Single-chain Multiple Antigen-binding Molecule, Its Preparation and Use
The present invention relates to a single-chain, multiple antigen-binding molecule with diverse variable domains of a heavy and of a light chain of an immunoglobulin, which are connected in the form of a VH-VL construct, which are in turn connected together via a peptide, and to the preparation and use thereof as pharmaceutical or diagnostic aid.
1 . A single-chain, multiple antigen-binding molecule comprising four variable domains, wherein said four variable domains are
(a) the variable domain of a heavy chain of an immunoglobulin (V H ) with a specificity A (V H -A), and functional parts thereof;
(b) the variable domain of a light chain of an immunoglobulin (V L ), with a specificity A (V L -A), and functional parts thereof,
(c) V H with a specificity B (V H -B), and functional parts thereof, and
(d) V L with a specificity B (V L -B), and functional parts thereof and V H -A is linked to VL-B via a linker (L) as V H -A-L-V L -B or V L -B-L-V H -A construct and V H -B is linked to V L -A as V H -B-L-V L -A or V L -A-L-V H -B construct, wherein L is 1 to 20 amino acids in length and the V H -A-L-V L -B and V H -B-L V L A constructs or the V L -B-L-V H -A and V L -A-L-V H -B are connected via a peptide (P) as V H -A-L-V L -B-P-V H -B-L-V L A or V L -B-L-V H -A-P-V L -A-L-V H -B constructs, wherein P is 12 to 40 amino acids in length, wherein V H -A and V L -A associate to form a binding site with specificity A and V H -B and V L -B associate to form a binding site with specificity B.
2 . The single-chain multiple antigen-binding molecule, according to claim 1 , wherein L is 1 to 5 amino acids.
3 . The single-chain multiple antigen-binding molecule, according to claim 1 , wherein P is 12 to 20 amino acids.
4 . The single-chain multiple antigen-binding molecule, according to claim 1 , wherein L is 1 to 5 amino acids and P is 12 to 20 amino acids.
5 . A bivalent monomeric F v antibody formed by one single-chain F v monomer having four variable domains, wherein said four variable domains are V H -A, V L -B, V H -B and V L -A, wherein V H -A and V L -A are V H and V L domains of an antibody specific for antigen A, respectively, and V H -B and V L -B are V H and V L domains of an antibody specific for antigen B, respectively; V H -A is linked to V L -B by peptide linker 1, V L -B is linked to V H -B by peptide linker 2 and V H -B is linked to V L -A by peptide linker 3; said peptide linker 1 and said peptide linker 3 are a peptide bond or have 1 to about 10 amino acids; and said peptide linker has about 10 to about 30 amino acids; V H -A and V L -A associate intramolecularly to form a binding site for antigen A; and V L -B and V H -B associate intramolecularly to form a binding site for antigen B.