Methods of modifying antibodies for purification of bispecific antibodies

Patent metadata

Field	Value
Publication number	US-9670269-B2
Application number	US-29507507-A
Country	US
Kind code	B2
Filing date	Mar 30, 2007
Priority date	Mar 31, 2006
Publication date	Jun 6, 2017
Grant date	Jun 6, 2017

Abstract

The present inventors devised methods for efficiently purifying bispecific antibodies using a chromatography column based on the difference in isoelectric points between the H chains of two types of antibodies, wherein the difference is introduced by modifying the amino acids present on the surface of the antibody variable regions of two types of antibodies that constitute a bispecific antibody. Furthermore, the inventors devised methods for efficiently purifying bispecific antibodies using a chromatography column by linking respective antigen binding sites (heavy chain variable regions) to the antibody constant regions having different isoelectric points, and then coexpressing these antibodies.

First claim

The invention claimed is:

1. A method for producing a modified multispecific human or humanized antibody comprising a first polypeptide and a second polypeptide, wherein the method comprises the steps of:
(a) providing a pair of nucleic acids respectively encoding a pair of polypeptides, wherein the pair of polypeptides is either a pair of human or humanized antibody heavy chains or a pair of human or humanized antibody light chains;
(b) modifying one or both of the nucleic acids to alter one or more surface amino acid residues of the encoded polypeptide(s), thereby generating a modified pair of nucleic acids encoding a modified pair of polypeptides, wherein the modification increases the difference between the respective isoelectric points of the pair of polypeptides;
(c) providing host cells containing the modified pair of nucleic acids;
(d) culturing the host cells so that they express the modified pair of nucleic acids; and
(e) using a method comprising ion exchange chromatography to purify, from the host cell culture, a modified human or humanized multispecific antibody comprising the modified pair of polypeptides, wherein ion exchange chromatography separates the modified human or humanized multispecific antibody from homomultimers formed from each polypeptide of the modified pair of polypeptides based on the difference between the respective isoelectric points of the modified pair of polypeptides,
wherein the one or more surface amino acid residues that are altered comprise at least one amino acid residue located at a position or positions selected from heavy chain variable region positions 1, 3, 5, 8, 10, 12, 13, 15, 16, 19, 23, 25, 26, 27, 39, 42, 43, 44, 46, 68, 71, 72, 73, 75, 76, 81, 82b, 83, 85, 86, 97, 105, 108, 110, and 112 (Kabat numbering), heavy chain constant region positions 137, 196, 203, 214, 217, 233, 268, 274, 276, 297, 355, 392, 419, and 435 (EU numbering), and light chain variable region positions 1, 3, 7, 8, 9, 11, 12, 16, 17, 18, 20, 22, 38, 39, 41, 42, 43, 45, 46, 49, 57, 60, 63, 65, 66, 68, 69, 70, 74, 76, 77, 79, 80, 81, 85, 100, 103, 105, 106, 107, and 108 (Kabat numbering).

2. The method of claim 1 , wherein the increased difference in isoelectric points resulting from the modification of step (b) is adequate to permit separation, in ion exchange chromatography, of a peak representing a homomultimer comprising two copies of the first polypeptide of the modified pair of polypeptides, a peak representing a homomultimer comprising two copies of the second polypeptide of the modified pair of polypeptides, and a peak representing a heteromultimer comprising the first and second polypeptides of the modified pair of polypeptides.

3. The method of claim 1 , wherein the pair of polypeptides is a pair of human or humanized antibody heavy chains.

4. The method of claim 1 , wherein each polypeptide of the modified pair of polypeptides comprises a heavy chain constant region, and the isoelectric point of the heavy chain constant region of the first polypeptide of the modified pair of polypeptides is different from the isoelectric point of the heavy chain constant region of the second polypeptide of the modified pair of polypeptides.

5. The method of claim 4 , wherein one of the heavy chain constant regions of the modified pair of polypeptides is a human IgG1 heavy chain constant region and the other is a human IgG4 or human IgG2 heavy chain constant region.

