Methods for producing polypeptides by regulating polypeptide association

Patent metadata

Field	Value
Publication number	US-10011858-B2
Application number	US-91012806-A
Country	US
Kind code	B2
Filing date	Mar 31, 2006
Priority date	Mar 31, 2005
Publication date	Jul 3, 2018
Grant date	Jul 3, 2018

Abstract

In the course of the present invention, it was discovered that one could regulate association between polypeptides by modifying amino acid residues that form the interface during the association to amino acids carrying the same type of charge. In this context, the present invention enables efficient formation of heterologous molecules. For example, the present invention can be suitably applied to the preparation of bispecific antibodies.

First claim

The invention claimed is:

1. A method for producing a desired multi-specific antibody, the method comprising
(a) identifying a first polypeptide that forms part of a multi-specific antibody by associating directly with either (i) a second polypeptide different from the first polypeptide or (ii) a third polypeptide different from the first and second polypeptides, wherein the first polypeptide comprises an antibody heavy chain CH3 region and an antibody heavy chain variable domain, wherein the second and third polypeptides respectively comprise different antibody light chain variable domains, wherein the amino acid residue at position 39 (Kabat numbering) (“H39”) of the heavy chain variable domain of the first polypeptide and the amino acid residue at position 38 (Kabat numbering) (“L38”) of the light chain variable domain of the second or third polypeptide can form part or all of an interface in the multi-specific antibody, wherein the residues at H39 of the first polypeptide and L38 of the second polypeptide may be charged or uncharged, but if both are charged they do not have the same charge, and wherein the multi-specific antibody contains a fourth polypeptide comprising an antibody heavy chain CH3 region and an antibody heavy chain variable domain different from the heavy chain variable domain of the first polypeptide;
(b) producing a nucleic acid encoding a modified first polypeptide in which the residue at H39 is substituted with a first substitute residue;
(c) producing a nucleic acid encoding a modified second polypeptide in which the residue at L38 is substituted with a second substitute residue, wherein the first and second substitute residues are either both positively charged or both negatively charged, wherein the like charges of the first and second substitute residues inhibit direct association between the modified first polypeptide and the modified second polypeptide;
(d) expressing the nucleic acids of (b) and (c), a nucleic acid encoding the third polypeptide, and a nucleic acid encoding the fourth polypeptide, thereby producing an expression product, wherein the residue at position 39 (Kabat numbering) of the heavy chain variable domain of the fourth polypeptide has either no charge or a charge that is opposite to that of the first substitute residue; and
(e) collecting the expression product of (d), the expression product comprising the desired multi-specific antibody comprising (1) the modified first polypeptide associated directly with the third polypeptide, and (2) the modified second polypeptide associated directly with the fourth polypeptide,
wherein any one or more of the following criteria (1) to (6) is true:
(1) amino acid residues at EU numbering positions 356 and 439 in the CH3 region of the first polypeptide either both have a positive charge or both have a negative charge, and if either of the amino acid residues at EU numbering positions 356 and 439 in the CH3 region of the fourth polypeptide is charged, it has a charge opposite to the charge of the amino acid residues at EU numbering positions 356 and 439 in the CH3 region of the first polypeptide;
(2) amino acid residues at EU numbering positions 357 and 370 in the CH3 region of the first polypeptide either both have a positive charge or both have a negative charge, and if either of the amino acid residues at EU numbering positions 357 and 370 in the CH3 region of the fourth polypeptide is charged, it has a charge opposite to the charge of the amino acid residues at EU numbering positions 357 and 370 in the CH3 region of the first polypeptide;
(3) amino acid residues at EU numbering positions 399 and 409 in the CH3 region of the first polypeptide either both have a positive charge or both have a negative charge, and if either of the amino acid residues at EU numbering positions 399 and 409 in the CH3 region of the fourth polypeptide is charged, it has a charge opposite to the charge of the amino acid residues at EU numbering positions 399 and 409 in the CH3 region of the first polypeptide;
(4) amino acid residues at EU numbering positions 356 and 439 in the CH3 region of the fourth polypeptide either both have a positive charge or both have a negative charge, and if either of the amino acid residues at EU numbering positions 356 and 439 in the CH3 region of the first polypeptide is charged, it has a charge opposite to the charge of the amino acid residues at EU numbering positions 356 and 439 in the CH3 region of the fourth polypeptide;
(5) amino acid residues at EU numbering positions 357 and 370 in the CH3 region of the fourth polypeptide either both have a positive charge or both have a negative charge, and if either of the amino acid residues at EU numbering positions 357 and 370 in the CH3 region of the first polypeptide is charged, it has a charge opposite to the charge of the amino acid residues at EU numbering positions 357 and 370 in the CH3 region of the fourth polypeptide;
(6) amino acid residues at EU numbering positions 399 and 409 in the CH3 region of the fourth polypeptide either both have a positive charge or both have a negative charge, and if either of the amino acid residues at EU numbering positions 399 and 409 in the CH3 region of the first polypeptide is charged, it has a charge opposite to the charge of the amino acid residues at EU numbering positions 399 and 409 in the CH3 region of the fourth polypeptide.

2. The method of claim 1 , wherein each of the first and second substitute amino acid residues is glutamic acid (E).

3. The method of claim 1 , wherein each of the first and second substitute amino acid residues is aspartic acid (D).

4. The method of claim 1 , wherein each of the first and second substitute amino acid residues is lysine (K).

5. The method of claim 1 , wherein each of the first and second substitute amino acid residues is arginine (R).

6. The method of claim 1 , wherein each of the first and second substitute amino acid residues is histidine (H).

7. The method of claim 1 , wherein the amino acid residue at position 38 (Kabat numbering) of the light chain variable domain of the third polypeptide has either no charge or a charge opposite to that of the first substitute residue.

8. The method of claim 1 , wherein the multi-specific antibody of (e) is a bispecific antibody.

9. The method of claim 1 , wherein criteria (1) and (4) are true.

10. The method of claim 1 , wherein criteria (2) and (5) are true.

11. The method of claim 1 , wherein criteria (3) and (6) are true.

12. A method for producing a desired multi-specific antibody, the method comprising
(a) identifying a first polypeptide that forms part of a multi-specific antibody by associating directly with either (i) a second polypeptide different from the first polypeptide or (ii) a third polypeptide different from the first and second polypeptides, wherein the first polypeptide comprises an antibody heavy chain CH3 region and an antibody heavy chain variable domain, wherein the second and third polypeptides respectively comprise different antibody light chain variable domains, wherein the amino acid residue at position 39 (Kabat numbering) (“H39”) of the heavy chain variable domain of the first polypeptide and the amino acid residue at position 38 (Kabat numbering) (“L38”) of the light chain variable domain of the second or third polypeptide can form part or all of an interface in the multi-specific antibody, wherein the residue at L38 of the second polypeptide is a charged residue and the residue at H39 of the first polypeptide is either uncharged or has a charge different from that of the residue at L38 of the second polypeptide, and wherein the multi-specific antibody contains a fourth polypeptide com

Assignees

• Igawa Tomoyuki
• Tsunoda Hiroyuki
• Chugai Pharmaceutical Co Ltd

Inventors

• Igawa Tomoyuki
• Tsunoda Hiroyuki

Classifications

• A61P35/00

  Antineoplastic agents · CPC title

• C07K16/18

  against material from animals or humans · CPC title

• C07K2317/31

  multispecific · CPC title

• C12P21/02 Primary

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title

• A61K2039/505

  comprising antibodies · CPC title