Materials and methods for producing endostatin fusion polypeptides in plant cells

Patent metadata

Field	Value
Publication number	US-10844392-B2
Application number	US-201615579690-A
Country	US
Kind code	B2
Filing date	Jun 3, 2016
Priority date	Jun 5, 2015
Publication date	Nov 24, 2020
Grant date	Nov 24, 2020

Abstract

This document provides materials and methods for producing endostatin fusion polypeptides having anti-fibrotic activity. For example, provided herein are fusion polypeptides having an IgG Fc domain and a portion of human endostatin that can form high molecular weight multimers, as well as methods for producing such fusion polypeptides in plant cells.

First claim

What is claimed is:

1. A method for producing a soluble fusion polypeptide having anti-fibrotic activity, wherein the fusion polypeptide comprises the sequence set forth in SEQ ID NO: 16, the method comprising:
introducing into a plant a plant viral vector that includes a polynucleotide encoding the fusion polypeptide having anti-fibrotic activity of SEQ ID NO:16.

2. The method of claim 1 , wherein the introducing comprises vacuum infiltration.

3. The method of claim 1 , further comprising harvesting the plant, wherein the plant comprises the fusion polypeptide having anti-fibrotic activity.

4. The method of claim 1 , further comprising extracting the fusion polypeptide of SEQ ID NO:16 from the plant.

5. The method of claim 4 , further comprising purifying the fusion polypeptide of SEQ ID NO:16 having antifibrotic activity extracted from the plant.

6. A method for producing high molecular weight multimer of a soluble fusion polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 16, wherein the fusion polypeptide has anti-fibrotic activity, the method comprising:
(a) introducing into a plant a plant viral vector that includes a polynucleotide encoding the fusion polypeptide of SEQ ID NO:16;
(b) maintaining the plant under conditions and for a time sufficient that the polynucleotide is expressed in at least some plant cells; and
(c) obtaining an extract from a plurality of plant cells.

7. The method of claim 1 , wherein the plant is a transgenic plant.

8. The method of claim 1 , wherein the plant is a Leguminosae, Gramineae, Solanaceae, Lycopersicon, Solanum, Capsium, Nicotiana, Umbelliferae, Compositae, or Brassicaceae plant.

9. The method of claim 1 , wherein the polynucleotide is operably linked to a heterologous promoter.

10. The method of claim 1 , wherein the polynucleotide is operably linked to a promoter that expresses in a mature plants, seedlings, sprouts, or seeds.

11. The method of claim 6 , wherein the plant is a transgenic plant.

12. The method of claim 6 , wherein the introducing comprises vacuum infiltration.

13. The method of claim 6 , further comprising extracting the fusion polypeptide of SEQ ID NO:16 from the plant cells.

14. The method of claim 6 , further comprising purifying the fusion polypeptide of SEQ ID NO:16 from the extract.

15. The method of claim 6 , wherein the polynucleotide is operably linked to a heterologous promoter.

16. The method of claim 6 , wherein the polynucleotide is operably linked to a promoter that expresses in a mature plants, seedlings, sprouts, or seeds.

17. The method of claim 6 , wherein the plant is a Leguminosae, Gramineae, Solanaceae, Lycopersicon, Solanum, Capsium, Nicotiana, Umbelliferae, Compositae, or Brassicaceae plant.

18. A transgenic plant comprising a nucleic acid expression vector that comprises a polynucleotide that encodes a fusion polypeptide comprising SEQ ID NO:16.

19. The transgenic of claim 18 , wherein the plant is a Leguminosae, Gramineae, Solanaceae, Lycopersicon, Solanum, Capsium, Nicotiana, Umbelliferae, Compositae, or Brassicaceae plant.

Assignees

• Ibio Inc
• Novici Biotech Llc
• Musc Found For Res Dev

Inventors

• Feghali-Bostwick Carol
• Ryan Terence E
• Padgett Hal S
• Mcgee Matthew

Classifications

• C07K14/78 Primary

  Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG] · CPC title

• C12N15/8257 Primary

  for the production of primary gene products, e.g. pharmaceutical products, interferon · CPC title

• C07K2319/30

  Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto · CPC title

• A61P11/00

  Drugs for disorders of the respiratory system · CPC title

• A61K2300/00

  Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00 · CPC title