title: 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.
authors.affiliation: Research Functions Unit, R&D Division, Kyowa Hakko Kirin Co., Ltd, Asahi-machi, Machida-shi, Tokyo, Japan.
authors.forename: Yuya
authors.initials: Y
authors.lastname: Isoda
authors.affiliation: Graduate School of Pharmaceutical Sciences, Nagoya City University, Tanabe-dori, Mizuho-ku, Nagoya, Japan.
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Yagi
authors.affiliation: Graduate School of Pharmaceutical Sciences, Nagoya City University, Tanabe-dori, Mizuho-ku, Nagoya, Japan; JST, PRESTO, Tanabe-dori, Mizuho-ku, Nagoya, Japan.
authors.forename: Tadashi
authors.initials: T
authors.lastname: Satoh
authors.affiliation: Immunology & Allergy R&D Unit, R&D Division, Kyowa Hakko Kirin Co., Ltd, Asahi-machi, Machida-shi, Tokyo, Japan.
authors.forename: Mami
authors.initials: M
authors.lastname: Shibata-Koyama
authors.affiliation: Research Functions Unit, R&D Division, Kyowa Hakko Kirin Co., Ltd, Asahi-machi, Machida-shi, Tokyo, Japan.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Masuda
authors.affiliation: Immunology & Allergy R&D Unit, R&D Division, Kyowa Hakko Kirin Co., Ltd, Asahi-machi, Machida-shi, Tokyo, Japan.
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Satoh
authors.affiliation: Graduate School of Pharmaceutical Sciences, Nagoya City University, Tanabe-dori, Mizuho-ku, Nagoya, Japan; Institute for Molecular Science and Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Higashiyama, Myodaiji, Okazaki, Aichi, Japan; GLYENCE Co., Ltd., Chikusa, Chikusa-ku, Nagoya, Japan; The Glycoscience Institute, Ochanomizu University, Ohtsuka, Bunkyo-ku, Tokyo, Japan.
authors.forename: Koichi
authors.initials: K
authors.lastname: Kato
authors.affiliation: Research Functions Unit, R&D Division, Kyowa Hakko Kirin Co., Ltd, Asahi-machi, Machida-shi, Tokyo, Japan.
authors.forename: Shigeru
authors.initials: S
authors.lastname: Iida
mesh_terms: D000818:Animals; D000920:Antibody-Dependent Cell Cytotoxicity; D018951:Antigens, CD20 / Q000276:immunology; D016466:CHO Cells; D002460:Cell Line; D003412:Cricetulus; D018360:Crystallography, X-Ray; D005643:Fucose / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D008958:Models, Molecular; D017354:Point Mutation; D017452:Receptors, IgG / Q000276:immunology*; D000069283:Rituximab / Q000737:chemistry / Q000235:genetics / Q000276:immunology
chemical_list: D018951:Antigens, CD20; C496124:FCGR3A protein, human; D007141:Immunoglobulin Fc Fragments; D017452:Receptors, IgG; D005643:Fucose; D000069283:Rituximab