Site-Selective Chemoenzymatic Modification on the Core Fucose of an Antibody Enhances Its Fcγ Receptor Affinity and ADCC Activity.

Fc glycosylation profoundly impacts the effector functions of antibodies and often dictates an antibody's pro- or anti-inflammatory activities. It is well established that core fucosylation of the Fc domain N-glycans of an antibody significantly reduces its affinity for FcγRIIIa receptors and antibody-dependent cellular cytotoxicity (ADCC). Previous structural studies have suggested that the presence of a core fucose remarkably decreases the unique and favorable carbohydrate-carbohydrate interactions between the Fc and the receptor N-glycans, leading to reduced affinity. We report here that in contrast to natural core fucose, special site-specific modification on the core fucose could dramatically enhance the affinity of an antibody for FcγRIIIa. The site-selective modification was achieved through an enzymatic transfucosylation with a novel fucosidase mutant, which was shown to be able to use modified α-fucosyl fluoride as the donor substrate. We found that replacement of the core l-fucose with 6-azide- or 6-hydroxy-l-fucose (l-galactose) significantly enhanced the antibody's affinity for FcγRIIIa receptors and substantially increased the ADCC activity. To understand the mechanism of the modified fucose-mediated affinity enhancement, we performed molecular dynamics simulations. Our data revealed that the number of glycan contacts between the Fc and the Fc receptor was increased by the selective core-fucose modifications, showing the importance of unique carbohydrate-carbohydrate interactions in achieving high FcγRIIIa affinity and ADCC activity of antibodies. Thus, the direct site-selective modification turns the adverse effect of the core fucose into a favorable force to promote the carbohydrate-carbohydrate interactions.