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.
Claims27
1-32. (canceled)
A method for producing a heteromultimer, the method comprising:
- (a) providing a first molecule comprising at least two copies of a first polypeptide, wherein the first polypeptide (i) comprises an antibody heavy chain constant region that comprises a Cys residue and has no core hinge region Cys residues, and (ii) has a first antigen-binding activity;
- (b) providing a second molecule comprising at least two copies of a second polypeptide different from the first polypeptide, wherein the second polypeptide (i) comprises an antibody heavy chain constant region that comprises a Cys residue and has no core hinge region Cys residues, and (ii) has a second antigen-binding activity that is different from the first antigen-binding activity;
- (c) incubating the first and second molecules together under a reducing condition that allows Cys-Cys disulfide bonds in the first molecule and in the second molecule to isomerize;
- and
- (d) obtaining a heteromultimer comprising the first polypeptide and the second polypeptide, joined by a single Cys-Cys disulfide bond, provided that the heteromultimer has no core hinge region disulfide bonds.
A method for producing a heteromultimer, the method comprising:
- (a) providing a first molecule comprising at least two copies of a first polypeptide, wherein the first polypeptide (i) comprises an antibody heavy chain constant region, and (ii) has a first antigen-binding activity;
- (b) providing a second molecule comprising at least two copies of a second polypeptide different from the first polypeptide, wherein the second polypeptide (i) comprises an antibody heavy chain constant region, and (ii) has a second antigen-binding activity that is different from the first antigen-binding activity;
- (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 the first and second polypeptides, wherein the heavy chain constant regions of the first and second polypeptides (i) both have an amino acid residue other than Cys at position 226 (EU numbering) and position 229 (EU numbering), and (ii) meet the criteria of one of the following (1) to (5):
- (1) the first polypeptide has a Cys at position 349 (EU numbering), and the second polypeptide has a Cys at position 356 (EU numbering);
- or
- (2) the first and second polypeptides both have a Cys at position 394 (EU numbering);
- or
- (3) the first and second polypeptides both have a Cys at position 351 (EU numbering);
- or
- (4) the first and second polypeptides both have a Cys at position 407 (EU numbering);
- or
- (5) the first polypeptide has a Cys at position 349 (EU numbering), and the second polypeptide has a Cys at position 354 (EU numbering).
Dependencies are inferred from the claim text.