Methods for producing modified glycoproteins

• US 7,029,872 B2
• Filed: 06/27/2001
• Issued: 04/18/2006
• Est. Priority Date: 06/28/2000
• Status: Expired due to Fees

• Current Assignee: GlycoFi, Inc. (Merck & Co., Inc.)
• Original Assignee: GlycoFi, Inc. (Merck & Co., Inc.)
• Inventors: Gerngross, Tillman U.
• Primary Examiner(s): QIAN, CELINE X
• Application Number:
  US09/892,591
• Publication Number:
  US 20020137134A1
• Time in Patent Office: 1,756 Days
• Field of Search: 530/395, 530/402, 435/320.1, 435/254.2, 435/69.1, 435/455
• US Class Current: 435/69.1
• 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 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: C07K 2319/04 containing an ER retention signal such as a C-terminal HDEL motif
• CPC Class Codes: C07K 2319/05 containing a GOLGI retention signal
• CPC Class Codes: C12N 1/14 Fungi culture of mushrooms A01G18/00; as new plants A01H15/00; Culture media therefor
• CPC Class Codes: C12N 15/79 Vectors or expression systems specially adapted for eukaryotic hosts
• CPC Class Codes: C12N 15/80 for fungi
• CPC Class Codes: C12N 15/81 for yeasts
• CPC Class Codes: C12N 9/1048 Glycosyltransferases (2.4)
• 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 302/01113 Mannosyl-oligosaccharide 1,2-alpha-mannosidase (3.2.1.113), i.e. alpha-1,2-mannosidase

Abstract

Cell lines having genetically modified glycosylation pathways that allow them to carry out a sequence of enzymatic reactions, which mimic the processing of glycoproteins in humans, have been developed. Recombinant proteins expressed in these engineered hosts yield glycoproteins more similar, if not substantially identical, to their human counterparts. Thelower eukaryotes, which ordinarily produce high-mannose containing N-glycans, including unicellular and multicellular fungi are modified to produce N-glycans such as Man₅GlcNAc₂ or other structures along human glycosylation pathways. This is achieved using a combination of engineering and/or selection of strains which: do not express certain enzymes which create the undesirable complex structures characteristic of the fungal glycoproteins, which express exogenous enzymes selected either to have optimal activity under the conditions present in the fungi where activity is desired, or which are targeted to an organelle where optimal activity is achieved, and combinations thereof wherein the genetically engineered eukaryote expresses multiple exogenous enzymes required to produce “human-like” glycoproteins.

Claims

1. A method for producing a recombinant glycoprotein comprising an N-glycan structure that comprises a Man₅GlcNAc₂ glycoform in a lower eukaryotic host cell that does not display alpha-1,6 mannosyltransferase activity with respect to the N-glycan on a glycoprotein, the method comprising the step of introducing into the host cell a nucleic acid encoding an alpha-1,2 mannosidase enzyme selected to have optimal activity in the ER or Golgi of said host cell, the enzyme comprising:

(a) an alpha-1,2 mannosidase catalytic domain having optimal activity in said ER or Golgi at a pH between 5.1 and 8.0;

fused to

(b) a cellular targeting signal peptide not normally associated with the catalytic domain selected to target the mannosidase enzyme to the ER or Golgi apparatus of the host cell;

whereby, upon passage of the recombinant glycoprotein through the ER or Golgi apparatus of the host cell, in excess of 30 mole % of the N-glycan structures attached thereto have a Man₅GlcNAc₂ glycoform that can serve as a substrate for GlcNAc transferase I in vivo.

6. The method of claim 1, wherein the mannosidase enzyme is targeted to the early, medial, late Golgi or the trans Golgi network of the host cell.

7. The method of claim 1, further comprising the step of introducing into the host cell one or more additional nucleic acids encoding one or more additional enzymes selected from the group consisting of mannosidases, glycosyltransferases and glycosidases.

8. The method of claim 1 or 7, wherein the recombinant glycoprotein comprising the N-glycan is further modified to comprise one or more sugars selected from the group consisting of N-acetylglucosamine, galactose, sialic acid and fucose.

9. The method of claim 1 or 7, wherein the recombinant glycoprotein comprising the N-glycan is further modified to comprise at least one oligosaccharide branch comprising the structure NeuNAc-Gal-GLcNAc-Man.

10. The method of any one of claims 1, 7, 2, 4 or 5, wherein the host is selected from the group consisting of Pichia pastoris, Pichiafinlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pyperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Candida albicans, Aspergillus nidulans, and Trichoderma reesei.

