Chemoenzymatic glycoengineering of antibodies and Fc fragments thereof
AssigneeUniversity of Maryland, Baltimore
InventorsLai-Xi Wang, Wei Huang
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Abstract
The present invention provides for recombinant Endo-S mutants that exhibit reduced hydrolysis activity and increased transglycosylation activity for the synthesis of glycoproteins wherein a desired sialylated oxazoline or synthetic oligosaccharide oxazoline is added to a core fucosylated or nonfucosylated GlcNAc-protein acceptor. Such recombinant Endo-S mutants are useful for efficient glycosylation remodeling of IgG1-Fc domain to provide different antibody glycoforms carrying structurally well-defined Fc N-glycans.
Claims11
A method of preparing a core fucosylated or nonfucosylated antibody or Fc fragment thereof having a predetermined oligosaccharide moiety, the method comprising:
- providing an antibody or Fc fragment comprising a core fucosylated or nonfucosylated GlcNAc-acceptor;
- and enzymatically reacting the core fucosylated or nonfucosylated GlcNAc-acceptor with an activated oligosaccharide donor using a Streptococcus pyogenes Endoglycosidase-S Asp233 substitution mutant, wherein the Endoglycosidase-S mutant is selected from a mutant comprising SEQ ID NO:2 (D233Q) or SEQ ID No:
- 3 (D233A) having decreased hydrolytic activity and increased transglycosylation activity relative to wild type S. pyogenes Endoglycosidase-S, wherein the activated oligosaccharide donor carries an oligosaccharide moiety comprising a predetermined number and type of sugar residues, wherein via an enzymatic reaction mediated by the Streptococcus pyogenes Endoglycosidase-S Asp233 substitution mutant, the activated oligosaccharide moiety is covalently linked to the core fucosylated or nonfucosylated GlcNAc-acceptor, thereby preparing the core fucosylated or nonfucosylated antibody or Fc fragment having the predetermined oligosaccharide moiety.
Dependencies are inferred from the claim text.