IMPROVED FIBRONECTIN-BASED BINDING MOLECULES AND USES THEREOF

• US 20110038866A1
• Filed: 05/04/2009
• Published: 02/17/2011
• Est. Priority Date: 05/02/2008
• Status: Active Grant

• Current Assignee: Novartis Ag
• Original Assignee: Novartis Ag
• Inventors: Hastewell, John, Loew, Andreas
• Granted Patent: US 9,296,810 B2
• Time in Patent Office: Days
• US Class Current: 424/134.1
• 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: A61K 47/62 the modifying agent being a protein, peptide or polyamino acid
• 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 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• 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: C07K 14/78 Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
• CPC Class Codes: C07K 16/005 constructed by phage libraries
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: C07K 16/2863 against receptors for growth factors, growth regulators
• CPC Class Codes: C07K 2317/31 multispecific
• 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 2319/31 fusions, other than Fc, for prolonged plasma life, e.g. albumin
• CPC Class Codes: C07K 2319/70 containing domain for protein-protein interaction
• CPC Class Codes: G01N 2333/78 Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
• CPC Class Codes: G01N 33/6845 Methods of identifying protein-protein interactions in protein mixtures

Abstract

The invention provides fibronectin type III (Fn3)-based binding molecules that bind to a specific target antigen. The invention further provides bispecific Fn3-based binding molecules that bind to two or more targets simultaneously. The Fn3-based binding molecules of the invention can also be linked together to form multispecific Fn3-based binding molecules, and/or can be conjugated to a non-Fn3 moiety, such as, Human Serum Albumin (HSA), for improved half life and stability. The invention also provides methods for generating, screening and using Fn3-based binding molecules in a variety of therapeutic and diagnostic applications.

Claims

1. A fibronectin type III (Fn3)-based binding molecule comprising an Fn3 domain, wherein at least one amino acid in one or more of the bottom AB, CD or EF loop regions or C-terminal of the Fn3 domain are altered compared to the wild-type Fn3 domain comprising SEQ ID NO:

1 to create a non-Fn3 binding sequence which binds to a specific target.

2. The Fn3-based binding molecule of claim 1, wherein said at least one amino acid is selected from the group consisting of the amino acids at position 15, 16, 38, 39, 40, 41, 42, 43, 44, 45, 60, 61, 62, 63, 64, 93, 95, and 96 of SEQ ID NO:

1.

3. The Fn3-based binding molecule of claim 1, wherein the non-Fn3 binding sequence comprises all or a portion of a complementarity determining region (CDR) of an antibody or a T-cell receptor.

4. The Fn3-based binding molecule of claim 1, wherein the specific target is a half-life extender.

5. The Fn3-based binding molecule of claim 4, wherein the half-life extender is human serum albumin.

6. The Fn3-based binding molecule of claim 1, wherein the specific target is lyzozyme.

42. A method of detecting a protein in a sample comprising labeling the Fn-3 based binding molecule or conjugate of any of the preceding claims, contacting the labeled binding molecule or conjugate with the sample, and detecting complex formation between the Fn3-based binding molecule or conjugate with the protein.

43. A variegated nucleic acid library encoding Fn3-based binding molecules of claim 1.

7. A Fn3-based binding molecule comprising a first Fn3 domain, wherein at least one amino acid in one or more of the bottom AB, CD or EF loop regions or C-terminal of the Fn3 domain are altered compared to the wild-type Fn3 domain comprising SEQ ID NO:

1 to create a non-Fn3 binding sequence which binds to a first target, and wherein at least one amino acid in one or more of the bottom AB, CD or EF loop regions or C-terminal of a second Fn3 domain are altered compared to the wild-type Fn3 domain comprising SEQ ID NO;

1 to create a non-Fn3 binding sequence which binds to a second target.

8. The Fn3-based binding molecule of claims 7, wherein said first and second targets are present on the same molecule.

9. The Fn3-based binding molecule of claim 7, wherein said first and second targets are present on different molecules.

44. A variegated nucleic acid library encoding Fn3-based binding molecules of claim 7.

10. A Fn3-based binding molecule comprising a first Fn3 domain, wherein at least one amino acid in one or more of the bottom AB, CD Or EF loop regions or C-terminal of the Fn3 domain are altered compared to the wild-type Fn3 domain comprising SEQ ID NO:

1 to create a non-Fn3 binding sequence which binds to a half-life extender, and wherein at least one amino acid in one or more of the bottom AB, CD or EF loop regions or C-terminal of a second Fn3 domain are altered compared to the wild-type Fn3 domain comprising SEQ ID NO;

1 to create a non-Fn3 binding sequence which binds to a second target.

11. The Fn3-based binding molecule of claim 10, wherein the half-life extender is human serum albumin.

12. The Fn3-based binding molecule of claim 10, wherein the second target is VEGFR2.

45. A variegated nucleic acid library encoding Fn3-based binding molecules of claim 10.

13. A bispecific Fn3-based binding molecule comprising an Fn3 domain, wherein at least one amino acid in one or more of the bottom AB, CD or EF loop regions or C-terminal of the Fn3 domain are altered compared to the wild-type Fn3 domain comprising SEQ ID NO:

1 to create a non-Fn3 binding sequence which binds to a first target, and wherein at least one amino acid in one or more of the top BC, DE or FG loop regions of the Fn3 domain are altered compared to the wild-type Fn3 domain comprising SEQ ID NO;

1 to create a non-Fn3 binding sequence which binds to a second target

14. The bispecific Fn3-based binding molecule of claim 13, wherein said at least one amino acid in said bottom AB, CD or EF loop regions is selected from the group consisting of the amino acids at position 15, 16, 38, 39, 40, 41, 42, 43, 44, 45, 60, 61, 62, 63, 64, 93, 95, and 96 of SEQ ID NO:

1, and wherein said at least one amino acid in said top BC, DE or FG loop regions is selected from the group consisting of amino acids at position 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 51, 52, 53, 54, 55, 56, 76, 77, 78, 79, 80, 80, 81, 82, 83, 84, 85, 86, 87, and 88 of SEQ ID NO;

1

15. The bispecific Fn3-based binding molecule of claim 13, wherein the non-Fn3 binding sequence comprises all or a portion of a complementarity determining region (CDR) of an antibody or a T-cell receptor.

