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 20130267473A1
• Filed: 09/29/2011
• Published: 10/10/2013
• 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 Noella
• Granted Patent: US 9,347,050 B2
• Time in Patent Office: Days
• US Class Current: 514/20.9
• 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 for uncapping mannose-1-phospho-6-mannose moieties and demannosylating phosphorylated N-glycans on a glycoprotein, said method comprising

a) providing said glycoprotein having phosphorylated N-glycans containing said mannose-1-phospho-6-mannose moiety;

and

b) contacting said glycoprotein with a mannosidase capable of (i) hydrolyzing a mannose-1-phospho-6-mannose moiety to mannose-6-phosphate and (ii) hydrolyzing terminal alpha-1,2 mannose, alpha-1,3 mannose and/or alpha-1,6 mannose linkages.

3. The method of claim 1 or 2, wherein said mannosidase is a family 38 glycosyl hydrolase.

4. The method of any one of claims 1-3, wherein said mannosidase is from Canavalia ensiformis.

5. The method of any one of claims 1-3, wherein said mannosidase is from Yarrowia lipolytica.

6. The method of any one of claims 1-5, said method further comprising, after steps (a) and (b), contacting a mammalian cell with said glycoprotein comprising said demannosylated phosphorylated N-glycans, wherein, after said contacting, said glycoprotein is transported to the interior of said mammalian cell.

7. The method of claim 6, wherein said mammalian cell is a human cell.

33. The method of any one of claims 1-32, wherein said glycoprotein is a human protein.

34. The method of any one of claims 1-32, 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.

35. The method of claim 34, wherein said lysosomal protein is a lysosomal enzyme.

36. The method of claim 35, wherein said lysosomal enzyme is acid alpha glucosidase or alpha galactosidase.

37. The method of claim 33, wherein said glycoprotein is associated with a LSD.

38. The method of claim 37, 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.

39. A glycoprotein capable of being transported to the interior of a mammalian cell, wherein said glycoprotein has been treated with the method of any of claims 1-38.

40. A mammalian cell comprising said glycoprotein of claim 39.

41. The mammalian cell of claim 40, wherein said cell is a human cell.

42. A method of treatment, said method comprising administering the glycoprotein of claim 39 to a subject in need thereof.

54. The fungal cell of claim 33, wherein said polypeptide capable of promoting mannosyl phosphorylation is a MNN4 polypeptide.

2. A method of demannosylating phosphorylated N-glycans, said method comprising

a) providing a glycoprotein comprising phosphorylated N-glycans;

and

b) contacting said glycoprotein with a mannosidase capable of (i) hydrolyzing a mannose-1-phospho-6-mannose moeity to mannose-6-phosphate and (ii) hydrolyzing terminal alpha-1,2 mannose, alpha-1,3 mannose and/or alpha-1,6 mannose linkages.

8. A method of directing a glycoprotein to the interior of a mammalian cell, the method comprising a) providing a glycoprotein wherein its mannose-6-phosphate moieties have been demannosylated, and b) contacting said cell with said demannosylated glycoprotein.

9. The method of claim 8, wherein said mannosidase is a family 47 glycosyl hydrolase.

10. The method of claim 8 or 9, wherein said glycoprotein is demannosylated with a mannosidase from Aspergillus satoi.

11. The method of claim 8, wherein said mannosidase is a family 92 glycosyl hydrolase.

12. The method of claim 8 or claim 11, wherein said glycoprotein is demannosylated with a mannosidase from Cellulosimicrobium cellulans.

13. The method of claim 8, wherein said mannosidase is a family 38 glycosyl hydrolase.

14. The method of claim 8 or claim 13, wherein said mannosidase is from Canavalia ensiformis or Yarrowia lipolytica.

15. A method of directing a glycoprotein to the interior of a mammalian cell, said method comprising

a) providing a glycoprotein having a phosphorylated N-glycan, wherein said glycoprotein does not substantially bind to a mannose-6-phosphate receptor on said cell;

b) contacting said glycoprotein with a mannosidase capable of hydrolyzing a terminal alpha-1,2 mannose linkage when the underlying mannose is phosphorylated to produce a demannosylated glycoprotein, wherein said glycoprotein after said demannosylation, substantially binds to said mannose-6-phosphate receptor on said cell; and

c) contacting said cell with said demannosylated glycoprotein.

