Patent ID: 8597911

Claim:
A method for producing an antibody, wherein the method comprises (a) providing a recombinant eukaryotic host cell comprising exogenous nucleic acid encoding a first light chain downstream of a signal sequence and exogenous nucleic acid encoding a first heavy chain downstream of a signal sequence and exogenous nucleic acid encoding a second light chain downstream of a signal sequence and exogenous nucleic acid encoding a second heavy chain downstream of a signal sequence, wherein the amino acid sequences of the first heavy chain and the second heavy chain are different, and the amino acid sequences of the first light chain and the second light chain are different, and wherein the CH3 domain of one of the first and second heavy chains carries one or more amino acid substitutions, and the CH3 domain of the other heavy chain carries one or more different amino acid substitutions, wherein the substitutions permit the first and second heavy chains to pair together into a knobs-into-holes structure, and wherein expression of the first light chain and the first heavy chain is induced by a first exogenous expression regulator, expression of the second light chain and the second heavy chain is induced by a second exogenous expression regulator, and the first and the second exogenous expression regulators are different; (b) inducing expression of the first light chain and first heavy chain in the cell; (c) causing induction of expression of the first light chain and first heavy chain to cease; (d) subsequent to step (c), inducing expression of the second light chain and second heavy chain in the cell, such that expression of the first light chain and first heavy chain is temporally separate from expression of the second light chain and second heavy chain in the cell; and (e) isolating a four-chain antibody comprising the first light and heavy chains and the second light and heavy chains, wherein the four-chain antibody is a bispecific antibody, wherein the first light chain and the first heavy chain together recognize a first antigen, and the second light chain and the second heavy chain together recognize a second antigen.