Methods for modifying human antibodies by glycan engineering
AssigneeAcademia Sinica
InventorsChi-Huey WONG, Chung-Yi WU
Life of this patent
Estimating the term…
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.
Claims42
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 an oxazoline 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 glycosylated IgG1 or IgG3 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 GlcNAc2Man3GlcNAc moiety joined to a leaving group, thereby coupling the terminal GlcNac unit of the synthetic oligosaccharide moiety to the single GlcNAc unit of the synthetic GlcNAc2Man3GlcNAc moiety to the defucosylated N-Acetylglucosamine (GlcNAc) unit to form the structure GlcNAc2Man3GlcNAc2 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 GlcNAc2Man3GlcNAc2 of step (c) with a β-1,4-galactosyltransferase, UDP galactose, and a set of UDP-galactose regeneration enzymes selected from the group consisting of a glucose kinase, a phosphoglucomutase, an N-acetyl glucosamine-1 phosphate uridyltransferase, a polyphosphate kinase, a pyrophosphatase, a nucleoside diphosphate kinase, a UDP-Glc/GlcNAc4 epimerase, a pyruvate kinase, a UDP kinase, a galactokinase, an ADP kinase, and a UDP-galactose pyrophosphorylase. thereby adding a terminal galactose to each terminal GlcNAc of the attached GlcNAc2Man3GlcNAc2 and wherein the UDP-galactose is regenerated following the addition of the terminal galactose;
- and
- (e) contacting the attached Gal2GlcNAc2Man3GlcNAc2 of step (d) 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 of 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 glycosylated antibody, wherein the glycosylated antibody comprises an oligosaccharide attached at Asn-297, wherein the oligosaccharide is selected from the group consisting of Gal2GlcNAc2Man3GlcNAc2, GalGlcNAc2Man3GlcNAc2, and GlcNAc2Man3GlcNAc2, the method comprising:
- (a) contacting the antibody with an alpha fucosidase and β-1,4-galactosyltransferase and UDP galactose thereby defucosylating the oligosaccharide and coupling the terminal GlcNac of the GalGlcNAc2Man3GlcNAc2, and the GlcNAc2Man3GlcNAc2 to the galactose to form the structure Gal2GlcNAc2Man3GlcNAc2 attached at each Asn 297 of the Fc region of the human, chimeric or humanized glycosylated antibody;
- and
- (b) contacting the antibody comprising the structure Gal2GlcNAc2Man3GlcNAc2 with an alpha-2,6-sialyltransferase and CMP-Neu5Ac, thereby adding a terminal Neu5Ac to each terminal galactose of the Gal2GlcNAc2Man3GlcNAc2, wherein the glycan engineered human, chimeric or humanized antibody has a Neu5Ac2Gal2GlcNAc2Man3GlcNAc2 attached at each Asn 297 in the Fc region.
A method for glycan engineering the Fc region of a human, chimeric, or humanized glycosylated antibody, wherein the glycosylated antibody comprises an oligosaccharide attached at Asn-297, wherein the oligosaccharide is selected from the group consisting of Gal2GlcNAc2Man3GlcNAc2, GalGlcNAc2Man3GlcNAc2, and GlcNAc2Man3GlcNAc2, the method comprising:
- (a) contacting the antibody with a β-1,4-galactosyltransferase and UDP galactose thereby coupling the terminal GlcNac of the GalGlcNAc2Man3GlcNAc2, and the GlcNAc2Man3GlcNAc2 to the galactose to form the structure Gal2GlcNAc2Man3GlcNAc2 attached at each Asn 297 of the Fc region of the human, chimeric or humanized glycosylated antibody;
- and
- (b) contacting the antibody comprising the structure Gal2GlcNAc2Man3GlcNAc2 with an alpha-2,6-sialyltransferase and CMP-Neu5Ac, thereby adding a terminal Neu5Ac to each terminal galactose of the Gal2GlcNAc2Man3GlcNAc2, wherein the glycan engineered human, chimeric or humanized antibody has a Neu5Ac2Gal2GlcNAc2Man3GlcNAc2 attached at each Asn 297 in the Fc region.
A method for glycan engineering the Fc region of a human, chimeric, or humanized glycosylated antibody, the method comprising:
- (a) providing an antibody comprising an N-glycan selected from the group consisting of Gal2GlcNAc2Man3GlcNAc, GalGlcNAc2Man3GlcNAc, and GlcNAc2Man3GlcNAc, attached at Asn-297;
- (b) contacting the antibody of step (a) with β-1,4-galactosyltransferase and UDP galactose (uridine diphosphate galactose) thereby coupling the terminal GlcNac of the GalGlcNAc2Man3GlcNAc, and the GlcNAc2Man3GlcNAc to the galactose to form the structure Gal2GlcNAc2Man3GlcNAc attached at each Asn 297 of the Fc region of the human, chimeric or humanized glycosylated antibody;
- and
- (c) contacting the antibody comprising the structure Gal2GlcNAc2Man3GlcNAc2 with an alpha-2,6-sialyltransferase and CMP-Neu5Ac, thereby adding a terminal Neu5Ac to each terminal galactose of the Gal2GlcNAc2Man3GlcNAc2, wherein the glycan engineered human, chimeric or humanized antibody has a Neu5Ac2Gal2GlcNAc2Man3GlcNAc2 attached at each Asn 297 in the Fc region.
Dependencies are inferred from the claim text.