Fibronectin based scaffold proteins

Abstract

Fibronectin type III ( 10 Fn3) binding domains having novel designs are provided. Also provided are 10 Fn3 domains having combinations of modified regions that can bind to a desired target.

1 . A polypeptide comprising a  10 Fn3 domain, wherein the  10 Fn3 domain comprises an amino acid sequence wherein
 the amino acids corresponding to residues 77-83 of SEQ ID NO: 1 or 6 are represented by x 3-12 ,   the amino acids corresponding to residues 77-86 of SEQ ID NO: 1 or 6 are represented by x 6-15 , or   the amino acids corresponding to residues 75-86 of SEQ ID NO: 1 or 6 are represented by x 4-8 Sx, or   the amino acids corresponding to residues 75-83 of SEQ ID NO: 1 or 6 are represented by x 8-14 , or   the amino acids corresponding to residues 73-89 of SEQ ID NO: 1 or 6 are represented by xAxxxx 3-12 xIx, or   the amino acids corresponding to residues 76-86 of SEQ ID NO: 1 or 6 are represented by xx 3-12 , or   the amino acids corresponding to residues 76-86 of SEQ ID NO: 1 or 6 are represented by xx 4-8 Sx, or   the amino acids corresponding to residues 76-83 of SEQ ID NO: 1 or 6 are represented by xx 8-14 , or   the amino acids corresponding to residues 23-29 of SEQ ID NO: 1 or 6 are represented by x 7 , or   the amino acids corresponding to residues 25-29 of SEQ ID NO: 1 or 6 are represented by x 5 , or   the amino acids corresponding to residues 26-29 of SEQ ID NO: 1 or 6 are represented by x 4 , or   the amino acids corresponding to residues 27-29 of SEQ ID NO: 1 or 6 are represented by x 3 , or   the amino acids corresponding to residues 24-28 of SEQ ID NO: 1 or 6 are represented by x 5 , or   the amino acids corresponding to residues 25-28 of SEQ ID NO: 1 or 6 are represented by x 4 , or   the amino acids corresponding to residues 26-28 of SEQ ID NO: 1 or 6 are represented by x 3  or   the amino acids corresponding to residues 17-26 of SEQ ID NO: 1 or 6 are represented by xLxIxWx 4 , or   the amino acids corresponding to residues 24-35 of SEQ ID NO: 1 or 6 are represented by x 4 YxIx, or   the amino acids corresponding to residues 1-9 of SEQ ID NO: 1 or 6 are represented by x 1-9 Lx,   wherein x is any amino acid, and wherein the  10 Fn3 domain binds specifically to a target that is not bound by a  10 Fn3 domain having an amino acid sequence consisting of SEQ ID NO: 1 or 6.   
     
     
         2 - 3 . (canceled) 
     
     
         4 . The polypeptide of  claim 1 , wherein the  10 Fn3 domain comprises at least one amino acid variation in the amino acid sequence corresponding to residues 17-30 of SEQ ID NO: 1 or 6. 
     
     
         5 . The polypeptide of  claim 1 , wherein the  10 Fn3 domain comprises at least one amino acid variation in the amino acid sequence corresponding to residues 1-9 of SEQ ID NO: 1 or 6. 
     
     
         6 - 7 . (canceled) 
     
     
         8 . The polypeptide of  claim 1 , wherein at least 50% of residues x are not the amino acid at the corresponding position in SEQ ID NO: 1 or 6. 
     
     
         9 . The polypeptide of  claim 1 , wherein the  10 Fn3 domain binds specifically to a target with a Kd of less than 500 nM. 
     
