Importance of the Side Chain at Position 296 of Antibody Fc in Interactions with FcγRIIIa and Other Fcγ Receptors

Abstract

Antibody-dependent cellular cytotoxicity (ADCC) is an important effector function determining the clinical efficacy of therapeutic antibodies. Core fucose removal from N-glycans on the Fc portion of immunoglobulin G (IgG) improves the binding affinity for Fcγ receptor IIIa (FcγRIIIa) and dramatically enhances ADCC. Our previous structural analyses revealed that Tyr–296 of IgG1-Fc plays a critical role in the interaction with FcγRIIIa, particularly in the enhanced FcγRIIIa binding of nonfucosylated IgG1. However, the importance of the Tyr–296 residue in the antibody in the interaction with various Fcγ receptors has not yet been elucidated. To further clarify the biological importance of this residue, we established comprehensive Tyr–296 mutants as fucosylated and nonfucosylated anti-CD20 IgG1s rituximab variants and examined their binding to recombinant soluble human Fcγ receptors: shFcγRI, shFcγRIIa, shFcγRIIIa, and shFcγRIIIb. Some of the mutations affected the binding of antibody to not only shFcγRIIIa but also shFcγRIIa and shFcγRIIIb, suggesting that the Tyr–296 residue in the antibody was also involved in interactions with FcγRIIa and FcγRIIIb. For FcγRIIIa binding, almost all Tyr–296 variants showed lower binding affinities than the wild-type antibody, irrespective of their core fucosylation, particularly in Y296K and Y296P. Notably, only the Y296W mutant showed improved binding to FcγRIIIa. The 3.00 Å-resolution crystal structure of the nonfucosylated Y296W mutant in complex with shFcγRIIIa harboring two N-glycans revealed that the Tyr-to-Trp substitution increased the number of potential contact atoms in the complex, thus improving the binding of the antibody to shFcγRIIIa. The nonfucosylated Y296W mutant retained high ADCC activity, relative to the nonfucosylated wild-type IgG1, and showed greater binding affinity for FcγRIIa. Our data may improve our understanding of the biological importance of human IgG1-Fc Tyr–296 in interactions with various Fcγ receptors, and have applications in the modulation of the IgG1-Fc function of therapeutic antibodies.

Conformational effects of N-glycan core fucosylation of immunoglobulin G Fc region on its interaction with Fcγ receptor IIIa

“…1b ). This is also in marked contrast to the observation of the same sFcγRIIIa in complex with the nonfucosylated Fc glycoform, in which the Asn162 glycan is extensively involved in interactions with the Fc glycan 13 – 15 . Because the sFcγRIIIa glycoprotein used in the crystallization had a homogeneous glycoform exclusively exhibiting the bi-antennary complex-type oligosaccharide Gal 2 GlcNAc 2 Man 3 GlcNAc 2 (Fuc), the ambiguous electron density of the Asn162 glycan of sFcγRIIIa indicates its conformational disorder, presumably because the intermolecular carbohydrate-carbohydrate interaction was weakened in the presence of the core fucose residue of the Fc glycan 4 , 6 – 12 .…”

“…In the non-fucosylated system, this innermost residue of the Fc chain A glycan is in close proximity to the innermost part, i.e., GlcNAc1 and GlcNAc2, of the Asn162 glycan. The previous crystallographic data indicated that the Asn162 glycan of sFcγRIIIa is involved in intermolecular interactions with the IgG1-Fc glycan in its nonfucosylated form 13 – 15 . The crystal structure, in which the core and the Man α1-3 arm of the Asn162 glycan of sFcγRIIIa are in contact with the Fc glycan, corresponds in terms of these distances to the most highly populated state.…”

“…This is consistent with the previously reported crystal structure formed between the fucosylated IgG1-Fc glycoform and sFcγRIIIa exclusively expressing high-mannose-type oligosaccharides, which gave no interpretable electron densities except for the reducing terminal GlcNAc1 and GlcNAc2 at Ans45 and GlcNAc1, GlcNAc2, Man3, Man4, and Man4 ′ at Asn162 13 . We also solved a 2.50-Å resolution crystal structure of the complex of sFcγRIIIa with a fucosylated IgG1-Fc with a Tyr-to-Trp mutation at position 296, which caused a two-fold increase in sFcγRIIIa-binding affinity, i.e., K D = 1.3 × 10 −7 (M) 15 . In this crystal structure, electron density of the Asn162 glycan was again largely unobservable.…”

“…Crystallographic studies have indicated that binding of IgG1-Fc to FcγRIIIa is mediated not only by protein-protein interactions but also by carbohydrate-protein and carbohydrate-carbohydrate interactions 13 – 15 . In this binding mode, one of two Fc glycans extensively contacts an N -glycan at Asn162 of FcγRIIIa, suggesting that core fucosylation causes steric hindrance against intermolecular carbohydrate-carbohydrate interactions and thereby negatively affects FcγRIIIa binding.…”

Engineering a human IgG2 antibody stable at low pH

“…Anti‐DNP antibody‐binding affinity for recombinant hFcγRIIIa (hFcγRIIIa‐158V, hFcγRIIIa‐158F) and hFcRn (pH 6.0 and pH 7.4) was tested with Biacore (GE Healthcare), as previously described, using human anti‐IgG2 isotype control antibody …”