Method for making multispecific antibodies having heteromultimeric and common components

A method of preparing a multispecific antibody comprising:

• (a) selecting a first nucleic acid encoding a first polypeptide comprising an amino acid residue in the interface of the first polypeptide is replaced with an amino acid residue on an additional polypeptide, and selecting at least one additional nucleic acid encoding at least one additional polypeptide so that the amino acid residue on the additional polypeptide specifically interacts with the amino acid residue on the first polypeptide, thereby generating a stable interaction between the first and additional polypeptides;
• (b) selecting a light chain encoding nucleic acid sequence, wherein the light chain is meant to associate with the binding region of each first and additional polypeptide of the multispecific antibody;
• (c) introducing into a host cell the first and additional nucleic acids and the light chain-encoding nucleic acid, and culturing the cell so that expression of the first and additional nucleic acids and the light chain-encoding nucleic acid occurs to form the bispecifc antibody;
• (d) recovering the multispecific antibody from the cell culture.

A method of measuring the formation of a heteromultimeric multispecific antibody comprising a first and at least one additional polypeptide from a mixture of polypeptides, wherein

• (a) the first and additional polypeptides meet at an interface of a multimerization domain of each of the first and additional polypeptides,
• (b) the interface of the first polypeptide comprises a free thiol-containing residue which is positioned to interact with a free thiol-containing residue of the interface of the additional polypeptide such that a disulfide bond is formed, the method comprising the steps of:
• (i) causing each of the multispecific antibodies to migrate in a gel matrix;
• and
• (ii) determining the relative amount of a band corresponding to the multispecific antibody having a non-naturally occurring disulfide bond between the first and additional polypeptides, and a slower migrating band corresponding to a heteromultimer lacking non-naturally occurring disulfide bonds between the first and additional polypeptide.