Fibronectin derivative Fc fusions

Abstract

Disclosed herein are proteins that include an immunoglobulin fold and that can be used as scaffolds. Also disclosed herein are nucleic acids encoding such proteins and the use of such proteins in diagnostic methods and in methods for evolving novel compound-binding species and their ligands.

1 . A pharmaceutical composition comprising a therapeutic protein comprising a tenth domain of fibronectin type III ( 10 Fn3) and a physiologically acceptable carrier, wherein the  10 Fn3 domain: (a) has at least one loop with a modified amino acid sequence relative to the sequence of the corresponding loop of a human  10 Fn3 domain, the loop selected from the group of the BC loop, the DE loop and the FG loop; and (b) binds to a target compound that is not bound by the corresponding human  10 Fn3 domain; wherein the therapeutic protein further comprises an Fc region of an antibody, and wherein the pharmaceutical composition is substantially free of endotoxin.  
     
     
         2 . The pharmaceutical composition of  claim 1 , wherein the  10 Fn3 domain has at least two loops with a modified amino acid sequence relative to the sequence of the corresponding loop of a human  10 Fn3 domain.  
     
     
         3 . The pharmaceutical composition of  claim 1 , wherein the  10 Fn3 domain has at least three loops with a modified amino acid sequence relative to the sequence of the corresponding loop of a human  10 Fn3 domain.  
     
     
         4 . The pharmaceutical composition of  claim 1 , wherein at least one of the modified loops in the  10 Fn3 domain is extended in length relative to the corresponding loop of a human  10 Fn3 domain.  
     
     
         5 . The pharmaceutical composition of  claim 1 , wherein the DE loop is extended by 10-13 amino acid residues relative to the corresponding loop of a human  10 Fn3 domain.  
     
     
         6 . The pharmaceutical composition of  claim 1 , wherein the integrin binding motif, RGD, of the  10 Fn3 domain is replaced by an amino acid sequence as follows: basic amino acid-neutral amino acid-acidic amino acid.  
     
     
         7 . The pharmaceutical composition of  claim 1 , wherein the Fc region is an IgG constant region.  
     
     
         8 . The pharmaceutical composition of  claim 1 , wherein the  10 Fn3 domain binds with a K D  of 500 nM or less to the target compound.  
     
     
         9 . The pharmaceutical composition of  claim 1 , wherein the  10 Fn3 domain binds with a K D  of 10 nM or less to the target compound.  
     
     
         10 . The pharmaceutical composition of  claim 1 , wherein the  10 Fn3 domain contains no free sulfhydryl moieties and no disulfide bonds.  
     
     
         11 . The pharmaceutical composition of  claim 1 , wherein the  10 Fn3 domain has an amino acid sequence that is at least 70% identical to the sequence of a human  10 Fn3 domain.  
     
     
         12 . The pharmaceutical composition of  claim 1 , wherein the at least one modified loop is randomized.  
     
     
         13 . The pharmaceutical composition of  claim 11 , wherein the  10 Fn3 domain is identified by a screening method comprising: (i) contacting the target compound with a candidate polypeptide having an  10 Fn3 domain comprising at least one randomized loop under conditions that allow target compound-polypeptide complex formation; and (ii) obtaining the polypeptide which binds to the compound.  
     
     
         14 . The pharmaceutical composition of  claim 13 , wherein the screening method further comprises: (iii) repeating (i) and (ii) using a further randomized  10 Fn3 domain.  
     
     
         15 . A pharmaceutical composition comprising a protein comprising a tenth domain of fibronectin type III ( 10 Fn3) and a physiologically acceptable carrier, wherein the  10 Fn3 domain: (a) has 5 or more amino acid changes from the amino acid sequence VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKS TATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT (b) binds to a target compound that is not bound by the corresponding human  10 Fn3 domain; wherein the protein further comprises an Fc region of an antibody, and wherein the pharmaceutical composition is substantially free of endotoxin.  
     
     
         16 . The pharmaceutical composition of  claim 15 , wherein the  10 Fn3 domain has 10 or more amino acid changes from the amino acid sequence  
       
         
           
                 
                 
               
                     
                 
                   VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTV 
                     
                 
                     
                 
                   PGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISTNYRT. 
                 
                     
                 
             
                
                
                
                
                
               
            
           
         
       
     
     
         17 . A pharmaceutical composition comprising a protein comprising a tenth domain of fibronectin type III ( 10 Fn3) and a physiologically acceptable carrier, wherein the  10 Fn3 domain: (a) is at least 70% identical to amino acid sequence VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKS TATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT (b) binds to a target compound that is not bound by the corresponding human  10 Fn3 domain; wherein the protein further comprises an Fc region of an antibody, and wherein the pharmaceutical composition is substantially free of endotoxin.