Pharmacokinetic modulation and compositions for modified Fn3 polypeptides

• US 20070160533A1
• Filed: 07/07/2006
• Published: 07/12/2007
• Est. Priority Date: 12/05/2003
• Status: Active Grant

• Current Assignee: Bristol-Myers Squibb Company
• Original Assignee: Bristol-Myers Squibb Company
• Inventors: Chen, Yan, Harris, Alan, Gokemeijer, Jochem, Getmanova, Elena, Wright, Martin, Lim, Ai
• Granted Patent: US 7,847,062 B2
• Time in Patent Office: Days
• US Class Current: 424/1.690
• 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 polypeptide comprising:

a) a fibronectin type III (Fn3) domain having from about 80 to about 150 amino acids, wherein the Fn3 domain binds to a target molecule with a K_D of less than 100 nM; and

b) a covalently bound polyoxyalkylene moiety.

2. The Fn3 polypeptide of claim 1, wherein the Fn3 polypeptide comprises a ¹⁰Fn3 domain.

3. The Fn3 polypeptide of claim 2, wherein the Fn3 polypeptide comprises an amino acid sequence that is at least 60% identical to SEQ NO:

5.

4. The Fn3 polypeptide of claim 3 wherein the Fn3 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.

5. The Fn3 polypeptide of claim 4, wherein the loop FG participates in target molecule binding.

6. The Fn3 polypeptide of claim 5, wherein the loops BC, DE and FG participate in target molecule binding.

7. The Fn3 polypeptide of claim 4, wherein each of the beta strands consists essentially of an amino acid sequence that is at least 80% identical to the sequence of a corresponding beta strand of SEQ ID NO:

5.

8. The Fn3 polypeptide of claim 4, wherein each of the loops AB, CD and EF consists essentially of an amino acid sequence that is at least 80% identical to the sequence of a corresponding loop of SEQ ID NO:

5.

9. The Fn3 polypeptide of claim 1, wherein the polyoxyalkylene moiety is a polyethylene glycol (PEG) moiety.

10. The Fn3 polypeptide of claim 3, wherein the polyoxyalkylene moiety is a polyethylene glycol (PEG) moiety.

11. The Fn3 polypeptide of claim 9, wherein the Fn3 polypeptide has a clearance rate of less than 30 mL/hr/kg in a mammal.

12. The Fn3 polypeptide of claim 11, wherein the Fn3 polypeptide binds to the target molecule with a K_D of less than 10 nM.

13. The Fn3 polypeptide of claim 2, wherein the polyoxyalkylene moiety is attached to a thiol group or an amine group.

14. The Fn3 polypeptide of claim 2, wherein the polyoxyalkylene moiety is attached to the Fn3 polypeptide by site directed conjugation.

15. The Fn3 polypeptide of claim 13, wherein the polyoxyalkylene moiety is conjugated to a cysteine residue.

16. The Fn3 polypeptide of claim 15, wherein the cysteine residue is not native to the Fn3 polypeptide.

17. The Fn3 polypeptide of claim 14, wherein the polyoxyalkylene moiety is conjugated to an amine.

18. The Fn3 polypeptide of claim 13, wherein the polyoxyalkylene moiety is conjugated to an ε

-amino group of a lysine residue.

19. The Fn3 polypeptide of claim 18, wherein the lysine residue is not native to the Fn3 polypeptide.

20. The Fn3 polypeptide of claim 3, wherein the polyoxyalkylene moiety does not substantially decrease the binding of the Fn3 polypeptide to the target molecule.

21. The Fn3 polypeptide of claim 20, wherein the polyoxyalkylene moiety is attached at a position on the Fn3 polypeptide selected from the group consisting a) the N-terminus;

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

c) a loop that does not participate in target binding;

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

e) at the C-terminus.

22. The Fn3 polypeptide of claim 9, wherein the PEG moiety has a molecular weight of between about 2 kDa and about 100 kDa.

