Glycoprotein synthesis and remodeling by enzymatic transglycosylation
A chemoenzymatic method for the preparation of a homogeneous glycoprotein or glycopeptide, including (a) providing an acceptor selected from the group consisting of GlcNAc-protein and GlcNAc-peptide; and (b) reacting the acceptor with a donor substrate including an activated oligosaccharide moiety, in the presence of a catalyst comprising endoglycosidase (ENGase), to transfer the oligosaccharide moiety to the acceptor and yield the homogeneous glycoprotein or glycopeptide. The donor substrate includes, in a specific implementation, a synthetic oligosaccharide oxazoline. A related method of glycoprotein or glycopeptide remodeling with a predetermined natural N-glycan or a tailor-made oligosaccharide moiety, and a method of remodeling an antibody including a heterogeneous sugar chain, are also described. The disclosed methodology enables glycoprotein drugs to be modified for prolonged half-life in vivo, reduced immunogenicity, and enhanced in vivo activity, and for targeting and drug delivery.
1. A modified antibody or modified fragment thereof having a predetermined number of saccharide mannose residues and glycosidic linkage types prepared by a method comprising:
providing an antibody or fragment thereof comprising heterogeneous distribution of saccharides and at least one N-acetylglucosamine (GlcNAc) moiety positioned on the Fc region of the antibody or Fc region of the fragment thereof;
removing the heterogeneous distribution of saccharides with an endoglycosidase enzyme to form a GlcNAc-peptide acceptor having a single N-acetylglucosamine (GlcNAc) moiety, wherein the single N-acetylglucosamine (GlcNAc) moiety is positioned on the Fc region of the antibody or Fc region of the fragment thereof; and
transglycosylating a synthetic oligosaccharide oxazoline comprising the predetermined number of saccharide mannose residues and glycosidic linkage types with the GlcNAc-peptide acceptor under the catalysis of an endoglycosidase (ENGase) enzyme selected from the group consisting of Endo A and Endo M to form the modified antibody or modified fragment thereof, wherein the synthetic oligosaccharide oxazoline is a di-, tri-, tetra-, penta-, hexyl-, hepta-, octyl-, nona-, deca-, or undeca-saccharide oxazoline.
2. A delivery device for delivering a tag, the delivery device comprising: a remodeled antibody or fragment thereof having a predetermined number of saccharide mannose residues and glycosidic linkage types, wherein the delivery device is prepared according to a method comprising:
(a) providing an antibody or fragment thereof comprising heterogeneous distribution of saccharides and at least one N-acetylglucosamine (GlcNAc) moiety positioned on the Fc region of the antibody or Fc region of the fragment thereof;
(b) removing the heterogeneous distribution of saccharides with an endoglycosidase enzyme to form a GlcNAc-peptide acceptor having a single N-acetylglucosamine (GlcNAc) moiety, wherein the single N-acetylglucosamine (GlcNAc) moiety is positioned on the Fc region of the antibody or Fc region of the fragment thereof;
(c) transglycosylating a synthetic oligosaccharide oxazoline comprising the predetermined number of saccharide mannose residues and glycosidic linkage types with the GlcNAc-peptide acceptor under the catalysis of an endoglycosidase (ENGase) enzyme selected from the group consisting of Endo A and Endo M to form the remodeled antibody or fragment thereof, wherein the synthetic oligosaccharide oxazoline is a di-, tri-, tetra-, penta-, hexyl-, hepta-, octyl-, nona-, deca-, or undeca-saccharide oxazoline; and
(d) adding the tag to a mannose saccharide residue of the synthetic oligosaccharide oxazoline either before reacting with the single N-acetylglucosamine (GlcNAc) moiety or to the remodeled antibody or fragment thereof of step (c) to form the deliver device, wherein the tag is an antigen, drug, toxin, fluorescent probe, biotin, a PEG species, lipid, nucleotides or polypeptide.
3. The delivery device of claim 2 , wherein the antibody is selected from the group consisting of 17b, 48d, A32, C11, 2G12, F240, IgG1b12, 19e, X5, TNX-355 and F91.
4. The modified antibody or modified fragment thereof of claim 1 , wherein the antibody is selected from the group consisting of 17b, 48d, A32, C11, 2G12, F240, IgG1b12, 19e, X5, TNX-355 and F91.