Optimal combination of beneficial mutations for improved ADCC effector function of aglycosylated antibodies

• Published in: Molecular immunology Vol. 114; pp. 62 - 71
• Main Authors: Yoon, Hyun Woung, Jo, Migyeong, Ko, Sanghwan, Kwon, Hyeong Sun, Lim, Chung Su, Ko, Byoung Joon, Lee, Ji Chul, Jung, Sang Taek
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.10.2019
• Subjects: Aglycosylated IgG; Antibodies, Monoclonal - immunology; Antibody-Dependent Cell Cytotoxicity - immunology; Antibody-dependent cell-mediated cytotoxicity; Cell Line; Effector functions; Fc engineering; FcγRIIIa; Glycosylation; HEK293 Cells; Humans; Immunoglobulin Fc Fragments - immunology; Immunoglobulin G - immunology; Mutation - immunology; Protein Binding - immunology; Protein Engineering; Receptors, IgG - immunology; GlycoT; MHC; FcγRIIIa; Aglycosylated IgG; PBMC; Fc engineering; Effector functions; FcRn; ADCC; Fc; FcγR; HEK293F; Antibody-dependent cell-mediated cytotoxicity
• ISSN: 0161-5890; 1872-9142
• DOI: 10.1016/j.molimm.2019.07.007

[Display omitted] •We constructed an aglycosylated Fc variant library containing combinations of previously identified beneficial mutations for enhanced FcγRs binding.•High-throughput library screening enabled the isolation of aglycosylated Fc variants with significantly improved FcγRIIIa binding affinity.•No mutation in the Fc variant with the greatest FcγRIIIa binding was previously found to enhance FcγRIIIa binding in glycosylated Fc variants.•The aglycosylated trastuzumab Fc variant featuring the identified combination of mutations showed higher ADCC than glycosylated trastuzumab. The Fc region of IgG antibodies is crucial for binding to Fc receptors expressed on the surfaces of various immune leukocytes and eliciting therapeutic effector functions such as clearance of antibody-opsonized tumor cells. Despite abrogated Fc gamma receptor (FcγR) binding and therapeutic effector function in the absence of N-linked glycosylation at Asn297, the aglycosylated Fc region of IgG antibodies has bioprocessing advantages such as the absence of glycan heterogeneity and simple bacterial antibody production. Therefore, these antibodies have been comprehensively engineered as effector functional units for human therapy. In this work, we constructed a huge library of Fc variants with combinations of 25 beneficial mutations that were previously identified to improve binding of glycosylated or aglycosylated Fc regions to human FcγRs in previous studies. High-throughput screening of the resulting library led to the identification of an aglycosylated Fc variant that exhibited almost double the antibody-dependent cell-mediated cytotoxicity than wild-type glycosylated Fc. All mutations in this aglycosylated Fc variant were derived from previously identified beneficial mutations for engineered aglycosylated Fc variants as opposed to glycosylated variants, suggesting that significantly different sets of beneficial mutations are necessary to improve the effector function of aglycosylated Fc.