Assembly of bispecific antibodies

Patent metadata

Field	Value
Publication number	US-11725065-B2
Application number	US-202016820660-A
Country	US
Kind code	B2
Filing date	Mar 16, 2020
Priority date	Oct 11, 2011
Publication date	Aug 15, 2023
Grant date	Aug 15, 2023

Abstract

Described herein are methods for the efficient production of a heteromultimeric protein, such as a bispecific antibody. Heteromultimeric proteins may be capable of specifically binding to more than one target molecule or different epitopes on a single target molecule. The methods modulate parameters to improve assembly of the heteromultimeric proteins at higher yield and efficiency than otherwise possible. Also described are compositions comprising a hinge-containing polypeptide, such as a half-antibody.

First claim

What is claimed is:

1. A method of producing a heteromultimeric protein, said method comprising:
a. obtaining a first half-antibody, wherein the first half-antibody comprises a heterodimerization domain;
b. obtaining a second half-antibody, wherein the second half-antibody comprises a heterodimerization domain;
c. adjusting pH of each half-antibody to between pH 4 and 9;
d. mixing the first and second half-antibodies to obtain an assembly mixture;
e. adding a 50-400x molar excess of glutathione (GSH) to the assembly mixture; and
f. incubating the assembly mixture to form a heteromultimeric protein comprising the first half-antibody and the second half-antibody.

2. The method of claim 1 , wherein the first and second half-antibodies each comprise an Fc component.

3. The method of claim 1 , wherein in step c the pH of the first and second half-antibodies are adjusted to pH 4-9 in the presence of a solubilizer.

4. The method of claim 3 , wherein the solubilizer is arginine that is added to a final concentration of between 20 mM to 1M prior to adjusting the pH.

5. The method of claim 1 , wherein the heterodimerization domain of the first and/or second half-antibodies comprises one or more of a knob, a hole, a leucine zipper, a coiled coil, or a polar amino acid residue capable of forming an electrostatic interaction.

6. The method of claim 1 , wherein the heterodimerization domain of the first half-antibody comprises a knob and the heterodimerization domain of the second half-antibody comprises a hole.

7. The method of claim 1 , wherein the pH is adjusted after mixing.

8. The method of claim 1 , further comprising incubating the assembly mixture at a temperature of between 15° C. and 39° C. for at least 30 minutes.

9. The method of claim 1 , wherein the assembly mixture in step f has an oxidation potential of between −200 to −600 mV.

10. The method of claim 1 , wherein incubating the assembly mixture is done at a temperature between 15° C. and 39° C. in the presence of Polyvinylpyrrolidone (PVP).

11. The method of claim 10 , wherein the PVP is added up to 40% (w/v).

12. The method of 1 , wherein the first or second half-antibody is produced by a bacterial cell, a yeast cell, a baculovirus, or a mammalian cell.

13. The method of claim 1 , wherein the first or second half-antibody is produced by a CHO cell.

14. The method of claim 1 , wherein the GSH is added in 100-300X molar excess.

15. The method of claim 1 , wherein the GSH is added in 200X molar excess.

16. The method of claim 6 , wherein the knob comprises a T366W substitution and the hole comprises T366S, L368A, and Y407V substitutions.

17. The method of claim 1 , further comprising adjusting the pH after the heteromultimeric protein is formed in step f.

18. The method of claim 17 , wherein the pH is lowered after the heteromultimeric protein is formed in step f.

19. The method of claim 1 , further comprising adjusting the temperature after the heteromultimeric protein is formed in step f.

20. The method of claim 19 , wherein the temperature is lowered after the heteromultimeric protein is formed in step f.

Assignees

• Genentech Inc

Inventors

• Giese Glen
• Lim Amy
• Persson Josefine
• Scheer Justin
• Williams Ambrose

Classifications

• C07K16/468 Primary

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

• C07K2317/31

  multispecific · CPC title

• C07K2317/522

  CH1 domain · CPC title

• C07K2317/524

  CH2 domain · CPC title

• C07K2317/526

  CH3 domain · CPC title