     
         10 . A library comprising a plurality of polypeptides comprising a  10 Fn3 domain, wherein the  10 Fn3 domain comprises an amino acid sequence wherein
 the amino acids corresponding to residues 77-83 of SEQ ID NO: 1 or 6 are represented by x 3-12 ,   the amino acids corresponding to residues 77-86 of SEQ ID NO: 1 or 6 are represented by x 6-15 , or   the amino acids corresponding to residues 75-86 of SEQ ID NO: 1 or 6 are represented by x 4-8 Sx, or   the amino acids corresponding to residues 75-83 of SEQ ID NO: 1 or 6 are represented by x 8-14 , or   the amino acids corresponding to residues 73-89 of SEQ ID NO: 1 or 6 are represented by xAxxxx 3-12 xIx, or   the amino acids corresponding to residues 76-86 of SEQ ID NO: 1 or 6 are represented by xx 3-12 , or   the amino acids corresponding to residues 76-86 of SEQ ID NO: 1 or 6 are represented by xx 4-8 Sx, or   the amino acids corresponding to residues 76-83 of SEQ ID NO: 1 or 6 are represented by xx 8-14 , or   the amino acids corresponding to residues 23-29 of SEQ ID NO: 1 or 6 are represented by x 7 , or   the amino acids corresponding to residues 25-29 of SEQ ID NO: 1 or 6 are represented by x 5 , or   the amino acids corresponding to residues 26-29 of SEQ ID NO: 1 or 6 are represented by x 4 , or   the amino acids corresponding to residues 27-29 of SEQ ID NO: 1 or 6 are represented by x 3 , or   the amino acids corresponding to residues 24-28 of SEQ ID NO: 1 or 6 are represented by x 5 , or   the amino acids corresponding to residues 25-28 of SEQ ID NO: 1 or 6 are represented by x 4 , or   the amino acids corresponding to residues 26-28 of SEQ ID NO: 1 or 6 are represented by x 3  or   the amino acids corresponding to residues 17-26 of SEQ ID NO: 1 or 6 are represented by xLxIxWx 4 , or   the amino acids corresponding to residues 24-35 of SEQ ID NO: 1 or 6 are represented by x 4 YxIx, or   the amino acids corresponding to residues 1-9 of SEQ ID NO: 1 or 6 are represented by x 1-9 Lx,   wherein x is any amino acid, and wherein the  10 Fn3 domain binds specifically to a target that is not bound by a  10 Fn3 domain having an amino acid sequence consisting of SEQ ID NO: 1 or 6.   
     
     
         11 - 12 . (canceled) 
     
     
         13 . A method for identifying a polypeptide that binds to a target comprising screening the library of  claim 10  to identify a polypeptide that binds to the target. 
     
     
         14 - 274 . (canceled) 
     
     
         275 . A polypeptide comprising a  10 Fn3 domain, wherein the  10 Fn3 domain comprises at least one variable amino acid sequence, wherein
 the amino acids corresponding to residues 1-9 of SEQ ID NO: 1 or 6 are represented by x 1-9 Lx; or   the amino acids corresponding to residues 1-9 of SEQ ID NO: 1 or 6 consist of the amino acid sequence GVSDVPGGSG, or a portion thereof, linked to an amino acid sequence represented by x 1-9 Lx;   and/or   the amino acids corresponding to residues 23-29 of SEQ ID NO: 1 or 6 are represented by x 7 ; or   the amino acids corresponding to residues 25-29 of SEQ ID NO: 1 or 6 are represented by x 5 ; or   the amino acids corresponding to residues 26-29 of SEQ ID NO: 1 or 6 are represented by x 4 ; or   the amino acids corresponding to residues 27-29 of SEQ ID NO: 1 or 6 are represented by x 3 ; or   the amino acids corresponding to residues 24-28 of SEQ ID NO: 1 or 6 are represented by x 5 ; or   the amino acids corresponding to residues 25-28 of SEQ ID NO: 1 or 6 are represented by x 4 ; or   the amino acids corresponding to residues 26-28 of SEQ ID NO: 1 or 6 are represented by x 3 ; or   the amino acids corresponding to residues 17-26 of SEQ ID NO: 1 or 6 are represented by xLxIxWx 4 ; or   the amino acids corresponding to residues 24-35 of SEQ ID NO: 1 or 6 are represented by x 6 YxIx;   and/or   the amino acids corresponding to residues 52-55 of SEQ ID NO: 1 or 6 are represented by x 4 ; or   the amino acids corresponding to residues 47-55 of SEQ ID NO: 1 or 6 are represented by xFxVx 4 ; or   the amino acids corresponding to residues 51-60 of SEQ ID NO: 1 or 6 are represented by x 6 AxIx;   and/or   the amino acids corresponding to residues 77-83 of SEQ ID NO: 1 or 6 are represented by x 3-12 ; or   the amino acids corresponding to residues 77-86 of SEQ ID NO: 1 or 6 are represented by x 6-15 ; or   the amino acids corresponding to residues 75-86 of SEQ ID NO: 1 or 6 are represented by x 4-8 Sx; or   the amino acids corresponding to residues 75-83 of SEQ ID NO: 1 or 6 are represented by x 8-14 ; or   the amino acids corresponding to residues 73-89 of SEQ ID NO: 1 or 6 are represented by xAxxx 3-12 xIx; or   the amino acids corresponding to residues 76-86 of SEQ ID NO: 1 or 6 are represented by xx 3-12 ; or   the amino acids corresponding to residues 76-86 of SEQ ID NO: 1 or 6 are represented by xx 4-8 Sx; or   the amino acids corresponding to residues 76-83 of SEQ ID NO: 1 or 6 are represented by xx 8-14 ;   and   
       x is any amino acid; and the  10 Fn3 domain binds specifically to a target that is not bound by a  10 Fn3 domain having an amino acid sequence consisting of SEQ ID NO: 1 or 6. 
     
