Fibronectin binding domains with reduced immunogenicity

• US 9,416,170 B2
• Filed: 02/01/2013
• Issued: 08/16/2016
• Est. Priority Date: 10/31/2011
• Status: Active Grant

• Current Assignee: Bristol-Myers Squibb Company
• Original Assignee: Bristol-Myers Squibb Company
• Inventors: Davis, Jonathan, Lipovsek, Dasa, Camphausen, Ray
• Primary Examiner(s): BOESEN, CHRISTIAN C
• Application Number:
  US13/757,664
• Publication Number:
  US 20130196871A1
• Time in Patent Office: 1,292 Days
• Field of Search: None
• US Class Current: 1/1
• 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: C07K 14/47 from mammals
• 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 16/241 Tumor Necrosis Factors
• CPC Class Codes: C07K 16/244 Interleukins [IL]
• CPC Class Codes: C07K 16/28 against receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: C07K 16/2833 against MHC-molecules, e.g. HLA-molecules
• CPC Class Codes: C07K 16/2857 against nuclear receptors, e.g. retinoic acid receptor [RAR], RXR, orphan receptor
• 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: 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
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: G01N 33/6887 from muscle, cartilage or connective tissue

Abstract

Fibronectin type III (¹⁰Fn3) binding domains having novel designs that are associated with reduced immunogenicity are provided. The application describes alternative ¹⁰Fn3 binding domains in which certain immunogenic regions are not modified when producing a binder in order to maintain recognition as a self antigen by the host organism. The application also describes ¹⁰Fn3 binding domains in which HLA anchor regions have been destroyed thereby reducing the immunogenic contribution of the adjoining region. Also provided are ¹⁰Fn3 domains having novel combinations of modified regions that can bind to a desired target with high affinity.

Claims

1. A polypeptide comprising a human fibronectin type 3 tenth (¹⁰Fn3) domain, which comprises AB, BC, CD, DE, EF, and FG loops and β

-strands A, B, C, D, E, F, and G, wherein the ¹⁰Fn3 domain comprises modifications only in the amino acid sequence of the FG loop relative to the FG loop of the wild-type human ¹⁰Fn3 domain as set forth in SEQ ID NO;

6, and in the amino acid sequence of the CD loop relative to the corresponding loop of the wild-type human ¹⁰Fn3 domain as set forth in SEQ ID NO;

6, wherein the modifications contribute to binding a target.

2. A library comprising the polypeptide of claim 1.

3. A method treating or diagnosing a disease or disorder in a subject in need thereof comprising administering to the subject the polypeptide of claim 1.

4. The polypeptide of claim 1, wherein the CD loop is 3 to 11 residues long and the FG loop is 1 to 10 residues long.

5. The polypeptide of claim 1, wherein the modification in the CD loop is in the amino acid residues corresponding to amino acids 39 to 45 of the wild-type human ¹⁰Fn3 domain as set forth in SEQ ID NO:

6.

6. The polypeptide of claim 1, wherein the modification in the FG loop is in the amino acid residues corresponding to amino acids 77 to 83 of the wild-type human ¹⁰Fn3 domain as set forth in SEQ ID NO:

6.

7. The polypeptide of claim 5, wherein the modification in the FG loop is in the amino acid residues corresponding to amino acids 77 to 83 of the wild-type human ¹⁰Fn3 domain as set forth in SEQ ID NO:

6.

8. The polypeptide of claim 1, wherein the modification in the CD loop is in the amino acid residues corresponding to amino acids 37 to 45 of the wild-type human ¹⁰Fn3 domain as set forth in SEQ ID NO:

6.

9. The polypeptide of claim 1, wherein the modification in the FG loop is in the amino acid residues corresponding to amino acids 76 to 86 of the wild-type human ¹⁰Fn3 domain as set forth in SEQ ID NO:

6.

10. The polypeptide of claim 8, wherein the modification in the PG loop is in the amino acid residues corresponding to amino acids 76 to 86 of the wild-type human ¹⁰Fn3 domain as set forth in SEQ ID NO:

6.

