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 Cγ2 domains. The presence of this glycan contributes to the structural organization of the Fc domain and determines its specificity for Fcγ 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 Cγ2 domain, making both protein-protein and protein-carbohydrate contacts and overlapping the binding site for Fcγ 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-FcγRIIIa interactions and rescue mice in a model of dengue virus infection.

Understanding antibody-dependent enhancement in dengue: Are afucosylated IgG1s a concern?

“…Using in vitro monocytic cell line, afucosylated DENV immune complexes engaged FcγRIIIa to increase immunoreceptor tyrosine-based afucosylated IgG motif (ITAM) signalling that enhanced infection [ 35 ]. In vivo studies revealed that afucosylated IgG1–FcγRIIIa interacts with splenic macrophages that promoted viral replication and anti-afucosylated IgG1 nanobodies protected against ADE pathogenicity in mice [ 36 , 37 ]. In SARS-CoV-2 infection, the uptake of afucosylated immune complexes by mononuclear cells was shown to trigger pro-inflammatory cytokine production such as TNF, IL-1β, and IL-6 [ 23 ].…”