A versatile design platform for glycoengineering therapeutic antibodies

Manipulation of glycosylation patterns, i.e., glycoengineering, is incorporated in the therapeutic antibody development workflow to ensure clinical safety, and this approach has also been used to modulate the biological activities, functions, or pharmacological properties of antibody drugs. Whereas most existing glycoengineering strategies focus on the canonical glycans found in the constant domain of immunoglobulin G (IgG) antibodies, we report a new strategy to leverage the untapped potential of atypical glycosylation patterns in the variable domains, which naturally occur in 15% to 25% of IgG antibodies. Glycosylation sites were added to the antigen-binding regions of two functionally divergent, interleukin-2-binding monoclonal antibodies. We used computational tools to rationally install various N-glycosylation consensus sequences into the antibody variable domains, creating glycovariants of these molecules. Strikingly, almost all the glycovariants were successfully glycosylated at their newly installed N-glycan sites, without reduction of the antibody's native function. Importantly, certain glycovariants exhibited modified activities compared to the parent antibody, showing the potential of our glycoengineering strategy to modulate biological function of antibodies involved in multi-component receptor systems. Finally, when coupled with a high-flux sialic acid precursor, a glycovariant with two installed glycosylation sites demonstrated superior in vivo half-life. Collectively, these findings validate a versatile glycoengineering strategy that introduces atypical glycosylation into therapeutic antibodies in order to improve their efficacy and, in certain instances, modulate their activity early in the drug development process.

Automated summary

This study introduces a new glycoengineering strategy, leveraging atypical glycosylation patterns in variable domains of antibodies, to modulate the biological activities of therapeutic antibodies. Glycosylation sites were added to two monoclonal antibodies and computational tools were used to install N-glycosylation consensus sequences, creating glycovariants. Almost all the glycovariants were successfully glycosylated at the new sites without affecting the antibody's native function. Certain glycovariants exhibited modified activities compared to the parent antibody, demonstrating the potential of this strategy to modulate the biological function of antibodies involved in multi-component receptor systems.