     
         276 . The polypeptide of  claim 275 , wherein the  10 Fn3 domain comprises at last one variable amino acid sequence, wherein
 the amino acids corresponding to residues 1-9 of SEQ ID NO: 1 or 6 are represented by x 1-9 Lx; or   the amino acids corresponding to residues 1-9 of SEQ ID NO: 1 or 6 consist of the amino acid sequence GVSDVPGGSG, or a portion thereof, linked to an amino acid sequence represented by x 1-9 Lx;   and   the amino acids corresponding to residues 23-29 of SEQ ID NO: 1 or 6 are represented by x 7 ; or   the amino acids corresponding to residues 25-29 of SEQ ID NO: 1 or 6 are represented by x 5 ; or   the amino acids corresponding to residues 26-29 of SEQ ID NO: 1 or 6 are represented by x 4 ; or   the amino acids corresponding to residues 27-29 of SEQ ID NO: 1 or 6 are represented by x 3 ; or   the amino acids corresponding to residues 24-28 of SEQ ID NO: 1 or 6 are represented by x 5 ; or   the amino acids corresponding to residues 25-28 of SEQ ID NO: 1 or 6 are represented by x 4 ; or   the amino acids corresponding to residues 26-28 of SEQ ID NO: 1 or 6 are represented by x 3 ; or   the amino acids corresponding to residues 17-26 of SEQ ID NO: 1 or 6 are represented by xLsIxWx 4 ; or   the amino acids corresponding to residues 24-35 of SEQ ID NO: 1 or 6 are represented by x 6 YxIx.   
     
     
         277 . The polypeptide of  claim 275 , wherein the  10 Fn3 domain comprises at last one variable amino acid sequence, wherein
 the amino acids corresponding to residues 1-9 of SEQ ID NO: 1 or 6 are represented by x 1-9 Lx; or   the amino acids corresponding to residues 1-9 of SEQ ID NO: 1 or 6 consist of the amino acid sequence GVSDVPGGSG, or a portion thereof, linked to an amino acid sequence represented by x 1-9 Lx;   and   the amino acids corresponding to residues 77-83 of SEQ ID NO: 1 or 6 are represented by x 3-12 ; or   the amino acids corresponding to residues 77-86 of SEQ ID NO: 1 or 6 are represented by x 6-15 ; or   the amino acids corresponding to residues 75-86 of SEQ ID NO: 1 or 6 are represented by x 4-8 Sx; or   the amino acids corresponding to residues 75-83 of SEQ ID NO: 1 or 6 are represented by x 8-14 ; or   the amino acids corresponding to residues 73-89 of SEQ ID NO: 1 or 6 are represented by xAxxx 3-12 xIx; or   the amino acids corresponding to residues 76-86 of SEQ ID NO: 1 or 6 are represented by xx 3-12 ; or   the amino acids corresponding to residues 76-86 of SEQ ID NO: 1 or 6 are represented by xx 4-8 Sx; or   the amino acids corresponding to residues 76-83 of SEQ ID NO: 1 or 6 are represented by xx 8-14 .   
     
