Facile Modulation of Antibody Fucosylation with Small Molecule Fucostatin Inhibitors and Cocrystal Structure with GDP-Mannose 4,6-Dehydratase.

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.

Telescoped Process to Manufacture 6,6,6-Trifluorofucose via Diastereoselective Transfer Hydrogenation: Scalable Access to an Inhibitor of Fucosylation Utilized in Monoclonal Antibody Production.

IgG1 monoclonal antibodies with reduced glycan fucosylation have been shown to improve antibody-dependent cellular cytotoxicity (ADCC) by allowing more effective binding of the Fc region of these proteins to T cells receptors. Increased in vivo efficacy in animal models and oncology clinical trials has been associated with the enhanced ADCC provided by these engineered mAbs. 6,6,6-Trifluorofucose (1) is a new inhibitor of fucosylation that has been demonstrated to allow the preparation of IgG1 monoclonal antibodies with lower fucosylation levels and thus improve the ADCC of these proteins. A new process has been developed to support the preparation of 1 on large-scale for wide mAb manufacture applications. The target fucosylation inhibitor (1) was synthesized from readily available d-arabinose in 11% overall yield and >99.5/0.5 dr (diastereomeric ratio). The heavily telescoped process includes seven steps, two crystallizations as purification handles, and no chromatography. The key transformation of the sequence involves the diastereoselective preparation of the desired trifluoromethyl-bearing alcohol in >9/1 dr from a trimethylsilylketal intermediate via a ruthenium-catalyzed tandem ketal hydrolysis-transfer hydrogenation process.

Protected peptide nanoparticles: experiments and brownian dynamics simulations of the energetics of assembly.

Soluble peptides, susceptible to degradation and clearance in therapeutic applications, have been formulated into protected nanoparticles for the first time through the process of kinetically controlled, block copolymer directed rapid precipitation using Flash NanoPrecipitation. Complementary Brownian dynamics simulations qualitatively model the nanoparticle formation process. The simulations corroborate the hypothesis that the size of nanoparticles decreases with increasing supersaturation. Additionally, the influence of the polymer-peptide interaction energy on the efficiency of nanoparticle protection by polymer surface coverage is elucidated in both experiments and simulations.