title: Effector-attenuating Substitutions That Maintain Antibody Stability and Reduce Toxicity in Mice.
abstract: The antibody Fc region regulates antibody cytotoxic activities and serum half-life. In a therapeutic context, however, the cytotoxic effector function of an antibody is often not desirable and can create safety liabilities by activating native host immune defenses against cells expressing the receptor antigens. Several amino acid changes in the Fc region have been reported to silence or reduce the effector function of antibodies. These earlier studies focused primarily on the interaction of human antibodies with human Fc-γ receptors, and it remains largely unknown how such changes to Fc might translate to the context of a murine antibody. We demonstrate that the commonly used N297G (NG) and D265A, N297G (DANG) variants that are efficacious in attenuating effector function in primates retain potent complement activation capacity in mice, leading to safety liabilities in murine studies. In contrast, we found an L234A, L235A, P329G (LALA-PG) variant that eliminates complement binding and fixation as well as Fc-γ-dependent, antibody-dependent, cell-mediated cytotoxity in both murine IgG2a and human IgG1. These LALA-PG substitutions allow a more accurate translation of results generated with an "effectorless" antibody between mice and primates. Further, we show that both human and murine antibodies containing the LALA-PG variant have typical pharmacokinetics in rodents and retain thermostability, enabling efficient knobs-into-holes bispecific antibody production and a robust path to generating highly effector-attenuated bispecific antibodies for preclinical studies.
authors.affiliation: From the Departments of Protein Chemistry.
authors.forename: Megan
authors.initials: M
authors.lastname: Lo
authors.affiliation: Antibody Engineering.
authors.forename: Hok Seon
authors.initials: HS
authors.lastname: Kim
authors.affiliation: From the Departments of Protein Chemistry.
authors.forename: Raymond K
authors.initials: RK
authors.lastname: Tong
authors.affiliation: From the Departments of Protein Chemistry.
authors.forename: Travis W
authors.initials: TW
authors.lastname: Bainbridge
authors.affiliation: Biochemical and Cellular Pharmacology.
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Vernes
authors.affiliation: Antibody Engineering.
authors.forename: Yin
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: BioAnalytical Sciences.
authors.forename: Yuwen Linda
authors.initials: YL
authors.lastname: Lin
authors.affiliation: BioAnalytical Sciences.
authors.forename: Shan
authors.initials: S
authors.lastname: Chung
authors.affiliation: Antibody Engineering.
authors.forename: Mark S
authors.initials: MS
authors.lastname: Dennis
authors.affiliation: Neuroscience, and.
authors.forename: Y Joy Yu
authors.initials: YJ
authors.lastname: Zuchero
authors.affiliation: Neuroscience, and.
authors.forename: Ryan J
authors.initials: RJ
authors.lastname: Watts
authors.affiliation: Neuroscience, and.|Safety Assessment, Genentech Inc., South San Francisco, California 94080.
authors.forename: Jessica A
authors.initials: JA
authors.lastname: Couch
authors.affiliation: Biochemical and Cellular Pharmacology.
authors.forename: Y Gloria
authors.initials: YG
authors.lastname: Meng
authors.affiliation: Neuroscience, and.
authors.forename: Jasvinder K
authors.initials: JK
authors.lastname: Atwal
authors.affiliation: Antibody Engineering.
authors.forename: Randall J
authors.initials: RJ
authors.lastname: Brezski
authors.affiliation: Antibody Engineering, spiess.christoph@gene.com.
authors.forename: Christoph
authors.initials: C
authors.lastname: Spiess
authors.affiliation: From the Departments of Protein Chemistry, ernst.james@gene.com.|Neuroscience, and.
authors.forename: James A
authors.initials: JA
authors.lastname: Ernst
mesh_terms: D000818:Animals; D018033:Antibodies, Bispecific / Q000276:immunology*; D000917:Antibody Formation; D000920:Antibody-Dependent Cell Cytotoxicity / Q000276:immunology; D015922:Complement C1q / Q000276:immunology; D006224:Cricetinae; D018360:Crystallography, X-Ray; D004797:Enzyme-Linked Immunosorbent Assay; D006031:Glycosylation; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000276:immunology; D007074:Immunoglobulin G / Q000737:chemistry* / Q000235:genetics; D051379:Mice; D011487:Protein Conformation; D017452:Receptors, IgG / Q000378:metabolism; D013696:Temperature
chemical_list: D018033:Antibodies, Bispecific; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D017452:Receptors, IgG; D015922:Complement C1q
keywords: Fc receptor; Fc-γ receptor; aglycosylation; antibody engineering; antibody-dependent, cell-mediated cytotoxicity (ADCC); bispecific antibody; complement; complement-dependent cytotoxicity (CDC); protein stability; recombinant protein expression