title: Stabilizing the CH2 Domain of an Antibody by Engineering in an Enhanced Aromatic Sequon.
abstract: Monoclonal antibodies (mAbs) exhibiting highly selective binding to a protein target constitute a large and growing proportion of the therapeutics market. Aggregation of mAbs results in the loss of their therapeutic efficacy and can result in deleterious immune responses. The CH2 domain comprising part of the Fc portion of Immunoglobulin G (IgG) is typically the least stable domain in IgG-type antibodies and therefore influences their aggregation propensity. We stabilized the CH2 domain by engineering an enhanced aromatic sequon (EAS) into the N-glycosylated C'E loop and observed a 4.8 °C increase in the melting temperature of the purified IgG1 Fc fragment. This EAS-stabilized CH2 domain also conferred enhanced stability against thermal and low pH induced aggregation in the context of a full-length monoclonal IgG1 antibody. The crystal structure of the EAS-stabilized (Q295F/Y296A) IgG1 Fc fragment confirms the design principle, i.e., the importance of the GlcNAc1•F295 interaction, and surprisingly reveals that the core fucose attached to GlcNAc1 also engages in an interaction with F295. Inhibition of core fucosylation confirms the contribution of the fucose-Phe interaction to the stabilization. The Q295F/Y296A mutations also modulate the binding affinity of the full-length antibody to Fc receptors by decreasing the binding to low affinity Fc gamma receptors (FcγRIIa, FcγRIIIa, and FcγRIIIb), while maintaining wild-type binding affinity to FcRn and FcγRI. Our results demonstrate that engineering an EAS into the N-glycosylated reverse turn on the C'E loop leads to stabilizing N-glycan-protein interactions in antibodies and that this modification modulates antibody-Fc receptor binding.
authors.affiliation: Department of Molecular and Experimental Medicine, The Scripps Research Institute , La Jolla, California 92037, United States.|Department of Chemistry, The Scripps Research Institute , La Jolla, California 92037, United States.
authors.forename: Wentao
authors.initials: W
authors.lastname: Chen
authors.affiliation: Department of Integrative Structural and Computational Biology, The Scripps Research Institute , La Jolla, California 92037, United States.
authors.forename: Leopold
authors.initials: L
authors.lastname: Kong
authors.affiliation: Department of Integrative Structural and Computational Biology, The Scripps Research Institute , La Jolla, California 92037, United States.
authors.forename: Stephen
authors.initials: S
authors.lastname: Connelly
authors.affiliation: Department of Molecular and Experimental Medicine, The Scripps Research Institute , La Jolla, California 92037, United States.|Department of Chemistry, The Scripps Research Institute , La Jolla, California 92037, United States.
authors.forename: Julia M
authors.initials: JM
authors.lastname: Dendle
authors.affiliation: Department of Molecular and Experimental Medicine, The Scripps Research Institute , La Jolla, California 92037, United States.|Department of Chemistry, The Scripps Research Institute , La Jolla, California 92037, United States.
authors.forename: Yu
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Department of Integrative Structural and Computational Biology, The Scripps Research Institute , La Jolla, California 92037, United States.|The Skaggs Institute for Chemical Biology, The Scripps Research Institute , La Jolla, California 92037, United States.
authors.forename: Ian A
authors.initials: IA
authors.lastname: Wilson
authors.affiliation: Department of Chemistry, The Scripps Research Institute , La Jolla, California 92037, United States.
authors.forename: Evan T
authors.initials: ET
authors.lastname: Powers
authors.affiliation: Department of Molecular and Experimental Medicine, The Scripps Research Institute , La Jolla, California 92037, United States.|Department of Chemistry, The Scripps Research Institute , La Jolla, California 92037, United States.|The Skaggs Institute for Chemical Biology, The Scripps Research Institute , La Jolla, California 92037, United States.
authors.forename: Jeffery W
authors.initials: JW
authors.lastname: Kelly
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism; D002152:Calorimetry, Differential Scanning; D015202:Protein Engineering / Q000379:methods*; D055550:Protein Stability; D017452:Receptors, IgG / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D000911:Antibodies, Monoclonal; D017452:Receptors, IgG