US4925796A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Method for enhancing glycoprotein stability
Assignee: Massachusetts Institute of Technology
•
Filing: 1986-03-07
•
Granted: 1990-05-15
•
Expires: 2006-03-07
Abstract
Abstract A method for modifying eukaryotic and prokaryotic proteins to extend their in vivo circulatory lifetimes. In the preferred embodiment, enzymatic and/or chemical treatments are used to produce a modified protein carrying one or more covalently attached trisaccharide, sialic acid→galactose→N-acetylglucosamine→(SA→Gal.fwdarw.GlcNac→), or tetrasaccharide (SA→Gal→GlcNAc→GlcNAc→) moieties. The method can be applied to any natural or recombinant protein possessing asparagine-linked oligosaccharides or to any non-glycosylated protein that can be chemically or enzymatically derivatized with the appropriate carbohydrate units. Following injection into an animal, the modified glycoproteins are protected from premature clearance by cells of the liver and reticulo-endothelial system which recognize and rapidly internalize circulating glycoproteins with carbohydrate chains containing terminal Gal, GlnNAc, fucose or mannose residues. The method can also be used to mask antigenic determinants on foreign proteins which would otherwise produce an immune response or to "target" a protein for recognition by sugar-specific cell surface receptors.
Inventors
- 👤 Michel L. E. Bergh
- 👤 S. Catherine Hubbard
- 👤 James R. Rasmussen
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-26 18:26
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/14; C12N9/24; C12N9/2402; C12N9/2405; C12N9/2408; C07; C07K; C07K1/00; C07K1/107; C07K1/1072; C07K1/1077; C07K14/00; C07K14/435; C07K14/76; C07K14/765; C12N9/16; C12N9/22; C12P; C12P21/00; C12P21/02
Claims Analysis
Total Claims: 29
Claim 1
Independent Claim
43 words
A method for modifying a glycoprotein including at least one asparagine linked oligosaccharide chain comprising: attaching a galactose residue to an N-acetylglucosamine residue which is linked to an asparagine through an N-glycosidic bond to form a Gal→GlcNAc→Asn(protein) sequence as part of a glycoprotein.
Claim 2
Independent Claim
43 words
A method for modifying a glycoprotein including at least one asparagine linked oligosaccharide chain comprising: attaching a galactose residue to an N-acetylglucosamine residue which is linked to an asparagine through an N-glycosidic bond to form a Gal→GlcNAc→Asn(protein) sequence as part of a glycoprotein.
Claim 3
Dependent Claim
28 words
The method of claim 1 further comprising first cleaving asparagine-linked oligosaccharide chains of the glycoprotein to remove all sugars other than core N-acetylglucosamine residues bound to the glycoprotein.
Claim 4
Dependent Claim
14 words
The method of claim 2 wherein the oligosaccharide chains are cleaved by an endoglycosidase.
Claim 5
Dependent Claim
30 words
The method of claim 3 wherein the endoglycosidase is selected from the group consisting of endo-β-N-acetylglucosaminidase H, endo-β-N-acetylglucosaminidase F, endo-β-N-acetylglucosaminidase C II , endo-β-N-acetylglucosaminidase D, endo-β-N-acetylglucosaminidase L, and combinations thereof.
Claim 6
Dependent Claim
25 words
The method of claim 4 further comprising cleaving O-linked oligosaccharide chains with an enzyme selected from the group consisting of α-mannosidase, endo-α-N-acetylgalactosaminidase, and combinations thereof.
Claim 7
Dependent Claim
25 words
The method of claim 1 further comprising cleaving O-linked oligosaccharide chains with an enzyme selected from the group consisting of α-mannosidase, endo-α-N-acetylgalactosaminidase, and combinations thereof.
Claim 8
Dependent Claim
16 words
The method of claim 2 wherein the oligosaccharide chain is cleaved by digestion with an exoglycosidase.
Claim 9
Dependent Claim
25 words
The method of claim 7 wherein the exoglycosidase is selected from the group consisting of sialidase, α-mannosidase, β-mannosidase, α-galactosidase, β-galactosidase, α-fucosidase, β-hexosaminidase, and combinations thereof.
Claim 10
Dependent Claim
36 words
The method of claim 8 wherein the oligosaccharide chains are sequentially cleaved by: (1) digesting the glycoprotein with α-mannosidase to remove α-mannose residues; (2) digesting the product of step (1) with β-mannosidase to remove β-mannose residues.
Claim 11
Dependent Claim
23 words
The method of claim 9 further comprising digesting the glycoprotein with an additional enzyme selected from the group consisting of exoglycosidases and phosphatases.
Claim 12
Dependent Claim
15 words
The method of claim 10 wherein the product of step 2 is digested with β-hexosaminidase.
Claim 13
Dependent Claim
25 words
The method of claim 2 wherein the oligosaccharide chains are cleaved by sequentially digesting the glycoprotein first with an exoglycosidase and secondly with an endoglycosidase.
