A versatile design platform for glycoengineering therapeutic antibodies

Seth D. Ludwig, Zachary J. Bernstein, Christian Agatemor, Kris Dammen-Brower, Jeffrey Ruffolo, Jonah M. Rosas, Jeremy D. Post, Robert N. Cole, Kevin J. Yarema, Jamie B. Spangler

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish (US)
Article number2095704
Issue number1
StatePublished - 2022


  • glycosylation
  • immunotherapy
  • interleukin-2
  • ManNAc analog
  • metabolic glycoengineering
  • Protein glycoengineering
  • sialylation
  • therapeutics

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology


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