Metabolic Engineering of Monoclonal Antibody Carbohydrates for Antibody–Drug Conjugation

Abstract

The role that carbohydrates play in antibody function and pharmacokinetics has made them important targets for modification. The terminal fucose of the N-linked glycan structure, which has been shown to be involved in modulation of antibody-directed cellular cytotoxicity, is a particularly interesting location for potential modification through incorporation of alternative sugar structures. A library of fucose analogues was evaluated for their ability to incorporate into antibody carbohydrates in place of the native fucose. A number of efficiently incorporated molecules were identified, demonstrating the ability of fucosyltransferase VIII to utilize a variety of non-natural sugars as substrates. Among these structures was a thiolated analogue, 6-thiofucose, which was incorporated into the antibody carbohydrate with good efficiency. This unnatural thio-sugar could then be used for conjugation using maleimide chemistry to produce antibody-drug conjugates with pronounced cytotoxic activities and improved homogeneity compared to drug attachment through hinge disulfides.

Antibody Drug Conjugates for Cancer Therapy

“…The resulting hydrazone trigger in the ADC is not particularly stable, though, exhibiting a half-life of about 18 hours at physiologic pH in phosphatebuffered saline. Carbohydrate as a site of antibody conjugation was also described by Okeley et al (2013), but in this scheme the reactive site was generated by metabolic incorporation of derivatized fucose into the N-linked glycan. The antibody is produced by Chinese hamster ovary cells in media containing 1 mM of a fucose analog substituting for native fucose as a substrate for fucosyltranseferase VIII.…”

Antibody Drug Conjugates: Design and Selection of Linker, Payload and Conjugation Chemistry

“…They first homogenize mAb glycoforms via galactosidase digestion and subsequently integrate keto-or azide-modified galactose residues using mutant galactosyltransferases (40,41). Similarly, a recent report by Senter and co-workers exploits the promiscuity of native fucosyltransferases to incorporate thiol-functional fucose analogues for thiolmaleimide conjugation to MMAE (42). This method, however, requires reduction/oxidation steps to selectively liberate active thiol nucleophiles from cysteine adducts and suffers from limited integration efficiency (60-70%).…”

RN927C, a Site-Specific Trop-2 Antibody–Drug Conjugate (ADC) with Enhanced Stability, Is Highly Efficacious in Preclinical Solid Tumor Models

“…One approach that can potentially overcome these limitations is to employ site-specific conjugation of antibodies, and many experimental approaches have been developed, including conjugation through engineered cysteine residues, nonnatural amino acids, antibody carbohydrates, aldehyde tags, and utilizing enzymes such as sortases and phosphopantetheinyl transferases (2,(27)(28)(29)(30)(31)(32)(33)(34)(35)(36). A site specifically conjugated anti-CD33 ADC, SGN-CD33A, has shown encouraging activities in targeting drug-resistant acute myeloid leukemia (37).…”