Multivalent fibronectin based scaffold domain proteins

• US 8,728,483 B2
• Filed: 06/26/2012
• Issued: 05/20/2014
• Est. Priority Date: 05/22/2008
• Status: Active Grant

• Current Assignee: Bristol-Myers Squibb Company
• Original Assignee: Bristol-Myers Squibb Company
• Inventors: Camphausen, Ray, Furfine, Eric, Carvajal, Irvith M., Marsh, H. Nicholas, Gottardis, Marco, Carboni, Joan, Attar, Ricardo
• Primary Examiner(s): Bunner, Bridget E
• Application Number:
  US13/533,382
• Publication Number:
  US 20130012435A1
• Time in Patent Office: 693 Days
• Field of Search: None
• US Class Current: 424/185.1
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 38/39 Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
• CPC Class Codes: A61K 39/3955 against proteinaceous materials, e.g. enzymes, hormones, lymphokines
• CPC Class Codes: A61K 47/60 the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 14/71 for growth factors; for growth regulators
• CPC Class Codes: C07K 14/715 for cytokines; for lymphokines; for 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 2317/31 multispecific
• CPC Class Codes: C07K 2317/73 Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
• CPC Class Codes: C07K 2317/76 Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• 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 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/74 containing a fusion for binding to a cell surface receptor

Abstract

The present invention relates to multivalent polypeptides comprising at least two fibronectin scaffold domains connected via a polypeptide linker. The invention also relates to multivalent polypeptides for use in diagnostic, research and therapeutic applications. The invention further relates to cells comprising such proteins, polynucleotide encoding such proteins or fragments thereof, and to vectors comprising the polynucleotides encoding the innovative proteins.

Claims

1. A polypeptide comprising:

(a) an N-terminal domain comprising a first fibronectin type III tenth domain (¹⁰Fn3), wherein the ¹⁰Fn3 domain (i) comprises a loop, AB;

a loop, BC;

a loop, CD;

a loop, DE;

a loop EF; and

a loop FG;

(ii) has at least one loop selected from loop BC, DE, and FG with an altered amino acid sequence relative to the sequence of the corresponding loop of the human ¹⁰Fn3 domain (SEQ ID NO;

     1), and (iii) binds to insulin-like growth factor-I receptor (IGF-IR) with a K_D of less than 500 nM; and

(b) a C-terminal domain comprising a second fibronectin type III tenth domain (¹⁰Fn3), wherein the ¹⁰Fn3 domain (i) comprises a loop, AB;

a loop, BC;

a loop, CD;

a loop, DE;

a loop EF; and

a loop FG;

(ii) has at least one loop selected from loop BC, DE, and FG with an altered amino acid sequence relative to the sequence of the corresponding loop of the human ¹⁰Fn3 domain (SEQ ID NO;

     1), and (iii) binds to vascular endothelial growth factor receptor 2 (VEGFR2) with a K_D of less than 500 nM;

wherein the first and second ¹⁰Fn3 domains are linked via a polypeptide selected from a glycine-serine based linker, a glycine-proline based linker, a proline-alanine based linker or the amino acid sequence of SEQ ID NO;

20, wherein the polypeptide comprises an amino acid sequence at least 85% identical to the amino acid sequence of any one of SEQ ID NOs;

8-11, 29-31, and 63-70.

2. The polypeptide of claim 1, wherein the first ¹⁰Fn3 domain binds to IGF-IR with a K_D of less than 100 nM and the second ¹⁰Fn3 domain binds to VEGFR2 with a K_D of less than 100 nM.

3. The polypeptide of claim 1, wherein the loop BC and loop FG of the first and second ¹⁰Fn3 domains have an altered amino acid sequence relative to the sequence of the corresponding loops of the human ¹⁰Fn3 domain.

4. The polypeptide of claim 1, wherein the first ¹⁰Fn3 domain is linked at its C-terminus to the amino acid sequence of SEQ ID NO:

19.

5. The polypeptide of claim 1, wherein the second ¹⁰Fn3 domain is linked at its C-terminus to the amino acid sequence of SEQ ID NO:

17 or 18.

6. The polypeptide of claim 1, selected from a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs:

8-11, 29-31, and 63-70.

7. The polypeptide of claim 1, further comprising one or more pharmacokinetic (PK) moieties selected from:

a polyoxyalkylene moiety, a human serum albumin binding protein, sialic acid, human serum albumin, IgG, an IgG binding protein, transferrin, and an Fc fragment.

8. The polypeptide of claim 7, wherein the PK moiety is the polyoxyalkylene moiety and said polyoxyalkylene moiety is polyethylene glycol.

9. The polypeptide of claim 7, wherein the PK moiety and the polypeptide are linked via at least one disulfide bond, a peptide bond, a polypeptide, a polymeric sugar, or a polyethylene glycol moiety.

