Method for producing polypeptide hetero-oligomer

Patent metadata

Field	Value
Publication number	US-2022010030-A1
Application number	US-202117483898-A
Country	US
Kind code	A1
Filing date	Sep 24, 2021
Priority date	Apr 1, 2015
Publication date	Jan 13, 2022
Grant date	—

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.

First claim

1 - 32 . (canceled)

33 . 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.

34 . The method of claim 33 , wherein
the first molecule of (a) comprises at least two copies of a third polypeptide, each copy of the third polypeptide being associated with a copy of the first polypeptide in the first molecule; the second molecule of (b) comprises at least two copies of a fourth polypeptide, each copy of the fourth polypeptide being associated with a copy of the second polypeptide in the second molecule; and the heteromultimer of (d) comprises the first polypeptide in association with the third polypeptide and comprises the second polypeptide in association with the fourth polypeptide.

35 . The method of claim 34 , wherein the first and second polypeptides are antibody heavy chains, and the third and fourth polypeptides are antibody light chains.

36 . The method of claim 33 , wherein, in each of the first and second polypeptides, both of positions 226 and 229 (EU numbering) are deleted or both are occupied by a residue other than Cys.

37 . The method of claim 33 , wherein each of the first and second polypeptides lacks all core hinge region positions.

38 . The method of claim 33 , wherein, in each of the first and second polypeptides, positions 220 to 225 (EU numbering) are occupied by the sequence Tyr-Gly-Pro-Pro.

39 . The method of claim 33 , wherein, in each of the first and second polypeptides, all of positions 219 to 229 (EU numbering) are deleted.

40 . The method of claim 33 , wherein
the heavy chain constant region of the first polypeptide comprises a CH3 region comprising at least one Cys residue; the heavy chain constant region of the second polypeptide comprises a CH3 region comprising at least one Cys residue; and in the heteromultimer, the CH3 region of the first polypeptide is linked to the CH3 region of the second polypeptide by the single disulfide bond.

41 . The method of claim 40 , wherein a Cys residue in the CH3 region of the first polypeptide is located at a position selected from positions 349, 351, 354, 356, 394, and 407 (EU numbering) of the first polypeptide; and a Cys residue in the CH3 region of the second polypeptide is located at a position selected from positions 349, 351, 354, 356, 394, and 407 (EU numbering) of the second polypeptide.

42 . The method of claim 40 , wherein a Cys residue in the CH3 region of the first polypeptide and a Cys residue in the CH3 region of the second polypeptide are located at a pair of positions selected from the following pairs (1) to (5) (all positions according to EU numbering):
(1) position 349 in one of the CH3 regions and position 356 in the other, (2) position 394 in one of the CH3 regions and position 394 in the other, (3) position 351 in one of the CH3 regions and position 351 in the other, (4) position 407 in one of the CH3 regions and position 407 in the other, and (5) position 349 in one of the CH3 regions and position 354 in the other.

43 . The method of claim 33 , wherein the heavy chain constant regions of the first and second polypeptides each comprise a CH3 region, and
(A) the CH3 region of the first polypeptide includes an amino acid substitution, compared to the sequence of a naturally-occurring IgG CH3 region, at a position or positions selected from positions 392, 397, and 409 (EU numbering); or (B) the CH3 region of the second polypeptide includes an amino acid substitution, compared to the sequence of a naturally-occurring IgG CH3 region, at a position or positions selected from positions 392, 397, and 409; or (C) both (A) and (B).

44 . The method of claim 33 , wherein the heavy chain constant regions of the first and second polypeptides each comprise a CH3 region, and one or more of (A) to (F) is true:
(A) amino acid residues at positions 356 and 439 (EU numbering) in the CH3 region of the first polypeptide have the same type of charge (+ or −), (B) amino acid residues at positions 357 and 370 (EU numbering) in the CH3 region of the first polypeptide have the same type of charge (+ or −), (C) amino acid residues at positions 399 and 409 (EU numbering) in the CH3 region of the first polypeptide have the same type of charge (+ or −), (D) amino acid residues at positions 356 and 439 (EU numbering) in the CH3 region of the second polypeptide have the same type of charge (+ or −), (E) amino acid residues at positions 357 and 370 (EU numbering) in the CH3 region of the second polypeptide have the same type of charge (+ or −), (F) amino acid residues at positions 399 and 409 (EU numbering) in the CH3 region of the second polypeptide have the same type of charge (+ or −), provided that, when both (A) and (D) are true, the type of charge in (A) is the opposite of the type of charge in (D), and when both (B) and (E) are true, the type of charge in (B) is the opposite of the type of charge in (E), and when both (C) and (F) are true, the type of charge in (C) is the opposite of the type of charge in (F).

45 . The method of claim 44 , wherein, when the type of charge is positive charge, the amino acid residues are selected from Lys, Arg, and His, and when the type of charge is negative charge, the amino acid residues are selected from Glu and Asp.

46 . The method of claim 44 , wherein the amino acid residues at positions 356, 399, 409, and 439 (EU numbering) of the first polypeptide are all positively charged, and the amino acid residues at positions 356, 399, 409, and 439 (EU numbering) of the second polypeptide are all negatively charged.

47 . The method of claim 33 , wherein the heavy chain constant region of at least one of the first and second polypeptides is selected from IgG1 type, IgG2 type, IgG3 type, or IgG4 type.

48 . The method of claim 33 , wherein the heavy chain constant region of at least one of the first and second polypeptides is a mouse-derived heavy chain constant region.

49 . The method of claim 48 , wherein the heavy chain constant regions of the first and second polypeptides each comprise a CH3 region, and one or more of (A) to (F) is true:
(A) amino acid residues at positions 356 and 439 (EU numbering) in the CH3 region of the first polypeptide have the same type of charge (+ or −), (B) amino acid residues at positions 360 and 371 (EU numbering) in the CH3 region of the first polypeptide have the same type of charge (+ or −), (C) amino acid residues at positions 399 and 409 (EU numbering) in the CH3 region of the first polypeptide have the same type o

Assignees

• Chugai Pharmaceutical Co Ltd

Inventors

• Igawa Tomoyuki
• Katada Hitoshi
• Hori Yuji
• Kadono Shojiro

Classifications

• G01N33/50 Primary

  Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing (measuring or testing processes involving enzymes or microorganisms, compositions or test papers therefor; processes for forming such compositions, condition responsive control in microbiological or enzymological processes C12Q) · CPC title

• C07K2317/31

  multispecific · CPC title

• C07K2317/56

  variable (Fv) region, i.e. VH and/or VL · CPC title

• C07K16/468

  Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies · CPC title

• C07K16/00

  Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies · CPC title