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.

1 - 76 . (canceled) 
     
     
         77 . A library comprising proteins, wherein the proteins are derived from a tenth module of the fibronectin type III domain ( 10 Fn3) molecule set forth as SEQ ID NO: 33 and comprise a first loop corresponding to amino acid residues 21 to 31 of SEQ ID NO: 33, a second loop corresponding to amino acid residues 51 to 56 of SEQ ID NO: 33, and a third loop corresponding to amino acid residues 76 to 88 of SEQ ID NO: 33,
 wherein the first loop comprises one or more alterations at amino acid residues corresponding to amino acids 23-29 of SEQ ID NO: 33; wherein the second loop comprises one or more alterations at amino acid residues corresponding to amino acids 52-55 of SEQ ID NO: 33; and wherein the third loop comprises one or more alterations at amino acid residues corresponding to amino acids 78-87 of SEQ ID NO: 33, and   wherein one or more of the proteins bind to a target.   
     
     
         78 . The library of  claim 77 , wherein the proteins are fused to a nucleic acid or wherein the proteins lack an integrin-binding motif. 
     
     
         79 . The library of  claim 77 , wherein the third loop has a complexity of 10 13 . 
     
     
         80 . The library of  claim 77 , wherein at least the first loop, the second loop, and the third loop is extended in length relative to a corresponding loop of a wild-type  10 Fn3 molecule set forth as SEQ ID NO: 33. 
     
     
         81 . The library of  claim 80 , wherein the second loop is extended in length relative to a corresponding loop of a wild-type  10 Fn3 molecule set forth as SEQ ID NO: 33. 
     
     
         82 . The library of  claim 77 , wherein each of the first loop, the second loop, and the third loop remains the length of a corresponding loop of a wild-type  10 Fn3 molecule set forth as SEQ ID NO: 33. 
     
     
         83 . The library of  claim 77 , wherein the first loop consists of alterations at seven amino acid residues, the second loop consists of alterations at four amino acid residues, and the third loop consists of alterations at ten amino acid residues. 
     
     
         84 . The library of  claim 77 , wherein the proteins comprise a CH 1 —CH 2 —CH 3  domain or a CH 2 —CH 3  domain, wherein the CH 1 , the CH 2 , and the CH 3  are domains of an IgG constant region and wherein the CH 1 —CH 2 —CH 3  domain or a CH 2 —CH 3  domain increases a half-life of the proteins. 
     
     
         85 . A non-antibody protein derived from a tenth module of the fibronectin type III domain ( 10 Fn3) molecule set forth as SEQ ID NO: 33 and comprising a first loop corresponding to amino acid residues 21 to 31 of SEQ ID NO: 33, a second loop corresponding to amino acid residues 51 to 56 of SEQ ID NO: 33, and a third loop corresponding to amino acid residues 76 to 88 of SEQ ID NO: 33, wherein the first loop, second loop, and the third loop comprise one or more amino acid alteration and wherein the protein is capable of binding to a target. 
     
     
         86 . The non-antibody protein of  claim 85 , wherein the non-antibody protein binds to the target with a K d  of at least 1 μM. 
     
     
         87 . The non-antibody protein of  claim 85 , wherein the one or more amino acid alterations in the first loop are at residues 23-29 of SEQ ID NO: 33, wherein the one or more amino acid alterations in the second loop are at residues 52-55 of SEQ ID NO: 33, and wherein the one or more amino acid alterations in the third loop are at residues 78-87 of SEQ ID NO: 33. 
     
     
         88 . The non-antibody protein of  claim 85 , which is conjugated to a heterologous protein, wherein the heterologous protein does not bind to the target. 
     
     
         89 . The non-antibody protein of  claim 85 , which is covalently bound to a nucleic acid. 
     
     
         90 . A nucleic acid encoding the non-antibody protein of  claim 85 . 
     
     
         91 . A method of obtaining a non-antibody protein, comprising:
 (a) generating a plurality of mutated derivatives of the non-antibody protein of  claim 85 , thereby producing a library of proteins;   (b) contacting the library of proteins with a target; and   (c) selecting from the library of proteins one or more mutated derivatives of the non-antibody protein that bind to the target with a K d  of at least 1 μM.   
     
     
         92 . A method of obtaining a non-antibody protein that binds to a target, comprising:
 (a) contacting the target with the non-antibody protein of  claim 85  under a condition that allows target-protein complex formation; and   (b) obtaining the non-antibody protein from the target-protein complex.   
     
     
         93 . A method of detecting a target in a sample, comprising:
 (a) contacting the sample with the non-antibody of  claim 85  under a condition that allows target-protein complex formation; and   (b) detecting the target-protein complex, thereby detecting the target in the sample.   
     
     
         94 . A method for improving the binding of a non-antibody protein to a target, comprising:
 (a) providing a non-antibody protein derived from a tenth module of the fibronectin type III domain ( 10 Fn3) molecule set forth as SEQ ID NO: 33;   (b) generating a plurality of mutated derivatives of the non-antibody protein, thereby producing a library of proteins;   (c) contacting the library of proteins with the target;   (d) selecting from the library of proteins one or more mutated derivatives of the non-antibody protein that bind to the target with a K d  of at least 1 μM; and   (e) optionally repeating steps (b)-(d) substituting for the plurality of mutated derivatives in repeated step (b) with a product from the previous step (d).