Fibronectin based scaffold proteins having improved stability

Abstract

The present application provides fibronectin based scaffold proteins associated with improved stability. The application also relates to stable formulations of fibronectin based scaffold proteins and the use thereof in diagnostic, research and therapeutic applications. The application further relates to cells comprising such proteins, polynucleotides encoding such proteins or fragments thereof, and to vectors comprising such polynucleotides.

1 - 46 . (canceled) 
     
     
         47 . A method of treating a hyperproliferative disease comprising administering to a subject in need thereof a therapeutically effective amount of a fibronectin-based protein dimer comprising a first fibronectin type III tenth ( 10 Fn3) domain and a second  10 Fn3 domain, wherein each of the first  10 Fn3 domain and the second  10 Fn3 domain:
 (i) comprises an AB loop, a BC loop, a CD loop, a DE loop, an EF loop, and a FG loop, wherein the first and second  10 Fn3 domains have at least one loop selected from the BC, DE, and FG loops with an altered amino acid sequence relative to the sequence of the corresponding loop of the human  10 Fn3 domain having the amino acid sequence of SEQ ID NO: 1;   (ii) comprises an amino acid sequence having at least 60% identity to SEQ ID NO: 1 and binds to insulin-like growth factor 1 receptor (IGF-IR), vascular endothelial growth factor receptor 2 (VEGFR2), or epidermal growth factor receptor (EGFR); and   (iii) comprises a C-terminal tail consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35.   
     
     
         48 . The method of  claim 47 , wherein one or both of the first  10 Fn3 domain and the second  10 Fn3 domain further comprised) an N-terminal extension comprising a sequence selected from the group consisting of: M, MG, G, and any of SEQ ID NOs: 19-21 and 26-31. 
     
     
         49 . The method of  claim 47 , wherein the first  10 Fn3 domain and the second  10 Fn3 domain bind to different targets. 
     
     
         50 . The method of  claim 47 , wherein the first  10 Fn3 domain and the second  10 Fn3 domain are connected by a polypeptide linker comprising 1-30 amino acids. 
     
     
         51 . The method of  claim 50 , wherein the linker is selected from the group consisting of: a glycine-serine based linker, a glycine-proline based linker, a proline-alanine linker, and an Fn-based linker. 
     
     
         52 . The method of  claim 47 , wherein the protein dimer has less than 4% fragmentation during storage in solution at pH 4.0 for at least 4 weeks. 
     
     
         53 . The method of  claim 47 , 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. 
     
     
         54 . The method of  claim 47 , wherein the protein dimer is administered by an intravenous, intramuscular, subcutaneous, or oral route. 
     
     
         55 . The method of  claim 47 , further comprising administering one or more additional therapeutic agents. 
     
     
         56 . The method of  claim 47 , wherein the hyperproliferative disorder is a cancer selected from the group consisting of: squamous cell carcinoma, bladder cancer, stomach cancer, liver cancer, kidney cancer, colorectal cancer, breast cancer, head cancer, neck cancer, esophageal cancer, gynecological cancer, thyroid cancer, lymphoma, chronic leukemia, and acute leukemia. 
     
     
         57 . A method of inhibiting tumor cell growth in a subject comprising administering to the subject a therapeutically effective amount of a fibronectin-based protein dimer comprising a first fibronectin type III tenth ( 10 Fn3) domain and a second  10 Fn3 domain, wherein each of the first  10 Fn3 domain and the second  10 Fn3 domain:
 (i) comprises an AB loop, a BC loop, a CD loop, a DE loop, an EF loop, and a FG loop, wherein the first and second  10 Fn3 domains have at least one loop selected from the BC, DE, and FG loops with an altered amino acid sequence relative to the sequence of the corresponding loop of the human  10 Fn3 domain having the amino acid sequence of SEQ ID NO: 1;   (ii) comprises an amino acid sequence having at least 60% identity to SEQ ID NO: 1 and binds to insulin-like growth factor 1 receptor (IGF-1R), vascular endothelial growth factor receptor 2 (VEGFR2), or epidermal growth factor receptor (EGFR); and   (iii) comprises a C-terminal tail consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35.   
     
     
         58 . The method of  claim 57 , wherein one or both of the first  10 Fn3 domain and the second  10 Fn3 domain further comprised) an N-terminal extension comprising a sequence selected from the group consisting of: M, MG, G, and any of SEQ ID NOs: 19-21 and 26-31. 
     
     
         59 . The method of  claim 57 , wherein the first  10 Fn3 domain and the second  10 Fn3 domain bind to different targets. 
     
     
         60 . The method of  claim 57 , wherein the first  10 Fn3 domain and the second  10 Fn3 domain are connected by a polypeptide linker comprising 1-30 amino acids. 
     
     
         61 . The method of  claim 60 , wherein the linker is selected from the group consisting of: a glycine-serine based linker, a glycine-proline based linker, a proline-alanine linker, and an Fn-based linker. 
     
     
         62 . The method of  claim 57 , wherein the protein dimer has less than 4% fragmentation during storage in solution at pH 4.0 for at least 4 weeks. 
     
     
         63 . The method of  claim 57 , 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. 
     
     
         64 . The method of  claim 57 , further comprising administering one or more additional therapeutic agents. 
     
     
         65 . The method of  claim 57 , wherein the tumor is selected from the group consisting of: brain tumor, tumor of the urogenital tract, tumor of the lymphatic system, stomach tumor, laryngeal tumor, monocytic leukemia, lung tumor, small-cell lung carcinoma, pancreatic tumor, glioblastoma, and breast tumor. 
     
     
         66 . A method of detection comprising (i) contacting a sample with a fibronectin-based protein dimer under conditions that allow the fibronectin-based protein dimer to form a complex with a target, and (ii) detecting the complex, wherein the fibronectin-based protein dimer comprises a first fibronectin type III tenth ( 10 Fn3) domain and a second  10 Fn3 domain, wherein each of the first  10 Fn3 domain and the second  10 Fn3 domain:
 (i) comprises an AB loop, a BC loop, a CD loop, a DE loop, an EF loop, and a FG loop, wherein the first and second  10 Fn3 domains have at least one loop selected from the BC, DE, and FG loops with an altered amino acid sequence relative to the sequence of the corresponding loop of the human  10 Fn3 domain having the amino acid sequence of SEQ ID NO: 1;   (ii) comprises an amino acid sequence having at least 60% identity to SEQ ID NO: 1 and binds to a target molecule;   (iii) comprises a C-terminal tail consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35.