Mechanism of glycoform specificity and protection against antibody dependent enhancement by an anti-afucosylated IgG nanobody

Abstract

Immunoglobulin G (IgG) antibodies contain a single, complex N-glycan on each IgG heavy chain protomer embedded in the hydrophobic pocket between its C{gamma}2 domains. The presence of this glycan contributes to the structural organization of the Fc domain and determines its specificity for Fc{gamma} receptors, thereby determining distinct cellular responses. On the Fc, the variable construction of this glycan structure leads to a family of highly-related, but non-equivalent glycoproteins known as glycoforms. We previously reported the development of synthetic nanobodies that distinguish IgG glycoforms without cross-reactivity to off-target glycoproteins or free glycans. Here, we present the X-ray crystal structure of one such nanobody, X0, in complex with its specific binding partner, the Fc fragment of afucosylated IgG1. Two X0 nanobodies bind a single afucosylated Fc homodimer at the upper C{gamma}2 domain, making both protein-protein and protein-carbohydrate contacts and overlapping the binding site for Fc{gamma} receptors. Upon binding, the elongated CDR3 loop of X0 undergoes a conformational shift to access the buried N-glycan and acts as a glycan sensor, forming hydrogen bonds with the afucosylated IgG N-glycan that would otherwise be sterically hindered by the presence of a core fucose residue. Based on this structure, we designed X0 fusion constructs that disrupt pathogenic afucosylated IgG1-Fc{gamma}RIIIa interactions and rescue mice in a model of dengue virus infection.