Unique carbohydrate-carbohydrate interactions are required for high affinity binding between FcgammaRIII and antibodies lacking core fucose

Abstract

Antibody-mediated cellular cytotoxicity (ADCC), a key immune effector mechanism, relies on the binding of antigen-antibody complexes to Fcγ receptors expressed on immune cells. Antibodies lacking core fucosylation show a large increase in affinity for FcγRIIIa leading to an improved receptor-mediated effector function. Although afucosylated IgGs exist naturally, a next generation of recombinant therapeutic, glycoenginereed antibodies is currently being developed to exploit this finding. In this study, the crystal structures of a glycosylated Fcγ receptor complexed with either afucosylated or fucosylated Fc were determined allowing a detailed, molecular understanding of the regulatory role of Fc-oligosaccharide core fucosylation in improving ADCC. The structures reveal a unique type of interface consisting of carbohydrate-carbohydrate interactions between glycans of the receptor and the afucosylated Fc. In contrast, in the complex structure with fucosylated Fc, these contacts are weakened or nonexistent, explaining the decreased affinity for the receptor. These findings allow us to understand the higher efficacy of therapeutic antibodies lacking the core fucose and also suggest a unique mechanism by which the immune system can regulate antibody-mediated effector functions.

Fig. 1.

Surface plasmon resonance analysis of the interaction between FcγRIIIa and hIgG1 glycovariants. (A) Overlay of sensorgrams for binding of 125 nM FcγRIIIa variants to fucosylated (dotted lines) and afucosylated (continuous lines) IgG1s. The association phase is represented by a solid bar above the curves. (B) The N-linked carbohydrate moiety contains a core pentasaccharide (gray box), shared among high mannose, hybrid and complex-type oligosaccharides, and variable core fucosylation, GlcNAc bisection and/or composition of outer arms. GlcNAc, N-acetylglucosamine; Fuc, fucose; Man, mannose; kif, kifunensine.

Fig. 2.

Structure of glycosylated Fc-FcγRIIIa. (A) Top and side views of the structure of the glycosylated Fc-FcγRIIIa complex. The Fc chains are shown in blue and magenta, the receptor in cyan. The oligosaccharides are depicted as ball and stick representations. (B) View on the interaction interface between afucosylated Fc fragment and glycosylated Fc receptor. Chain A of the Fc fragment is shown in blue, the Fc receptor in cyan. Hydrogen bonds are presented as dashed lines with distance between donor and acceptor shown. (C) View on the interaction interface between fucosylated Fc fragment and glycosylated Fc receptor. Chain A of the Fc fragment is shown in magenta, the Fc receptor in dark violet. Core fucose of fucosylated Fc is highlighted in yellow.

Fig. 3.

Close-up view on the Tyr296 loop. (A) View on the region surrounding Tyr296 of the afucosylated Fc fragment (blue) in complex with glycosylated FcγRIIIa (cyan). (B) Close-up view on the loop regions around Tyr296 of the Fc fragment and Lys128 (125 in FcγRIIIb). In blue the afucosylated complex structure is shown, in magenta the fucosylated Fc complex, in yellow 1T83 (22), in gray 1E4K (4).

Fig. 4.

Overlay of view on the interaction interface between glycosylated Fc receptor and Fc fragment. Chain A of the afucosylated Fc fragment is shown in blue, its complexed Fc receptor in cyan. Chain A of the fucosylated Fc fragment is shown in magenta, with core fucose highlighted in yellow; its complexed Fc receptor is in dark violet.