Method for making multispecific antibodies having heteromultimeric and common components
First Claim
1. A method of preparing a multispecific antibody comprising a first polypeptide and at least one additional polypeptide, wherein (a) the first polypeptide comprises a multimerization domain forming an interface positioned to interact with an interface of a multimerization domain of the additional polypeptide, (b) the first and additional polypeptides each comprise a binding domain, the binding domain comprising a heavy chain and a light chain, wherein the variable light chains of the first and additional polypeptides comprise a common sequence, the method comprising the steps of:
- (I) culturing a host cell comprising nucleic acid encoding the first polypeptide and additional polypeptide, and the variable light chain, wherein the culturing is such that the nucleic acid is expressed; and
(ii) recovering the multispecific antibody from the host cell culture.
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Abstract
The invention relates to a method of preparing heteromultimeric polypeptides such as bispecific antibodies, bispecific immunoadhesins and antibody-immunoadhesin chimeras. The invention also relates to the heteromultimers prepared using the method. Generally, the method provides a multispecific antibody having a common light chain associated with each heteromeric polypeptide having an antibody binding domain. Additionally the method futher involves introducing into the multispecific antibody a specific and complementary interaction at the interface of a first polypeptide and the interface of a second polypeptide, so as to promote heteromultimer formation and hinder homomultimer formation; and/or a free thiol-containing residue at the interface of a first polypeptide and a corresponding free thiol-containing residue in the interface of a second polypeptide, such that a non-naturally occurring disulfide bond is formed between the first and second polypeptide. The method allows for the enhanced formation of the desired heteromultimer relative to undesired heteromultimers and homomultimers.
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Citations
29 Claims
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1. A method of preparing a multispecific antibody comprising a first polypeptide and at least one additional polypeptide, wherein
(a) the first polypeptide comprises a multimerization domain forming an interface positioned to interact with an interface of a multimerization domain of the additional polypeptide, (b) the first and additional polypeptides each comprise a binding domain, the binding domain comprising a heavy chain and a light chain, wherein the variable light chains of the first and additional polypeptides comprise a common sequence, the method comprising the steps of: -
(I) culturing a host cell comprising nucleic acid encoding the first polypeptide and additional polypeptide, and the variable light chain, wherein the culturing is such that the nucleic acid is expressed; and
(ii) recovering the multispecific antibody from the host cell culture. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, 20)
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13. A multispecific antibody comprising a first polypeptide and at least one additional polypeptide which meet at an interface, wherein
(a) the first polypeptide comprises a multimerization domain forming an interface positioned to interact with an interface of a multimerization domain of the additional polypeptide; - and
(b) the first and additional polypeptides each comprise a binding domain, the binding domain comprising a variable heavy chain and a variable light chain, wherein the variable light chain of the first and additional polypeptides comprise a common sequence.
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21. A method of preparing a multispecific antibody comprising:
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(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. - View Dependent Claims (22, 23, 24, 25, 26, 27, 29)
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28. 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.
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Specification