TARGETED THERAPEUTICS BASED ON ENGINEERED PROTEINS FOR TYROSINE KINASES RECEPTORS, INCLUDING IGF-IR

• US 20130310317A1
• Filed: 05/13/2013
• Published: 11/21/2013
• Est. Priority Date: 11/22/2006
• Status: Abandoned Application

• Current Assignee: Bristol-Myers Squibb Company
• Original Assignee: Bristol-Myers Squibb Company
• Inventors: CAMPHAUSEN, Ray, FABRIZIO, David, WRIGHT, Martin C., GAGE, Patrick, MENDLEIN, John
• Application Number:
  US13/892,418
• Publication Number:
  US 20130310317A1
• Time in Patent Office: Days
• US Class Current: 514/8.7
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 38/179 for growth factors; for growth regulators
• CPC Class Codes: A61K 38/18 Growth factors; Growth regulators
• 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 47/10 Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
• 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: A61K 47/643 Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
• CPC Class Codes: A61K 47/644 Transferrin, e.g. a lactoferrin or ovotransferrin
• CPC Class Codes: A61K 47/68 the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 14/00 Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C07K 14/71 for growth factors; for growth regulators
• CPC Class Codes: C07K 14/765 Serum albumin, e.g. HSA
• 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 14/79 Transferrins, e.g. lactoferrins, ovotransferrins
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/2863 against receptors for growth factors, growth regulators
• CPC Class Codes: C07K 19/00 : Hybrid peptides , i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
• CPC Class Codes: C07K 2317/92 : Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: C07K 2319/30 : Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• CPC Class Codes: C07K 2319/74 : containing a fusion for binding to a cell surface receptor
• CPC Class Codes: C12N 15/1062 : mRNA-Display, e.g. polypeptide and encoding template are connected covalently
• CPC Class Codes: C40B 30/04 : by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
• 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

Abstract

The present invention provides innovative proteins that bind to insulin-like growth factor-I receptor (IGF-IR), as well as other important proteins. The invention also provides innovative proteins in pharmaceutical preparations and derivatives of such proteins and the uses of same in diagnostic, research and therapeutic applications. The invention further provides cells comprising such proteins, polynucleotide encoding such proteins or fragments thereof, and vectors comprising the polynucleotides encoding the innovative proteins.

Claims

1. A polypeptide comprising an altered tenth fibronectin type III (¹⁰Fn3) domain, wherein the altered ¹⁰Fn3 domain (i) comprises an AB loop, BC loop, CD loop, DE loop, EF loop, and FG loop, wherein the amino acid sequences of the BC, DE, and FG loops of the ¹⁰Fn3 domain are at least 80% identical to the amino acid sequences of the respective BC, DE, and FG loops of an ¹⁰Fn3 domain with an amino acid sequence selected from the group consisting of:

SEQ ID NOs;

2-125, 184-203, or 226, and (ii) binds human insulin-like growth factor-I receptor (IGF-IR) with a disassociation constant of about 1 μ

M or less.

2. The polypeptide of claim 1, wherein the amino acid sequences of the BC, DE, and FG loops of the ¹⁰Fn3 domain are identical to the amino acid sequences of the respective BC, DE and FG loops of an ¹⁰Fn3 domain with an amino acid sequence selected from the group consisting of SEQ ID NOs:

2-125, 184-203, or 226.

4. The polypeptide of claim 1, wherein the altered ¹⁰Fn3 domain comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs:

2-125, 184-203, or 226.

5. The polypeptide of claim 1, wherein the altered ¹⁰Fn3 domain comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs:

2-125, 184-203, or 226.

6. The polypeptide of claim 1, wherein the altered ¹⁰Fn3 domain comprises an amino acid sequence selected from any one of SEQ ID NOs:

2-125, 184-203, or 226.

7. The polypeptide of claim 1 or 3, wherein the altered ¹⁰Fn3 domain binds human IGF-IR with a disassociation constant of about 10 nM or less.

