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Chemoenzymatic Glyco-engineering of Monoclonal Antibodies

  • John P. Giddens
  • Lai-Xi Wang
Part of the Methods in Molecular Biology book series (MIMB, volume 1321)

Abstract

Monoclonal antibodies (mAbs) are an important class of therapeutic glycoproteins widely used for the treatment of cancer, inflammation, and infectious diseases. Compelling data have shown that the presence and fine structures of the conserved N-glycans at the Fc domain can profoundly affect the effector functions of antibodies. However, mAbs are usually produced as mixtures of Fc glycoforms and the control of glycosylation to a favorable, homogeneous status in various host expression systems is still a challenging task. In this chapter, we describe a detailed procedure of chemoenzymatic glyco-engineering of monoclonal antibodies, using rituximab (a therapeutic monoclonal antibody) as a model system. The protocol includes the deglycosylation of a mAb by an endoglycosidase (such as wild type EndoS) to remove the heterogeneous Fc N-glycans, leaving only the innermost GlcNAc or the core-fucosylated GlcNAc at the glycosylation site. Then the deglycosylated IgG serves as an acceptor for an endoglycosidase-catalyzed transglycosylation to add a desired N-glycan to the GlcNAc acceptor to reconstitute a defined, homogeneous natural glycoform of IgG, using a glycosynthase mutant as the enzyme and activated glycan oxazoline as the donor substrate. A semi-synthesis of sialylated and asialylated biantennary N-glycan oxazolines is also described. This detailed procedure can be used for the Fc glycosylation remodeling of other mAbs to provide homogeneous Fc glycoforms for various applications.

Key words

Monoclonal antibody Fc glycosylation Glyco-engineering Chemoenzymatic synthesis Transglycosylation ADCC 

Notes

Acknowledgements

This work was supported by the National Institutes of Health (NIH grant R01GM096973).

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • John P. Giddens
    • 1
  • Lai-Xi Wang
    • 1
  1. 1.Department of Chemistry and BiochemistryUniversity of MarylandCollege ParkUSA

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