Targeted therapeutics based on engineered proteins that bind EGFR

• US 8,524,244 B2
• Filed: 02/10/2009
• Issued: 09/03/2013
• Est. Priority Date: 02/14/2008
• Status: Active Grant

• Current Assignee: Bristol-Myers Squibb Company
• Original Assignee: Bristol-Myers Squibb Company
• Inventors: Camphausen, Ray, Morse, Brent, Emanuel, Stuart, Fabrizio, David
• Primary Examiner(s): Bunner, Bridget E
• Application Number:
  US12/867,406
• Publication Number:
  US 20110053842A1
• Time in Patent Office: 1,666 Days
• Field of Search: None
• US Class Current: 424/185.1
• 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: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• 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 16/2863 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 2317/31 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 2319/30 Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Abstract

The present invention relates to single domain proteins that bind to epidermal growth factor receptor (EGFR). The invention also relates to single domain proteins 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 tenth fibronectin type III (¹⁰Fn3 ) domain, 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, (iii) binds human epidermal growth factor receptor (EGFR) with a disassociation constant of less than 10<sup>−

4</sup>M, and (iv) comprises an amino acid sequence at least 80% identical to any one of SEQ ID NOs;

207-231.

2. The polypeptide of claim 1, wherein the ¹⁰Fn3 domain binds human EGFR with a disassociation constant of less than 10⁻

6M.

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, wherein the ¹⁰Fn3 domain comprises

a polypeptide comprising an amino acid sequence at least 90% identical to any of one of SEQ ID NOs:

207-231.

5. The polypeptide of claim 4, wherein the ¹⁰Fn3 domain comprises a polypeptide comprising the amino acid sequence of any of one of SEQ ID NOs:

207-231.

6. 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.

7. The polypeptide of claim 6, wherein the PK moiety is the polyoxyalkylene moiety and said polyoxyalkylene moiety is polyethylene glycol.

8. The polypeptide of claim 6, wherein the PK moiety and the ¹⁰Fn3 domain are operably linked via at least one disulfide bond, a peptide bond, a polypeptide, a polymeric sugar, or a polyethylene glycol moiety.

9. The polypeptide of claim 8, wherein the PK moiety and the ¹⁰Fn3 domain are operably linked via a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs:

232-235.

10. The polypeptide of claim 1, further comprising a second domain selected from:

an antibody moiety;

a derivative of lipocalin;

a derivative of tetranectin;

an avimer;

a derivative of ankyrin; and

a second fibronectin type III (Fn3) domain, wherein the second domain binds to a human protein, and wherein the second Fn3 domain (i) comprises a loop, AB;

a loop, BC;

a loop, CD;

a loop, DE; 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 Fn3 domain, and (iii) binds a human protein that is not bound by the human Fn3 domain.

11. The polypeptide of claim 10, wherein the second domain is a second Fn3 domain, and wherein the second Fn3 domain binds the human protein with a disassociation constant of less than 10⁻

4M.

12. The polypeptide of claim 10, wherein the second domain is a second Fn3 domain, and wherein the second Fn3 domain is a tenth fibronectin type III domain (¹⁰Fn3 ).

13. The polypeptide of claim 12, wherein the second ¹⁰Fn3 domain comprises

a polypeptide comprising an amino acid sequence at least 90% identical to any of one of SEQ ID NOs:

2-231 and 236.

14. The polypeptide of claim 13, wherein the second ¹⁰Fn3 domain comprises a polypeptide comprising the amino acid sequence of any of one of SEQ ID NOs:

2-231 and 236.

15. The polypeptide of claim 10, wherein the human protein bound by the second domain is selected from insulin-like growth factor-I receptor (IGF-IR), epidermal growth factor receptor (EGFR), or vascular endothelial growth factor receptor-2 (VEGFR2).

16. The polypeptide of claim 10, wherein the ¹⁰Fn3 domain and the second domain are operably linked via at least one disulfide bond, a peptide bond, a polypeptide, a polymeric sugar, or a polyethylene glycol moiety.

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

18. The polypeptide of claim 1, wherein said polypeptide inhibits the binding of transforming growth factor alpha (TGF-alpha) or epidermal growth factor (EGF) to EGFR and does not activate human EGFR at sub IC₅₀ concentrations in a cell-based assay.

19. The polypeptide of claim 1, wherein said polypeptide competes with an anti-EGFR antibody for binding to EGFR.

20. The polypeptide of claim 1, wherein said polypeptide inhibits total EGF-stimulated phosphotyrosine activation of EGFR with an IC₅₀ of less than 10 μ

M.

21. The polypeptide of claim 1, wherein said polypeptide inhibits ERK phosphorylation with an IC₅₀ of less than 10 μ

M.

22. The polypeptide of claim 1, wherein said polypeptide inhibits AKT phosphorylation with an IC₅₀ of less than 10 μ

M.

23. The polypeptide of claim 1, wherein said 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-¹⁰Fn3fusions;

c) contacting said population of candidate RNA-¹⁰Fn3 fusions with EGFR; and

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

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