Recombinant Production of Polyanionic Polymers, and Uses Thereof

• US 20080176288A1
• Filed: 10/30/2007
• Published: 07/24/2008
• Est. Priority Date: 03/21/2001
• Status: Abandoned Application

• Current Assignee: Cell Therapeutics, Inc.
• Original Assignee: Cell Therapeutics, Inc.
• Inventors: Leung, David W., Waggoner, David W., Pietz, Gregory E., Bergman, Philip A., Tompkins, Christopher K., Lofquist, Alan
• Application Number:
  US11/928,737
• Publication Number:
  US 20080176288A1
• Time in Patent Office: Days
• US Class Current: 435/69.7
• CPC Class Codes: A61K 47/645 Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
• CPC Class Codes: C07K 14/001 by chemical synthesis
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 7/06 having 5 to 11 amino acids
• CPC Class Codes: C07K 7/08 having 12 to 20 amino acids gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68

Abstract

A polyanionic polymer can improve the bioactivity and water-solubility properties of a drug to which it is joined. The inventive method provides a monodispersed preparation of a recombinantly-produced polyanionic polymer that can be easily manipulated, such as lengthened. An active moiety may be chemically or recombinantly joined to a polyanionic polymer to increase its biological half-life and/or solubility. The instant invention also provides a method for targeting the delivery of a polyanionic polymer conjugate or fusion protein to a specific cell type or tissue.

Claims

1. A recombinant fusion protein, comprising (i) a polyanionic polypeptide and (ii) a granulocyte colony stimulating factor at either one end or at both ends thereof, wherein the size of the polyanionic polypeptide is between 10 kD and 100 kD.

2. The recombinant fusion protein of claim 1, wherein the granulocyte colony stimulating factor is attached to the amino-terminal end of the polyanionic polypeptide and a second polypeptide is attached at the carboxyl-terminal end of the polyanionic polypeptide.

3. The recombinant fusion protein of claim 2, wherein the second polypeptide is a targeting polypeptide.

5. The recombinant fusion protein of claim 2, wherein the second polypeptide is selected from the group consisting of an interferon, interferon-α

, interferon-β

, interferon-γ

, granulocyte colony stimulating factor, granulocyte-macrophage colony stimulating factor, macrophage colony stimulating factor, interleukin-18, FLT3 ligand, stem cell factor, stromal cell-derived factor-1 alpha, human growth hormone, the extracellular domain of tumor necrosis factor receptor, the extracellular domain of tumor necrosis factor-related apoptosis-inducing ligand, Apo2 ligand, the extracellular domain of vascular endothelial growth factor receptor (VEGF) that includes the first 330 amino acids of the kinase domain receptor of VEGF, a region that includes the first 656 amino acids of VEGF receptor 1, the extracellular domain of transforming growth factor b type III receptor, the extracellular domain of transforming growth factor b type II receptor that includes the first 159 amino acids of the receptor, herstatin, the extracellular domain of HER-2/neu receptor, a secreted form of human ErbB3 receptor isoform, the secreted form of human fibroblast growth factor receptor 4 isoform, β

-glucocerebrosidase, basic fibroblast growth factor, human interleukin-1 receptor antagonist, osteoprotegerin, osteoclastogenesis inhibitory factor, and erythropoietin.

6. The recombinant fusion protein of claim 2, wherein the second polypeptide is an anti-angiogenic protein selected from the group consisting of a pigment epithelium-derived factor, vascular endothelial growth inhibitor, the domain 5 region of high molecular weight kininogen, endostatin, restin, plasminogen kringle 1 domain, plasminogen kringle 5 domain, and angiostatin.

7. The recombinant fusion protein of claim 3, wherein the targeting polypeptide comprises a recognition motif, selected from the group consisting of an antibody, an antibody fragment, folate, AGCKNFFWKTFTSC, ALNGREESP, CNGRC, ATWLPPR and CTTHWGFTLC.

11. The recombinant fusion protein of claim 1, wherein the polyanionic polymer is polyglutamic acid or polyaspartic acid.

12. The recombinant fusion protein of claim 1, wherein the polyanionic polymer is polyglutamic acid.

13. The recombinant fusion protein of claim 1, wherein the polyanionic polymer is polyaspartic acid.

15. The recombinant fusion protein of claim 1, further comprising a spacer amino acid, selected from the group consisting of glycine, an alanine, a β

-alanine, a glutamate and leucine.

4. (canceled)

8-10. -10. (canceled)

14. (canceled)

16. A vector, comprising a cassette which comprises a nucleotide sequence that encodes (i) a polyanionic polymer, wherein the size of the polyanionic polypeptide when expressed is between 10 kD and 100 kD and (ii) granulocyte colony stimulating factor.

17. The vector of claim 16, wherein the nucleotide sequence encodes a polyanionic polymer that is polyglutamic acid or polyaspartic acid.

18. The vector of claim 16, wherein the nucleotide sequence encodes a polyglutamic acid polyanionic polymer.

19. The vector of claim 16, wherein the nucleotide sequence encodes a polyaspartic acid polyanionic polymer.

24. The vector of claim 16, further comprising a a nucleotide sequence that encodes at least one spacer amino acid between the sequences encoding the polyanionic polymer and the granulocyte colony stimulating factor.

25. A method for producing a polyanionic fusion protein, comprising (1) expressing in a host cell the cassette of the vector of claim 16, (2) isolating the protein product of the cassette, (3) purifying the protein product and (4) screening the protein product for activity, wherein the protein product is the polyanionic fusion protein that comprises a polyanionic polymer joined to another protein.

32. A cell comprising the vector of claim 16.

20-23. -23. (canceled)

26-31. -31. (canceled)

33-62. -62. (canceled)