Methods for modifying human antibodies by glycan engineering
AssigneeAcademia Sinica
InventorsChi-Huey Wong, Chung-Yi Wu
Life of this patent
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Abstract
Modified Fc regions of antibodies and antibody fragments, both human and humanized, and having enhanced stability and efficacy, are provided. Fc regions with core fucose residues removed, and attached to oligosaccharides comprising terminal sialyl residues, are provided. Antibodies comprising homogeneous glycosylation of Fc regions with specific oligosaccharides are provided. Fc regions conjugated with homogeneous glycoforms of monosaccharides and trisaccharides, are provided. Methods of preparing human antibodies with modified Fc using glycan engineering, are provided.
Claims8
A method for glycan engineering the Fc region of a human, chimeric or humanized IgG1 or IgG3 glycosylated antibody, the method comprising the steps of:
- (a) providing a human, chimeric or humanized IgG1 or IgG3 glycosylated antibody comprising an oligosaccharide attached at each Asn 297 in the Fc region;
- (b) contacting the human, chimeric or humanized IgG1 or IgG3 glycosylated antibody with an endo-β-N-acetylglucosaminidase (endo-NAG) and alpha-fucosidase thereby digesting the oligosaccharide to a defucosylated N-Acetylglucosamine (GlcNAc) unit attached at each Asn 297 in the Fc region;
- (c) contacting the defucosylated N-Acetylglucosamine (GlcNAc) unit with an endo-β-N-acetylglucosaminidase (endo-NAG) and a synthetic Gal2GlcNAc2Man3GlcNAc moiety joined to a leaving group, thereby coupling the terminal GlcNac of the synthetic Gal2GlcNAc2Man3GlcNAc moiety to the defucosylated N-Acetylglucosamine (GlcNAc) unit to form the structure Gal2GlcNAc2Man3GlcNAc2 attached at each Asn 297 of the Fc region of the human, chimeric or humanized IgG1 or IgG3 glycosylated antibody;
- and
- (d) contacting the attached Gal2GlcNAc2Man3GlcNAc2 of step (c) with an alpha-2,6-sialyltransferase, CMP-Neu5Ac and a set of CMP-Neu5Ac regeneration enzymes comprising a pyrophosphatase and a cytidine monophosphate kinase, thereby adding a terminal Neu5Ac to each terminal galactose of the attached Gal2GlcNAc2Man3GlcNAc2, wherein the glycan engineered human, chimeric or humanized IgG1 or IgG3 antibody has a Neu5Ac2Gal2GlcNAc2Man3GlcNAc2 attached at each Asn 297 in the Fc region, and wherein the CMP-Neu5Ac is regenerated following the addition of the terminal Neu5Ac.
A method for glycan engineering the Fc region-of a human, chimeric or humanized IgG1 or IgG3 glycosylated antibody, the method comprising the steps of:
- (a) providing a human, chimeric or humanized IgG1 or IgG3 glycosylated antibody comprising an oligosaccharide attached at each Asn 297in the Fc region;
- and
- (b) contacting the human, chimeric or humanized IgG1 or IgG3 glycosylated antibody with an endo-β-N-acetylglucosaminidase (endo-NAG) and alpha-fucosidase thereby digesting the oligosaccharide to a defucosylated N-Acetylglucosamine (GlcNAc) unit attached at each Asn 297 in the Fc region;
- and;
- (c) contacting the defucosylated N-Acetylglucosamine (GlcNAc) unit with an endo-β-N-acetylglucosaminidase (endo-NAG) and a synthetic Neu5Ac2Gal2GlcNAc2Man3GlcNAc moiety joined to a leaving group, thereby coupling the terminal GlcNac of the synthetic moiety to the defucosylated N-Acetylglucosamine (GlcNAc) unit to form the structure Neu5Ac2Gal2G1cNAc2Man3G1cNAc2 attached at each Asn 297 of the Fc region of the human, chimeric or humanized IgG1 or IgG3 glycosylated antibody.
Dependencies are inferred from the claim text.