Multivalent fibronectin based scaffold domain proteins

• US 9,902,762 B2
• Filed: 03/28/2014
• Issued: 02/27/2018
• 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:
  US14/229,415
• Publication Number:
  US 20140349929A1
• Time in Patent Office: 1,432 Days
• 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:

(I) (a) an N-terminal domain comprising a first fibronectin type III tenth domain (¹⁰Fn3) which binds to IGF-1R, wherein the ¹⁰Fn3 domain comprises BC, DE, and FG loops comprising the amino acid sequence set forth in SEQ ID NOs;

2, 3, and 4, respectively; and

(b) a C-terminal domain comprising a second ¹⁰Fn3 domain which binds to vascular endothelial growth factor receptor 2 (VEGFR2), wherein the second ¹⁰Fn3 domain comprises BC, DE, and FG loops comprising the amino acid sequence set forth in SEQ ID NOs;

5, 6, and 7, respectively;

or

(II) (a) an N-terminal domain comprising a first ¹⁰Fn3 domain which binds to VEGFR2, wherein the ¹⁰Fn3 domain comprises BC, DE, and FG loops comprising the amino acid sequence set forth in SEQ ID NOs;

5, 6, and 7, respectively; and

(b) a C-terminal domain comprising a second ¹⁰Fn3 domain which binds to IGF-1R, wherein the second ¹⁰Fn3 domain comprises BC, DE, and FG loops comprising the amino acid sequence set forth in SEQ ID NOs;

2, 3, and 4, respectively.

2. The polypeptide of claim 1, wherein the first and/or second ¹⁰Fn3 domain comprises an N-terminal extension sequence selected from the group consisting of M, MG, G, or SEQ ID NO:

45, 46, or 48.

3. The polypeptide of claim 1, wherein the first and/or second ¹⁰Fn3 domain is linked at its C-terminus to the amino acid sequence of E, EI, ES, EC, EGS, EGC, EID, SEQ ID NO:

17, SEQ ID NO;

18, SEQ ID NO;

19, SEQ ID NO;

50, SEQ ID NO;

51, SEQ ID NO;

52, SEQ ID NO;

71, or SEQ ID NO;

72.

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

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

5. The polypeptide of claim 4, 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.

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

7. A pharmaceutically acceptable composition comprising the polypeptide of claim 1.

8. The polypeptide of claim 1, wherein the IGF-IR binding ¹⁰Fn3 domain binds to IGF-IR with a K_D of less than 500 nM.

9. The polypeptide of claim 1, wherein the VEGFR2 binding ¹⁰Fn3 domain binds to VEGFR2 with a K_D of less than 500 nM.

10. The polypeptide of claim 1, wherein the first ¹⁰Fn3 domain and second ¹⁰Fn3 domain are linked via a polypeptide selected from a glycine-serine based linker, a glycine-proline based linker, a proline-alanine based linker, or a polypeptide comprising the amino acid sequence of SEQ ID NO:

20.