Protein Scaffolds for Antibody Mimics and Other Binding Proteins

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.

What is claimed is: 
     
         1 - 119 . (canceled) 
     
     
         120 . A non-antibody protein comprising a domain having an immunoglobulin-like fold, said non-antibody protein deriving from a reference protein by having a mutated amino acid sequence wherein said non-antibody protein binds with a Kd at least as tight as 1 μM to a compound that is not bound as tightly by said reference protein. 
     
     
         121 . The non-antibody protein of  claim 120 , wherein said non-antibody protein contains one, two, or three mutated loops and wherein at least one of said loops contributes to the binding of said non-antibody protein to said compound. 
     
     
         122 . The non-antibody protein of  claim 121 , wherein at least two of said loops contribute to said binding of said non-antibody protein to said compound. 
     
     
         123 . The non-antibody protein of  claim 121 , wherein three of said loops contribute to said binding of said non-antibody protein to said compound. 
     
     
         124 . The non-antibody protein of  claim 120 , wherein said domain having an immunoglobulin-like fold is mutated and comprises up to 34% mutated amino acids as compared to the immunoglobulin-like fold of said reference protein. 
     
     
         125 . The non-antibody protein of  claim 120 , wherein said domain having an immunoglobulin-like fold consists of approximately 50-150 amino acids. 
     
     
         126 . The non-antibody protein of  claim 120 , said non-antibody protein being immobilized on a solid support. 
     
     
         127 . The non-antibody protein of  claim 120 , wherein said non-antibody protein is joined to a heterologous protein, said heterologous protein being unable to bind to said compound. 
     
     
         128 . The non-antibody protein of  claim 127 , wherein said heterologous protein is an antibody or an antibody domain. 
     
     
         129 . The non-antibody protein of  claim 127 , wherein said heterologous protein is selected from the group consisting of a complement protein, a toxin protein, and an albumin protein. 
     
     
         130 . The non-antibody protein of  claim 120 , wherein said non-antibody protein is covalently bound to a nucleic acid. 
     
     
         131 . A nucleic acid encoding the non-antibody protein of  claim 120 . 
     
     
         132 . A method of obtaining a non-antibody protein of  claim 120 , said method comprising:
 (a) providing a non-antibody protein comprising an immunoglobulin-like fold, wherein the protein does not bind to the compound with a Kd as tight as 1 μM;   (b) generating mutated derivatives of said protein, thereby producing a library of mutated proteins;   (c) contacting said library with said compound;   (d) selecting from said library at least one derivative protein that binds to said compound with a Kd at least as tight as 1 μM; and   (e) optionally repeating steps (b)-(d) substituting for the protein in repeated step (b) the product from the previous step (d).   
     
     
         133 . A method for obtaining a non-antibody protein which binds to a compound, said method comprising:
 (a) contacting a compound with a non-antibody protein of  claim 120 , wherein said contacting is carried out under conditions that allow compound-protein complex formation; and   (b) obtaining, from said complex, said protein which binds to said compound.   
     
     
         134 . A method for obtaining a compound which binds to a non-antibody protein comprising:
 (a) contacting a non-antibody protein of  claim 120  with a candidate compound, wherein said contacting is carried out under conditions that allow compound-protein complex formation; and   (b) obtaining, from said complex, said compound which binds to said derivative protein.   
     
     
         135 . A method for detecting a compound, said method comprising:
 (a) contacting a compound with a non-antibody protein of  claim 120  that binds to the compound, wherein said contacting is carried out under conditions that allow compound-protein complex formation; and   (b) detecting said complex, thereby detecting said compound.   
     
     
         136 . A method for detecting a compound in a sample, said method comprising:
 (a) contacting said sample with a non-antibody protein of  claim 120  that binds to the compound, wherein said contacting is carried out under conditions that allow compound-protein complex formation; and   (b) detecting said complex, thereby detecting said compound in said sample.   
     
     
         137 . A method for improving the binding of a non-antibody protein to a compound, said method comprising:
 (a) providing a non-antibody protein comprising an immunoglobulin-like fold, wherein the protein is derived from a  10 Fn3 molecule;   (b) generating mutated derivatives of said protein, thereby producing a library of mutated proteins;   (c) contacting said library with said compound;   (d) selecting from said library at least one derivative protein that binds to said compound with a Kd at least as tight as 1 μM; and   (e) optionally repeating steps (b)-(d) substituting for the protein in repeated step (b) the product from the previous step (d).   
     
     
         138 . A library of proteins, wherein the proteins are derived from a  10 Fn3 molecule and comprise at least one amino acid alteration in each of three loops relative to the  10 Fn3 molecule. 
     
     
         139 . A library of nucleic acids encoding the proteins of  claim 138 .