用，触发效应功能。IgG1-Fc N-糖链影响与 FcγRs 的相互作用，被认为是一个关键的质量属性。FcγR N-糖链的开创性研究揭示了一个额外的复杂性，即 FcγRIIIa 上的 Asn-162 连接的 N-糖链被证明直接参与了对去岩藻糖基化 IgG1 的强亲和力。因此，在过去的几年中，出现了许多研究，通过糖基化位点或糖型研究了 FcγRIIIa N-糖链对与 IgG1 相互作用的复杂影响。在这种情况下，我们对 FcγRs（FcγRI、FcγRIIa/b 和 FcγRIIIa）进行了定点突变和糖工程改造，以阐明 FcγRs N-糖链对与 IgG1 相互作用的影响。此外，我们使用增强的表面等离子体共振测定法评估了它们与各种曲妥珠单抗糖型的结合。FcγRIIIa N-糖链对与 IgG1 的相互作用影响最大。具体来说，N162 糖链正向影响了去岩藻糖基化 IgG1 的亲和力（15 倍），而 N45 糖链无论 IgG1 糖型如何都产生了负向影响（2 倍）。有趣的是，只有当 FcγRIIIa 显示高甘露糖糖型时，FcγRIIIa 糖型才会对与 IgG1 的相互作用产生影响，亲和力增加 1.5 倍。这些结果为 N-糖基化对 FcγR/IgG1 结合的复杂而强烈的影响提供了宝贵的见解，并有助于进一步了解 FcγR N-糖基化在其自然形式下的影响。