Claim 14
Dependent Claim
44 words
The method of claim 12 wherein the oligosaccharide chains are cleaved by (1) digesting the glycoprotein with α-mannosidase to remove α-mannose residues; and (2) digesting the product of step (1) with an endoglycosidase selected from the group consisting of endo-β-N-acetylglucosaminidase L and endo-β-N-acetylglucosaminidase D.
Claim 15
Dependent Claim
52 words
The method of claim 12 for modifying glycoproteins wherein the oligosaccharide chains are cleaved by digesting the glycoprotein with an enzyme selected from the group consisting of sialidase, α-galactosidase, β-galactosidase, β-hexosaminidase, α-fucosidase, and combinations thereof followed by digestion with an enzyme selected from the group consisting of endo-β-N-acetylglucosaminidase D and endo-β-N-acetylglucosaminidase F.
Claim 16
Dependent Claim
17 words
The method of claim 2 further comprising cleaving high-mannose oligosaccharide chains with α-mannosidase to remove mannose residues.
Claim 17
Dependent Claim
14 words
The method of claim 2 wherein the oligosaccharide chains are cleaved by chemical treatment.
Claim 18
Dependent Claim
26 words
The method of claim 16 wherein the oligosaccharide chains are cleaved with a compound selected from the group consisting of trifluromethane sulfonic acid and hydrofluoric acid.
Claim 19
Dependent Claim
19 words
The method of claim 1 further comprising first producing the glycoproteins in cells in the presence of glycosidase inhibitor.
Claim 20
Dependent Claim
21 words
The method of claim 18 wherein the glycosidase inhibitor is selected from the group consisting of deoxymannojirimycin, swainsonine, castanospermine and deoxynojirimycin.
Claim 21
Dependent Claim
23 words
The method of claim 1 further comprising first producing the glycoprotein in cells with one or more mutations in the oligosaccharide processing pathway.
Claim 22
Dependent Claim
18 words
The method of claim 1 wherein the galactose residue is attached to the N-acetylglucosamine residue by a galactosyltransferase.
Claim 23
Dependent Claim
22 words
The method of claim 21 wherein the galactosyltransferase is selected from the group consisting of UDP-Gal:GlcNAc-R β1→4 galactosyltransferase and UDP-Gal:GlcNAc-R β1→3 galactosyltransferase.
Claim 24
Dependent Claim
94 words
The method of claim 1 wherein the galactose is attached to the N-acetylglucosamine by: (1) incubating UDP-Gal:GlcNAc-R β1→4 galactosyltransferase with the derivatized protein to attach a terminal galactose to the GlcNAc; (2) incubating the product of step 1 with UDP-GlcNAc:-Galβ1→4GlcNAc-R β1→3 N-acetylglucosaminyltransferase to attach a terminal GlcNAc to the terminal galactose; (3) incubating the product of step 2 with UDP-Gal:GlcNAc-R 1→4 galactosyltransferase to attach a terminal galactose to the terminal GlcNAc; and (4) repeating steps 2 and 3 until an oligosaccharide chain (Galβ1→4GlcNAcβ1→3) n units, wherein n is between 1 and 10, is produced.
Claim 25
Dependent Claim
26 words
The method of claim 1 or 23 further comprising attaching a saccharide selected from the group consisting of fucose and alpha galactose to a Gal→GlcNAc→ sequence.
Claim 26
Dependent Claim
34 words
The method of claim 24 wherein the saccharide is attached to the Gal-GlcNAc→ sequence with an enzyme selected from the group consisting of GPD-Fuc:GlcNAc α 1 → fucosyltransferase and UDP-Gal:GalNAc α 1 →3 galactosyltransferase.
Claim 27
Dependent Claim
43 words
The method of claim 1 further comprising attaching the galactose residues to the N-acetylglucosamine in a solution containing a non-ionic detergent, a chaotropic agent, an organic solvent, urea, a protease inhibitor, an exoglycosidase inhibitor, a disulfide bond reducing agent, or a combination thereof.
Claim 28
Dependent Claim
23 words
The method of claim 1 wherein the sialic acid residue is attached to the Gal-GlcNAc sequence in an α linkage by a sialyltransferase.
Claim 29
Dependent Claim
25 words
The method of claim 27 wherein the sialyltransferase is selected from the group consisting of CMP-SA:Galβ1→4GlcNAc-R β2→6 sialyltransferase, CMP-SA:Galβ1→3(4)GlcNAc α2→3 sialyltransferase, and CMP-SA:Galβ1→GlcNAc α2→3 sialyltransferase.
References Cited (Backward Citations)
US4184917A
Cited
US4326033A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1993-05-14 | 1993-11-13 | 1994-05-15 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1997-05-14 | 1997-11-13 | 1998-05-15 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2001-05-14 | 2001-11-13 | 2002-05-15 | $7700.00 | Window Closed / Lapsed |