11. The method of claim 1 or 7, wherein the host further lacks the activity of one or more enzymes selected from the group consisting of mannosyltransferases and phosphomannosyltransferases.

12. The method of claim 11, wherein the host lacks an enzyme activity with respect to the N-glycan on a glycoprotein, the activity selected from the group consisting of 1,6 mannosyltransferase;

1,3 mannosyltransferase; and

1,2 mannosyltransferase.

13. The method of claim 1 or 7, wherein the host is an OCHi mutant of P. pastoris.

14. The method of claim 1 or 7, wherein the host is genetically modified to express one or more enzymes selected from:

GnTI;

a UDP-specific diphosphatase;

a GDP-specific diphosphatase; and

a UDP-GlcNAc transporter.

15. The method of claim 1 or 7, further comprising the step of isolating the recombinant glycoprotein subsequent to passage of the recombinant glycoprotein through the ER or Golgi apparatus of the host cell.

16. The method of claim 15, further comprising the step of subjecting the isolated glycoprotein to at least one further glycosylation reaction in vitro, subsequent to its isolation from the host.

17. The method of claim 1, wherein the mannosidase enzyme comprises an alpha-1,2-mannosidase catalytic domain from mouse, human, Lepidoptera, Aspergillus nidulans, Xanthomonas man ihotas or Bacillus sp.

18. The method of claim 7, wherein at least one of the additional enzymes is localized in the host by forming a fusion protein between a catalytic domain of the enzyme and a cellular targeting signal peptide.

19. The method of claim 18, wherein the fusion protein is encoded by at least one genetic construct formed by the in-frame ligation of a DNA fragment encoding a cellular targeting signal peptide with a DNA fragment encoding a glycosylation enzyme or catalytically active fragment thereof.

20. The method of claim 18, wherein the catalytic domain encodes a glycosidase or glycosyltransferase selected from the group consisting of GnT I, GnT II, GnT III, GnT IV, GnT V, GnT VI, GalT, Fucosyltransferase and ST, and wherein the catalytic domain has optimal activity at a pH between 5.1 and 8.0.

21. The method of claim 1, further comprising the step of introducing into the host cell one or more additional nucleic acids encoding one or more additional enzymes selected from the group consisting of UDP-GlcNAc transferase, UDP-galactosyltransferase, GDP-fucosyltransferase, CMP-sialyltransferase, UDP-GlcNAc transporter, UDP-galactose transporter, GDP-fucose transporter, CMP-sialic acid transporter, and nucleotide diphosphatases.

22. The method of claim 7, wherein the host is genetically modified to express GnTI and a UDP-GlcNac transporter.

23. The method of claim 7, wherein the host is genetically modified to express a UDP- or GDP-specific diphosphatase.

24. The method of claim 7, wherein the one or more additional enzymes is targeted to the endoplasmic reticulum, the early, medial or late Golgi, or the trans Golgi network of the host cell.

25. The method of claim 24, wherein the one or more additional enzymes is targeted by means of a cellular targeting signal peptide not normally associated with the enzyme.

26. The method of claim 7, wherein the one or more additional enzymes has optimal activity at a pH between 5.1 and 8.0.

27. The method of claim 1 or 7, wherein a nucleic acid encoding one or more enzymes is introduced into the host cell by integration into the host cell chromosome.

28. The method of claim 7 or 27, wherein at least one of the encoded enzymes is GnTI.

30. The method of any one of claims 1, 7, 2, 3, 4 or 5, further comprising the step of analyzing a glycosylated protein or isolated N-glycan produced in the host cell by one or more methods selected from the group consisting of:

(a) mass spectroscopy;

(b) liquid chromatography;

(c) characterizing cells using a fluorescence activated cell sorter, spectrophotometer, fluorimeter, or scintillation counter;

(d) exposing host cells to a lectin or antibody having a specific affinity for a desired oligosaccharide moiety; and

(e) exposing cells to a cytotoxic or radioactive molecule selected from the group consisting of sugars, antibodies and lectins.

