Enhanced Effector Functions Due to Antibody Defucosylation Depend on the Effector Cell Fcγ Receptor Profile

Abstract

Abs of the IgG isotype are glycosylated in their Fc domain at a conserved asparagine at position 297. Removal of the core fucose of this glycan greatly increases the affinity for FcγRIII, resulting in enhanced FcγRIII-mediated effector functions. Normal plasma IgG contains ∼94% fucosylated Abs, but alloantibodies against, for example, Rhesus D (RhD) and platelet Ags frequently have reduced fucosylation that enhances their pathogenicity. The increased FcγRIII-mediated effector functions have been put to use in various afucosylated therapeutic Abs in anticancer treatment. To test the functional consequences of Ab fucosylation, we produced V-gene-matched recombinant anti-RhD IgG Abs of the four different subclasses (IgG1-4) with and without core fucose (i.e., 20% fucose remaining). Binding to all human FcγR types and their functional isoforms was assessed with surface plasmon resonance. All hypofucosylated anti-RhD IgGs of all IgG subclasses indeed showed enhanced binding affinity for isolated FcγRIII isoforms, without affecting binding affinity to other FcγRs. In contrast, when testing hypofucosylated anti-RhD Abs with FcγRIIIa-expressing NK cells, a 12- and 7-fold increased erythrocyte lysis was observed with the IgG1 and IgG3, respectively, but no increase with IgG2 and IgG4 anti-RhD Abs. Notably, none of the hypofucosylated IgGs enhanced effector function of macrophages, which, in contrast to NK cells, express a complex set of FcγRs, including FcγRIIIa. Our data suggest that the beneficial effects of afucosylated biologicals for clinical use can be particularly anticipated when there is a substantial involvement of FcγRIIIa-expressing cells, such as NK cells.

Red pulp macrophages in the human spleen are a distinct cell population with a unique expression of Fc-γ receptors

“…However, we have previously shown that different antibodies and antigens showed similar results in erythrophagocytosis by monocyte-derived macrophages. 29 Our finding of FcgRIIIa being constitutively expressed on all red pulp macrophages is in line with several older publications showing FcgRIII in the red pulp of the spleen, but is in direct contrast with an immunohistochemistry study by Wu et al that proposed that FcgRIII is only expressed on splenic macrophages in a minority of individuals. 30 However, we consistently observed FcgRIIIa on red pulp macrophages of 82 individuals, as tested by immunofluorescence of spleen sections and flow cytometry of freshly isolated single-cell suspensions.…”

“…Our data suggest that, despite differences in FcgR expression levels, monocyte-derived macrophages are an equally suitable model as isolated red pulp macrophages when studying the in vitro effects of IVIg on macrophage phagocytosis. 8,29 Concluding, we describe the splenic architecture and FcgR expression on splenic macrophages both in situ and with a novel method for the specific isolation of splenic red pulp macrophages. These red pulp macrophages have an expression profile that is highly distinct from monocytes and monocytederived macrophages.…”

Antigen binding allosterically promotes Fc receptor recognition

“…25,26 While glycan truncation may affect antibody stability, 27 defucosylation may enhance effector functions. 28 N-Glycan optimization can also be used to maximize ADCC. 29 Most intriguing, glycan may also regulate antigen recognition, and it has been reported that core fucosylation of IgG B cell receptor is required for antigen recognition and antibody production.…”

Unique patterns of glycosylation in immunoglobulin subclass G4‐related disease and primary sclerosing cholangitis

“…We observed increased IgG4‐specific Fc fucosylation in both IgG4‐RD and PSC. Non‐fucosylated IgG1 (and IgG4) antibodies bind FcγRIIIa with a 20–30‐fold higher affinity than fucosylated antibodies, and the small increase in fucosylation is in fact an almost twofold decrease in the relative amount of these non‐fucosylated antibodies . This interesting observation may suggest novel FcR binding and reduced antibody‐dependent cytotoxicity in patients with IgG4‐RD and PSC.…”