     
         278 . The polypeptide of  claim 275 , wherein the  10 Fn3 domain comprises at last one variable amino acid sequence, wherein
 the amino acids corresponding to residues 23-29 of SEQ ID NO: 1 or 6 are represented by x 7 ; or   the amino acids corresponding to residues 25-29 of SEQ ID NO: 1 or 6 are represented by x 5 ; or   the amino acids corresponding to residues 26-29 of SEQ ID NO: 1 or 6 are represented by x 4 ; or   the amino acids corresponding to residues 27-29 of SEQ ID NO: 1 or 6 are represented by x 3 ; or   the amino acids corresponding to residues 24-28 of SEQ ID NO: 1 or 6 are represented by x 5 ; or   the amino acids corresponding to residues 25-28 of SEQ ID NO: 1 or 6 are represented by x 4 ; or   the amino acids corresponding to residues 26-28 of SEQ ID NO: 1 or 6 are represented by x 3 ; or   the amino acids corresponding to residues 17-26 of SEQ ID NO: 1 or 6 are represented by xLxIxWx 4 ; or   the amino acids corresponding to residues 24-35 of SEQ ID NO: 1 or 6 are represented by x 6 YxIx;   and/or   the amino acids corresponding to residues 77-83 of SEQ ID NO: 1 or 6 are represented by x 3-12 ; or   the amino acids corresponding to residues 77-86 of SEQ ID NO: 1 or 6 are represented by x 6-15 ; or   the amino acids corresponding to residues 75-86 of SEQ ID NO: 1 or 6 are represented by x 4-8 Sx; or   the amino acids corresponding to residues 75-83 of SEQ ID NO: 1 or 6 are represented by x 8-14 ; or   the amino acids corresponding to residues 73-89 of SEQ ID NO: 1 or 6 are represented by xAxxx 3-12 xIx; or   the amino acids corresponding to residues 76-86 of SEQ ID NO: 1 or 6 are represented by xx 3-12 ; or   the amino acids corresponding to residues 76-86 of SEQ ID NO: 1 or 6 are represented by xx 4-8 Sx; or   the amino acids corresponding to residues 76-83 of SEQ ID NO: 1 or 6 are represented by xx 8-14 .   
     
