GlycINATOR® in Peptide Mapping of Fc Domain-Drug Conjugates

The field of antibody-drug conjugates (ADCs) is rapidly emerging as a new, important class of cancer therapeutics. ADCs consist of monoclonal antibodies (mAbs) covalently linked to cytotoxic drugs, and after selective surface receptor binding on cancer cells, the drug is delivered into the intracellular compartment and can exert its cytotoxic effect.
 

Besides conventional ADCs generated from full-length mAbs, drug conjugates generated from smaller antibody fragments have been explored for enhanced tumor penetration and improved efficacy. Recently, the scientists behind this paper have described Fc fragments engineered to form an antigen binding site in the CH3 domain of human IgG1, called Fcabs, as a promising format for ADC-generation. With success, HER2-binding drug conjugates were generated.
 

In the present study, the concept of Fcab-based generation of site-specific, stable and highly potent ADCs is further investigated, and expanded to epidermal growth factor receptor (EGFR) binding Fcab ADCs carrying a higher drug load. The GlycINATOR (EndoS2) enzyme was used to enable identification of conjugation sites via LC-MS peptide mapping. GlycINATOR is an IgG-specific endoglycosidase that successfully hydrolyzes all Fc N-glycans and, in this case, reduces the complexity prior to the LC-MS analysis.

It was found that the Fcab-drug conjugates exhibited a high stability in human and mouse serum and the conjugates showed EGFR-mediated cytotoxicity at sub-nanomolar concentrations similar to cetuximab-based conjugates. Whether Fcab’s smaller size and intermediate plasma half-life are beneficial for ADC exposure to solid tumors remain to be evaluated.
 

Reference

Jäger et al., 2021. EGFR binding Fc domain-drug conjugates: stable and highly potent cytotoxic molecules mediate selective cell killing. Biol. Chem. https://doi.org/10.1515/hsz-2021-0321.

GlycINATOR (EndoS2) – Hydrolysis of all Fc glycans