Crystal structure of a novel asymmetrically engineered Fc variant with improved affinity for FcγRs

• Published in: Molecular immunology Vol. 58; no. 1; pp. 132 - 138
• Main Authors: Mimoto, F., Kadono, S., Katada, H., Igawa, T., Kamikawa, T., Hattori, K.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.03.2014
• Subjects: ADCC; Amino Acid Substitution - genetics; Antibodies, Monoclonal - genetics; Antibodies, Monoclonal - immunology; Antibody Affinity; Antibody-Dependent Cell Cytotoxicity - immunology; Crystallography, X-Ray; Effector function; Fc engineering; FcγR interactions; Humans; Immunoglobulin Fc Fragments - chemistry; Immunoglobulin Fc Fragments - immunology; Immunoglobulin Fc Fragments - ultrastructure; Protein Binding - genetics; Protein Binding - immunology; Protein Engineering; Receptors, IgG - immunology; Recombinant Proteins - genetics; Recombinant Proteins - immunology; X-ray structure; A/I ratio; PBMC; ADCP; Fc engineering; mAb; PDB; SPR; asym-mAb23; rms; FcγR interactions; ADCC; FcγR; Effector function; X-ray structure; peripheral blood mononuclear cells; antibody-dependent cell-mediated cytotoxicity; Fcγ receptor; activating FcγR binding to inhibitory FcγR binding; antibody-dependent cell-mediated phagocytosis; a novel asymmetrically engineered Fc; root-mean-square; surface plasmon resonance; monoclonal antibody; Protein Data Bank
• ISSN: 0161-5890; 1872-9142
• DOI: 10.1016/j.molimm.2013.11.017

•We created an asymmetric Fc with the highest affinity for FcγRIIIa and FcγRIIa.•An asymmetric Fc showed comparable ADCC with afucosylated antibody.•We solved the crystal structure of a novel asymmetrically engineered Fc.•The structural analysis reveals the unique features of the asymmetric Fc. Enhancing the effector function by optimizing the interaction between Fc and Fcγ receptor (FcγR) is a promising approach to enhance the potency of anticancer monoclonal antibodies (mAbs). To date, a variety of Fc engineering approaches to modulate the interaction have been reported, such as afucosylation in the heavy chain Fc region or symmetrically introducing amino acid substitutions into the region, and there is still room to improve FcγR binding and thermal stability of the CH2 domain with these approaches. Recently, we have reported that asymmetric Fc engineering, which introduces different substitutions into each Fc region of heavy chain, can further improve the FcγR binding while maintaining the thermal stability of the CH2 domain by fine-tuning the asymmetric interface between the Fc domain and FcγR. However, the structural mechanism by which the asymmetrically engineered Fc improved FcγR binding remained unclear. In order to elucidate the mechanism, we solved the crystal structure of a novel asymmetrically engineered Fc, asym-mAb23, in complex with FcγRIIIa. Asym-mAb23 has enhanced binding affinity for both FcγRIIIa and FcγRIIa at the highest level of previously reported Fc variants. The structural analysis reveals the features of the asymmetrically engineered Fc in comparison with symmetric Fc and how each asymmetrically introduced substitution contributes to the improved interaction between asym-mAb23 and FcγRIIIa. This crystal structure could be utilized to enable us to design a more potent asymmetric Fc.