Glycosylation of molecules

• US 8,597,906 B2
• Filed: 04/26/2011
• Issued: 12/03/2013
• Est. Priority Date: 04/03/2007
• Status: Active Grant

• Current Assignee: Universiteit Gent Vzw, VIB VZW
• Original Assignee: Oxyrane UK Limited, Universiteit Gent Vzw, VIB VZW
• Inventors: Callewaert, Nico Luc Marc, Vervecken, Wouter, De Pourcq, Karen Jacqueline Marcel, Geysens, Steven Christian Jozef, Guerfal, Mouna
• Primary Examiner(s): Brunner, Bridget E
• Assistant Examiner(s): HAMUD, FOZIA M
• Application Number:
  US13/094,606
• Publication Number:
  US 20110201540A1
• Time in Patent Office: 952 Days
• Field of Search: None
• US Class Current: 435/68.1
• 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: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 3/06 Antihyperlipidemics
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/06 Antianaemics
• CPC Class Codes: C07K 14/00 Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• CPC Class Codes: C12N 9/1051 Hexosyltransferases (2.4.1)
• CPC Class Codes: C12N 9/24 acting on glycosyl compounds (3.2)
• CPC Class Codes: C12N 9/2402 hydrolysing O- and S- glycosyl compounds (3.2.1)
• CPC Class Codes: C12N 9/2465 acting on alpha-galactose-glycoside bonds, e.g. alpha-galactosidase (3.2.1.22)
• CPC Class Codes: C12N 9/2488 Mannanases
• CPC Class Codes: C12P 21/005 Glycopeptides, glycoproteins
• CPC Class Codes: C12P 21/06 produced by the hydrolysis of a peptide bond, e.g. hydrolysate products preparing foodstuffs by protein hydrolysis A23J3/00
• CPC Class Codes: C12Y 204/01232 Initiation-specific alpha-1,6-mannosyltransferase (2.4.1.232)
• CPC Class Codes: C12Y 302/01024 Alpha-mannosidase (3.2.1.24)
• CPC Class Codes: C12Y 302/01045 : Glucosylceramidase (3.2.1.45), i.e. beta-glucocerebrosidase
• CPC Class Codes: C12Y 302/01046 : Galactosylceramidase (3.2.1.46)
• CPC Class Codes: C12Y 302/01084 : Glucan 1,3-alpha-glucosidase (3.2.1.84), i.e. mutanase
• CPC Class Codes: C12Y 302/01113 : Mannosyl-oligosaccharide 1,2-alpha-mannosidase (3.2.1.113), i.e. alpha-1,2-mannosidase
• CPC Class Codes: Y02P 20/52 : using catalysts, e.g. selective catalysts

Abstract

Described herein are methods and genetically engineered cells useful for producing an altered N-glycosylation form of a target molecule. Also described are methods and molecules with altered N-glycosylation useful for treating a variety of disorders such as metabolic disorders.

Claims

1. A method of producing an altered N-glycosylation form of a target protein, the method comprising:

providing a Yarrowia lipolytica or an Arxula adeninivorans cell genetically engineered to express a protein capable of removing glucose residues from GlcMan₅GlcNAc₂ or Glc₂Man₅GlcNac₂ and to comprise a deficiency in an Asparagine Linked Glycosylation 3 (ALG3) activity; and

introducing into the cell a nucleic acid encoding a target protein, wherein the cell produces the target protein in an altered N-glycosylation form relative to the N-glycosylation form of the target protein produced in a corresponding non-genetically engineered cell.

2. The method of claim 1, further comprising isolating the altered N-glycosylation form of the target protein.

3. The method of claim 1, wherein the target protein is an exogenous protein.

4. The method of claim 1, wherein the target protein is an endogenous protein.

5. The method of claim 1, wherein the target protein is a human protein.

6. The method of claim 1, wherein the target protein is a pathogen protein, a growth factor, a cytokine, a chemokine, an antibody or antigen-binding fragment thereof, or a fusion protein.

7. The method of claim 6, wherein said antigen-binding fragment is selected from the group consisting of Fab, F(ab′

)₂, Fv, and single chain Fv (scFv) fragments.

8. The method of claim 1, wherein the altered N-glycosylation form comprises Man₅GlcNAc₂.

9. The method of claim 1, wherein the altered glycosylation is Man₅GlcNAc₂.

10. The method of claim 1, wherein the cell is further genetically engineered to be deficient in Outer Chain Elongation (OCH1)activity.

11. The method of claim 1, wherein the cell is further genetically engineered to express a protein having glucosyltransferase activity.

12. The method of claim 11, wherein the protein having glucosyltransferase activity is Asparagine Linked Glycosylation 6 (ALG6).

