Targeted therapeutics based on engineered proteins for tyrosine kinases receptors, including IGF-IR

• US 8,470,332 B2
• Filed: 11/21/2007
• Issued: 06/25/2013
• Est. Priority Date: 11/22/2006
• Status: Active Grant

• Current Assignee: Bristol-Myers Squibb Company
• Original Assignee: Bristol-Myers Squibb Company
• Inventors: Camphausen, Ray, Fabrizio, David, Wright, Martin C., Gage, Patrick, Mendlein, John
• Primary Examiner(s): Bunner, Bridget E
• Application Number:
  US12/312,725
• Publication Number:
  US 20100121033A1
• Time in Patent Office: 2,043 Days
• Field of Search: None
• US Class Current: 424/185.1
• 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 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 having the amino acid sequence of SEQ ID NO;

1, and (iii) binds human insulin-like growth factor-I receptor (IGF-IR) with a disassociation constant of about 1 μ

M or less;

wherein the altered ¹⁰Fn3 domain comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs;

184-203.

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

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

4. 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, transferrin, and an Fc fragment.

5. The polypeptide of claim 4, wherein the PK moiety is the polyoxyalkylene moiety and said polyoxyalkylene moiety is polyethylene glycol.

6. 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.

7. The polypeptide of claim 1, wherein said altered ¹⁰Fn3 domain is selected by the method comprising the steps of a) producing a population of candidate RNA molecules, each comprising a candidate tenth fibronectin type III (¹⁰Fn3) domain sequence which differs from human ¹⁰Fn3 domain coding sequence, said RNA molecules each comprising a translation initiation sequence and a start codon operably linked to said candidate ¹⁰Fn3 domain coding sequence and each being operably linked to a nucleic acid-puromycin linker at the 3′

end;

b) in vitro translating said candidate ¹⁰Fn3 domain coding sequences to produce a population of candidate RNA-¹⁰Fn3 fusions;

c) contacting said population of candidate RNA-Fn3 fusions with IGF-IR; and

d) selecting an RNA-¹⁰Fn3 fusion, the protein portion of which has a binding affinity or specificity for IGF-IR that is altered relative to the binding affinity or specificity of said human ¹⁰Fn3 for IGF-IR.

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

9. The polypeptide of claim 1, wherein the altered ¹⁰Fn3 domain comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs:

184-203.

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

184-203.

11. The polypeptide of claim 1, wherein the altered ¹⁰Fn3 domain comprises an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs:

184-203.

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

184-203.

13. The polypeptide of claim 1, wherein the altered ¹⁰Fn3 domain comprises the amino acid sequence of SEQ ID NO:

184.