Fibronectin-based binding molecules and uses thereof

Abstract

The invention provides fibronectin type III (Fn3)-based binding molecules that bind to a specific target antigen. The invention further provides bispecific Fn3-based binding molecules that bind to two or more targets simultaneously. The Fn3-based binding molecules of the invention can also be linked together to form multispecific Fn3-based binding molecules, and/or can be conjugated to a non-Fn3 moiety, such as, Human Serum Albumin (HSA), for improved half life and stability. The invention also provides methods for generating, screening and using Fn3-based binding molecules in a variety of therapeutic and diagnostic applications.

1 - 12 . (canceled) 
     
     
         13 . A bispecific Fn3-based binding molecule comprising an Fn3 domain, wherein at least one amino acid in an EF loop and at least one amino acid in one or more of the bottom AB loop region, CD loop region or C-terminal of the Fn3 domain are altered compared to the wild-type Fn3 domain comprising SEQ ID NO:1 to create a non-Fn3 binding sequence which binds to a first target, and wherein at least one amino acid in one or more of the top BC, DE or FG loop regions of the Fn3 domain are altered compared to the wild-type Fn3 domain comprising SEQ ID NO:1 to create a non-Fn3 binding sequence which binds to a second target. 
     
     
         14 . The bispecific Fn3-based binding molecule of  claim 13 , wherein said at least one amino acid in said bottom EF loop regions is selected from the group consisting of the amino acids at position 60, 61, 62, 63, and of SEQ ID NO:1, and wherein said at least one amino acid in said top DE loop is selected from the group consisting of amino acids at position 51, 52, 53, 54, 55, and of SEQ ID NO:1 
     
     
         15 . The bispecific Fn3-based binding molecule of  claim 13 , wherein the non-Fn3 binding sequence comprises all or a portion of a complementarity determining region (CDR) of an antibody or a T-cell receptor. 
     
     
         16 . The bispecific Fn3-based binding molecule of  claim 13 , wherein said first and second targets are present on the same molecule. 
     
     
         17 . The bispecific Fn3-based binding molecule of  claim 13 ,
 wherein said first and second targets are present on different molecules.   
     
     
         18 - 31 . (canceled) 
     
     
         32 . An Fn3-based binding molecule comprising SEQ ID NO: 119 (clone 87) 
     
     
         33 . (canceled) 
     
     
         34 . A conjugate comprising a bispecific Fn3-based binding molecule of  claim 13  linked to one or more non-Fn3 moieties. 
     
     
         35 . The conjugate of  claim 34 , wherein the non-Fn3 moiety comprises or binds a molecule which increases the half-life of the Fn3-based binding molecule. 
     
     
         36 . The conjugate of  claim 34 , wherein the non-Fn3 moiety comprises a molecule selected from the group consisting of an antibody Fc region, Human Serum Albumin (HSA) and polyethylene glycol (PEG). 
     
     
         37 . The conjugate of  claim 34  which further comprises at least one modified amino acid residue compared to the wild-type Fn3 domain comprising SEQ ID NO: 1 for attaching a functional moiety. 
     
     
         38 . The conjugate of  claim 37 , wherein
 the modified amino acid residue comprises the addition or substitution of a cysteine residue or non-natural amino acid residue.   
     
     
         39 . The Fn3-based binding molecule or conjugate of  claim 34 , comprising beta strands from two or more different Fn3 domains. 
     
     
         40 . A composition comprising the bispecific Fn3-based binding molecule of  claim 13  and a carrier. 
     
     
         41 - 43 . (canceled)