Binding agent
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. A binding agent comprising Formula: A-a′:a-S-b:b′-B:X(n), wherein A is a monovalent antibody Fab′ fragment comprising a heavy chain variable region (VH) and a light chain variable region (VL) wherein (a) the VH comprises SEQ ID NO:1 and the VL comprises SEQ ID NO:2 or (b) the VH comprises SEQ ID NO:3 and the VL comprises SEQ ID NO:4; B is a monovalent antibody Fab′ fragment comprising a heavy chain variable region (VH) and a light chain variable region (VL) wherein (a) the VH comprises SEQ ID NO:1 and the VL comprises SEQ ID NO:2 or (b) the VH comprises SEQ ID NO:3 and the VL comprises SEQ ID NO:4; S is a spacer of at least 1 nm in length; (n) is an integer and at least 1; represents a covalent bond; a′ is a single-stranded DNA oligonucleotide comprising SEQ ID NO:5; b′ is a single-stranded DNA oligonucleotide comprising SEQ ID NO:6; wherein the first Fab′ is conjugated to a single-stranded DNA oligonucleotide of SEQ ID NO:5, wherein the second Fab′ is conjugated to a single-stranded DNA oligonucleotide of SEQ ID NO:6; a-S-b is a biotinylated linker wherein a is SEQ ID NO:7, b is SEQ ID NO:8, and S is selected from the group consisting of (T40), Tn(biotin-dT)Tn wherein Tn is 0 to 30, Spacer C18-(biotin-dT)-Spacer C18, (Spacer C18)2-(biotin-dT)-(Spacer C18)2, (Spacer C18)3-(biotin-dT)-(Spacer C18)3, (Spacer C18)4-(biotin-dT)-(Spacer C18)4, Tn(Dig-dT)Tn wherein Tn is 0 to 20, and (GGGGS)n (SEQ ID NO:13) wherein n is at least 1; a′:a is a binding pair; b:b′ is a binding pair, wherein a′ and a do not interfere with the binding of b to b′ and b′ and b do not interfere with the binding of a′ to a; (: X) denotes a functional moiety bound either covalently or via a binding pair to at least one of a′, a, b, b′ or S; and linker a-S-b has a length of 6 to 100 nm.
2. The binding agent of claim 1 , wherein the spacer S is 1 to 95 nm in length.
3. The binding agent of claim 1 , wherein the a′:a binding pair and the b:b′ binding pair are hybridizing nucleic acid sequences and wherein the different hybridizing nucleic acid sequences of the a′:a binding pair does not hybridize with the b:b′ binding pair.
4. The binding agent of claim 1 , wherein X is a functional moiety selected from the group consisting of a labeling group, a binding group and an effector group.
5. The binding agent of claim 1 , wherein the functional moiety X is bound to a, b, or S.
6. The binding agent of claim 1 , wherein the functional moiety X is bound to the spacer S.
7. The binding agent of claim 1 , wherein the functional moiety X is covalently bound to the spacer S.
8. The binding agent of claim 1 , wherein the functional moiety X is bound to the spacer S via a hybridizing nucleic acid.