Multispecific antibodies

A multispecific antibody comprising at least three antigen binding sites, wherein two antigen binding sites are formed by a first Fab fragment comprising a constant heavy chain domain (CH1) and a constant light chain domain (CL) and a second Fab fragment comprising a constant heavy chain domain (CH1) and a constant light chain domain (CL), wherein

• a) a third antigen binding site is formed by a variable heavy chain domain (VH3) and a variable light chain domain (VL3), wherein
• the N-terminus of the VH3 domain is connected to the C-terminus of the CH1 or CL of the first Fab fragment via a first peptide connector, and
• the N-terminus of the VL3 domain is connected to the C-terminus of the CH1 or CL of the second Fab fragment via a second peptide connector,
• wherein the third binding site is disulfide stabilized by introduction of cysteine residues at the following positions to form a disulfide bond between the VH3 and VL3 domains, according to Kabat numbering:
• (i) VH3 at position 44, and VL3 at position 100;
• (ii) VH3 at position 105, and VL3 at position 43;
• or
• (iii) VH3 at position 101, and VL3 at position 100;
• b) the multispecific antibody comprises a first and a second constant heavy chain domains 3 (CH3), which are altered to promote heterodimerization by
• i) generation of a protuberance in the first CH3 domain by substituting at least one original amino acid residue with an amino acid residue having a larger side chain volume than the original amino acid residue, and generation of a cavity in the second CH3 domain by substituting at least one original amino acid residue with an amino acid residue having a smaller side chain volume than the original amino acid residue, such that the protuberance generated in the first CH3 domain is positionable in the cavity generated in the second CH3 domain;
• or substituting at least one original amino acid residue in the first CH3 domain with a positively charged amino acid, and substituting at least one original amino acid residue in the second CH3 domain with a negatively charged amino acid;
• ii) introduction of at least one cysteine residue in each CH3 domain such that a disulfide bond is formed between the CH3 domains, or
• iii) both modifications of i) and ii);
• c) the C-terminus of the VH3 domain of the third antigen binding site is connected to the N-terminus of one of the CH3 domains, wherein the N-terminus of the CH3 domain comprises the amino acid sequence set forth in SEQ ID NO:2, and the C-terminus of the VL3 domain of the third antigen binding site is connected to the N-terminus of the other one of the CH3 domains, wherein the N-terminus of the other CH3 domain comprises the amino acid sequence set forth in SEQ ID NO:2, and
• d) the multispecific antibody is devoid of constant heavy chain domains 2 (CH2).