FIBRONECTIN BASED SCAFFOLD DOMAIN PROTEINS THAT BIND TO PCSK9

• US 20120094909A1
• Filed: 04/13/2011
• Published: 04/19/2012
• 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., Saeed-Kothe, Amna, Lipovsek, Dasa, Lo, Ching-Hsiung Frederick, Miao, Bowman, Parker, Rex A., Sitkoff, Doree F., Denhez, Fabienne M., Low, Chee Meng, Mitchell, Tracy S., Rakestraw, Ginger C., Russo, Katie A.
• Granted Patent: US 8,420,098 B2
• Time in Patent Office: Days
• US Class Current: 514/9.3
• 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. A polypeptide comprising a fibronectin type III tenth domain (¹⁰Fn3) wherein the ¹⁰Fn3 has at least one loop selected from loop BC, DE and FG with an altered amino acid sequence relative to the sequence of the corresponding loop of the human ¹⁰Fn3 domain, and wherein the polypeptide binds (proprotein convertase subtilisin kexin type 9) PCSK9.

2. The polypeptide of claim 1, wherein the polypeptide binds PCSK9 with a K_D of less than 500 nM.

3. The polypeptide of claim 1, wherein the BC loop comprises a sequence according to the formula (X₁)_ZX₂G (SEQ ID NO:

449), wherein X₁ is any amino acid, Z is a number from 6-9, and X₂ is Y or H.

4. The polypeptide of claim 1, wherein the BC loop comprises the shaded portion of any one of SEQ ID NOs:

2-17, 107-135, and 301-303, as shown in Table 3.

5. The polypeptide of claim 3, wherein the BC loop is selected from SEQ ID NO:

2-17, 107-135 and 301-303.

6. The polypeptide of claim 1, wherein the DE loop comprises a sequence according to the formula X₁X₁X₁X₃ (SEQ ID NO:

450), wherein X₁ is any amino acid; and

X₃ is G or S.

7. The polypeptide of claim 1, wherein the DE loop comprises the shaded portion of any one of SEQ ID NOs:

18-27 and 136-141, as shown in Table 3.

8. The polypeptide of claim 6, wherein the DE loop is selected from SEQ ID NO:

18-27 and 136-141.

9. The polypeptide of claim 1, wherein the FG loop comprises a sequence according to the formula EX₄X₁X₅X₁X₁X₆GYX₄HR (SEQ ID NO:

451), wherein X₁ is any amino acid;

X₄ is Y or F;

X₅ is Y, F, or W; and

X₆ is S or A.

10. The polypeptide of claim 1, wherein the FG loop comprises the shaded portion of any one of SEQ ID NOs:

28-38 and 142-172, as shown in Table 3.

11. The polypeptide of claim 9, wherein the FG loop is selected from SEQ ID NO:

28-38 and 142-172.

12. The polypeptide of claim 1, wherein the BC, DE or FG loop amino acid sequence is at least 80% identical to any one of SEQ ID NOs:

2-38, 106-172 and 301-303.

13. The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs:

39-76, 173-290 and 304-309.

14. The polypeptide of claim 1, further comprising one or more pharmacokinetic (PK) moieties selected from the group consisting of polyethylene glycol, sialic acid, Fc, Fc fragment, transferrin, serum albumin, a serum albumin binding protein, and a serum immunoglobulin binding protein.

15. The polypeptide of claim 14, wherein the serum albumin binding protein comprises a fibronectin type III tenth domain (¹⁰Fn3).

16. The polypeptide of claim 15, wherein the¹⁰Fn3 domain binds HSA.

17. The polypeptide of claim 14, wherein the PK moiety is polyethylene glycol.

18. A pharmaceutically acceptable composition comprising the polypeptide of claim 1, wherein the composition is essentially endotoxin free.