Chemoenzymatic glycoengineering of antibodies and Fc fragments thereof

Patent metadata

Field	Value
Publication number	US-9434786-B2
Application number	US-201314376248-A
Country	US
Kind code	B2
Filing date	Feb 11, 2013
Priority date	Feb 10, 2012
Publication date	Sep 6, 2016
Grant date	Sep 6, 2016

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.

First claim

That which is claimed is:

1. 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.

2. The method of claim 1 , wherein the activated oligosaccharide component is a synthetic oligosaccharide oxazoline or sialylated oxazoline.

3. The method of claim 1 , wherein the synthetic oligosaccharide oxazoline is a di-, tri-, tetra-, penta-, hexyl-, hepta-, octyl-, nona-, deca- or undeca-saccharide oxazoline.

4. The method of claim 1 , wherein the activated oligosaccharide component further comprises an additional biologically active agent or a tag.

5. The method of claim 4 , wherein the additional biologically active agent or tag is a drug, toxin, fluorescent probe, biotin, a PEG, lipid, or polypeptide.

6. The method of claim 1 , wherein the fucosylated GlcNAc-acceptor is an alpha-1-6-fucosyl-GlcNAc-protein.

7. The method of claim 1 , wherein the core fucosylated or nonfucosylated antibody is a monoclonal antibody selected from the group consisting of 17b, 48d, A32, C11, 2G12, F240, IgG1b12, 19e, X5, TNX-355, cetuximab, rituximab, muromonab-CD3, abciximab, daclizumab, basiliximab, palivizumab, infliximab, trastuzumab, gemtuzumab ozogamicin, alemtuzumab, ibritumomab tiuxetan, adalimumab, omalizumab, tositumomab, I-131 tositumomab, efalizumab, bevacizumab, panitumumab, pertuzumab, natalizumab, etanercept, IGN101, volociximab, Anti-CD80 mAb, Anti-CD23 mAb, CAT-3888, CDP-791, eraptuzumab, MDX-010, MDX-060, MDX-070, matuzumab, CP-675,206, CAL, SGN-30, zanolimumab, adecatumumab, oregovomab, nimotuzumab, ABT-874, denosumab, AM 108, AMG 714, fontolizumab, daclizumab, golimumab, CNTO 1275, ocrelizumab, HuMax-CD20, belimumab, epratuzumab, MLN1202, visilizumab, tocilizumab, ocrerlizumab, certolizumab pegol, eculizumab, pexelizumab, abciximab, ranibizimumab, mepolizumab and MYO-029.

8. The method of claim 1 , wherein the antibody further comprises an additional moiety selected from a group consisting of a therapeutic agent for treating cancer, a therapeutic agent for HIV, a toxin, an antibody different from the modified antibody which is reactive to another receptor, an antigen, a chemokine and a cytokine.

9. The method of claim 1 , wherein the core fucosylated or nonfucosylated antibody or Fc fragment thereof having a predetermined oligosaccharide moiety is homogeneous and optionally the core fucosylated or nonfucosylated IgG glycoprotein or IgG-Fc fragment comprises heterogeneous or undesired N-glycans that are removed by an enzyme selected from the group Endo-H, Endo-F3, Endo S or Endo-A thereby providing the antibody or Fc fragment comprising the core fucosylated or nonfucosylated GlcNAc-acceptor.

10. The method of claim 9 , wherein the oligosaccharide containing oxazoline is a di-, tri-, tetra-, penta-, hexyl-, hepta-, octyl-, nona-, deca-, or undeca-saccharide oxazoline.

11. The method of claim 10 , wherein the core-fucosylated GlcNAc containing protein is an alpha-1-6-fucosyl-GlcNAc-protein.

Assignees

• Univ Maryland

Inventors

• Wang Lai-Xi
• Huang Wei

Classifications

• A61K47/6849

  the antibody targeting a receptor, a cell surface antigen or a cell surface determinant · CPC title

• A61P31/18

  for HIV · CPC title

• C07K2317/10

  characterized by their source of isolation or production · CPC title

• C07K16/18

  against material from animals or humans · CPC title

• C07K2317/94

  Stability, e.g. half-life, pH, temperature or enzyme-resistance · CPC title