23. The Fn3 polypeptide of claim 22, wherein the PEG moiety is a branched PEG moiety.

24. The Fn3 polypeptide of claim 22, wherein the Fn3 polypeptide comprises a ¹⁰Fn3 domain.

25. The Fn3 polypeptide of claim 24, wherein the Fn3 polypeptide comprises a ¹⁰Fn3 domain and has an amino acid sequence that is at least 60% identical to SEQ NO:

5.

44. A pharmaceutical preparation comprising a polypeptide of claim 1.

26. A polypeptide comprising:

a) a fibronectin type III (Fn3) domain of about 80 to about 150 amino acids, wherein the Fn3 domain binds to a target molecule with a K_D of less than 100 nM; and

, wherein the Fn3 domain comprises a cysteine residue at a position where a cysteine residue does not naturally occur, wherein the position is 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 that does not contact the target molecule;

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

e) at the C-terminus.

27. The polypeptide of claim 26, wherein the cysteine is conjugated to an active moiety.

28. The polypeptide of claim 27, wherein the active moiety is selected from the group consisting of:

a radioactive moiety, a chemotherapeutic agent, and a label.

29. The polypeptide of claim 26, wherein the cysteine is a surface-exposed residue.

30. The polypeptide of claim 26, wherein the polypeptide binds to the target molecule with a K_D of less than 10 nM.

45. A pharmaceutical preparation comprising a polypeptide of claim 26.

31. A polypeptide comprising a tenth fibronectin type III (¹⁰Fn3) domain of about 80 to about 150 amino acids and comprising a) a loop, BC;

b) a loop, DE; and

c) a loop FG;

wherein, wherein the ¹⁰Fn3 domain binds to a target molecule with a K_D of less than 100 nM, and wherein the ¹⁰Fn3 domain comprises a cysteine residue at a position where a cysteine residue does not naturally occur, wherein the position is not within loop BC, loop DE or loop FG.

32. The polypeptide of claim 31, wherein the ¹⁰Fn3 domain comprises an amino acid sequence that is at least 60% identical to SEQ NO:

5.

33. The polypeptide of claim 31 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.

34. The polypeptide of claim 31, wherein the loop FG participates in target molecule binding.

35. The polypeptide of claim 33, wherein each of the beta strands consists essentially of an amino acid sequence that is identical to the sequence of a corresponding beta strand of SEQ ID NO:

5.

36. The Fn3 polypeptide of claim 33, wherein each of the loops AB, CD and EF consists essentially of an amino acid sequence that is identical to the sequence of a corresponding loop of SEQ ID NO:

5.

37. The polypeptide of claim 31, wherein the cysteine is conjugated to an active moiety.

38. The polypeptide of claim 37, wherein the active moiety is selected from the group consisting of:

a radioactive moiety, a chemotherapeutic agent, and a label.

39. The polypeptide of claim 31, wherein the cysteine is located in a loop that does not contact the target molecule.

40. The polypeptide of claim 39, wherein the cysteine is a surface-exposed residue.

41. The polypeptide of claim 31, wherein the polypeptide binds to target molecule with a K_D of less than 10 nM.

46. A pharmaceutical preparation comprising a polypeptide of claim 36.

42. A polypeptide comprising a fibronectin type III (Fn3) domain of about 80 to about 150 amino acids, wherein at least one loop of the Fn3 domain participates in binding to a target molecule, wherein the Fn3 domain comprises a non-native lysine residue at a position 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 that does not contact the target molecule;

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

e) at the C-terminus.

43. A polypeptide comprising:

a ¹⁰Fn3 domain of about 80 to about 150 amino acids and comprising a) a loop, BC;

b) a loop, DE; and

c) a loop FG;

wherein at least one loop of the ¹⁰Fn3 domain participates in binding to a target molecule, and wherein the ¹⁰Fn3 domain comprises a non-native lysine residue at a position that is not within loop BC, loop DE or loop FG.