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

PLoS One. 2015 Oct 7;10(10):e0140120. doi: 10.1371/journal.pone.0140120. eCollection 2015.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibody-Dependent Cell Cytotoxicity*
  • Antigens, CD20 / immunology
  • CHO Cells
  • Cell Line
  • Cricetulus
  • Crystallography, X-Ray
  • Fucose / chemistry
  • Fucose / genetics
  • Fucose / immunology*
  • Humans
  • Immunoglobulin Fc Fragments / chemistry
  • Immunoglobulin Fc Fragments / genetics
  • Immunoglobulin Fc Fragments / immunology*
  • Models, Molecular
  • Point Mutation
  • Receptors, IgG / immunology*
  • Rituximab / chemistry
  • Rituximab / genetics
  • Rituximab / immunology

Substances

  • Antigens, CD20
  • FCGR3A protein, human
  • Immunoglobulin Fc Fragments
  • Receptors, IgG
  • Fucose
  • Rituximab

Associated data

  • PDB/5BW7

Grants and funding

This work was supported in part by JSPS KAKENHI (Grant Numbers 25102008, 24249002 to K.K.), the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO) (to K.K.), and the Okazaki ORION project. Yuya Isoda, Mami Shibata-Koyama, Kazuhiro Masuda, Mitsuo Satoh, and Shigeru Iida are from Kyowa Hakko Kirin Co., Ltd. Koichi Kato is a scientific advisor of Medical & Biological Laboratories Co. Ltd. These funders provided support in the form of salaries for authors (Y.I., M.S., K.M., M.S., S.I., K.K.), but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of the authors are articulated in the ‘author contributions’ section.