10. The polypeptide of claim 1, wherein said polypeptide has been deimmunized to remove one or more T-cell epitopes.

11. The polypeptide of claim 1, wherein the first and second ¹⁰Fn3 domains are linked via a glycine-serine based linker.

12. The polypeptide of claim 11, wherein the glycine-serine based linker is selected from the amino acid sequence of SEQ ID NOs:

21 and 22.

13. A pharmaceutically acceptable composition comprising the polypeptide claim 1, wherein the composition is essentially endotoxin free.

14. The polypeptide of claim 1, wherein the first and second ¹⁰Fn3 domains are linked via a glycine-proline based linker.

15. The polypeptide of claim 14, wherein the glycine-proline based linker is selected from the amino acid sequence of SEQ ID NOs:

32, 33, and 34.

16. The polypeptide of claim 1, wherein the BC, DE and FG loops of the first ¹⁰Fn3 domain comprise the amino acid sequences set forth in SEQ ID NOs:

2, 3 and 4, respectively.

17. The polypeptide of claim 1, wherein the BC, DE and FG loops of the second ¹⁰Fn3 domain comprise the amino acid sequences set forth in SEQ ID NOs:

5, 6 and 7, respectively.

18. A polypeptide comprising:

(a) an N-terminal domain comprising a first fibronectin type III tenth domain (¹⁰Fn3), wherein the ¹⁰Fn3 domain (i) comprises a loop, AB;

a loop, BC;

a loop, CD;

a loop, DE;

a loop EF; and

a loop FG;

(ii) has at least one loop selected from loop BC, DE, and FG with an altered amino acid sequence relative to the sequence of the corresponding loop of the human ¹⁰Fn3 domain (SEQ ID NO;

     1), (iii) binds to vascular endothelial growth factor receptor 2 (VEGFR2) with a K_D of less than 500 nM, and (iv) is at least 85% identical to SEQ ID NO;

40; and

(b) a C-terminal domain comprising a second fibronectin type III tenth domain (¹⁰Fn3), wherein the ¹⁰Fn3 domain (i) comprises a loop, AB;

a loop, BC;

a loop, CD;

a loop, DE;

a loop EF; and

a loop FG;

(ii) has at least one loop selected from loop BC, DE, and FG with an altered amino acid sequence relative to the sequence of the corresponding loop of the human ¹⁰Fn3 domain (SEQ ID NO;

     1), (iii) binds to insulin-like growth factor-I receptor (IGF-IR) with a K_D of less than 500 nM, and (iv) is at least 85% identical to SEQ ID NO;

35;

wherein the first and second ¹⁰Fn3 domains are linked via a polypeptide selected from a glycine-serine based linker, a glycine-proline based linker, a proline-alanine based linker or the amino acid sequence of SEQ ID NO;

20.

19. The polypeptide of claim 18, selected from a polypeptide comprising an amino acid sequence at least 85% identical to the amino acid sequence of any one of SEQ ID NOs:

12-15.

20. The polypeptide of claim 19, selected from a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs:

12-15.

21. The polypeptide of claim 18, wherein the first ¹⁰Fn3 domain binds to VEGFR2 with a K_D of less than 100 nM and the second ¹⁰Fn3 domain binds to IGFR-IR with a K_D of less than 100 nM.

22. The polypeptide of claim 18, wherein the loop BC and loop FG of the first and second ¹⁰Fn3 domains have an altered amino acid sequence relative to the sequence of the corresponding loops of the human ¹⁰Fn3 domain.

23. The polypeptide of claim 18, further comprising one or more pharmacokinetic (PK) moieties selected from:

a polyoxyalkylene moiety, a human serum albumin binding protein, sialic acid, human serum albumin, IgG, an IgG binding protein, transferrin, and an Fc fragment.

24. The polypeptide of claim 23, wherein the PK moiety is the polyoxyalkylene moiety and said polyoxyalkylene moiety is polyethylene glycol.

25. The polypeptide of claim 23, wherein the PK moiety and the polypeptide are linked via at least one disulfide bond, a peptide bond, a polypeptide, a polymeric sugar, or a polyethylene glycol moiety.

26. The polypeptide of claim 18, wherein said polypeptide has been deimmunized to remove one or more T-cell epitopes.

27. A pharmaceutically acceptable composition comprising the polypeptide claim 18, wherein the composition is essentially endotoxin free.

28. The polypeptide of claim 18, wherein the BC, DE and FG loops of the first ¹⁰Fn3 domain comprise the amino acid sequences set forth in SEQ ID NOs:

5, 6 and 7, respectively.

29. The polypeptide of claim 18, wherein the BC, DE and FG loops of the second ¹⁰Fn3 domain comprise the amino acid sequences set forth in SEQ ID NOs:

2, 3 and 4, respectively.