Methods for introducing mannose 6-phosphate and other oligosaccharides onto glycoproteins

• US 7,001,994 B2
• Filed: 01/17/2002
• Issued: 02/21/2006
• Est. Priority Date: 01/18/2001
• Status: Active Grant

• Current Assignee: Genzyme Corporation (Sanofi )
• Original Assignee: Genzyme Corporation (Sanofi )
• Inventors: Zhu, Yunxiang
• Primary Examiner(s): Wilson, James O.
• Assistant Examiner(s): Khare, Devesh
• Application Number:
  US10/051,711
• Publication Number:
  US 20020137125A1
• Time in Patent Office: 1,496 Days
• Field of Search: 536/4.1, 536/18.5, 536/18.6, 536/123.1, 536/124, 536/23.2, 536/23.5, 536/23.1, 435/68.1, 435/206, 435/195, 435/6, 435/69.1, 435/18, 435/193, 435/196, 435/208, 435/69.7, 435/97, 424/94.61, 424/155.1, 424/179.1, 424/183.1, 530/350
• US Class Current: 536/4.1
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: C12P 21/005 Glycopeptides, glycoproteins

Abstract

Methods to introduce highly phosphorylated mannopyranosyl oligosaccharide derivatives containing mannose 6-phosphate (M6P), or other oligosaccharides bearing other terminal hexoses, to carbonyl groups on oxidized glycans of glycoproteins while retaining their biological activity are described. The methods are useful for modifying glycoproteins, including those produced by recombinant protein expression systems, to increase uptake by cell surface receptor-mediated mechanisms, thus improving their therapeutic efficacy in a variety of applications.

Claims

1. A method for coupling a oligosaccharide comprising a phosphorylated hexose to a lysosomal enzyme, the method comprising the steps of:

(a) derivatizing the oligosaccharide comprising a phosphorylated hexose with a compound containing a carbonyl-reactive group;

(b) oxidizing the lysosomal enzyme to generate at least one carbonyl group on the lysosomal enzyme; and

(c) reacting the derivatized oligosaccharide with the oxidized lysosomal enzyme,

thereby coupling the oligosaccharide to the lysosomal enzyme.

2. The method according to claim 1, wherein the phosphate group is linked to a terminal mannose.

3. The method according to claim 1, wherein the phosphate group is linked to a penultimate mannose.

4. The method according to claim 1, wherein the oligosaccharide comprises two or more mannose-6-phosphate (M6P) groups.

5. The method according to claim 1, wherein the oxidizing step is carried out with periodate or galactose oxidase.

6. The method according to claim 1, wherein the lysosomal enzyme is chosen from beta-glucocerebrosidase, alpha-galactosidase A, acid alpha-glucosidase, alpha-N-acetylglucosaminidase, beta-N-acetyl-hexosaminidase, and beta-glucuronidase.

7. The method according to claim 1, wherein the oligosaccharide is chosen from a biantennary mannopyranosyl oligosaccharide and a triantennary mannopyranosyl oligosaccharide.

8. The method according to claim 7, wherein the biantennary mannopyranosyl oligosaccharide comprises bis-M6P.

9. The method according to claim 7, wherein the triantennary mannopyrannosyl oligoscacharide comprises bis-M6P or tri-M6P.

10. The method according to claim 1, wherein the oligosaccharide comprises:

wherein M is mannose or a mannopyranosyl group.

11. The method according to claim 1, wherein the derivatized oligosaccharide has a formula chosen from 6-P-M_n-R and (6P-M_x)_mL_n-R,

wherein M is mannose or a mannopyranosyl group,

P is a phosphate group linked to the C-6 position of M,

L is a hexose,

R is a compound containing at least one carbonyl-reactive group,

m is an integer ranging from 2 to 3,

n is an integer ranging from 1 to 15, wherein if n>

1, the M_n are linked to one another by alpha (1,2), alpha (1,3), alpha (1,4), or alpha (1,

     6), and

x is an integer ranging from 1 to 15.

12. The method according to claim 11, wherein at least one L is mannose.

13. The method according to claim 11, wherein at least one L is chosen from galactose, N-acetylglucosamine, and fucose.

14. The method according to claim 1 or claim 11, wherein the carbonyl-reactive group is chosen from a hydrazine, a hydrazide, an aminooxy, a semicarbazide.

15. The method according to claim 1, further comprising the step of adding a reducing agent to the coupled lysosomal enzyme.

16. The method according to claim 15, wherein the reducing agent comprises cyanoborohydride.

17. The method of claim 1, wherein the lysosomal enzyme is acid alpha-glucosidase and the carbonyl-reactive group is chosen from a hydrazine, a hydrazide, an aminooxy, a semicarbazide.

18. The method of claim 1, wherein the lysosomal enzyme is acid alpha-glucosidase.

19. The method of claim 1, wherein the carbonyl-reactive group is aminooxy.