Binding agent

Abstract

A binding agent of the Formula A-a′:a-S-b:b′-B:X(n), wherein A as well as B is a monovalent binder, a′:a as well as b:b′ is a binding pair wherein a′ and a do not interfere with the binding of b to b′ and vice versa, S is a spacer of at least 1 nm in length, :X denotes a functional moiety bound either covalently or via a binding pair to at least one of a′, a, b, b′ or S, (n) is an integer and at least 1, — represents a covalent bond, and the linker a-S-b has a length of 6 to 100 nm. Also disclosed are methods of producing such binding agent and certain uses thereof.

1 .- 20 . (canceled) 
     
     
         21 . A method for preparing a binding agent of Formula I
   A-a′:a-S-b:b′-B:X(n)  (I)
   wherein A is selected from the group consisting of a monovalent antibody and a monovalent antibody fragment;   wherein B is selected from the group consisting of a monovalent antibody and a monovalent antibody fragment;   wherein S is a spacer of at least 1 nm in length;   wherein (n) is an integer and at least 1;   wherein — is a covalent bond;   wherein a′:a is an oligonucleotide binding pair of hybridizing nucleic acid sequences;   wherein b:b′ is an oligonucleotide binding pair of hybridizing nucleic acid sequences,   wherein a′ and a do not interfere with the hybridizing of b to b′ and b′ and b do not interfere with the hybridizing of a′ to a;   wherein (: X) denotes a functional moiety bound either covalently or via a binding pair to at least one of a′, a, b, b′ and S;   and wherein linker a-S-b has a length of 6 to 100 nm,   the method comprising:
 contacting A-a′ and b′-B with linker a-S-b to form A-a′:a-S-b:b′-B; and 
 contacting X with at least one of A-a′, b′-B, linker a-S-b, and A-a′:a-S-b:b′-B wherein X binds to at least one of a′, a, b, b′ and S to result in the binding agent of Formula I. 
   
     
     
         22 . The method of  claim 21 , further comprising analyzing the binding agent for a Kdiss of 10 −5 /second or less. 
     
     
         23 . The method of  claim 21 , further comprising analyzing the binding agent for a Kdiss ranging from 10 −5 /second to 10 −7 /second. 
     
     
         24 . The method of  claim 21 , further comprising analyzing each of A and B for a Kdiss ranging from 5×10 −3 /sec to 10 −4 /sec. 
     
     
         25 . The method of  claim 21  wherein the monovalent antibody fragment A and the monovalent antibody fragment B are independently selected from the group consisting of a Fab; a Fab′-SH; a single domain antibody; a Fv; and a scFv. 
     
     
         26 . The method of  claim 21 , wherein the monovalent antibody fragment A and the monovalent antibody fragment B are independently produced by enzymatic digestion of a monoclonal antibody. 
     
     
         27 . The method of  claim 21 , wherein the monovalent antibody fragment A and the monovalent antibody fragment B are independently produced by recombinant expression. 
     
     
         28 . The method of  claim 21 , wherein X is a functional moiety selected from the group consisting of a labeling group, a binding group, and an effector group. 
     
     
         29 . The method of  claim 21 , wherein S is a nucleic acid. 
     
     
         30 . A method of detecting a target molecule in a sample, the method comprising:
 obtaining a sample;   contacting a portion of the sample with a binding agent of Formula I
   A-a′:a-S-b:b′-B:X(n)  (I)
 
 wherein A is selected from the group consisting of a monovalent antibody and a monovalent antibody fragment; 
 wherein B is selected from the group consisting of a monovalent antibody and a monovalent antibody fragment; 
 wherein S is a spacer of at least 1 nm in length; 
 wherein (n) is an integer and at least 1; 
 wherein — is a covalent bond; 
 wherein a′:a is an oligonucleotide binding pair of hybridizing nucleic acid sequences; 
 wherein b:b′ is an oligonucleotide binding pair of hybridizing nucleic acid sequences, 
 wherein a′ and a do not interfere with the hybridizing of b to b′ and b′ and b do not interfere with the hybridizing of a′ to a; 
 wherein (: X) denotes a functional moiety bound either covalently or via a binding pair to at least one of a′, a, b, b′ and S; 
 and wherein linker a-S-b has a length of 6 to 100 nm; 
 wherein the target molecule and the binding agent of Formula I form a complex; 
   detecting the complex formed between the target molecule and the binding agent of Formula I.   
     
     
         31 . The method of  claim 30 , wherein the method is an immunoassay. 
     
     
         32 . The method of  claim 30 , wherein the detecting is a determination of a concentration of the target molecule. 
     
     
         32 . The method of  claim 30 , wherein the detecting is a determination of a location of the target molecule in a tissue.