title: Knobs-into-holes antibody production in mammalian cell lines reveals that asymmetric afucosylation is sufficient for full antibody-dependent cellular cytotoxicity.
abstract: Knobs-into-holes is a well-validated heterodimerization technology for the third constant domain of an antibody. This technology has been used to produce a monovalent IgG for clinical development (onartuzumab) and multiple bispecific antibodies. The most advanced uses of this approach, however, have been limited to E. coli as an expression host to produce non-glycosylated antibodies. Here, we applied the technology to mammalian host expression systems to produce glycosylated, effector-function competent heterodimeric antibodies. In our mammalian host system, each arm is secreted as a heavy chain-light chain (H-L) fragment with either the knob or hole mutations to allow for preferential heterodimer formation in vitro with low levels of homodimer contaminants. Like full antibodies, the secreted H-L fragments undergo Fc glycosylation in the endoplasmic reticulum. Using a monospecific anti-CD20 antibody, we show that full antibody-dependent cell-mediated cytotoxicity (ADCC) activity can be retained in the context of a knobs-into-holes heterodimer. Because the knobs-into-holes mutations convert the Fc into an asymmetric heterodimer, this technology was further used to systematically explore asymmetric recognition of the Fc. Our results indicate that afucosylation of half the heterodimer is sufficient to produce ADCC-enhancement similar to that observed for a fully afucosylated antibody with wild-type Fc. However, the most dramatic effect on ADCC activity is observed when two carbohydrate chains are present rather than one, regardless of afucosylation state.
authors.affiliation: Department of Protein Chemistry; Genentech, Inc; San Francisco, CA USA.
authors.forename: Whitney
authors.initials: W
authors.lastname: Shatz
authors.affiliation: Department of BioAnalytical Sciences; Genentech, Inc; San Francisco, CA USA.
authors.forename: Shan
authors.initials: S
authors.lastname: Chung
authors.affiliation: Department of Antibody Engineering; Genentech, Inc; San Francisco, CA USA.
authors.forename: Bing
authors.initials: B
authors.lastname: Li
authors.affiliation: Department of Biological Technologies; Genentech, Inc; San Francisco, CA USA.
authors.forename: Brett
authors.initials: B
authors.lastname: Marshall
authors.affiliation: Department of Biological Technologies; Genentech, Inc; San Francisco, CA USA.
authors.forename: Max
authors.initials: M
authors.lastname: Tejada
authors.affiliation: Department of Protein Chemistry; Genentech, Inc; San Francisco, CA USA.
authors.forename: Wilson
authors.initials: W
authors.lastname: Phung
authors.affiliation: Department of Protein Chemistry; Genentech, Inc; San Francisco, CA USA.
authors.forename: Wendy
authors.initials: W
authors.lastname: Sandoval
authors.affiliation: Department of Antibody Engineering; Genentech, Inc.; San Francisco, CA USA.
authors.forename: Robert F
authors.initials: RF
authors.lastname: Kelley
authors.affiliation: Department of Protein Chemistry; Genentech, Inc; San Francisco, CA USA.
authors.forename: Justin M
authors.initials: JM
authors.lastname: Scheer
mesh_terms: D000818:Animals; D000917:Antibody Formation / Q000276:immunology*; D000920:Antibody-Dependent Cell Cytotoxicity / Q000276:immunology*; D016466:CHO Cells; D002460:Cell Line; D002470:Cell Survival / Q000276:immunology; D002478:Cells, Cultured; D003412:Cricetulus; D019281:Dimerization; D005643:Fucose / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D015202:Protein Engineering
chemical_list: D005643:Fucose
keywords: ADCC; afucosylation; asymmetric antibody; effector function; glycosylation; heterodimer; knobs-into-holes; symmetry