Inhibitors of type 2 vascular endothelial growth factor receptors

• US 7,858,739 B2
• Filed: 06/05/2006
• Issued: 12/28/2010
• Est. Priority Date: 12/05/2003
• Status: Active Grant

• Current Assignee: Bristol-Myers Squibb Company
• Original Assignee: Bristol-Myers Squibb Company
• Inventors: Gokemeijer, Jochem, Lim, Ai Ching, Chen, Yan, Sun, Lin, Getmanova, Elena, Wittekind, Michael, Wright, Martin C., Harris, Alan S.
• Primary Examiner(s): Bunner; Bridget E
• Assistant Examiner(s): Howard; Zachary C
• Application Number:
  US11/448,171
• Publication Number:
  US 20070148126A1
• Time in Patent Office: 1,667 Days
• Field of Search: None
• US Class Current: 530/324
• 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. An isolated polypeptide comprising a fibronectin type III tenth (¹⁰Fn3) domain having between about 80 and about 150 amino acids wherein the ¹⁰Fn3 domain 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, wherein the ¹⁰Fn3 domain comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO;

192, and wherein the polypeptide is at least 85% pure.

2. The polypeptide of claim 1, wherein the ¹⁰Fn3 domain comprises, in order from N-terminus to C-terminus,

a) a beta strand or beta-like strand, A;

b) a loop, AB;

c) a beta strand, B;

d) a loop, BC;

e) a beta strand C;

f) a loop CD;

g) a beta strand D;

h) a loop DE;

i) a beta strand F;

j) a loop FG; and

k) a beta strand or beta-like strand, G.

3. The polypeptide of claim 2, wherein the loop FG participates in KDR binding.

4. The polypeptide of claim 2, wherein the loops BC, DE and FG participate in KDR binding.

5. The polypeptide of claim 1, wherein the ¹⁰Fn3 domain comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:

192.

6. The polypeptide of claim 1, wherein a loop that participates in KDR binding has a sequence selected from the group consisting of:

SEQ ID NO;

1, SEQ ID NO;

2, SEQ ID NO;

3 and SEQ ID NO;

4.

7. The polypeptide of claim 1, 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.

8. The polypeptide of claim 7, wherein the moiety that reduces the clearance rate is a polyethylene glycol moiety.

9. The polypeptide of claim 8, wherein the polyethylene glycol moiety has a molecular weight of between 2 and 100 kDa.

10. The polypeptide of claim 8, wherein the polyethylene glycol moiety is covalently bonded to a thiol moiety or an amine moiety in the polypeptide.

11. The polypeptide of claim 1, wherein the polypeptide is attached to a label moiety.

12. The polypeptide of claim 1, 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>−

7</sup>M and inhibits KDR-mediated VEGF activity.

13. The polypeptide of claim 1, wherein the polypeptide binds KDR competitively with the VEGF₁₆₅ isoform of VEGF-A.

14. The polypeptide of claim 1, wherein the polypeptide binds KDR competitively with VEGF-A and VEGF-D.

15. The polypeptide of claim 1, wherein the polypeptide also binds to an extracellular domain of the mouse Flk1 with a K_D of less than 1×

10<sup>−

6</sup>.

16. The polypeptide of claim 1, wherein the ¹⁰Fn3 domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

192.

17. The polypeptide of claim 1, wherein the ¹⁰Fn3 domain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:

192.

18. The polypeptide of claim 16, wherein said polypeptide inhibits a biological activity of VEGF.

19. The polypeptide of claim 16, wherein said polypeptide binds said KDR with a K_D of 50 nM or less.

20. The polypeptide of claim 16, 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>−

7</sup>M and inhibits KDR-mediated VEGF activity.

21. The polypeptide of claim 1, wherein the polypeptide is covalently bound to a polyethylene glycol (PEG) moiety, and wherein said polypeptide binds to said extracellular domain of the human KDR protein with a K_D of less than 100 nM and has a clearance rate of less than 30 mL/hr/kg in a mammal.

