title: Facile Modulation of Antibody Fucosylation with Small Molecule Fucostatin Inhibitors and Cocrystal Structure with GDP-Mannose 4,6-Dehydratase.
abstract: The efficacy of therapeutic antibodies that induce antibody-dependent cellular cytotoxicity can be improved by reduced fucosylation. Consequently, fucosylation is a critical product attribute of monoclonal antibodies produced as protein therapeutics. Small molecule fucosylation inhibitors have also shown promise as potential therapeutics in animal models of tumors, arthritis, and sickle cell disease. Potent small molecule metabolic inhibitors of cellular protein fucosylation, 6,6,6-trifluorofucose per-O-acetate and 6,6,6-trifluorofucose (fucostatin I), were identified that reduces the fucosylation of recombinantly expressed antibodies in cell culture in a concentration-dependent fashion enabling the controlled modulation of protein fucosylation levels. 6,6,6-Trifluorofucose binds at an allosteric site of GDP-mannose 4,6-dehydratase (GMD) as revealed for the first time by the X-ray cocrystal structure of a bound allosteric GMD inhibitor. 6,6,6-Trifluorofucose was found to be incorporated in place of fucose at low levels (<1%) in the glycans of recombinantly expressed antibodies. A fucose-1-phosphonate analog, fucostatin II, was designed that inhibits fucosylation with no incorporation into antibody glycans, allowing the production of afucosylated antibodies in which the incorporation of non-native sugar is completely absent-a key advantage in the production of therapeutic antibodies, especially biosimilar antibodies. Inhibitor structure-activity relationships, identification of cellular and inhibitor metabolites in inhibitor-treated cells, fucose competition studies, and the production of recombinant antibodies with varying levels of fucosylation are described.
authors.affiliation: Therapeutic Discovery, Amgen Inc. , One Amgen Center Drive, Thousand Oaks, California 91320, United States.
authors.forename: John G
authors.initials: JG
authors.lastname: Allen
authors.affiliation: Process Development - Drug Substance Technologies, Amgen Inc. , 1201 Amgen Court W., Seattle, Washington 98119, United States.
authors.forename: Mirna
authors.initials: M
authors.lastname: Mujacic
authors.affiliation: Therapeutic Discovery, Amgen Inc. , One Amgen Center Drive, Thousand Oaks, California 91320, United States.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Frohn
authors.affiliation: Therapeutic Discovery, Amgen Inc. , One Amgen Center Drive, Thousand Oaks, California 91320, United States.
authors.forename: Alex J
authors.initials: AJ
authors.lastname: Pickrell
authors.affiliation: Process Development - Drug Substance Technologies, Amgen Inc. , 1201 Amgen Court W., Seattle, Washington 98119, United States.
authors.forename: Paul
authors.initials: P
authors.lastname: Kodama
authors.affiliation: Therapeutic Discovery, Amgen Inc. , One Amgen Center Drive, Thousand Oaks, California 91320, United States.
authors.forename: Dhanashri
authors.initials: D
authors.lastname: Bagal
authors.affiliation: Therapeutic Discovery, Amgen Inc. , One Amgen Center Drive, Thousand Oaks, California 91320, United States.
authors.forename: Tisha
authors.initials: T
authors.lastname: San Miguel
authors.affiliation: Therapeutic Discovery, Amgen Inc. , One Amgen Center Drive, Thousand Oaks, California 91320, United States.
authors.forename: E Allen
authors.initials: EA
authors.lastname: Sickmier
authors.affiliation: Process Development - Attribute Sciences, Amgen Inc. , One Amgen Center Drive, Thousand Oaks, California 91320, United States.
authors.forename: Steve
authors.initials: S
authors.lastname: Osgood
authors.affiliation: Process Development - Attribute Sciences, Amgen Inc. , One Amgen Center Drive, Thousand Oaks, California 91320, United States.
authors.forename: Aleksander
authors.initials: A
authors.lastname: Swietlow
authors.affiliation: Therapeutic Discovery, Amgen Inc. , One Amgen Center Drive, Thousand Oaks, California 91320, United States.
authors.forename: Vivian
authors.initials: V
authors.lastname: Li
authors.affiliation: Therapeutic Discovery, Amgen Inc. , One Amgen Center Drive, Thousand Oaks, California 91320, United States.
authors.forename: John B
authors.initials: JB
authors.lastname: Jordan
authors.affiliation: Cardiometabolic Disorders, Amgen Inc. , One Amgen Center Drive, Thousand Oaks, California 91320, United States.
authors.forename: Ki-Won
authors.initials: KW
authors.lastname: Kim
authors.affiliation: Therapeutic Innovations Unit, Amgen Inc. , 1201 Amgen Court W., Seattle, Washington 98119, United States.
authors.forename: Anne-Marie C
authors.initials: AC
authors.lastname: Rousseau
authors.affiliation: Therapeutic Discovery, Amgen Inc. , One Amgen Center Drive, Thousand Oaks, California 91320, United States.
authors.forename: Yong-Jae
authors.initials: YJ
authors.lastname: Kim
authors.affiliation: Process Development - Drug Substance Technologies, Amgen Inc. , One Amgen Center Drive, Thousand Oaks, California 91320, United States.
authors.forename: Seb
authors.initials: S
authors.lastname: Caille
authors.affiliation: Process Development - Drug Substance Technologies, Amgen Inc. , One Amgen Center Drive, Thousand Oaks, California 91320, United States.
authors.forename: Mike
authors.initials: M
authors.lastname: Achmatowicz
authors.affiliation: Process Development - Drug Substance Technologies, Amgen Inc. , One Amgen Center Drive, Thousand Oaks, California 91320, United States.
authors.forename: Oliver
authors.initials: O
authors.lastname: Thiel
authors.affiliation: Therapeutic Discovery, Amgen Inc. , One Amgen Center Drive, Thousand Oaks, California 91320, United States.
authors.forename: Christopher H
authors.initials: CH
authors.lastname: Fotsch
authors.affiliation: Process Development - Drug Substance Technologies, Amgen Inc. , 1201 Amgen Court W., Seattle, Washington 98119, United States.
authors.forename: Pranhitha
authors.initials: P
authors.lastname: Reddy
authors.affiliation: Therapeutic Discovery, Amgen Inc. , One Amgen Center Drive, Thousand Oaks, California 91320, United States.
authors.forename: John D
authors.initials: JD
authors.lastname: McCarter
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D018360:Crystallography, X-Ray; D005643:Fucose / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006155:Guanosine Diphosphate Mannose / Q000378:metabolism; D006836:Hydro-Lyases / Q000378:metabolism*; D013058:Mass Spectrometry; D015394:Molecular Structure; D054852:Small Molecule Libraries; D020349:Surface Plasmon Resonance
chemical_list: D054852:Small Molecule Libraries; D005643:Fucose; D006155:Guanosine Diphosphate Mannose; D006836:Hydro-Lyases