title: Engineering an IgG Scaffold Lacking Effector Function with Optimized Developability.
abstract: IgG isotypes can differentially bind to Fcγ receptors and complement, making the selection of which isotype to pursue for development of a particular therapeutic antibody important in determining the safety and efficacy of the drug. IgG2 and IgG4 isotypes have significantly lower binding affinity to Fcγ receptors. Recent evidence suggests that the IgG2 isotype is not completely devoid of effector function, whereas the IgG4 isotype can undergo in vivo Fab arm exchange leading to bispecific antibody and off-target effects. Here an attempt was made to engineer an IgG1-based scaffold lacking effector function but with stability equivalent to that of the parent IgG1. Care was taken to ensure that both stability and lack of effector function was achieved with a minimum number of mutations. Among the Asn297mutants that result in lack of glycosylation and thus loss of effector function, we demonstrate that the N297G variant has better stability and developability compared with the N297Q or N297A variants. To further improve the stability of N297G, we introduced a novel engineered disulfide bond at a solvent inaccessible location in the CH2 domain. The resulting scaffold has stability greater than or equivalent to that of the parental IgG1 scaffold. Extensive biophysical analyses and pharmacokinetic (PK) studies in mouse, rat, and monkey further confirmed the developability of this unique scaffold, and suggest that it could be used for all Fc containing therapeutics (e.g. antibodies, bispecific antibodies, and Fc fusions) requiring lack of effector function or elimination of binding to Fcγ receptors.
authors.affiliation: From the Biologics Optimization-Therapeutic Discovery, Clinical Immunology, and Process Development, Amgen Inc., Thousand Oaks, California 91320. Electronic address: rjacobse@amgen.com.
authors.forename: Frederick W
authors.initials: FW
authors.lastname: Jacobsen
authors.affiliation: From the Biologics Optimization-Therapeutic Discovery, Clinical Immunology, and Process Development, Amgen Inc., Thousand Oaks, California 91320.
authors.forename: Riki
authors.initials: R
authors.lastname: Stevenson
authors.affiliation: From the Biologics Optimization-Therapeutic Discovery, Clinical Immunology, and Process Development, Amgen Inc., Thousand Oaks, California 91320.
authors.forename: Cynthia
authors.initials: C
authors.lastname: Li
authors.affiliation: From the Biologics Optimization-Therapeutic Discovery, Clinical Immunology, and Process Development, Amgen Inc., Thousand Oaks, California 91320.
authors.forename: Hossein
authors.initials: H
authors.lastname: Salimi-Moosavi
authors.affiliation: From the Biologics Optimization-Therapeutic Discovery, Clinical Immunology, and Process Development, Amgen Inc., Thousand Oaks, California 91320.
authors.forename: Ling
authors.initials: L
authors.lastname: Liu
authors.affiliation: From the Biologics Optimization-Therapeutic Discovery, Clinical Immunology, and Process Development, Amgen Inc., Thousand Oaks, California 91320.
authors.forename: Jie
authors.initials: J
authors.lastname: Wen
authors.affiliation: From the Biologics Optimization-Therapeutic Discovery, Clinical Immunology, and Process Development, Amgen Inc., Thousand Oaks, California 91320.
authors.forename: Quanzhou
authors.initials: Q
authors.lastname: Luo
authors.affiliation: From the Biologics Optimization-Therapeutic Discovery, Clinical Immunology, and Process Development, Amgen Inc., Thousand Oaks, California 91320.
authors.forename: Kristine
authors.initials: K
authors.lastname: Daris
authors.affiliation: From the Biologics Optimization-Therapeutic Discovery, Clinical Immunology, and Process Development, Amgen Inc., Thousand Oaks, California 91320.
authors.forename: Lynette
authors.initials: L
authors.lastname: Buck
authors.affiliation: From the Biologics Optimization-Therapeutic Discovery, Clinical Immunology, and Process Development, Amgen Inc., Thousand Oaks, California 91320.
authors.forename: Sterling
authors.initials: S
authors.lastname: Miller
authors.affiliation: From the Biologics Optimization-Therapeutic Discovery, Clinical Immunology, and Process Development, Amgen Inc., Thousand Oaks, California 91320.
authors.forename: Shu-Yin
authors.initials: SY
authors.lastname: Ho
authors.affiliation: From the Biologics Optimization-Therapeutic Discovery, Clinical Immunology, and Process Development, Amgen Inc., Thousand Oaks, California 91320.
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.affiliation: From the Biologics Optimization-Therapeutic Discovery, Clinical Immunology, and Process Development, Amgen Inc., Thousand Oaks, California 91320.
authors.forename: Qing
authors.initials: Q
authors.lastname: Chen
authors.affiliation: From the Biologics Optimization-Therapeutic Discovery, Clinical Immunology, and Process Development, Amgen Inc., Thousand Oaks, California 91320.
authors.forename: Kenneth
authors.initials: K
authors.lastname: Walker
authors.affiliation: From the Biologics Optimization-Therapeutic Discovery, Clinical Immunology, and Process Development, Amgen Inc., Thousand Oaks, California 91320.
authors.forename: Jette
authors.initials: J
authors.lastname: Wypych
authors.affiliation: From the Biologics Optimization-Therapeutic Discovery, Clinical Immunology, and Process Development, Amgen Inc., Thousand Oaks, California 91320.
authors.forename: Linda
authors.initials: L
authors.lastname: Narhi
authors.affiliation: From the Biologics Optimization-Therapeutic Discovery, Clinical Immunology, and Process Development, Amgen Inc., Thousand Oaks, California 91320. Electronic address: guna.kannan@yahoo.com.
authors.forename: Kannan
authors.initials: K
authors.lastname: Gunasekaran
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics*; D007141:Immunoglobulin Fc Fragments / Q000235:genetics*; D007074:Immunoglobulin G / Q000235:genetics*; D008252:Macaca fascicularis; D051379:Mice; D020125:Mutation, Missense; D051381:Rats
chemical_list: D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G
keywords: Fc receptor; aglycosylated IgG; antibody developability; antibody engineering; antibody-dependent cellular cytotoxicity; complement-dependent cytotoxicity; disulfide engineering; drug development; immunoglobulin G (IgG); protein stability