Fibronectin binding domains with reduced immunogenicity

Abstract

Fibronectin type III (10Fn3) binding domains having novel designs that are associated with reduced immunogenicity are provided. The application describes alternative 10Fn3 binding domains in which certain immunogenic regions are not modified when producing a binder in order to maintain recognition as a self antigen by the host organism. The application also describes 10Fn3 binding domains in which HLA anchor regions have been destroyed thereby reducing the immunogenic contribution of the adjoining region. Also provided are 10Fn3 domains having novel combinations of modified regions that can bind to a desired target with high affinity.

1 - 27 . (canceled) 
     
     
         28 . A polypeptide comprising a human fibronectin type 3 tenth ( 10 Fn3) domain, wherein the  10 Fn3 domain comprises (i) a modification in an amino acid sequence of at least one of loops AB, BC, CD, DE, EF, or FG relative to a corresponding loop of a wild-type human  10 Fn3 domain (SEQ ID NO:1 or 6), and (ii) a modification in an amino acid sequence of at least one of β-strands A, B, C, D, E, F, or G relative to a corresponding β-strand of a wild-type human  10 Fn3 domain (SEQ ID NO:1 or 6), wherein the at least one modified loop and the at least one modified β-strand contribute to binding a target. 
     
     
         29 . The polypeptide of  claim 28 , wherein the at least one modified β-strand is adjacent to the at least one modified loop in a linear sequence of the  10 Fn3 domain. 
     
     
         30 . The polypeptide of  claim 28 , wherein two of the loops have modified amino acid sequences relative to the corresponding loops of the wild-type human 10Fn3 domain (SEQ ID NO:1 or 6) and wherein two of the β-strands have modified amino acid sequences relative to the corresponding β-strands of the wild-type human  10 Fn3 domain (SEQ ID NO:1 or 6). 
     
     
         31 . The polypeptide of  claim 30 , wherein each of the two modified β-strands is adjacent to each side of the two modified loops in the linear sequence of the  10 Fn3 domain. 
     
     
         32 . The polypeptide of  claim 30 , wherein each of the two modified loops and each of the two modified β-strands contribute to binding to the target. 
     
     
         33 . (canceled) 
     
     
         34 . The polypeptide of  claim 30 , wherein the amino acid sequences of the BC loop and the CD loop are modified relative to the corresponding loops of the wild-type human  10 Fn3 domain (SEQ ID NO:1 or 6) and wherein the amino acid sequences of the β-strand C and the β-strand D are modified relative to the corresponding β-strands of the wild-type human  10 Fn3 domain (SEQ ID NO: 1 or 6). 
     
     
         35 . (canceled) 
     
     
         36 . The polypeptide of  claim 34 , wherein at least one north pole loop and at least one of the south pole loop have the amino acid sequences of the corresponding loops of the wild-type human  10 Fn3 domain (SEQ ID NO:1 or 6). 
     
     
         37 . The polypeptide of  claim 34 , wherein at least a portion of the BC loop has the amino acid sequence of the corresponding loop of the wild-type human  10 Fn3 domain (SEQ ID NO:1 or 6). 
     
     
         38 . The polypeptide of  claim 37 , wherein at least the first 3 residues of the BC loop are the same as the corresponding residues in the BC loop of the wild-type human  10 Fn3 domain (SEQ ID NO:1 or 6). 
     
     
         39 . The polypeptide of  claim 37 , wherein at least the first 4 residues of the BC loop are the same as the corresponding residues in the BC loop of the wild-type human  10 Fn3 domain (SEQ ID NO:1 or 6). 
     
     
         40 . The polypeptide of  claim 37 , wherein at least the first 5 residues of the BC loop are the same as the corresponding residues in the BC loop of the wild-type human  10 Fn3 domain (SEQ ID NO:1 or 6). 
     
     
         41 . (canceled) 
     
     
         42 . The polypeptide of  claim 37 , wherein the polypeptide has reduced immunogenicity relative to an equivalent polypeptide that has additional modifications in the BC loop. 
     
     
         43 . (canceled) 
     
     
         44 . The polypeptide of  claim 34 , wherein the hydrophobic core residues have not been modified relative to the wild-type human  10 Fn3 domain (SEQ ID NO:1 or 6). 
     
     
         45 . The polypeptide of  claim 34 , wherein the amino acid sequence of at least one modified loop has been extended in length relative to the amino acid sequence of the corresponding loop of the wild-type human  10 Fn3 domain (SEQ ID NO:1 or 6). 
     
     
         46 . The polypeptide of  claim 34 , wherein the amino acid sequence of at least one modified loop has been reduced in length relative to the amino acid sequence of the corresponding loop of the wild-type human  10 Fn3 domain (SEQ ID NO:1 or 6). 
     
     
         47 - 49 . (canceled) 
     
     
         50 . The polypeptide of  claim 34 , wherein the polypeptide has at least 50% identity to the amino acid sequence of the wild-type human  10 Fn3 domain (SEQ ID NO:1 or 6). 
     
     
         51 . The polypeptide of  claim 34 , wherein the polypeptide has at least 65% identity to the amino acid sequence of the wild-type human  10 Fn3 domain (SEQ ID NO:1 or 6). 
     
     
         52 . A library comprising a plurality of polypeptides comprising a human fibronectin type 3 tenth ( 10 Fn3) domain, wherein the  10 Fn3 domain comprises (i) a modification in an amino acid sequence of at least one of loops AB, BC, CD, DE, EF, or FG relative to a corresponding loop of a wild-type human  10 Fn3 domain (SEQ ID NO:1), and (ii) a modification in an amino acid sequence of at least one of β-strands A, B, C, D, E, F, or G relative to a corresponding β-strand of a wild-type human  10 Fn3 domain (SEQ ID NO:1 or 6). 
     
     
         53 . (canceled) 
     
     
         54 . A method for identifying a polypeptide that binds to a target comprising screening the library of  claim 52  to identify a polypeptide that binds to the target. 
     
     
         55 . An isolated polypeptide identified by the method of  claim 54 . 
     
     
         56 - 224 . (canceled)