Yeast strains producing mammalian-like complex N-glycans

• US 10,287,557 B2
• Filed: 03/06/2015
• Issued: 05/14/2019
• Est. Priority Date: 11/19/2009
• Status: Active Grant

• Current Assignee: Oxyrane UK Limited
• Original Assignee: Oxyrane UK Limited
• Inventors: Geysens, Steven Christian Jozef, Vervecken, Wouter
• Primary Examiner(s): Kolker, Daniel E
• Assistant Examiner(s): Seharaseyon, Jegatheesan
• Application Number:
  US14/641,002
• Publication Number:
  US 20150337273A1
• Time in Patent Office: 1,530 Days
• Field of Search: None
• 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 35/00 Antineoplastic agents
• CPC Class Codes: C07K 16/32 against translation products of oncogenes
• CPC Class Codes: C07K 2317/14 Specific host cells or culture conditions, e.g. components, pH or temperature
• CPC Class Codes: C07K 2317/41 Glycosylation, sialylation, or fucosylation
• CPC Class Codes: C12N 15/81 for yeasts
• CPC Class Codes: C12N 9/1051 Hexosyltransferases (2.4.1)
• 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: C12N 9/60 from yeast
• CPC Class Codes: C12P 21/005 Glycopeptides, glycoproteins
• CPC Class Codes: C12Y 204/01 Hexosyltransferases (2.4.1)
• CPC Class Codes: C12Y 204/01133 Xylosylprotein 4-beta-galactosyltransferase (2.4.1.133)
• CPC Class Codes: C12Y 302/01024 Alpha-mannosidase (3.2.1.24)
• CPC Class Codes: C12Y 302/01084 Glucan 1,3-alpha-glucosidase (3.2.1.84), i.e. mutanase
• CPC Class Codes: C12Y 302/01114 Mannosyl-oligosaccharide 1,3-1,6-alpha-mannosidase (3.2.1.114)

Abstract

Described herein are methods and genetically engineered fungal cells useful for producing target molecules containing mammalian-like complex N-glycans or containing intermediates in a mammalian glycosylation pathway.

Claims

1. A substantially pure culture of Yarrowia lipolytica cells, a substantial number of which are genetically engineered to produce glycoproteins comprising GalGlcNAc₂Man₃GlcNAc₂ or Gal₂GlcNAc₂Man₃GlcNAc₂ N-glycans, wherein said cells are genetically engineered to be deficient in Outer CHain elongation (OCH1) activity and in Asparagine Linked Glycosylation 3 (ALG3) activity and to comprise one or more nucleic acids encoding an α

-1,2-mannosidase, a nucleic acid encoding Asparagine Linked Glycosylation 6 (ALG6), and one or more nucleic acids encoding each of a GlcNAc-transferase I, a GlcNAc-transferase II, and a galactosyltransferase, wherein each of said one or more nucleic acids encoding said α

-1,2-mannosidase, said GlcNAc-transferase I, said GlcNAc-transferase II, and said galactosyltransferase comprises nucleotide sequences encoding targeting sequences to target each encoded protein to an intracellular compartment, wherein said cells produce proteins comprising GalGlcNAc₂Man₃GlcNAc₂ or Gal₂GlcNAc₂Man₃GlcNAc₂ N-glycans.

2. The culture of claim 1, wherein each of said cells further comprises a nucleic acid encoding a target protein, wherein each of said cells produces said target protein comprising GalGlcNAc₂Man₃GlcNAc₂ or Gal₂GlcNAc₂Man₃GlcNAc₂ N-glycans.

3. The culture of claim 2, wherein the target protein is a protein selected from the group consisting of a therapeutic glycoprotein, an antibody or fragment thereof, interferon-β

, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-γ

, and erythropoietin.

4. The culture of claim 2, wherein said target protein is an antibody or fragment thereof comprising an Fc part that can interact with an Fc receptor.

5. The culture of claim 1, wherein said intracellular compartment is the Golgi apparatus.

6. The culture of claim 1, wherein said coding sequence encoding said α

-1,2-mannosidase comprises an endoplasmic reticulum targeting sequence to target the encoded α

-1,2-mannosidase to the endoplasmic reticulum.

7. The culture of claim 6, wherein said endoplasmic reticulum targeting sequence is an HDEL endoplasmic reticulum-retention (HDEL) sequence (SEQ ID NO:

21).

8. The culture of claim 1, wherein each of said cells further comprise a nucleic acid encoding the α

and β

subunits of a Glucosidase II.

9. An isolated Yarrowia lipolytica cell genetically engineered to produce proteins comprising GalGlcNAc₂Man₃GlcNAc₂ or Gal₂GlcNAc₂Man₃GlcNAc₂ N-glycans, wherein said cell is genetically engineered to be deficient in Outer CHain elongation (OCH1) activity and in Asparagine Linked Glycosylation 3 (ALG3) activity and to comprise one or more nucleic acids encoding an α

-1,2-mannosidase, a nucleic acid encoding Asparagine Linked Glycosylation 6 (ALG6), and one or more nucleic acids encoding each of a GlcNAc-transferase I, a GlcNAc-transferase II, and a galactosyltransferase, wherein each of said one or more nucleic acids encoding said α

-1,2-mannosidase, said GlcNAc-transferase I, said GlcNAc-transferase II, and said galactosyltransferase comprises nucleotide sequences encoding targeting sequences to target each encoded protein to an intracellular compartment, wherein said cell produces proteins comprising GalGlcNAc₂Man₃GlcNAc₂ or Gal₂GlcNAc₂Man₃GlcNAc₂ N-glycans.

10. The Yarrowia lipolytica cell of claim 9, wherein said cell further comprises a nucleic acid encoding a target protein, wherein each of said cells produces said target protein comprising GalGlcNAc2Man3GlcNAc2 or Gal2GlcNAc2Man3GlcNAc2 N-glycans.

11. The Yarrowia lipolytica cell of claim 10, wherein the target protein is a protein selected from the group consisting of a therapeutic glycoprotein, an antibody or fragment thereof, interferon-β

, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-γ

, and erythropoietin.

12. The Yarrowia lipolytica cell of claim 10, wherein said target protein is an antibody or fragment thereof comprising an Fc part that can interact with an Fc receptor.

13. The Yarrowia lipolytica cell of claim 9, wherein said intracellular compartment is the Golgi apparatus.

14. The Yarrowia lipolytica cell of claim 9, wherein said coding sequence encoding said α

-1,2-mannosidase is linked to an endoplasmic reticulum targeting sequence to target the encoded α

-1,2-mannosidase to the endoplasmic reticulum.

15. The Yarrowia lipolytica cell of claim 14, wherein said endoplasmic reticulum targeting sequence is an HDEL endoplasmic reticulum-retention (HDEL) sequence (SEQ ID NO:

21).

16. The Yarrowia lipolytica cell of claim 9, wherein said cell further comprises a nucleic acid encoding the α

and β

subunits of a Glucosidase II.