TARGETED THERAPEUTICS BASED ON ENGINEERED PROTEINS THAT BIND EGFR

• US 20140038893A1
• Filed: 08/01/2013
• Published: 02/06/2014
• Est. Priority Date: 02/14/2008
• Status: Active Grant

• Current Assignee: Bristol-Myers Squibb Company
• Original Assignee: Bristol-Myers Squibb Company
• Inventors: CAMPHAUSEN, Ray, EMANUEL, Stuart, FABRIZIO, David, MORSE, Brent
• Granted Patent: US 9,234,028 B2
• Time in Patent Office: Days
• US Class Current: 514/9.3
• 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

1-23. -23. (canceled)

24. A polypeptide comprising a tenth fibronectin type III (¹⁰Fn3) domain, wherein the ¹⁰Fn3 domain (i) comprises an AB loop, a BC loop, a CD loop, a DE loop, an EF loop, and an FG loop, wherein the amino acid sequences of the BC, DE, and FG loops 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:

207-231; and

(ii) binds human epidermal growth factor receptor (EGFR) with a disassociation constant of less than 10<sup>−

4</sup>M.

25. The polypeptide of claim 24, wherein the ¹⁰Fn3 domain binds human EGFR with a dissociation constant of less than 10⁻

6M.

27. The polypeptide of claim 24, 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.

28. The polypeptide of claim 27, wherein the PK moiety is the polyoxyalkylene moiety and said polyoxyalkylene moiety is polyethylene glycol.

29. The polypeptide of claim 27, 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.

30. The polypeptide of claim 27, wherein the PK moiety and the ¹⁰Fn3 domain are operably linked via a polypeptide comprising the amino acid sequence of SEQ ID NOs:

232-235.

31. The polypeptide of claim 24, 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 an AB loop, a BC loop, a CD loop, a DE loop, an EF loop, and an FG loop;

(ii) has at least one loop selected from loop BC, DE, and FG with a randomized 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.

32. The polypeptide of claim 31, 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.

33. The polypeptide of claim 31, wherein the second domain is a second Fn3 domain, and wherein the second Fn3 domain is a tenth fibronectin type III domain (¹⁰Fn3).

34. The polypeptide of claim 33, wherein the second ¹⁰Fn3 domain comprises a polypeptide selected from:

(i) a polypeptide comprising the amino acid sequence of any of one of SEQ ID NOS;

2-231 and 236; and

(ii) a polypeptide comprising the amino acid sequence at least 90% identical to any of one of SEQ ID NOS;

2-231 and 236.

35. The polypeptide of claim 31, 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.

36. The polypeptide of claim 31, 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.

37. The polypeptide of claim 24, wherein said polypeptide has been deimmunized to remove one or more T-cell epitopes.

38. The polypeptide of claim 24, wherein said polypeptide inhibits total EGF-stimulated phosphotyrosine activation of EGFR with an IC₅₀ of less than 10 μ

M.

39. The polypeptide of claim 24, wherein said polypeptide inhibits ERK phosphorylation with an IC₅₀ of less than 10 μ

M.

40. The polypeptide of claim 24, wherein said polypeptide inhibits AKT phosphorylation with an IC₅₀ of less than 10 μ

M.

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

44. A nucleic acid encoding the polypeptide of claim 24.

45. A method of treating cancer comprising administering to a subject in need thereof an effective amount of the polypeptide of claim 24.

26. A polypeptide comprising a tenth fibronectin type III (¹⁰Fn3) domain, wherein the Fn3 domain (i) comprises an AB loop, a BC loop, a CD loop, a DE loop, an EF loop, and an FG loop, wherein the amino acid sequence of one or more of the BC, DE, and FG loops has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids substituted relative to the respective BC, DE, and FG loops of a ¹⁰Fn3 domain with an amino acid sequence selected from the group consisting of SEQ ID NOs:

207-231; and

(ii) binds human epidermal growth factor receptor (EGFR) with a disassociation constant of less than 10<sup>−

4</sup>M.

42. A polypeptide comprising a tenth fibronectin type III (¹⁰Fn3) domain, wherein the ¹⁰Fn3 domain (i) comprises an AB loop, a BC loop, a CD loop, a DE loop, an EF loop, and an FG loop;

(ii) comprises an amino acid sequence at least 80% identical to any one of SEQ ID NOs;

207-231; and

does not comprise an RGD sequence.

43. The polypeptide of claim 42, wherein the polypeptide comprises an amino acid sequence at least 90% identical to any one of SEQ ID NOs:

207-231.