Claims for Patent: 9,434,786

Summary for Patent: 9,434,786

Title:	Chemoenzymatic glycoengineering of antibodies and Fc fragments thereof
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.
Inventor(s):	Wang; Lai-Xi (Ellicott City, MD), Huang; Wei (Shanghai, CN)
Assignee:	UNIVERSITY OF MARYLAND, BALTIMORE (Baltimore, MD)
Application Number:	14/376,248
Patent Claims:	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.

Details for Patent 9,434,786

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Centocor Ortho Biotech Products, L.p.	ORTHOCLONE OKT3	muromanab-cd3	Injection	103463	09/14/1992		2032-02-10
Janssen Biotech, Inc.	REOPRO	abciximab	Injection	103575	12/22/1994		2032-02-10
Genentech, Inc.	RITUXAN	rituximab	Injection	103705	11/26/1997		2032-02-10
Idec Pharmaceuticals Corp.	RITUXAN	rituximab	Injection	103737	02/19/2002		2032-02-10
Hoffmann-la Roche Inc.	ZENAPAX	daclizumab	Injection	103749	12/10/1997		2032-02-10