Fibronectin binding domains with reduced immunogenicity

• US 9,765,132 B2
• Filed: 02/01/2013
• Issued: 09/19/2017
• 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:
  US13/757,668
• Publication Number:
  US 20130245238A1
• Time in Patent Office: 1,691 Days
• Field of Search: None
• 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 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 a modification only in the amino acid sequence of the CD loop and β

-strands C and D relative to the wild-type human ¹⁰Fn3 domain as set forth in SEQ ID NO;

6, wherein the modification in the CD loop is in the amino acid residues corresponding to amino acids 37 to 45 or 47 of the wild-type human ¹⁰Fn3 domain as set forth in SEQ ID NO;

6, wherein the polypeptide does not comprise amino acids Arg-Glv-Asp, wherein the polypeptide is capable of binding to a target, and wherein the modification contributes to binding to the target.

2. A library comprising the polypeptide of claim 1.

3. A method of 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 at least 7 amino acid residues of the CD loop are modified relative to the wild-type human ¹⁰Fn3 domain as set forth in SEQ ID NO:

6.

5. The polypeptide of claim 1, wherein at least 8 amino acid residues of the CD loop are modified relative to the wild-type human ¹⁰Fn3 domain as set forth in SEQ ID NO:

6.

6. The polypeptide of claim 1, wherein at least 9 amino acid residues of the CD loop axe modified relative to the wild-type human ¹⁰Fn3 domain as set forth in SEQ ID NO:

6.

7. The polypeptide of claim 1, wherein 10 amino acid residues of the CD loop are modified relative to the wild-type human ¹⁰Fn3 domain as set forth SEQ ID NO:

6.

8. The polypeptide of claim 1, wherein the CD loop corresponds to residues 37 to 47 of SEQ ID NO:

6.

9. The polypeptide of claim 1, wherein at most 7 amino acid residues of the CD loop axe modified relative to the wild-type human ¹⁰Fn3 domain as set forth in SEQ ID NO:

6.

10. The polypeptide of claim 1, wherein at most 8 amino acid residues of the CD loop are modified relative to the wild-type human ¹⁰Fn3 domain as set forth in SEQ ID NO:

6.

11. The polypeptide of claim 1, wherein at most 9 amino acid residues of the CD loop are modified relative to the wild-type human ¹⁰Fn3 domain as set forth in SEQ ID NO:

6.

12. The polypeptide of claim 1, wherein the amino acid sequence of the CD loop is extended in length or reduced in length relative to the CD loop of the wild-type human ¹⁰Fn3 domain as set forth in SEQ ID NO:

6.

13. The polypeptide of any one of claims 1 and 4 to 12, wherein the modification comprises a substitution of an amino acid residue.

14. The polypeptide of any one of claims 1 and 4 to 12, wherein the modification comprises a deletion of an amino acid residue.

15. The polypeptide of any one of claims 1 and 4 to 12, wherein the modification comprises an insertion of an amino acid residue.

16. The polypeptide of any one of claims 1 and 4 to 12, wherein the polypeptide has at least 90% sequence identity to the amino acid sequence of the wild-type human ¹⁰Fn3 domain as set forth in SEQ ID NO:

6.

17. The polypeptide of any one of claims 1 and to 12, wherein the polypeptide binds to the target with a K_d of less than 500 nM.

18. The polypeptide of any one of claims 1 and 4 to 12, wherein the polypeptide binds to the target with a K_d of less than 100 nM.

19. The polypeptide of any one of claims 1 and 4 to 12, wherein the polypeptide binds to the target with a K_d of less than 50 nM.

20. The polypeptide of any one of claims 1 and to 12, wherein the target is a human protein.

21. The polypeptide of any one of claims 1 and 4 to 12, wherein the polypeptide binds to tumor necrosis factor alpha (TNF-alpha), delta-like protein 4 (DLL4), interleukin 17 (IL-17), or pregnane X receptor (PXR).

22. The polypeptide of any one of claims 1 and 4 to 12, wherein the polypeptide binds to TNF-alpha.

23. The polypeptide of any one of claims 1 and to 12, wherein the polypeptide binds to DLL4.

24. The polypeptide of any one of claims 1 and 4 to 12, wherein the polypeptide binds to IL-17.

25. The polypeptide of any one of claims 1 and to 12, wherein the polypeptide binds to PXR.

26. The polypeptide of any one of claims 1 and 4 to 12, wherein the ¹⁰Fn3 domain further comprises a pharmacokinetic moiety.

27. The polypeptide of claim 26, 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.

28. The polypeptide of claim 26, wherein the pharmacokinetic moiety reduces a clearance rate of the polypeptide in a mammal relative to an unmodified polypeptide.

29. The polypeptide of claim 28, wherein the clearance rate is reduced by greater than three-fold relative to the unmodified polypeptide.

30. The polypeptide of claim 16, wherein the ¹⁰Fn3 domain further comprises a pharmacokinetic moiety.

31. The polypeptide of claim 17, wherein the ¹⁰Fn3 domain further comprises a pharmacokinetic moiety.

32. The polypeptide of claim 18, wherein the ¹⁰Fn3 domain further comprises a pharmacokinetic moiety.

33. The polypeptide of claim 19, wherein the ¹⁰Fn3 domain further comprises a pharmacokinetic moiety.

34. The polypeptide of claim 20, wherein the ¹⁰Fn3 domain further comprises a pharmacokinetic moiety.

35. The polypeptide of claim 21, wherein the ¹⁰Fn3 domain further comprises a pharmacokinetic moiety.

36. The polypeptide of claim 22, wherein the ¹⁰Fn3 domain further comprises a pharmacokinetic moiety.

37. The polypeptide of claim 23, wherein the ¹⁰Fn3 domain further comprises a pharmacokinetic moiety.

38. The polypeptide of claim 24, wherein the ¹⁰Fn3 domain further comprises a pharmacokinetic moiety.

39. The polypeptide of claim 25, wherein the ¹⁰Fn3 domain further comprises a pharmacokinetic moiety.

40. The polypeptide of any one of claims 1 and 4 to 12, wherein the ¹⁰Fn3 domain further comprises a detectable moiety.

41. A library comprising the polypeptide of airy one of claims 1 to 12.

42. A molecule comprising two or more polypeptides of any one of claims 1 and 4 to 12.

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

32-43.

44. The molecule of claim 42, wherein each of the polypeptides binds to a single target molecule.

45. The molecule of claim 42, wherein two or more of the polypeptides bind to different target molecules.

46. 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 to 12.