Transglycosylation of endo-S and endo-S mutants for antibody glycosylation remodeling

Patent metadata

Field	Value
Publication number	US-11459380-B2
Application number	US-201816022978-A
Country	US
Kind code	B2
Filing date	Jun 29, 2018
Priority date	Jun 29, 2017
Publication date	Oct 4, 2022
Grant date	Oct 4, 2022

Abstract

The present invention provides for a one-pot enzymatic approach which does not require removal of the enzyme and purification of the intermediate after deglycosylation step, and the Endo-S treatment is able to do both deglycosylation and transglycosylation. The one-pot strategy of the present invention enables chemoenzymatic synthesis of an azido-tagged N-glycoform of monocloncal antibodies which could be further modified through orthogonal chemical ligation for various applications.

First claim

That which is claimed is:

1. A glycosylation remodeling method for a therapeutic antibody or Fc fragment thereof with a Streptococcus pyogenes wild-type endoglycosidase-S (Endo-S), the method comprising:
providing a single container;
introducing into the single container the Streptococcus pyogenes wild-type Endo-S, a core fucosylated or non-fucosylated antibody or Fc fragment thereof comprising Fc N-glycans including two GlcNAc residues positioned closest to a peptide domain of the antibody;
introducing into the single container and treating the core fucosylated antibody or non- fucosylated antibody or Fc fragment thereof with the Streptococcus pyogenes wild-type Endo-S in the single container and having an amino acid sequence of SEQ ID NO: 1 to hydrolyze a bond between the two GlcNAc residues thereby forming a deglycosylated core fucosylated or nonfucosylated GlcNAc-acceptor to yield an Asn-linked GlcNAc moiety; and
introducing into the single container a high-mannose oxazoline or hybrid glycan oxazoline having a predetermined oligosaccharide component with a defined number and type of sugar residues and with specific linkage types for reacting with the Asn-linked GlcNAc moiety wherein a transglycosylation step is carried out by the Streptococcus pyogenes wild-type Endo-S, thereby adding the predetermined oligosaccharide component to provide a remodeled glycosylated therapeutic antibody or Fc fragment thereof in the single container.

2. The glycosylation remodeling method of claim 1 , wherein the Streptococcus pyogenes wild-type Endo-S remains in the single container during the glycosylation remodeling method for both deglycosylation and transglycosylati on.

3. The glycosylation remodeling method of claim 1 , wherein the high-mannose oxazoline is selected from the group consisting of penta-, hexyl-, hepta-, octyl-, nona-, deca-, and undeca-saccharide oxazolines.

4. The glycosylation remodeling method of claim 3 , wherein the hybrid glycan oxazoline comprises a Man3 glycan oxazoline protected by addition of an extra moiety to resist hydrolysis by the Streptococcus pyogenes wild-type Endo-S.

5. The glycosylation remodeling method of claim 1 , wherein the hybrid glycan oxazoline comprises at least one compound selected from the group consisting of mannose, sialic acid, D-galactose, and L-fucose.

Assignees

• Univ Maryland

Inventors

• Wang Lai-Xi
• Tong Xin
• Li Tiezheng

Classifications

• C07K16/2887 Primary

  against CD20 · CPC title

• C07K16/32

  against translation products of oncogenes · CPC title

• A61K47/61

  the organic macromolecular compound being a polysaccharide or a derivative thereof · CPC title

• C12R2001/46

  Streptococcus {; Enterococcus; Lactococcus} · CPC title

• C12N1/205

  Bacterial isolates · CPC title