8. The polypeptide of claim 1 or 3, further comprising one or more pharmacokinetic (PK) moieties selected from:

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

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

10. The polypeptide of claim 1, wherein said polypeptide inhibits the binding of insulin-like growth factor-I (IGF-I) or insulin-like growth factor-II (IGF-II) to IGF-IR and does not activate human IGF-IR at sub IC50 concentrations in a cell-based assay.

11. A pharmaceutically acceptable composition comprising the polypeptide of claim 1 or 3, wherein the composition is essentially endotoxin free.

12. The polypeptide of claim 1, further comprising a second altered ¹⁰Fn3 domain.

13. The polypeptide of claim 12, wherein the second altered ¹⁰Fn3 domain binds to a target selected from the group consisting of EGFR, c-Met, c-kit, Her2, FGFR1, VEGFR2, VEGF-A, VEGF-B, VEGF-C, VEGF-D and folate receptor.

14. The polypeptide of claim 13, wherein the second altered ¹⁰Fn3 domain binds to the target with a disassociation constant of about 1 μ

M or less.

15. The polypeptide of claim 14, wherein the second altered ¹⁰Fn3 domain binds to the target with a disassociation constant of about 10 nM or less.

16. A nucleic acid encoding the polypeptide of claim 1 or 3.

17. A vector comprising the nucleic acid of claim 16.

3. A polypeptide comprising an altered tenth fibronectin type III (¹⁰Fn3) domain, wherein the altered ¹⁰Fn3 domain (i) comprises an AB loop, BC loop, CD loop, DE loop, EF loop, and FG loop, wherein at least one loop of the BC, DE, and FG loops of the ¹⁰Fn3 domain has 1, 2, or 3 amino acid substitutions relative to the respective BC, DE, and FG loops of an ¹⁰Fn3 domain with an amino acid sequence selected from the group consisting of SEQ ID NOs:

3-125, 184-203, and 226, and (ii) wherein the ¹⁰Fn3 domain binds to human insulin-like growth factor-I receptor (IGF-IR) with a disassociation constant of about 1 μ

M or less.

18. A method of treating cancer associated with increased insulin-like growth factor (IGF) activity comprising administering to a subject in need thereof an effective amount of a polypeptide comprising an altered tenth fibronectin type III (¹⁰Fn3) domain, wherein the altered ¹⁰Fn3 domain (i) comprises an AB loop, BC loop, CD loop, DE loop, EF loop, and FG loop, wherein at least one loop of the BC, DE, and FG loops of the ¹⁰Fn3 domain has 1, 2, or 3 amino acid substitutions relative to the respective BC, DE, and FG loops of an ¹⁰Fn3 domain with an amino acid sequence selected from the group consisting of SEQ ID NOs:

3-125, 184-203, and 226, and (ii) wherein the ¹⁰Fn3 domain binds to human insulin-like growth factor-I receptor (IGF-IR) with a disassociation constant of about 1 μ

M or less.

19. The method of claim 18, wherein the cancer is selected from the group consisting of breast cancer, lung cancer, colon cancer, ovarian cancer, synovial sarcoma, osteosarcoma, cervical cancer, prostate cancer, and pancreatic cancer.

20. A method of inhibiting insulin-like growth factor (IGF)-mediated proliferation of tumor cells comprising contacting the cell with an effective amount of a polypeptide comprising an altered tenth fibronectin type III (¹⁰Fn3) domain, wherein the altered ¹⁰Fn3 domain (i) comprises an AB loop, BC loop, CD loop, DE loop, EF loop, and FG loop, wherein at least one loop of the BC, DE, and FG loops of the ¹⁰Fn3 domain has 1, 2, or 3 amino acid substitutions relative to the respective BC, DE, and FG loops of an ¹⁰Fn3 domain with an amino acid sequence selected from the group consisting of SEQ ID NOs:

3-125, 184-203, and 226, and (ii) wherein the ¹⁰Fn3 domain binds to human insulin-like growth factor-I receptor (IGF-IR) with a disassociation constant of about 1 μ

M or less.