     
         279 . The polypeptide of  claim 275 , wherein the  10 Fn3 domain comprises at last one variable amino acid sequence, wherein
 the amino acids corresponding to residues 1-9 of SEQ ID NO: 1 or 6 are represented by x 1-9 Lx; or   the amino acids corresponding to residues 1-9 of SEQ ID NO: 1 or 6 consist of the amino acid sequence GVSDVPGGSG, or a portion thereof, linked to an amino acid sequence represented by x 1-9 Lx;   and   the amino acids corresponding to residues 23-29 of SEQ ID NO: 1 or 6 are represented by x 7 ; or   the amino acids corresponding to residues 25-29 of SEQ ID NO: 1 or 6 are represented by x 5 ; or   the amino acids corresponding to residues 26-29 of SEQ ID NO: 1 or 6 are represented by x 4 ; or   the amino acids corresponding to residues 27-29 of SEQ ID NO: 1 or 6 are represented by x 3 ; or   the amino acids corresponding to residues 24-28 of SEQ ID NO: 1 or 6 are represented by x 5 ; or   the amino acids corresponding to residues 25-28 of SEQ ID NO: 1 or 6 are represented by x 4 ; or   the amino acids corresponding to residues 26-28 of SEQ ID NO: 1 or 6 are represented by x 3 ; or   the amino acids corresponding to residues 17-26 of SEQ ID NO: 1 or 6 are represented by xLxIxWx 4 ; or   the amino acids corresponding to residues 24-35 of SEQ ID NO: 1 or 6 are represented by x 6 YxIx;   and   the amino acids corresponding to residues 77-83 of SEQ ID NO: 1 or 6 are represented by x 3-12 ; or   the amino acids corresponding to residues 77-86 of SEQ ID NO: 1 or 6 are represented by x 6-15 ; or   the amino acids corresponding to residues 75-86 of SEQ ID NO: 1 or 6 are represented by x 4-8 Sx; or   the amino acids corresponding to residues 75-83 of SEQ ID NO: 1 or 6 are represented by x 8-14 ; or   the amino acids corresponding to residues 73-89 of SEQ ID NO: 1 or 6 are represented by xAxxx 3-12 xIx; or   the amino acids corresponding to residues 76-86 of SEQ ID NO: 1 or 6 are represented by xx 3-12 ; or   the amino acids corresponding to residues 76-86 of SEQ ID NO: 1 or 6 are represented by xx 4-8 Sx; or   the amino acids corresponding to residues 76-83 of SEQ ID NO: 1 or 6 are represented by xx 8-14 .   
     
     
         280 . The polypeptide of  claim 275 , wherein the  10 Fn3 domain comprises at last one variable amino acid sequence, wherein
 the amino acids corresponding to residues 23-29 of SEQ ID NO: 1 or 6 are represented by x 7 ; or   the amino acids corresponding to residues 25-29 of SEQ ID NO: 1 or 6 are represented by x 5 ; or   the amino acids corresponding to residues 26-29 of SEQ ID NO: 1 or 6 are represented by x 4 ; or   the amino acids corresponding to residues 27-29 of SEQ ID NO: 1 or 6 are represented by x 3 ; or   the amino acids corresponding to residues 24-28 of SEQ ID NO: 1 or 6 are represented by x 5 ; or   the amino acids corresponding to residues 25-28 of SEQ ID NO: 1 or 6 are represented by x 4 ; or   the amino acids corresponding to residues 26-28 of SEQ ID NO: 1 or 6 are represented by x 3 ; or   the amino acids corresponding to residues 17-26 of SEQ ID NO: 1 or 6 are represented by xLxIxWx 4 ; or   the amino acids corresponding to residues 24-35 of SEQ ID NO: 1 or 6 are represented by x 6 YxIx;   and   the amino acids corresponding to residues 52-55 of SEQ ID NO: 1 or 6 are represented by x 4 ; or   the amino acids corresponding to residues 47-55 of SEQ ID NO: 1 or 6 are represented by xFxVx 4 ; or   the amino acids corresponding to residues 51-60 of SEQ ID NO: 1 or 6 are represented by x 6 AxIx.   
     
     
         281 . The polypeptide of  claim 275 , wherein the  10 Fn3 domain comprises at last one variable amino acid sequence, wherein
 the amino acids corresponding to residues 52-55 of SEQ ID NO: 1 or 6 are represented by x 4 ; or   the amino acids corresponding to residues 47-55 of SEQ ID NO: 1 or 6 are represented by xFxVx 4 ; or   the amino acids corresponding to residues 51-60 of SEQ ID NO: 1 or 6 are represented by x 6 AxIx;   and   the amino acids corresponding to residues 77-83 of SEQ ID NO: 1 or 6 are represented by x 3-12 ; or   the amino acids corresponding to residues 77-86 of SEQ ID NO: 1 or 6 are represented by x 6-15 ; or   the amino acids corresponding to residues 75-86 of SEQ ID NO: 1 or 6 are represented by x 4-8 Sx; or   the amino acids corresponding to residues 75-83 of SEQ ID NO: 1 or 6 are represented by x 8-14 ; or   the amino acids corresponding to residues 73-89 of SEQ ID NO: 1 or 6 are represented by xAxxx 3-12 xIx; or   the amino acids corresponding to residues 76-86 of SEQ ID NO: 1 or 6 are represented by xx 3-12 ; or   the amino acids corresponding to residues 76-86 of SEQ ID NO: 1 or 6 are represented by xx 4-8 Sx; or   the amino acids corresponding to residues 76-83 of SEQ ID NO: 1 or 6 are represented by xx 8-14 .   
     