6. The method of claim 1 , wherein the modified multispecific antibody of step (e) is a bispecific antibody.

7. The method of claim 3 , wherein the modified multispecific antibody comprises two copies of an antibody light chain, and wherein each polypeptide of the modified pair of polypeptides associates with one of the copies of the antibody light chain.

8. The method of claim 3 , wherein the one or more surface amino acid residues that are altered comprise at least one amino acid residue located at a position or positions selected from heavy chain variable region positions 1, 3, 5, 8, 10, 12, 13, 15, 16, 19, 23, 25, 26, 27, 39, 42, 43, 44, 46, 68, 71, 72, 73, 75, 76, 81, 82b, 83, 85, 86, 97, 105, 108, 110, and 112 (Kabat numbering).

9. The method of claim 3 , wherein the one or more surface amino acid residues that are altered comprise at least one amino acid residue located at a position or positions selected from heavy chain constant region positions 137, 196, 203, 214, 217, 233, 268, 274, 276, 297, 355, 392, 419, and 435 (EU numbering).

10. The method of claim 1 , wherein the pair of polypeptides is a pair of human or humanized antibody light chains.

11. The method of claim 10 , wherein the one or more surface amino acid residues that are altered comprise at least one amino acid residue located at a position or positions selected from light chain variable region positions 1, 3, 7, 8, 9, 11, 12, 16, 17, 18, 20, 22, 38, 39, 41, 42, 43, 45, 46, 49, 57, 60, 63, 65, 66, 68, 69, 70, 74, 76, 77, 79, 80, 81, 85, 100, 103, 105, 106, 107, and 108 (Kabat numbering).

12. The method of claim 1 , wherein the difference between the respective isoelectric points of the pair of polypeptides post-modification is 0.5 or more.

13. The method of claim 1 , wherein the difference between the respective isoelectric points of the pair of polypeptides post-modification is 0.7 or more.

14. The method of claim 1 , wherein the difference between the respective isoelectric points of the pair of polypeptides post-modification is 0.9 or more.

15. The method of claim 3 , wherein one or more of the surface amino acid residues that are altered in one or both polypeptides are located at a position or positions selected from heavy chain variable region positions 10, 12, 23, 39, 43, and 105 (Kabat numbering).

16. The method of claim 15 , wherein one of the polypeptides of the modified pair of polypeptides has a positively charged amino acid residue at a position selected from heavy chain variable region positions 10, 12, 23, 39, 43, and 105 (Kabat numbering), and the other polypeptide of the modified pair of polypeptides has a negatively charged amino acid residue at the same position.

17. The method of claim 15 , wherein one of the polypeptides of the modified pair of polypeptides has a charged amino acid residue at a position selected from heavy chain variable region positions 10, 12, 23, 39, 43, and 105 (Kabat numbering), and the other polypeptide of the modified pair of polypeptides has an uncharged amino acid residue at the same position.

18. The method of claim 16 , wherein the positively charged amino acid residue is selected from the group consisting of lysine (K), arginine (R), and histidine (H), and the negatively charged amino acid residue is selected from the group consisting of glutamic acid (E) and aspartic acid (D).

19. The method of claim 17 , wherein the charged amino acid residue is selected from the group consisting of lysine (K), arginine (R), histidine (H), glutamic acid (E), and aspartic acid (D).

20. The method of claim 1 , wherein the modification results in one of the polypeptides of the modified pair of polypeptides being modified to have an isoelectric point higher than prior to the modification, and the other polypeptide of the modified pair of polypeptides being modified to have an isoelectric point lower than prior to the modification.

21. The method of claim 1 , wherein, after the modification of (b), all of the altered surface amino acid residues of one of the polypeptides of the modified pair of polypeptides are positively charged amino

Assignees

• Igawa Tomoyuki
• Tsunoda Hiroyuki
• Chugai Pharmaceutical Co Ltd

Inventors

• Igawa Tomoyuki
• Tsunoda Hiroyuki

Classifications

• C07K2317/56

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

• A61P35/00

  Antineoplastic agents · CPC title

• A61P43/00

  Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 · CPC title

• C07K2317/76

  Antagonist effect on antigen, e.g. neutralization or inhibition of binding · CPC title

• C07K16/00 Primary

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