22. The polypeptide of claim 21, wherein the PEG moiety is attached to a thiol group or an amine group.

23. The polypeptide of claim 21, wherein the PEG moiety is attached to the polypeptide by site directed pegylation.

24. The polypeptide of claim 23, wherein the PEG moiety is attached to a Cys residue.

25. The polypeptide of claim 21, wherein the PEG moiety is attached at a position on the polypeptide selected from the group consisting of:

(a) the N-terminus;

(b) between the N-terminus and the most N-terminal beta strand or beta-like strand;

(c) a loop positioned on a face of the polypeptide opposite the target-binding site;

(d) between the C-terminus and the most C-terminal beta strand or beta-like strand; and

(e) at the C-terminus.

26. A combination product comprising (a) a polypeptide of claim 1 and (b) a second agent, wherein the second agent is selected from the group consisting of (i) a microtubule disruptor, (ii) a mTOR inhibitor, (iii) a pyrimidine analog, (iv) an anti-angiogenic agent, (v) an alkylating agent, and (vi) an antibody;

wherein the polypeptide and the second agent are formulated to be administered in combination.

38. A therapeutic formulation comprising a polypeptide of any of claims 1, 7, 8 and 27, and a pharmaceutically acceptable carrier.

27. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO:

192.

28. The polypeptide of claim 27, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

193.

29. The polypeptide of claim 27, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

194.

30. The polypeptide of claim 29, wherein a polyethylene glycol moiety is covalently bound to the cysteine residue at position 93.

31. The polypeptide of claim 30, wherein the polyethylene glycol moiety has a molecular weight ranging from about 10 kDa to about 60 kDa.

32. A therapeutic formulation comprising a polypeptide of claim 28, and a pharmaceutically acceptable carrier.

33. A therapeutic formulation comprising a polypeptide of claim 29, and a pharmaceutically acceptable carrier.

34. A therapeutic formulation comprising a polypeptide of claim 30, and a pharmaceutically acceptable carrier.

35. A therapeutic formulation comprising a polypeptide of claim 31, and a pharmaceutically acceptable carrier.

36. The polypeptide of claim 31, wherein the polyethylene glycol moiety has a molecular weight of 40 kDa and is branched.

37. A therapeutic formulation comprising a polypeptide of claim 36, and a pharmaceutically acceptable carrier.

39. An isolated polypeptide comprising the amino acid sequence of any of SEQ ID NOs:

6-183, 186-197, and 199, wherein said polypeptide binds to an extracellular domain of the human KDR, and wherein said polypeptide is at least 85% pure.

40. A therapeutic formulation comprising a polypeptide consisting of the amino acid sequence of SEQ ID NO:

194, a branched 40 kDa polyethylene glycol moiety that is covalently bound to the cysteine residue at position 93 of the polypeptide, and a pharmaceutically acceptable carrier.

41. An isolated polypeptide comprising a fibronectin type III tenth (¹⁰Fn3) domain having between about 80 and about 150 amino acids, wherein the ¹⁰Fn3 domain 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, wherein the ¹⁰Fn3 domain comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO;

193, and wherein the polypeptide is at least 85% pure.

42. An isolated polypeptide comprising a fibronectin type III tenth (¹⁰Fn3) domain having between about 80 and about 150 amino acids, wherein the ¹⁰Fn3 domain 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, wherein the ¹⁰Fn3 domain comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO;

194, and wherein the polypeptide is at least 85% pure.

43. An isolated polypeptide comprising a fibronectin type III tenth (¹⁰Fn3) domain having between about 80 and about 150 amino acids wherein the ¹⁰Fn3 domain 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, wherein the polypeptide also binds to an extracellular domain of the mouse Flk1 with a K_D of less than 1×

10<sup>−

6</sup>, and wherein the ¹⁰Fn3 domain comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO;

192.

44. The polypeptide of claim 43, wherein the ¹⁰Fn3 domain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:

192.

45. The polypeptide of claim 43, wherein the ¹⁰Fn3 domain comprises the amino acid sequence of SEQ ID NO:

192.

46. The polypeptide of claim 43, wherein the ¹⁰Fn3 domain comprises the amino acid sequence of SEQ ID NO:

193.

47. The polypeptide of claim 43, wherein the ¹⁰Fn3 domain comprises the amino acid sequence of SEQ ID NO:

194.

48. The polypeptide of claim 43, 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.

49. The polypeptide of claim 48, wherein the moiety that reduces the clearance rate is a polyethylene glycol moiety.

50. The polypeptide of claim 49, wherein the polyethylene glycol moiety has a molecular weight of between 2 and 100 kDa.

51. The polypeptide of claim 50, wherein the polyethylene glycol moiety has a molecular weight ranging from about 10 kDa to about 60 kDa.

