DataSheet_1_Role of N-Glycosylation in FcγRIIIa interaction with IgG.pdf

Immunoglobulins G (IgG) and their Fc gamma receptors (FcγRs) play important roles in our immune system. The conserved N-glycan in the Fc region of IgG1 impacts interaction of IgG with FcγRs and the resulting effector functions, which has led to the design of antibody therapeutics with greatly improved antibody-dependent cell cytotoxicity (ADCC) activities. Studies have suggested that also N-glycosylation of the FcγRIII affects receptor interactions with IgG, but detailed studies of the interaction of IgG1 and FcγRIIIa with distinct N-glycans have been hindered by the natural heterogeneity in N-glycosylation. In this study, we employed comprehensive genetic engineering of the N-glycosylation capacities in mammalian cell lines to express IgG1 and FcγRIIIa with different N-glycan structures to more generally explore the role of N-glycosylation in IgG1:FcγRIIIa binding interactions. We included FcγRIIIa variants of both the 158F and 158V allotypes and investigated the key N-glycan features that affected binding affinity. Our study confirms that afucosylated IgG1 has the highest binding affinity to oligomannose FcγRIIIa, a glycan structure commonly found on Asn162 on FcγRIIIa expressed by NK cells but not monocytes or recombinantly expressed FcγRIIIa.

免疫球蛋白G（Immunoglobulins G, IgG）及其Fcγ受体（Fc gamma receptors, FcγRs）在机体免疫系统中发挥关键作用。IgG1 Fc区的保守N-聚糖（N-glycan）可影响IgG与FcγRs的相互作用及其后续的效应功能，这一特性推动了抗体依赖性细胞介导的细胞毒性（antibody-dependent cell cytotoxicity, ADCC）活性大幅提升的治疗性抗体的研发。已有研究表明，FcγRIII的N-糖基化修饰同样会影响其与IgG的相互作用，但由于N-糖基化的天然异质性，针对IgG1与FcγRIIIa结合不同N-聚糖的相互作用的深入研究长期受到阻碍。本研究通过对哺乳动物细胞系（mammalian cell lines）的N-糖基化能力进行全面基因工程改造，成功表达出携带不同N-聚糖结构的IgG1与FcγRIIIa，从而更系统地探究N-糖基化在IgG1与FcγRIIIa结合相互作用中的调控作用。本研究纳入了158F与158V两种同种异型（allotypes）的FcγRIIIa变体，并考察了影响结合亲和力（binding affinity）的关键N-聚糖特征。实验结果证实，去岩藻糖基化（afucosylated）的IgG1与高甘露糖型（oligomannose）FcγRIIIa的结合亲和力最高；该聚糖结构常见于自然杀伤细胞（NK cells）表达的FcγRIIIa的Asn162位点，但单核细胞以及重组表达的FcγRIIIa并不携带此类聚糖。