Fibronectin binding domains with reduced immunogenicity

Abstract

Fibronectin type III ( 10 Fn3) binding domains having novel designs that are associated with reduced immunogenicity are provided. The application describes alternative 10 Fn3 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 10 Fn3 binding domains in which HLA anchor regions have been destroyed thereby reducing the immunogenic contribution of the adjoining region. Also provided are 10 Fn3 domains having novel combinations of modified regions that can bind to a desired target with high affinity.

1 . A polypeptide comprising a human fibronectin type 3 tenth ( 10 Fn3) domain, wherein the  10 Fn3 domain comprises (i) a modification in the amino acid sequence of at least one north pole loop selected from the BC, DE and FG loops relative to the corresponding loop of the wild-type human  10 Fn3 domain (SEQ ID NO:1), and (ii) a modification in the amino acid sequence of at least one south pole loop selected from the AB, CD and EF loops relative to the corresponding loop of the wild-type human  10 Fn3 domain (SEQ ID NO:1 or 6), wherein the at least one modified north pole loop and the at least one modified south pole loop contribute to binding the same target. 
     
     
         2 - 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 the amino acid sequence of at least one of loops AB, BC, CD, DE, EF, or FG relative to the corresponding loop of the wild-type human  10 Fn3 domain (SEQ ID NO:1 or 6), and (ii) a modification in the amino acid sequence of at least one β-strand relative to the corresponding β-strand of the 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 the same target. 
     
     
         29 - 224 . (canceled) 
     
     
         225 . A polypeptide comprising a human  10 Fn3 domain, wherein the  10 Fn3 domain comprises an AG binder, which comprises a modification in the amino acid sequence of β-strand A and β-strand G relative to the wild-type human  10 Fn3 domain (SEQ ID NO: 1 or 6), wherein the modifications contribute to binding the same target. 
     
     
         226 . The polypeptide of  claim 225 , wherein the  10 Fn3 domain comprises modifications in the amino acid sequences of the EF and FG loops relative to the sequences of the corresponding loops of the wild-type human  10 Fn3 domain (SEQ ID NO:1), and wherein the EF and FG loops contribute to binding to the same target. 
     
     
         227 . The polypeptide of  claim 225 , wherein the  10 Fn3 domain comprises modifications in the amino acid sequence of the AB loop is modified relative to the sequence of AB loop of the wild-type human  10 Fn3 domain (SEQ ID NO:1 or 6), and wherein the AB loop contributes to binding to the target. 
     
     
         228 . The polypeptide of  claim 225 , wherein the  10 Fn3 domain comprises modifications in the amino acid sequences of the AB, EF and FG loops relative to the corresponding loops of the wild-type human  10 Fn3 domain (SEQ ID NO:1 or 6), and wherein the modified loops and strands contribute to binding to the same target. 
     
     
         229 . The polypeptide of  claim 225 , wherein the  10 Fn3 domain comprises modifications in the amino acid sequences of the AB, CD and EF loops relative to the corresponding loops of the wild-type human  10 Fn3 domain (SEQ ID NO:1 or 6), and wherein the modified loops and strands contribute to binding to the same target. 
     
     
         230 . The polypeptide of  claim 225 , wherein the  10 Fn3 domain comprises modifications in the amino acid sequences of β-strand A, loop AB, β-strand B, loop FG, and β-strand G relative to the sequences of the corresponding β-strands and loops of the wild-type human  10 Fn3 domain (SEQ ID NO:1 or 6), and wherein the modified loops and strands contribute to binding to the same target. 
     
     
         231 . A library comprising a plurality of polypeptides of  claim 225 . 
     
     
         232 . A nucleic acid encoding a polypeptide of  claim 225 . 
     
     
         233 . A vector comprising the nucleic acid of  claim 232 . 
     
     
         234 . A host cell comprising the nucleic acid of  claim 232 . 
     
     
         235 . A method of treating or diagnosing a disease or disorder in a subject in need thereof, comprising administering to the subject the polypeptide of  claim 225 . 
     
     
         236 . The polypeptide of  claim 225 , wherein the modifications are insertions, substitutions, or deletions. 
     
     
         237 . The polypeptide of  claim 236 , wherein the modifications are substitutions. 
     
     
         238 . The polypeptide of  claim 225 , wherein the  10 Fn3 domain further comprises a pharmacokinetic moiety. 
     
     
         239 . The polypeptide of  claim 238 , wherein the pharmacokinetic moiety is selected from the group consisting of a polyoxyalkylene moiety, a human serum albumin binding protein, a sialic acid, a human serum albumin, a transferring, an IgG, an IgG binding protein, and an Fc fragment. 
     
     
         240 . The polypeptide of  claim 225 , comprising a second  10 Fn3 domain. 
     
     
         241 . The polypeptide of  claim 240 , wherein the  10 Fn3 domains are connected by a polypeptide linker selected from any one of SEQ ID NOs: 32-43. 
     
     
         242 . The polypeptide of  claim 240 , wherein each of the  10 Fn3 domains binds to the same target molecule. 
     
     
         243 . The polypeptide of  claim 240 , wherein each of the  10 Fn3 domains binds to a different target molecule.