52. The polypeptide of claim 51, wherein the polyethylene glycol moiety has a molecular weight of 40 kDa and is branched.

53. The polypeptide of claim 49, wherein the polyethylene glycol moiety is covalently bonded to a thiol moiety or an amine moiety in the polypeptide.

54. The polypeptide of claim 43, 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>−

7</sup>M and inhibits KDR-mediated VEGF activity.

55. A therapeutic formulation comprising a polypeptide of any of claims 43-45 and 48-49, and a pharmaceutically acceptable carrier.

56. An isolated polypeptide comprising a fibronectin type III tenth (¹⁰Fn3) domain having between about 80 and about 150 amino acids wherein the ¹⁰Fn3 domain 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 95% identical to the amino acid sequence of SEQ ID NO;

192.

57. The polypeptide of claim 56, wherein the ¹⁰Fn3 domain comprises the amino acid sequence of SEQ ID NO:

192.

58. The polypeptide of claim 56, wherein the ¹⁰Fn3 domain comprises the amino acid sequence of SEQ ID NO:

193.

59. The polypeptide of claim 56, wherein the ¹⁰Fn3 domain comprises the amino acid sequence of SEQ ID NO:

194.

60. The polypeptide of claim 56, 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.

61. The polypeptide of claim 60, wherein the moiety that reduces the clearance rate is a polyethylene glycol moiety.

62. The polypeptide of claim 61, wherein the polyethylene glycol moiety has a molecular weight of between 2 and 100 kDa.

63. The polypeptide of claim 62, wherein the polyethylene glycol moiety has a molecular weight ranging from about 10 kDa to about 60 kDa.

64. The polypeptide of claim 63, wherein the polyethylene glycol moiety has a molecular weight of 40 kDa and is branched.

65. The polypeptide of claim 61, wherein the polyethylene glycol moiety is covalently bonded to a thiol moiety or an amine moiety in the polypeptide.

66. The polypeptide of claim 56, 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>−

7</sup>M and inhibits KDR-mediated VEGF activity.

67. The polypeptide of claim 56, wherein the polypeptide also binds to an extracellular domain of the mouse Flk1 with a K_D of less than 1×

10<sup>−

6</sup>.

68. A therapeutic formulation comprising a polypeptide of any of claims 56 and 60-61, and a pharmaceutically acceptable carrier.

69. An isolated polypeptide comprising a fibronectin type III tenth (¹⁰Fn3) domain having between about 80 and about 150 amino acids wherein the ¹⁰Fn3 domain binds to an extracellular domain of the human KDR protein with a dissociation constant (K_D) of less than 100 nM, wherein the ¹⁰Fn3 domain comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:

192, wherein the polypeptide is covalently bound to a polyethylene glycol (PEG) moiety, wherein the PEG moiety is attached to a Cys residue of the polypeptide by site directed pegylation, and wherein the polypeptide has a clearance rate of less than 30 mL/hr/kg in a mammal.

70. The polypeptide of claim 69, wherein the ¹⁰Fn3 domain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:

192.

71. The polypeptide of claim 69, wherein the ¹⁰Fn3 domain comprises the amino acid sequence of SEQ ID NO:

192.

72. The polypeptide of claim 69, wherein the ¹⁰Fn3 domain comprises the amino acid sequence of SEQ ID NO:

193.

73. The polypeptide of claim 69, wherein the ¹⁰Fn3 domain comprises the amino acid sequence of SEQ ID NO:

194.

74. The polypeptide of claim 69, wherein the PEG moiety has a molecular weight of between 2 and 100 kDa.

75. The polypeptide of claim 74, wherein the PEG moiety has a molecular weight ranging from about 10 kDa to about 60 kDa.

76. The polypeptide of claim 75, wherein the PEG moiety has a molecular weight of 40 kDa and is branched.

77. The polypeptide of claim 69, wherein the polypeptide inhibits KDR-mediated VEGF activity.

78. The polypeptide of claim 69, wherein the polypeptide also binds to an extracellular domain of the mouse Flk1 with a K_D of less than 1×

10<sup>−

6</sup>.

79. A therapeutic formulation comprising a polypeptide of any of claims 69-71, and a pharmaceutically acceptable carrier.