title: An "Fc-Silenced" IgG1 Format With Extended Half-Life Designed for Improved Stability.
abstract: Multiple mutation combinations in the IgG Fc have been characterized to tailor immune effector function or IgG serum persistence to fit desired biological outcomes for monoclonal antibody therapeutics. An unintended consequence of introducing mutations in the Fc (particularly the CH2 domain) can be a reduction in biophysical stability which can correlate with increased aggregation propensity, poor manufacturability, and lower solubility. Herein, we characterize the changes in IgG conformational and colloidal stability when 2 sets of CH2 mutations "TM" (L234F/L235E/P331S) and "YTE" (M252Y/S254T/T256E) are combined to generate an antibody format lacking immune receptor binding and exhibiting extended half-life. In addition to significantly lowered thermostability, we observe greater conformational flexibility for TM-YTE in CH2, increased self-association, and poorer solubility and aggregation profiles. To improve these properties, we dissected the contributions of individual mutations within TM-YTE on thermostability and substituted destabilizing mutations with new mutations that raise thermostability. One novel combination, FQQ-YTE (L234F/L235Q/K322Q/M252Y/S254T/T256E), had significantly improved conformational and colloidal stability, and was found to retain the same biological activities as TM-YTE (extended half-life and lack of antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity activity). Our engineering approach offers a way to improve the developability of antibodies containing Fc mutations while retaining tailored biological activity.
authors.affiliation: Department of Antibody Discovery and Protein Engineering, MedImmune LLC, Gaithersburg, Maryland 20878.
authors.forename: M Jack
authors.initials: MJ
authors.lastname: Borrok
authors.affiliation: Department of Formulation Sciences, MedImmune LLC, Gaithersburg, Maryland 20878.
authors.forename: Neil
authors.initials: N
authors.lastname: Mody
authors.affiliation: Department of Analytical Biotechnology, MedImmune LLC, Gaithersburg, Maryland 20878.
authors.forename: Xiaojun
authors.initials: X
authors.lastname: Lu
authors.affiliation: Department of Analytical Biotechnology, MedImmune LLC, Gaithersburg, Maryland 20878.
authors.forename: Megan L
authors.initials: ML
authors.lastname: Kuhn
authors.affiliation: Department of Antibody Discovery and Protein Engineering, MedImmune LLC, Gaithersburg, Maryland 20878.
authors.forename: Herren
authors.initials: H
authors.lastname: Wu
authors.affiliation: Department of Antibody Discovery and Protein Engineering, MedImmune LLC, Gaithersburg, Maryland 20878.
authors.forename: William F
authors.initials: WF
authors.lastname: Dall'Acqua
authors.affiliation: Department of Antibody Discovery and Protein Engineering, MedImmune LLC, Gaithersburg, Maryland 20878. Electronic address: tsuip@medimmune.com.
authors.forename: Ping
authors.initials: P
authors.lastname: Tsui
mesh_terms: D000818:Animals; D020868:Gene Silencing; D057809:HEK293 Cells; D006207:Half-Life; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry* / Q000235:genetics*; D007074:Immunoglobulin G / Q000737:chemistry* / Q000235:genetics*; D057507:Mice, 129 Strain; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009154:Mutation / Q000235:genetics; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017452:Receptors, IgG / Q000737:chemistry / Q000235:genetics
chemical_list: C080568:Fc gamma receptor IIA; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D017452:Receptors, IgG
keywords: C(H)2; Fc-silenced; IgG antibody; benign blocker; biotechnology; calorimetry (DSC); developability; immunology; light scattering (dynamic); pharmacokinetics; protein aggregation; thermal analysis