Mannosidases capable of uncapping mannose-1-phospho-6-mannose linkages and demannosylating phosphorylated N-glycans and methods of facilitating mammalian cellular uptake of glycoproteins

• US 9,347,050 B2
• Filed: 09/29/2011
• Issued: 05/24/2016
• Est. Priority Date: 09/29/2010
• Status: Active Grant

• Current Assignee: VIB VZW
• Original Assignee: Oxyrane UK Limited
• Inventors: Piens, Kathleen Camilla Telesphore Alida Maria, Vervecken, Wouter, Valevska, Albena Vergilieva, Pynaert, Gwenda Nolla
• Primary Examiner(s): Ha, Julie
• Assistant Examiner(s): Hellman, Kristina M
• Application Number:
  US13/876,769
• Publication Number:
  US 20130267473A1
• Time in Patent Office: 1,699 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 38/16 Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof enzyme inhibitors A61K38/005
• CPC Class Codes: A61K 38/1709 from mammals
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 9/2402 hydrolysing O- and S- glycosyl compounds (3.2.1)
• CPC Class Codes: C12N 9/2488 Mannanases
• CPC Class Codes: C12P 21/005 Glycopeptides, glycoproteins
• CPC Class Codes: C12Y 302/01 Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
• CPC Class Codes: C12Y 302/01024 Alpha-mannosidase (3.2.1.24)
• CPC Class Codes: Y02P 20/52 using catalysts, e.g. selective catalysts

Abstract

The invention provides mannosidases capable of uncapping mannose-1-phospho-6-mannose moieties and demannosylating phosphorylated N-glycans, methods of using such mannosidases, glycoproteins produced using the methods, as well as methods of facilitating mammalian cellular uptake of glycoproteins.

Claims

1. A method of directing a glycoprotein to the interior of a mammalian cell, the glycoprotein comprising a mannose-1-phospho-6-mannose moiety, wherein the mannose residue that has a phosphate residue bound at the 6 position is linked to a terminal mannose residue at the 1 position of the terminal mannose residue, the method comprising contacting the cell with the glycoprotein after it has undergone:

(a) uncapping of the mannose-1-phospho-6-mannose moiety to mannose-6-phosphate on the glycoprotein; and

(b) removal of the terminal mannose residue,

wherein the glycoprotein that has undergone (a) and not (b) or (b) but not (a) does not substantially bind to a mannose-6-phosphate receptor on the cell,

wherein the glycoprotein that has undergone (a) and (b) does substantially bind to a mannose-6-phosphate receptor on the cell, and

wherein steps (a) and (b) are catalyzed by a single mannosidase enzyme that is a family 38 glycosyl hydrolase (GH38 family) mannosidase.

2. The method of claim 1, wherein said glycoprotein is a human protein.

3. The method of claim 1, wherein said glycoprotein is a pathogen protein, a lysosomal protein, a growth factor, a cytokine, a chemokine, an antibody or antigen-binding fragment thereof, or a fusion protein.

4. The method of claim 3, wherein said lysosomal protein is a lysosomal enzyme.

5. The method of claim 4, wherein said lysosomal enzyme is acid alpha glucosidase or alpha galactosidase.

6. The method of claim 1, wherein said glycoprotein is associated with a lysosomal storage disease (LSD).

7. The method of claim 6, wherein said LSD is Fabry'"'"'s disease, mucopolysaccharidosis I, Farber disease, Gaucher disease, GM1-gangliosidosis, Tay-Sachs disease, Sandhoff disease, GM2 activator disease, Krabbe disease, metachromatic leukodystrophy, Niemann-Pick disease, Scheie disease, Hunter disease, Sanfilippo disease, Morquio disease, Maroteaux-Lamy disease, hyaluronidase deficiency, aspartylglucosaminuria, fucosidosis, mannosidosis, Schindler disease, sialidosis type 1, Pompe disease, Pycnodysostosis, ceroid lipofuscinosis, cholesterol ester storage disease, Wolman disease, Multiple sulfatase deficiency, galactosialidosis, mucolipidosis, cystinosis, sialic acid storage disorder, chylomicron retention disease with Marinesco-Sjö

gren syndrome, Hermansky-Pudlak syndrome, Chediak-Higashi syndrome, Danon disease, or Geleophysic dysplasia.