High affinity fibronectin derivatives

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 protein comprising a tenth domain of fibronectin type III ( 10 Fn3), 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, wherein the loop is selected from the group of BC loop, the DE loop and the FG loop; (b) binds to a target compound that is not bound by the corresponding human  10 Fn3 domain, and (c) binds to said target compound with a K D  of 10 nM or less.  
     
     
         2 . The protein  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 protein 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 protein 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 protein of  claim 4 , wherein the DE loop is extended by 10-13 amino acid residues relative to the corresponding loop of a human Fn3 domain.  
     
     
         6 . The protein 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 protein 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.  
     
     
         8 . The protein of  claim 1 , wherein the  10 Fn3 domain contains no free sulfhydryl moieties and no disulfide bonds.  
     
     
         9 . The protein of  claim 1 , wherein the at least one loop is randomized relative to the sequence of the corresponding loop of a human  10 Fn3 domain.  
     
     
         10 . The protein of  claim 3 , wherein the at least three loops are randomized relative to the sequence of the corresponding loop of a human  10 Fn3 domain.  
     
     
         11 . The protein of  claim 9 , 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.  
     
     
         12 . The protein of  claim 11 , wherein the screening method further comprises: (iii) repeating (i) and (ii) using a further randomized  10 Fn3 domain.  
     
     
         13 . The protein of  claim 1 , wherein the protein is substantially free of endotoxin.  
     
     
         14 . 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 and binds to said target compound with a K D  of 10 nM or less.  
     
     
         15 . The protein of  claim 14 , wherein the  10 Fn3 domain has 10 or more amino acid changes from the amino acid sequence  
       
         
           
                 
                 
               
                     
                 
                   VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTV 
                     
                 
                     
                 
                   PGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT. 
                 
                     
                 
             
                
                
                
                
                
               
            
           
         
       
     
     
         16 . 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 and binds to said target compound with a K D  of 10 nM or less.