Targeted therapeutics based on engineered proteins that bind egfr

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.

1 - 23 . (canceled) 
     
     
         24 . A method of detecting human epidermal growth factor receptor (EGFR) in a sample comprising:
 (a) contacting the sample with a polypeptide comprising a tenth fibronectin type III ( 10 Fn3) domain, wherein the  10 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 sequences of the BC, DE, and FG loops are identical to the amino acid sequences of the respective BC, DE, and FG loops of a  10 Fn3 domain with an amino acid sequence selected from the group consisting of SEQ ID NOs: 207-231, and (ii) binds to human EGFR with a disassociation constant of less than 10 −6 M, under conditions that allow polypeptide-EGFR complex formation, and   (b) detecting the complex.   
     
     
         25 . The method of  claim 24 , wherein the  10 Fn3 domain comprises an amino acid sequence which is at least 90% identical to any one of SEQ ID NOs: 207-231. 
     
     
         26 . The method of  claim 24 , wherein the  10 Fn3 domain comprises the amino acid sequence of any one of SEQ ID NOs: 207-231. 
     
     
         27 . The method of  claim 24 , wherein the polypeptide further comprises 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 method of  claim 27 , wherein the PK moiety is a polyoxyalkylene moiety. 
     
     
         29 . The method of  claim 28 , wherein the polyoxyalkylene moiety is polyethylene glycol. 
     
     
         30 . The method of  claim 27 , wherein the PK moiety and the  10 Fn3 domain are operably linked via at least one disulfide bond, a peptide bond, a polypeptide, a polymeric sugar, or a polyethylene glycol moiety. 
     
     
         31 . The method of  claim 27 , wherein the PK moiety and the  10 Fn3 domain are operably linked via a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 232-235. 
     
     
         32 . The method of  claim 24 , wherein the polypeptide comprises a detectable moiety. 
     
     
         33 . The method of  claim 32 , wherein the detectable moiety is selected from the group consisting of: a radioactive moiety, a fluorescent moiety, a chromogenic moiety, a chemiluminescent moiety, an enzyme moiety, and a hapten moiety. 
     
     
         34 . The method of  claim 24 , wherein the detecting of the complex is performed by radiography, immunological assay, fluorescence detection, mass spectrometry, or surface plasmon resonance. 
     
     
         35 . The method of  claim 24 , wherein the sample is a biological sample. 
     
     
         36 . The method of  claim 35 , wherein the biological sample is a human tumor sample. 
     
     
         37 . A method for in vivo imaging of human epidermal growth factor receptor (EGFR) comprising:
 (a) administering to a subject a polypeptide comprising a tenth fibronectin type III ( 10 Fn3) domain, wherein the  10 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 sequences of the BC, DE, and FG loops are identical to the amino acid sequences of the respective BC, DE, and FG loops of a  10 Fn3 domain with an amino acid sequence selected from the group consisting of SEQ ID NOs: 207-231, and (ii) binds to human EGFR with a disassociation constant of less than 10 −6 M, and wherein the polypeptide comprises a detectable moiety, and   (b) detecting the presence and location of the labeled polypeptide in the subject.   
     
     
         38 . A method of treating an epidermal growth factor (EGFR)-associated disease comprising administering to a subject in need thereof an effective amount of a polypeptide comprising a tenth fibronectin type III ( 10 Fn3) domain, wherein the  10 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 sequences of the BC, DE, and FG loops are identical to the amino acid sequences of the respective BC, DE, and FG loops of a  10 Fn3 domain with an amino acid sequence selected from the group consisting of SEQ ID NOs: 207-231, and (ii) binds to human EGFR with a disassociation constant of less than 10 −6 M.