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US 11,142,587 B2Granted
Method for producing polypeptide hetero-oligomer
AssigneeChugai Seiyaku Kabushiki Kaisha
InventorsTomoyuki Igawa, Hitoshi Katada, Yuji Hori, Shojiro Kadono
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Abstract
It is intended to provide a method for efficiently and stably producing a heteromultimer by incubating homo variants of plural types of heavy chain constant region-containing polypeptides differing in antigen-binding activity under a reducing condition that reorganize the inter-polypeptide disulfide bond between cysteine residues outside of core hinge regions. A feature of the production method of the present invention is that amino acid residues in the core hinge regions do not form any disulfide bond.
Claims14
A method for producing a heteromultimer, the method comprising:
- (a) providing a first molecule comprising two copies of a first antibody heavy chain and two copies of a first antibody light chain,
- wherein the first antibody heavy chain comprises a heavy chain constant region of human IgG1 type that has no core hinge region Cys residues and does not have a Cys at position 220 (EU numbering),
- wherein the first antibody light chain comprises a light chain constant region comprising one or more Cys residues, including a Cys at position 214 (EU numbering),
- wherein the first molecule has a first antigen-binding activity, and
- wherein the two copies of the first antibody light chain are linked to each other by a Cys-Cys disulfide bond linking their respective positions 214 (EU numbering);
- (b) providing a second molecule comprising two copies of a second antibody heavy chain different from the first antibody heavy chain and two copies of a second antibody light chain different from the first antibody light chain,
- wherein the second antibody heavy chain comprises a heavy chain constant region of human IgG1 type that has no core hinge region Cys residues and does not have a Cys at position 220 (EU numbering),
- wherein the second antibody light chain comprises a light chain constant region comprising one or more Cys residues including a Cys at position 214 (EU numbering),
- wherein the second molecule has a second antigen-binding activity different from the first antigen-binding activity, and
- wherein the two copies of the second antibody light chain are linked to each other by a Cys-Cys disulfide bond linking their respective positions 214 (EU numbering);
- (c) incubating the first and second molecules together under a reducing condition that allows Cys-Cys disulfide bonds in the first molecule and second molecule to isomerize;
- and
- (d) obtaining a heteromultimer comprising a first arm comprising the first antibody heavy and light chains associated together and a second arm comprising the second antibody heavy and light chains associated together,
- wherein the only covalent bond linking the first and second arms together is a Cys-Cys disulfide bond linking position 214 (EU numbering) of the first antibody light chain and position 214 (EU numbering) of the second antibody light chain.
11 more elements
A method for producing a heteromultimer, the method comprising:
- (a) providing a first molecule comprising two copies of a first antibody heavy chain and two copies of a first antibody light chain,
- wherein the first antibody heavy chain comprises a heavy chain constant region of human IgG4 type that has no core hinge region Cys residues and does not have a Cys at position 131 (EU numbering),
- wherein the first antibody light chain comprises a light chain constant region comprising one or more Cys residues, including a Cys at position 214 (EU numbering),
- wherein the first molecule has a first antigen-binding activity, and
- wherein the two copies of the first antibody light chain are linked to each other by a Cys-Cys disulfide bond linking their respective positions 214 (EU numbering);
- (b) providing a second molecule comprising two copies of a second antibody heavy chain different from the first antibody heavy chain and two copies of a second antibody light chain different from the first antibody light chain,
- wherein the second antibody heavy chain comprises a heavy chain constant region of human IgG4 type that has no core hinge region Cys residues and does not have a Cys at position 131 (EU numbering),
- wherein the second antibody light chain comprises a light chain constant region comprising one or more Cys residues including a Cys at positions 214 (EU numbering),
- wherein the second molecule has a second antigen-binding activity different from the first antigen-binding activity, and
- wherein the two copies of the second antibody light chain are linked to each other by a Cys-Cys disulfide bond linking their respective positions 214 (EU numbering);
- (c) incubating the first and second molecules together under a reducing condition that allows Cys-Cys disulfide bonds in the first molecule and second molecule to isomerize;
- and
- (d) obtaining a heteromultimer comprising a first arm comprising the first antibody heavy and light chains associated together and a second arm comprising the second antibody heavy and light chains associated together,
- wherein the only covalent bond linking the first and second arms together is a Cys-Cys disulfide bond linking position 214 (EU numbering) of the first antibody light chain and position 214 (EU numbering) of the second antibody light chain.
11 more elements
Dependencies are inferred from the claim text.