title: Efficient production of bispecific IgG of different isotypes and species of origin in single mammalian cells.
abstract: Bispecific IgG production in single host cells has been a much sought-after goal to support the clinical development of these complex molecules. Current routes to single cell production of bispecific IgG include engineering heavy chains for heterodimerization and redesign of Fab arms for selective pairing of cognate heavy and light chains. Here, we describe novel designs to facilitate selective Fab arm assembly in conjunction with previously described knobs-into-holes mutations for preferential heavy chain heterodimerization. The top Fab designs for selective pairing, namely variants v10 and v11, support near quantitative assembly of bispecific IgG in single cells for multiple different antibody pairs as judged by high-resolution mass spectrometry. Single-cell and in vitro-assembled bispecific IgG have comparable physical, in vitro biological and in vivo pharmacokinetics properties. Efficient single-cell production of bispecific IgG was demonstrated for human IgG1, IgG2and IgG4thereby allowing the heavy chain isotype to be tailored for specific therapeutic applications. Additionally, a reverse chimeric bispecific IgG2awith humanized variable domains and mouse constant domains was generated for preclinical proof-of-concept studies in mice. Efficient production of a bispecific IgG in stably transfected mammalian (CHO) cells was shown. Individual clones with stable titer and bispecific IgG composition for >120 days were readily identified. Such long-term cell line stability is needed for commercial manufacture of bispecific IgG. The single-cell bispecific IgG designs developed here may be broadly applicable to biotechnology research, including screening bispecific IgG panels, and to support clinical development.
authors.affiliation: a Department of Antibody Engineering , Genentech, Inc. , South San Francisco , CA , USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Dillon
authors.affiliation: a Department of Antibody Engineering , Genentech, Inc. , South San Francisco , CA , USA.
authors.forename: Yiyuan
authors.initials: Y
authors.lastname: Yin
authors.affiliation: a Department of Antibody Engineering , Genentech, Inc. , South San Francisco , CA , USA.
authors.forename: Jianhui
authors.initials: J
authors.lastname: Zhou
authors.affiliation: b Department of Protein Chemistry , Genentech, Inc. , South San Francisco , CA , USA.
authors.forename: Luke
authors.initials: L
authors.lastname: McCarty
authors.affiliation: b Department of Protein Chemistry , Genentech, Inc. , South San Francisco , CA , USA.
authors.forename: Diego
authors.initials: D
authors.lastname: Ellerman
authors.affiliation: c Department of Translational Oncology , Genentech, Inc. , South San Francisco , CA , USA.
authors.forename: Dionysos
authors.initials: D
authors.lastname: Slaga
authors.affiliation: c Department of Translational Oncology , Genentech, Inc. , South San Francisco , CA , USA.
authors.forename: Teemu T
authors.initials: TT
authors.lastname: Junttila
authors.affiliation: d Department of Microchemistry, Proteomics and Lipidomics , Genentech, Inc. , South San Francisco , CA , USA.
authors.forename: Guanghui
authors.initials: G
authors.lastname: Han
authors.affiliation: d Department of Microchemistry, Proteomics and Lipidomics , Genentech, Inc. , South San Francisco , CA , USA.
authors.forename: Wendy
authors.initials: W
authors.lastname: Sandoval
authors.affiliation: e Department of Preclinical and Translational Pharmacokinetics , Genentech, Inc. , South San Francisco , CA , USA.
authors.forename: Meric A
authors.initials: MA
authors.lastname: Ovacik
authors.affiliation: e Department of Preclinical and Translational Pharmacokinetics , Genentech, Inc. , South San Francisco , CA , USA.
authors.forename: Kedan
authors.initials: K
authors.lastname: Lin
authors.affiliation: f Department of Early Stage Cell Culture , Genentech, Inc. , South San Francisco , CA , USA.
authors.forename: Zhilan
authors.initials: Z
authors.lastname: Hu
authors.affiliation: f Department of Early Stage Cell Culture , Genentech, Inc. , South San Francisco , CA , USA.
authors.forename: Amy
authors.initials: A
authors.lastname: Shen
authors.affiliation: g Department of Early Discovery Biochemistry, Genentech, Inc. , South San Francisco , CA , USA.
authors.forename: Jacob E
authors.initials: JE
authors.lastname: Corn
authors.affiliation: a Department of Antibody Engineering , Genentech, Inc. , South San Francisco , CA , USA.
authors.forename: Christoph
authors.initials: C
authors.lastname: Spiess
authors.affiliation: a Department of Antibody Engineering , Genentech, Inc. , South San Francisco , CA , USA.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Carter
mesh_terms: D000818:Animals; D018033:Antibodies, Bispecific / Q000096:biosynthesis*; D006801:Humans; D007074:Immunoglobulin G; D015202:Protein Engineering / Q000379:methods*
chemical_list: D018033:Antibodies, Bispecific; D007074:Immunoglobulin G
keywords: Bispecific antibody; bispecific IgG; orthogonal Fab engineering; single-cell production; stable CHO cell lines