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.

An Alkynyl-Fucose Halts Hepatoma Cell Migration and Invasion by Inhibiting GDP-Fucose-Synthesizing Enzyme FX, TSTA3

“…Thus, to effectively and selectively manipulate cellular fucosylation, it is reasonable to target these proteins (GMDS, FX, GDP-fucose transporter, and FUTs), rather than interfere with the early glycan biosynthetic pathway as in the case for tunicamycin, swainsonine, and deoxymannojirimycin. Many previous attempts to develop fucosylation inhibitors targeted these proteins and found that several fucose analogs (fluoro-and thio-fucose) inhibit these enzymes in vitro (Allen et al, 2016;Burkart et al, 2000) and/or reduce cellular fucosylation levels (Okeley et al, 2013;Rillahan et al, 2012;Zandberg et al, 2012). This suggests that chemical modification of fucose is a reasonable strategy to develop new fucosylation inhibitors.…”

Interaction of cell culture process parameters for modulating mAb afucosylation

“…genetic engineering of recombinant cell lines (Louie et al, 2016;Yamane-Ohnuki et al, 2004); (b) addition of enzyme inhibitors (Allen et al, 2016;Okeley et al, 2015); (c) modification of the levels of cofactors and substrates involved in glycosylation, including supplementation with alternative sugars (Hossler et al, 2014;Hossler et al, 2017); and (d) fine-tuning of cell culture process parameters (Konno et al, 2012). Of the many cell culture process parameters known to impact glycosylation previously (Hossler, Khattak, & Li, 2009), osmolality was shown to affect afucosylation (Konno et al, 2012).…”

Real-time product attribute control to manufacture antibodies with defined N-linked glycan levels

“…Recent studies have indicated that Mn, a cofactor for glycosyltransferase reactions, can impact glycosylation of antibodies depending on the culture conditions. Interestingly, recent work in synthetic chemistry has produced specific inhibitors of cellular fucosylation. When antibody producing cell lines are exposed to these inhibitors, they produce antibodies with reduced fucosylation, so specific inhibitors could potentially be used in a control loop to control antibody fucosylation levels.…”