11. The polypeptide of claim 1 or 4, wherein the modifications are insertions, substitutions, or deletions.

12. The polypeptide of claim 5, wherein the modifications are insertions, substitutions, or deletions.

13. The polypeptide of claim 6, wherein the modifications are insertions substitutions, or deletions.

14. The polypeptide of claim 7, wherein the modifications are insertions, substitutions, or deletions.

15. The polypeptide of claim 8, wherein the modifications are insertions, substitutions, or deletions.

16. The polypeptide of claim 9, wherein the modifications are insertions, substitutions, or deletions.

17. The polypeptide of claim 10, wherein the modifications are insertions, substitutions, or deletions.

18. The polypeptide of claim 1 or 4, wherein the modifications are substitutions.

19. The polypeptide of claim 5, wherein the modifications are substitutions.

20. The polypeptide of claim 6, wherein the modifications are substitutions.

21. The polypeptide of claim 7, wherein the modifications are substitutions.

22. The polypeptide of claim 8, wherein the modifications are substitutions.

23. The polypeptide of claim 9, wherein the modifications are substitutions.

24. The polypeptide of claim 10, wherein the modifications are substitutions.

25. The polypeptide of any one of claims 1 and 4 to 7, wherein the ¹⁰Fn3 domain binds to the target with a K_d of less than 500 nM.

26. The polypeptide of any one of claims 1 and 4 to 7, wherein the ¹⁰Fn3 domain binds to the target with a K_d of less than 100 nM.

27. The polypeptide of any one of claims 1 and 4 to 7, wherein the ¹⁰Fn3 domain binds to the target with a K_d of less than 50 nM.

28. The polypeptide of any one of claims 1 and 4 to 7, wherein the ¹⁰Fn3 domain binds to the target with a K_d of less than 1 nM.

29. The polypeptide of any one of claims 1 and 4 to 7, wherein the ¹⁰Fn3 domain binds to the target with a K_d of less than 500 pM.

30. The polypeptide of any one of claims 1 and 4 to 7, wherein the target is a human protein.

31. The polypeptide of any one of claims 1 and 4 to 7, wherein the ¹⁰Fn3 domain binds to tumor necrosis factor alpha (TNF-alpha), delta-like protein 4 (DLL4), interleukin 17 (IL-17), or pregnane X receptor (PXR).

32. The polypeptide of any one of claims 1 and 4 to 7, wherein the ¹⁰Fn3 domain binds to TNF-alpha.

33. The polypeptide of any one of claims 1 and 4 to 7, wherein the ¹⁰Fn3 domain binds to DLL4.

34. The polypeptide of any one of claims 1 and 4 to 7, wherein the ¹⁰Fn3 domain binds to IL-17.

35. The polypeptide of any one of claims 1 and 4 to 7, wherein the ¹⁰Fn3 domain binds to PXR.

36. The polypeptide of any one of claims 1 and 4 to 7, wherein the ¹⁰Fn3 domain further comprises a pharmacokinetic moiety.

37. The polypeptide of claim 36, wherein the pharmacokinetic moiety is selected from a polyoxyalkylene moiety, a human serum albumin binding protein, a sialic acid, a human serum albumin, a transferrin, an IgG, an IgG binding protein, and an Fc fragment.

38. The polypeptide of claim 36, wherein the pharmacokinetic moiety reduces a clearance rate of the polypeptide in a mammal relative to an unmodified polypeptide.

39. The polypeptide of claim 38, wherein the clearance rate is reduced by greater than three-fold relative to the unmodified polypeptide.

40. A library comprising the polypeptide of any one of claims 4 to 7.

41. A molecule comprising two or more polypeptides of any one of claims 1 and 4 to 7.

42. The molecule of claim 41, wherein each of the polypeptides are connected by a polypeptide linker selected from any one of SEQ ID NOs:

32-43.

43. The molecule of claim 41, wherein each of the polypeptides binds to a single target molecule.

44. The molecule of claim 43, wherein two or more of the polypeptides bind to different target molecules.

45. A method of treating or diagnosing a disease or disorder in a subject in need thereof comprising administering to the subject the polypeptide of any one of claims 1 and 4 to 7.