Glycosylation of Molecules

• US 20110201540A1
• Filed: 04/26/2011
• Published: 08/18/2011
• Est. Priority Date: 04/03/2007
• Status: Active Grant

• Current Assignee: Oxyrane UK Limited, Universiteit Gent Vzw, VIB VZW
• Original Assignee: Oxyrane UK Limited, Universiteit Gent Vzw, VIB VZW
• Inventors: De Pourcq, Karen Jacqueline Marcel, Geysens, Steven Christian Jozef, Callewaert, Nico Luc Marc, Vervecken, Wouter, Guerfal, Mouna
• Granted Patent: US 8,597,906 B2
• Time in Patent Office: Days
• US Class Current: 514/1.1
• 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: 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 comprise a deficiency in an 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 one or more N-glycan structures selected from group consisting of Man₃GlcNAc₂, Glc₁Man₅GlcNAc₂, and Glc₂Man₅GlcNAc₂.

9. The method of claim 1, wherein the altered glycosylation is Man₃GlcNAc₂, Glc₁Man₅GlcNAc₂, or Glc₂Man₅GlcNAc₂.

10. The method of claim 1, wherein the cell further is deficient in OCH1 activity.

11. The method of claim 1, wherein the cell further comprises a nucleic acid encoding a protein having glucosyltransferase activity.

12. The method of claim 11, wherein the protein having glucosyltransferase activity is ALG6.

13. The method of claim 1, wherein the cell further comprises a nucleic acid comprising a nucleotide sequence encoding 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-mannosidase is MNS1.

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

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

18. The method of claim 1, wherein the cell further comprises a nucleic acid encoding a protein capable of removing glucose residues from Man₅GlcNAc₂.

19. The method of claim 18, wherein the protein is a glucosidase II.

20. The method of claim 18, wherein the protein is a mutanase.

21. The method of claim 18, wherein the protein comprises one or both of the alpha and beta subunits of a glucosidase II.

22. The method of claim 1, wherein the cell further comprises an elevated level of an ALG6 activity;

and an elevated level of a glucosidase II activity.

23. The method of claim 1, wherein the cell comprises an elevated level of an ALG6 activity;

an elevated level of a glucosidase II activity; and

an elevated level of an alpha-mannosidase activity.

24. The method of claim 1, wherein the cell comprises a deficiency in an OCH1 activity;

an elevated level of an ALG6 activity;

an elevated level of a glucosidase II activity; and

an elevated level of an alpha-mannosidase activity.

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

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

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

28. An isolated protein having altered N-glycosylation, wherein the protein is produced by the method of claim 1.

29. The isolated protein of claim 28, wherein said protein is an antibody or antigen-binding fragment thereof.

30. The isolated protein of claim 29, wherein said antigen-binding fragment is selected from the group consisting of Fab, F(ab′

)2, Fv, and scFv fragments.

34. A method of treating a disorder treatable by administration of a protein having altered N-glycosylation, the method comprising:

administering to a subject a protein of claim 28,

wherein the subject is one having, or suspected of having, a disease treatable by administration of a protein having altered N-glycosylation.

31. An isolated Yarrowia lipolytica or Arxula adeninivorans cell genetically engineered to comprise a deficiency in an ALG3 activity.

32. The isolated cell of claim 31, said cell further comprising a nucleic acid encoding a protein having glucosyltransferase activity.

33. The isolated cell of claim 32, 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.

35. 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 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.

36. The method of claim 35, said cell further comprising a nucleic acid encoding a protein having glucosyltransferase activity.

37. A substantially pure culture of Yarrowia lipolytica or Arxula adeninivorans cells, a substantial number of which are genetically engineered to comprise a deficiency in an ALG3 activity.

38. The substantially pure culture of claim 37, said cells further comprising a nucleic acid encoding a protein having glucosyltransferase activity.

39. The substantially pure culture of claim 38, said cells 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.