Methods of inhibiting type 2 vascular endothelial growth factor receptors using polypeptides

• US 8,609,613 B2
• Filed: 07/18/2012
• Issued: 12/17/2013
• Est. Priority Date: 12/05/2003
• Status: Active Grant

• Current Assignee: Bristol-Myers Squibb Company
• Original Assignee: Bristol-Myers Squibb Company
• Inventors: Chen, Yan, Getmanova, Elena, Wright, Martin C., Harris, Alan S., Lim, Ai Ching, Gokemeijer, Jochem, Sun, Lin, Wittekind, Michael
• Primary Examiner(s): HOWARD, ZACHARY C
• Application Number:
  US13/552,398
• Publication Number:
  US 20120283184A1
• Time in Patent Office: 517 Days
• Field of Search: None
• US Class Current: 514/8.1
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 38/39 Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
• CPC Class Codes: A61P 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 1/04 for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 11/06 Antiasthmatics
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 15/00 Drugs for genital or sexual disorders for disorders of sex hormones A61P5/24; Contraceptives
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 17/06 Antipsoriatics
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 27/06 Antiglaucoma agents or miotics
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: A61P 35/04 : specific for metastasis
• CPC Class Codes: A61P 37/00 : Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 37/02 : Immunomodulators
• CPC Class Codes: A61P 37/06 : Immunosuppressants, e.g. drugs for graft rejection
• 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: C07H 21/04 : with deoxyribosyl as saccharide radical
• CPC Class Codes: C07K 14/4703 : Inhibitors; Suppressors
• CPC Class Codes: C07K 14/52 : Cytokines; Lymphokines; Interferons
• 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 16/2863 : against receptors for growth factors, growth regulators
• CPC Class Codes: C07K 16/30 : from tumour cells
• CPC Class Codes: C07K 19/00 : Hybrid peptides , i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
• CPC Class Codes: C07K 2317/34 : Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
• CPC Class Codes: C07K 2317/73 : Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
• CPC Class Codes: C07K 2317/92 : Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• 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 2319/21 : containing a His-tag
• CPC Class Codes: C07K 2319/23 : containing a GST-tag
• 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 2319/41 : containing a Myc-tag
• CPC Class Codes: C07K 2319/42 : containing a HA(hemagglutinin)-tag
• CPC Class Codes: C07K 2319/43 : containing a FLAG-tag
• CPC Class Codes: C12N 15/09 : Recombinant DNA-technology
• CPC Class Codes: C12N 15/1062 : mRNA-Display, e.g. polypeptide and encoding template are connected covalently
• CPC Class Codes: C40B 40/08 : Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
• CPC Class Codes: C40B 40/10 : Libraries containing peptides or polypeptides, or derivatives thereof

Abstract

The present disclosure relates to novel vascular endothelial growth factor receptor (VEGFR)-binding polypeptides and methods for using these polypeptides to inhibit biological activities mediated by vascular endothelial growth factors (VEGFs). The present disclosure also provides various improvements relating to single domain binding polypeptides.

Claims

1. A method of inhibiting vascular endothelial growth factor (VEGF) biological activity in a cell comprising contacting said cell with a polypeptide that binds human kinase insert domain receptor (KDR) in an amount and for a time sufficient to inhibit said VEGF biological activity;

wherein the polypeptide comprises a ¹⁰Fn3 domain between about 80 and about 150 amino acids that has a structural organization comprising;

a) at least five to seven beta strands or beta-like strands distributed among at least two beta sheets, and

b) at least one loop portion connecting two strands that are beta strands or beta-like strands, which loop portion participates in binding to KDR,

wherein the polypeptide binds to an extracellular domain of the human KDR protein with a dissociation constant (K_D) of less than 1×

10<sup>−

6</sup> M, and wherein the ¹⁰Fn3 domain comprises an amino acid sequence that is at least 60% identical to SEQ ID NO;

5.

7. The method of claim 1 or 2, wherein the polypeptide comprises an amino acid sequence at least 90% identical to the sequence of SEQ ID NO:

192.

8. The method of claim 1 or 2, wherein the polypeptide comprises an amino acid sequence at least 95% identical to the sequence of SEQ ID NO:

192.

9. The method of claim 1 or 2, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

192.

10. The method of claim 9, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

193.

11. The method of claim 9, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

194.

18. The method of claim 1 or 2, wherein the polypeptide comprises an amino acid sequence that is at least 65% identical to SEQ ID NO:

5.

19. The method of claim 1 or 2, wherein the polypeptide comprises an amino acid sequence that is at least 70% identical to SEQ ID NO:

5.

20. The method of claim 1 or 2, wherein the polypeptide comprises an amino acid sequence that is at least 75% identical to SEQ ID NO:

5.

2. A method for treating a subject having a condition which responds to the inhibition of inhibiting vascular endothelial growth factor (VEGF), said method comprising administering to said subject an effective amount of a polypeptide that binds human kinase insert domain receptor (KDR) and inhibits a biological activity of VEGF;

wherein the polypeptide comprises between about 80 and about 150 amino acids that have a structural organization comprising;

a) at least five to seven beta strands or beta-like strands distributed among at least two beta sheets, and

b) at least one loop portion connecting two strands that are beta strands or beta-like strands, which loop portion participates in binding to KDR,

wherein the polypeptide binds to an extracellular domain of the human KDR protein with a dissociation constant (K_D) of less than 1×

10<sup>−

6</sup> M, and wherein the polypeptide comprises an amino acid sequence that is at least 60% identical to SEQ ID NO;

5.

3. The method of claim 2, wherein the condition is a condition characterized by inappropriate angiogenesis.

4. The method of claim 3, wherein the condition is a hyperproliferative condition.

5. The method of claim 3, wherein said condition is selected from the group consisting of:

an autoimmune disorder, an inflammatory disorder, a retinopathy, and a cancer.

6. The method of claim 2, wherein the polypeptide comprises an amino acid sequence at least 85% identical to the sequence of any of SEQ ID NOs:

6-175, 178-186, 188 and 192-194.

12. The method of claim 2, wherein the polypeptide is attached to a moiety that reduces the clearance rate of the polypeptide in a mammal by greater than three-fold relative to the unmodified polypeptide.

13. The method of claim 12, wherein the moiety that reduces the clearance rate is a polyethylene glycol moiety.

14. The method of claim 13, wherein the polyethylene glycol moiety has a molecular weight of between 2 and 100 kDa.

15. The method of claim 13, wherein the polyethylene glycol moiety has a molecular weight ranging from about 10 kDa to about 60 kDa.

16. The method of claim 13, wherein the polyethylene glycol moiety has a molecular weight of 40 kDa and is branched.

17. The method of claim 13, wherein the polyethylene moiety is covalently bonded to a thiol moiety or an amine moiety in the polypeptide.