13. The method of claim 1, wherein the cell is further genetically engineered to express an alpha-mannosidase.

14. The method of claim 13, wherein the alpha-mannosidase is an alpha-1,2-mannosidase.

15. The method of claim 14, wherein the alpha-1,2-mannosidase is mannosidase 1 (MNS1).

16. The method of claim 14, wherein the alpha-1,2-mannosidase is targeted to the endoplasmic reticulum.

17. The method of claim 16, wherein the alpha-1,2-mannosidase is a fusion protein comprising an alpha-1,2-mannosidase polypeptide and an HDEL endoplasmic reticulum retention peptide.

18. The method of claim 1, wherein the protein capable of removing glucose residues from GlcMan₅GlcNAc₂ or Glc₂Man₅GlcNAc₂ is a glucosidase II.

19. The method of claim 18, wherein the cell is further genetically engineered to express ALG6 activity.

20. The method of claim 18, wherein the cell is further genetically engineered to express ALG6 activity;

and alpha-mannosidase activity.

21. The method of claim 18, wherein the cell is further genetically engineered to be deficient in OCH1 activity;

to express ALG6 activity; and

to express alpha-mannosidase activity.

22. The method of claim 1, wherein the protein capable of removing glucose residues from Man₅GlcNAc₂ is a mutanase.

23. The method of claim 1, wherein the protein capable of removing glucose residues from GlcMan₅GlcNAc₂ or Glc₂Man₅GlcNAc₂ comprises one or both of the alpha and beta subunits of a glucosidase II.

24. The method of claim 1, wherein the cell is not genetically engineered to be deficient in OCH1 activity.

25. The method of claim 1, further comprising additional processing of the glycoprotein.

26. The method of claim 25, wherein the additional processing comprises enzymatic or chemical treatment of the altered N-glycosylation form of the target protein.

27. The method of claim 1, wherein the protein capable of removing glucose residues from GlcMan₅GlcNAc₂ or Glc₂Man₅GlcNAc₂ comprises an endoplasmic reticulum retention peptide.

28. An isolated Yarrowia lipolytica or Arxula adeninivorans cell genetically engineered to express a protein capable of removing glucose residues from GlcMan₅GlcNAc₂ or Glc₂Man₅GlcNAc₂ and to comprise a deficiency in an ALG3 activity.

29. The isolated cell of claim 28, said cell being further genetically engineered to express a protein having glucosyltransferase activity.

30. The isolated cell of claim 28, said cell further comprising a nucleic acid encoding a target protein, wherein the cell produces the target protein in an altered N-glycosylation form relative to the N-glycosylation form of the target protein produced in a corresponding non-genetically engineered cell.

31. The isolated cell of claim 28, wherein the protein capable of removing glucose residues from GlcMan₅GlcNAc₂ or Glc₂Man₅GlcNAc₂ comprises an endoplasmic reticulum retention peptide.

32. A method of producing an altered N-glycosylation form of a target protein, the method comprising contacting a target protein with a cell lysate prepared from a Yarrowia lipolytica or an Arxula adeninivorans cell genetically engineered to express a protein capable of removing glucose residues from GlcMan₅GlcNAc₂ or Glc₂Man₅GlcNAc₂ and to comprise a deficiency in an ALG3 activity, wherein the contacting of the target protein with the cell lysate results in an altered N-glycosylation form of the target protein relative to the N-glycosylation form of the target protein produced from the cell lysate of a corresponding non-genetically engineered cell.

33. The method of claim 32, said cell being further genetically engineered to express a protein having glucosyltransferase activity.

34. The method of claim 32, wherein the protein capable of removing glucose residues from GlcMan₅GlcNAc₂ or Glc₂Man₅GlcNAc₂ comprises an endoplasmic reticulum retention peptide.

35. A substantially pure culture of Yarrowia lipolytica or Arxula adeninivorans cells, wherein the culture comprises Yarrowia lipolytica or Arxula adeninivorans cells which are genetically engineered to express a protein capable of removing glucose residues from GlcMan₅GlcNAc₂ or Glc₂Man₅GlcNAc₂ and to comprise a deficiency in an ALG3 activity.

36. The substantially pure culture of claim 35, said genetically engineered cells being further genetically engineered to express a protein having glucosyltransferase activity.

37. The substantially pure culture of claim 36, said genetically engineered cells further comprising a nucleic acid encoding a target protein, wherein the genetically engineered cells comprising a nucleic acid encoding a target protein produce the target protein in an altered N-glycosylation form relative to the N-glycosylation form of the target protein produced in a corresponding non-genetically engineered cell.

38. The substantially pure culture of claim 35, wherein the protein capable of removing glucose residues from GlcMan₅GlcNAc₂ or Glc₂Man₅GlcNAc₂ comprises an endoplasmic reticulum retention peptide.