16. The bispecific Fn3-based binding molecule of claim 13, wherein said first and second targets are present on the same molecule.

17. The bispecific Fn3-based binding molecule of claim 13, wherein said first and second targets are present on different molecules.

18. A bispecific Fn3-based binding molecule comprising an Fn3 domain, wherein at least one amino acid in one or more of the bottom AB, CD or EF loop regions or C-terminal of the Fn3 domain are altered compared to the wild-type Fn3 domain comprising SEQ ID NO:

1 to create a non-Fn3 binding sequence which binds to a half-life extender, and wherein at least one amino acid in one or more of the top BC, DE or FG loop regions of the Fn3 domain are altered compared to the wild-type Fn3 domain comprising SEQ ID NO;

1 to create a non-Fn3 binding sequence which binds to a second target.

19. The bispecific Fn3-based binding molecule of claim 18, wherein the half-life extender is human serum albumin

20. The bispecific Fn3-based binding molecule of claim 18, wherein the second target is VEGFR2.

46. A variegated nucleic acid library encoding Fn3-based binding molecules of claim 18.

21. A multispecific Fn3-based binding molecule comprising two or more Fn3-based binding molecules.

22. The multispecific Fn3-based binding molecule of claim 21, wherein each of said Fn3-based binding molecules binds to a different target.

23. The multispecific Fn3-based binding molecule of claim 22, wherein said different targets are present on the same molecule.

24. The multispecific Fn3-based binding molecule of claim 22, wherein said different targets are present on different molecule

47. A variegated nucleic acid library encoding Fn3-based binding molecules of claim 21.

25. The Fn3-based binding molecule which binds to a non-Fn3 moiety that increases the half-life of the Fn3-based binding molecule.

26. A multispecific Fn3-based binding molecule comprising two or more monospecific Fn3-based binding molecules, wherein at least one monospecific Fn3-based binding molecule comprises at least one amino acid in one or more of the bottom AB, CD or EF loop regions or C-terminal of the Fn3 domain that is altered compared to the wild-type Fn3 domain comprising SEQ ID NO:

1 to create a non-Fn3 binding sequence which binds to a first target; and

a second monospecific Fn3-based binding molecule having at least one amino acid in one or more of the top BC, DE or FG loop regions of the Fn3 domain that is altered compared to the wild-type Fn3 domain comprising SEQ ID NO;

1 to create a non-Fn3 binding sequence which binds to a second target, wherein the first and second monospecific Fn3-based binding molecules are connected by a linker sequence.

27. The multispecific Fn3-based binding molecule of claim 26, further comprising a third monospecific Fn3-based binding molecule having at least one amino acid in one or more of the top BC, DE or FG loop regions of the Fn3 domain that is altered compared to the wild-type Fn3 domain comprising SEQ ID NO:

1 to create a non-Fn3 binding sequence which binds to one or more targets, and wherein the second and third monospecific Fn3-based binding molecules are connected by a linker sequence.

28. The multispecific Fn3-based binding molecule of claim 26, wherein the first target is a half-life extender.

29. The multispecific Fn3-based binding molecule of claim 28, wherein the half-life extender is human serum albumin.

30. The multispecific Fn3-based binding molecule of claim 26, wherein the second target is VEGFR2.

31. The multispecific Fn3-based binding molecule of claim 26, wherein the linker sequence is a G-S linker sequence.

48. A variegated nucleic acid library encoding Fn3-based binding molecules of claim 26.

32. An Fn3-based binding molecule comprising SEQ ID NO:

119 (clone

     87)

33. An Fn3-based binding molecule comprising SEQ ID NO:

120 (clone

     89)

34. A conjugate comprising an Fn3-based binding molecule linked to one or more non-Fn3 moieties.

35. The conjugate of claim 34, wherein the non-Fn3 moiety comprises or binds to a molecule which increases the half-life of the Fn3-based binding molecule.

36. The Fn3-based binding molecule or conjugate of claim 34, wherein the non-Fn3 moiety comprises a molecule selected from the group consisting of an antibody Fc region, Human Serum Albumin (HSA) and polyethylene glycol (PEG).

37. The Fn3-based binding molecule or conjugate which further comprises at least one modified amino acid residue compared to the wild-type Fn3 domain comprising SEQ ID NO:

1 for attaching a functional moiety.

38. The Fn3-based binding molecule or conjugate of claim 37, wherein the modified amino acid residue comprises the addition or substitution of a cysteine residue or non-natural amino acid residue.

39. The Fn3-based binding molecule or conjugate comprising beta strands from two or more different Fn3 domains.

40. A composition comprising the Fn3-based binding molecule or conjugate and a carrier.

41. A method of treating a subject for a disease selected from the group consisting of an autoimmune disease, a cancer, and an infectious disease, the method comprising administering to the subject a Fn3-based binding molecule, conjugate, or composition comprising an Fn3-based binding molecule.