16. The method of claim 15, wherein said mannosidase is a family 47 glycosyl hydrolase.

17. The method of claim 15 or claim 16, wherein said mannosidase is from Aspergillus satoi.

18. The method of claim 15, wherein said mannosidase is a family 92 glycosyl hydrolase.

19. The method of claim 15 or claim 18, wherein said mannosidase is from Cellulosimicrobium cellulans.

20. The method of claim 15, wherein said mannosidase is a family 38 glycosyl hydrolase.

21. The method of claim 15 or 20, wherein said mannosidase is from Canavalia ensiformis or Yarrowia lipolytica.

22. A method of converting a glycoprotein from a first form that does not substantially bind to a mannose-6-phosphate receptor on a mammalian cell to a second form that does substantially bind to a mannose-6-phosphate receptor on a mammalian cell, wherein in the first form, the glycoprotein comprises one or more N-glycans containing one or more mannose residues that are linked at the 1 position to a mannose residue that contains a phosphate residue at the 6 position, the method comprising contacting the first form of the glycoprotein with a mannosidase that demannosylates said terminal mannose residues.

23. The method of claim 22, wherein said mannosidase has uncapping and demannosylating activity.

24. The method of claim 22 or claim 23, wherein said mannosidase is a family 38 glycosyl hydrolase.

25. The method of any one of claims 22-24, wherein said mannosidase is from Canavalia ensiformis or Yarrowia lipolytica.

26. The method of claim 22, wherein said mannosidase does not have uncapping activity.

27. The method of claim 22 or claim 26, wherein said mannosidase is a family 47 or family 92 glycosyl hydrolase.

28. The method of claim 22, claim 26, or claim 27, wherein said mannosidase is from Aspergillus satoi or Cellulosimicrobium cellulans.

29. A method of directing a glycoprotein to the interior of a mammalian cell, the glycoprotein comprising one or more mannose-1-phospho-6-mannose moieties, the method comprising contacting the cell with the glycoprotein after (a) uncapping the one or more mannose-1-phospho-6-mannose moieties to mannose-6-phosphate on the glycoprotein, wherein, after uncapping, the glycoprotein does not substantially bind to a mannose-6-phosphate receptor on the cell and, after step (a), (b) demannosylating phosphorylated N-glycans on the glycoprotein, wherein after both the uncapping and the demannosylation, the glycoprotein does substantially bind to a mannose-6-phosphate receptor on the cell.

30. The method of claim 29, wherein step (a) and step (b) are catalyzed by two different enzymes.

31. The method of claim 29, wherein step (a) and step (b) are catalyzed by a single enzyme.

32. A method of directing a glycoprotein to the interior of a mammalian cell, said method comprising providing a glycoprotein having uncapped and demannosylated phosphorylated N-glycans, and contacting said mammalian cell with said glycoprotein.

53. The fungal cell of any one of claims 32 to 41, wherein said fungal cell is a Yarrowia lipolytica or Arxula adeninivorans cell.

43. An isolated fungal cell genetically engineered to produce glycoproteins comprising demannosylated phosphorylated N-glycans, said fungal cell comprising a nucleic acid encoding a mannosidase, said mannosidase capable of (i) hydrolyzing a mannose-1-phospho-6-mannose moiety to mannose-6-phosphate and (ii) hydrolyzing a terminal alpha-1,2 mannose, alpha-1,3 mannose and/or alpha-1,6 mannose linkage.

44. The fungal cell of claim 43, said fungal cell further comprising a nucleic acid encoding a polypeptide capable of promoting mannosyl phosphorylation.

45. The fungal cell of claim 43 or claim 44, wherein said fungal cell is genetically engineered to be deficient in OCH1 activity.

46. The fungal cell of any one of claims 43 to 45, said fungal cell further comprising a nucleic acid encoding a target protein, wherein said target protein is a glycoprotein.

47. The fungal cell of claim 46, wherein said target protein is a human protein.

48. The fungal cell of claim 46, wherein said target protein 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.

49. The fungal cell of claim 48, wherein said lysosomal protein is a lysosomal enzyme.

50. The fungal cell of claim 49, wherein said lysosomal enzyme is acid alpha glucosidase or alpha galactosidase.

51. The fungal cell of claim 47, wherein said glycoprotein is a protein associated with a LSD.

52. The fungal cell of claim 51, 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.

55. The fungal cell of claim 43, wherein said MNN4 polypeptide is a Yarrowia lipolytica, S. cerevisiae, Ogataea minuta, Pichia pastoris, or C. albicans polypeptide.

56. The fungal cell of claim 44, wherein said polypeptide capable of promoting mannosyl phosphorylation is a P. pastoris PNO1 polypeptide.

57. The fungal cell of any one of claims 43 to 56, wherein said mannosidase comprises a secretion signal.

58. The fungal cell of any one of claims 43 to 56, wherein said mannosidase comprises a targeting signal to target said mannosidase to an intracellular compartment.