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.
Highlights
To date, more than 30 monoclonal antibodies have been approved as drugs for the treatment of cancers, chronic diseases, and autoimmune diseases, and over 500 clinical trials investigating the application of monoclonal antibodies are currently ongoing
Glycosylation of Fcγ receptors (FcγRs) is known to influence the affinities of these molecules for antibodies, and removal of core fucoses from N-linked oligosaccharides in the IgG1-Fc region can increase FcγRIIIa binding and dramatically enhance Antibody-dependent cellular cytotoxicity (ADCC) activity [12, 15,16,17,18,19,20,21]
We solved the structure of the complex formed between nonfucosylated IgG1-Fc and shFcγRIIIa with a minimal two N-glycans at Asn–45 and Asn–162 and showed that the Asn–162 N-glycan of shFcγRIIIa mediates the interaction with nonfucosylated Fc, thereby stabilizing the complex [24]
Summary
More than 30 monoclonal antibodies have been approved as drugs for the treatment of cancers, chronic diseases, and autoimmune diseases, and over 500 clinical trials investigating the application of monoclonal antibodies are currently ongoing. Recent structural analyses of the IgG1-Fc/shFcγRIIIa complex have revealed that the aromatic ring of Tyr–296 in nonfucosylated antibody is involved in interactions with N-glycans at Asn–162 and Lys–128 of FcγRIIIa through a hydrogen bond and van der Waals contacts [24, 25]. These findings demonstrate the structural importance of IgG1-Fc Tyr–296 in interactions with FcγRIIIa, for the enhanced binding of nonfucosylated antibodies to FcγRIIIa. a detailed analysis of the importance of the Tyr–296 residue of the antibody in the interactions with various Fcγ receptors has not been reported. Our findings provide new insights into the biological significance of IgG1-Fc Tyr–296 and the potential for modulation of the effector function of therapeutic antibodies
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