2. A method for producing a recombinant glycoprotein comprising an N-glycan structure that comprises a GlcNAcMan₅GlcNAc₂ glycoform in a lower eukaryotic host cell, the cell genetically modified to produce N-glycan structures having an excess of 30 mole % of a Man₅GlcNAc₂ glycoform that can serve as a substrate for GlcNAc transferase I in vivo, the method comprising the step of introducing into said host cell a nucleic acid encoding a GlcNAc transferase I enzyme selected to have optimal activity in the ER or Golgi of said host cell. the enzyme comprising:

(a) a GlcNAc transferase I catalytic domain having optimal activity in said ER or Golgi at a pH between 5.1 and 8.0;

fused to

(b) a cellular targeting signal peptide not normally associated with the catalytic domain selected to target the GlcNAc transferase I enzyme to the ER or Golgi apparatus of the host cell;

whereby, upon passage of the recombinant glycoprotein through the ER or Golgi apparatus of the host cell, a recombinant glycoprotein comprising a GlcNAcMan₅GlcNAc₂ glycoform is produced.

29. The method of any one of claims 2, 3, 4 and 5, further comprising the step of introducing into the host cell a nucleic acid encoding a UDP-GlcNAc transporter.

3. A method for producing a recombinant glycoprotein comprising an N-glycan structure that comprises a GlcNAcMan₅GlcNAc₂ glycoform in a lower eukaryotic host cell that does not display alpha-1,6 mannosyltransferase activity with respect to the N-glycan on a glycoprotein, the method comprising the step or steps of introducing into the host cell one or more nucleic acids encoding at least two enzymes, the first enzyme selected to have optimal activity in the ER or Golgi of said host cell, the enzyme compnsing:

(a) an alpha-1,2 mannosidase catalytic domain having optimal activity in said ER or Golgi at a pH between 5.1 and 8.0;

fused to

(b) a cellular targeting signal peptide not normally associated with the catalytic domain of (a) and selected to target the first enzyme to the ER or Golgi apparatus of the host cell;

the second enzyme selected to have optimal activity in the ER or Golgi of said host cell, the enzyme comprising;

(c) a GlcNAc transferase I catalytic domain having optimal activity in said ER or Golgi at a pH between 5.1 and 8.0;

fused to

(d) a cellular targeting signal peptide not normally associated with the catalytic domain of (c) and selected to target the second enzyme to the ER or Golgi apparatus of the host cell;

whereby, upon passage of the recombinant glycoprotein through the ER or Golgi apparatus of the host cell, a recombinant glycoprotein comprising a GlcNAcMan₅GlcNAc₂ glycoform is produced.

4. A method for producing a recombinant glycoprotein comprising an N-glycan comprising a GlcNAcMan₃GlcNAc₂ glycoform in a lower eukaryotic host cell genetically modified to produce N-glycan structures having an excess of 30 mole % of a Man₅GlcNAc₂ glycoform that are converted in vivo to a GIcNAcMan₅GlcNAc₂ glycoform by GlcNAc transferase I activity localized in the ER or Golgi apparatus of the host cell, the method comprising the step of introducing into the host cell a nucleic acid encoding a mannosidase II enzyme selected to have optimal activity in the ER or Golgi of said host cell, the enzyme comprising:

(a) a mannosidase II catalytic domain having optimal activity in said ER or Golgi at a pH between 5.1 and 8.0;

fused to

(b) a cellular targeting signal peptide not normally associated with the catalytic domain selected to target the mannosidase II enzyme to the ER or Golgi apparatus of the host cell;

whereby, upon passage of the recombinant glycoprotein through the ER or Golgi apparatus of the host cell, a recombinant glycoprotein comprising a GlcNAcMan₃GlcNAc₂ glycoform is produced.

5. A method for producing a recombinant glycoprotein comprising an N-glycan comprising a GlcNAc₂Man₃GlcNAc₂ glycoform in a lower eukaryotic host cell genetically modified to produce N-glycan structures having an excess of 30 mole % of a Man₅GlcNAc₂ glycoform that are converted in vivo to a GlcNAcMan₃GlcNAc₂ glycoform by GlcNAc transferase I and mannosidase II localized in the ER or Golgi apparatus of the host cell, the method comprising the step of introducing into the host cell a nucleic acid encoding a GlcNAc transferase II enzyme selected to have optimal activity in the ER or Golgi of said host cell, the enzyme comprising:

(a) a GlcNAc transferase II catalytic domain having optimal activity in said ER or Golgi at a pH between 5.1 and 8.0;

fused to

(b) a cellular targeting signal peptide not normally associated with the catalytic domain selected to target the GlcNAc transferase II enzyme to the ER or Golgi apparatus of the host cell;

whereby, upon passage of the recombinant glycoprotein through the ER or Golgi apparatus of the host cell, a recombinant glycoprotein comprising a GlcNAc₂Man₃GlcNAc₂ glycoform is produced.