     
         282 . The polypeptide of  claim 275 , wherein the  10 Fn3 domain comprises at last one variable amino acid sequence, wherein
 the amino acids corresponding to residues 23-29 of SEQ ID NO: 1 or 6 are represented by x 7 ; or   the amino acids corresponding to residues 25-29 of SEQ ID NO: 1 or 6 are represented by x 5 ; or   the amino acids corresponding to residues 26-29 of SEQ ID NO: 1 or 6 are represented by x 4 ; or   the amino acids corresponding to residues 27-29 of SEQ ID NO: 1 or 6 are represented by x 3 ; or   the amino acids corresponding to residues 24-28 of SEQ ID NO: 1 or 6 are represented by x 5 ; or   the amino acids corresponding to residues 25-28 of SEQ ID NO: 1 or 6 are represented by x 4 ; or   the amino acids corresponding to residues 26-28 of SEQ ID NO: 1 or 6 are represented by x 3 ; or   the amino acids corresponding to residues 17-26 of SEQ ID NO: 1 or 6 are represented by xLxIxWx 4 ; or   the amino acids corresponding to residues 24-35 of SEQ ID NO: 1 or 6 are represented by x 6 YxIx;   and   the amino acids corresponding to residues 52-55 of SEQ ID NO: 1 or 6 are represented by x 4 ; or   the amino acids corresponding to residues 47-55 of SEQ ID NO: 1 or 6 are represented by xFxVx 4 ; or   the amino acids corresponding to residues 51-60 of SEQ ID NO: 1 or 6 are represented by x 6 AxIx;   and   the amino acids corresponding to residues 77-83 of SEQ ID NO: 1 or 6 are represented by x 3-12 ; or   the amino acids corresponding to residues 77-86 of SEQ ID NO: 1 or 6 are represented by x 6-15 ; or   the amino acids corresponding to residues 75-86 of SEQ ID NO: 1 or 6 are represented by x 4-8 Sx; or   the amino acids corresponding to residues 75-83 of SEQ ID NO: 1 or 6 are represented by x 8-14 ; or   the amino acids corresponding to residues 73-89 of SEQ ID NO: 1 or 6 are represented by xAxxx 3-12 xIx; or   the amino acids corresponding to residues 76-86 of SEQ ID NO: 1 or 6 are represented by xx 3-12 ; or   the amino acids corresponding to residues 76-86 of SEQ ID NO: 1 or 6 are represented by xx 4-8 Sx; or   the amino acids corresponding to residues 76-83 of SEQ ID NO: 1 or 6 are represented by xx 8-14 .   
     
     
         283 . The polypeptide of  claim 275 , wherein the  10 Fn3 domain comprises at most 20 additional amino acid variations relative to SEQ ID NO: 1 or 6. 
     
     
         284 . The polypeptide of  claim 275 , wherein the amino acid sequence of the  10 Fn3 domain that is located outside of the variable regions is at least 90% identical to the corresponding regions in SEQ ID NO: 1 or 6. 
     
     
         285 . The polypeptide of  claim 275 , wherein the  10 Fn3 domain does not comprise any other amino acid variation relative to SEQ ID NO: 1 or 6. 
     
     
         286 . The polypeptide of  claim 285 , wherein the  10 Fn3 domain comprises an amino acid sequence selected from the group of SEQ ID NOs: 76-131. 
     
     
         287 . The polypeptide of  claim 283 , wherein the  10 Fn3 domain comprises an amino acid sequence that differs from an amino acid sequence selected from SEQ ID NOs: 76-131 outside of the variable regions in at most 10 amino acids. 
     
     
         288 . (canceled)