Hepatitis c virus E1/E2 heterodimers and methods of producing same

Patent metadata

Field	Value
Publication number	US-12202859-B2
Application number	US-202117509808-A
Country	US
Kind code	B2
Filing date	Oct 25, 2021
Priority date	Oct 8, 2015
Publication date	Jan 21, 2025
Grant date	Jan 21, 2025

Abstract

The present disclosure provides affinity tagged heterodimeric polypeptides comprising a hepatitis C virus (HCV) E1 polypeptide and an HCV E2 polypeptide, where one or both of the E1 and E2 polypeptides comprises an affinity tag. The present disclosure provides a method of producing an affinity tagged E1/E2 heterodimer of the present disclosure. The present disclosure provides methods of producing untagged HCV E1/E2 heterodimers. The present disclosure provides HCV E1/E2 heterodimers, compositions comprising same, and methods of inducing an immune response to HCV.

First claim

What is claimed is:

1. A method of purifying a hepatitis C virus (HCV) E1/E2 heterodimer from a lysate, the method comprising:
a) contacting a lysate of a genetically modified host cell with an affinity tag-binding polypeptide immobilized on an insoluble support, wherein the genetically modified host cell is genetically modified with a nucleic acid comprising a nucleotide sequence encoding a polyprotein comprising, in order from N-terminus to C-terminus:
a) an HCV E1 polypeptide; and
b) an HCV E2-affinity tag fusion polypeptide comprising, in order from N-terminus to C-terminus: i) an affinity tag polypeptide; ii) a proteolytically cleavable linker; and iii) an HCV E2 polypeptide
i) a first nucleotide sequence encoding an HCV E1 polypeptide; and
ii) a second nucleotide sequence encoding an HCV E2 polypeptide,

wherein the polyprotein is processed in the host cell to produce the affinity tagged HCV E1/E2 heterodimer and the lysate comprises the affinity tagged HCV E1/E2 heterodimer,
wherein the affinity tagged HCV E1/E2 heterodimer present in the lysate binds to the immobilized affinity tag-binding polypeptide, generating an immobilized affinity tagged HCV E1/E2 heterodimer;
b) contacting the immobilized HCV E1/E2 heterodimer with a protease that cleaves the proteolytically cleavable linker present in the immobilized affinity tagged HCV E1/E2 heterodimer, thereby releasing the HCV E1/E2 heterodimer from the affinity tag; and
c) collecting the HCV E1/E2 heterodimer.

2. The method of claim 1 , wherein the affinity tag polypeptide is an Ig Fc polypeptide, and wherein the affinity tag-binding polypeptide is an immunoglobulin (Ig) Fc-binding polypeptide.

3. The method of claim 2 , wherein the Ig Fc-binding polypeptide is Protein A, Protein G, or a Protein A/G fusion.

4. The method of claim 1 , wherein the proteolytically cleavable linker comprises an amino acid sequence selected from the group consisting of LEVLFQGP as set forth in SEQ ID NO:1, ENLYFQS as set forth in SEQ ID NO:2, DDDDK as set forth in SEQ ID NO:3, I(E/D)GR as set forth in SEQ ID NO:45, and LVPRGS as set forth in SEQ ID NO:5.

5. The method of claim 1 , wherein the proteolytically cleavable linker comprises the amino acid sequence ENLYFQS as set forth in SEQ ID NO:2, and wherein the protease is a tobacco etch virus protease.

6. The method of claim 1 , wherein the proteolytically cleavable linker comprises the amino acid sequence LEVLFQGP as set forth in SEQ ID NO:1, and wherein the protease is a human rhinovirus 3C protease.

7. The method of claim 1 , wherein the genetically modified host cell is a genetically modified Chinese hamster ovary (CHO) cell.

8. The method of claim 1 , comprising removing the protease from a composition comprising the collected HCV E1/E2 heterodimer.

9. The method of claim 1 , comprising further purifying the collected HCV E1/E2 heterodimer on a hydroxyapatite column.

10. The method of claim 1 , wherein the nucleotide sequence encoding the polyprotein is operably linked to a promoter.

11. The method of claim 1 , wherein the affinity tagged E1/E2 polypeptide comprises:
a) an HCV E1 polypeptide having a length of from about 175 amino acids to about 195 amino acids; and
b) an affinity-tagged HCV E2 polypeptide comprising, in order from N-terminus to C-terminus:
i) an Ig Fc polypeptide;
ii) a proteolytically cleavable linker, wherein the proteolytically cleavable linker comprises an amino acid sequence selected from the group consisting of LEVLFQGP as set forth in SEQ ID NO:1, ENLYFQS as set forth in SEQ ID NO: 2, DDDDK as set forth in SEQ ID NO:3, I(E/D)GR as set forth in SEQ ID NO: 45, and LVPRGS as set forth in SEQ ID NO:5; and
iii) an HCV E2 polypeptide having a length of from about 350 amino acids to about 370 amino acids.

12. The method of claim 1 , wherein the HCV E2 polypeptide and the HCV E1 polypeptide are of the same genotype.

13. The method of claim 1 , wherein the HCV E2 polypeptide and the HCV E1 polypeptide are of different genotypes.

14. The method of claim 1 , wherein the released HCV E1/E2 heterodimer is at least 50% pure.

15. The method of claim 1 , wherein the released HCV E1/E2 heterodimer is at least 90% pure.

Assignees

• Univ Alberta

Inventors

• Houghton Michael
• Hockman Darren
• Law John L
• Chen Chao
• Logan Michael

Classifications

• C12N2770/28122

  New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes · CPC title

• C12N7/00

  Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00) · CPC title

• A61K39/29

  Hepatitis virus · CPC title

• A61P31/14

  for RNA viruses · CPC title

• A61K2039/55572

  Lipopolysaccharides; Lipid A; Monophosphoryl lipid A · CPC title