FIBRONECTIN BASED SCAFFOLD DOMAIN PROTEINS THAT BIND PCSK9

• US 20130210703A1
• Filed: 03/15/2013
• Published: 08/15/2013
• Est. Priority Date: 04/13/2010
• Status: Active Grant

• Current Assignee: Bristol-Myers Squibb Company
• Original Assignee: Bristol-Myers Squibb Company
• Inventors: Camphausen, Ray, Davis, Jonathan H., Cload, Sharon T., Denhez, Fabienne M., Saeed-Kothe, Amna, Lipovsek, Dasa, Lo, Ching-Hsiung Frederick, Parker, Rex A., Russo, Katie A., Sitkoff, Doree F., Low, Cheen Meng, Miao, Bowman, Mitchell, Tracy S., Rackestraw, Ginger C.
• Granted Patent: US 9,234,027 B2
• Time in Patent Office: Days
• US Class Current: 514/1.9
• CPC Class Codes: A61K 38/39 Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
• CPC Class Codes: A61K 47/549 Sugars, nucleosides, nucleotides or nucleic acids
• CPC Class Codes: A61K 47/60 the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• CPC Class Codes: A61P 3/06 Antihyperlipidemics
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C07K 14/78 Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: C07K 16/40 against enzymes
• CPC Class Codes: C07K 2318/20 Antigen-binding scaffold molecules wherein the scaffold is not an immunoglobulin variable region or antibody mimetics
• CPC Class Codes: C07K 2319/30 Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• CPC Class Codes: C07K 2319/31 fusions, other than Fc, for prolonged plasma life, e.g. albumin
• CPC Class Codes: C07K 2319/70 containing domain for protein-protein interaction
• CPC Class Codes: C12N 9/6424 Serine endopeptidases (3.4.21)

Abstract

The present invention relates to fibronectin based scaffold domain proteins that bind PCSK9. The invention also relates to the use of the innovative proteins in therapeutic applications to treat atherosclerosis, hypercholesterolemia and other cholesterol related diseases. The invention further relates to cells comprising such proteins, polynucleotides encoding such proteins or fragments thereof, and to vectors comprising the polynucleotides encoding the innovative protein.

Claims

1. An isolated nucleic acid molecule encoding a polypeptide comprising a fibronectin type III tenth (¹⁰Fn3) domain wherein the ¹⁰Fn3 has a BC, DE and FG loop, and wherein the BC loop comprises SEQ ID NO:

14, the DE comprises SEQ ID NO;

25 and the FG loop comprises SEQ ID NO;

37, and wherein the polypeptide binds PCSK9.

2. The isolated nucleic acid molecule of claim 1, wherein the BC loop comprises nucleotides 67-96 of SEQ ID NO:

83, the DE loop comprises nucleotides 157-174 of SEQ ID NO;

83 and the FG loop comprises nucleotides 232-270 of SEQ ID NO;

83.

5. An expression vector comprising the isolated nucleic acid sequence of claim 1.

7. A cell comprising the isolated nucleic acid sequence of claim 1.

9. A method of producing a PCSK9-binding polypeptide comprising culturing the cell of claim 7 under conditions suitable for expressing the polypeptide and purifying the polypeptide.

3. An isolated nucleic acid molecule encoding a polypeptide comprising a ¹⁰Fn3 domain wherein the polypeptide comprises SEQ ID NO:

82, and wherein the polypeptide binds PCSK9.

4. The isolated nucleic acid molecule of claim 3, wherein the isolated nucleic acid molecule comprises SEQ ID NO:

83.

6. An expression vector comprising the isolated nucleic acid sequence of claim 3.

8. A cell comprising the isolated nucleic acid sequence of claim 3.

10. A method of producing a PCSK9-binding polypeptide comprising culturing the cell of claim 8 under conditions suitable for expressing the polypeptide and purifying the polypeptide.

11. A method of treating a PCSK9-related disease or disorder in a subject comprising administering an effective amount of a polypeptide comprising a fibronectin type III tenth (¹⁰Fn3) domain wherein the ¹⁰Fn3 has a BC, DE and FG loop, and wherein the BC loop comprises SEQ ID NO:

14, the DE comprises SEQ ID NO;

25 and the FG loop comprises SEQ ID NO;

37, and wherein the polypeptide binds PCSK9.

12. The method of claim 11, wherein the disease or disorder is selected from the group consisting of atherosclerosis, hypercholesterolemia and other cholesterol related diseases.

13. The method of claim 12, wherein the disease or disorder is atherosclerosis.

14. The method of claim 12, wherein the disease or disorder is hypercholesterolemia.

15. The method of claim 12, wherein the disease or disorder is a cholesterol related disease.

16. A method of treating a PCSK9-related disease or disorder in a subject comprising administering an effective amount of a polypeptide comprising a ¹⁰Fn3 domain wherein the isolated polypeptide comprises SEQ ID NO:

82, and wherein the isolated polypeptide binds PCSK9.

17. The method of claim 16, wherein the disease or disorder is selected from the group consisting of atherosclerosis, hypercholesterolemia and other cholesterol related diseases.

18. The method of claim 17, wherein the disease or disorder is atherosclerosis.

19. The method of claim 17, wherein the disease or disorder is hypercholesterolemia.

20. The method of claim 17, wherein the disease or disorder is a cholesterol related disease.