Assembly of bispecific antibodies

Patent metadata

Field	Value
Publication number	US-9862778-B2
Application number	US-201414251428-A
Country	US
Kind code	B2
Filing date	Apr 11, 2014
Priority date	Oct 11, 2011
Publication date	Jan 9, 2018
Grant date	Jan 9, 2018

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 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 comprising the steps of:
a. providing a first half-antibody at pH 5-9 in the presence of a first solubilizer, wherein the first half-antibody comprises a heteromultimerization domain;
b. providing a second half-antibody at pH 5-9 in the presence of a second solubilizer, wherein the second half-antibody comprises a heteromultimerization domain;
c. mixing the first and second half-antibodies to form an assembly mixture in a reducing condition comprising 50-400× molar excess of glutathione (GSH) with respect to the total amount of half-antibodies; and
d. incubating the assembly mixture to produce a heteromultimeric protein comprising the first and second half-antibodies, wherein the first half-antibody interacts with the second half-antibody at the heteromultimerization domain.

2. The method of claim 1 , wherein the first solubilizer or the second solubilizer is selected from the group consisting of arginine, histidine and sucrose.

3. The method of claim 1 , wherein the first solubilizer or the second solubilizer is arginine, an arginine salt or arginine derivative.

4. The method of claim 1 , wherein the first solubilizer or the second solubilizer is histidine, a histidine salt or histidine derivative.

5. The method of claim 2 , wherein the first solubilizer or the second solubilizer is present at a concentration of between 20 mM and 1M.

6. The method of claim 5 , wherein the first solubilizer or the second solubilizer is present at a concentration of between 20 mM and 400 mM.

7. The method of claim 1 wherein the first and second solubilizers are arginine HCl.

8. The method of claim 1 , wherein the first half-antibody and the second half-antibody are purified before mixing.

9. The method of claim 1 , wherein the first half-antibody and the second half-antibody are co-purified.

10. The method of claim 1 , wherein the step a is preceded by the step of purifying the first half-antibody or the step b is preceded by the step of purifying the second half-antibody.

11. The method of claim 10 , wherein the first half-antibody and the second half-antibody are purified by protein A.

12. The method of claim 1 , wherein the first and second half-antibodies are produced by a bacterial cell, a yeast cell, a baculovirus, or a mammalian cell.

13. The method of claim 12 , wherein the first and second half-antibodies are produced by a mammalian cell.

14. The method of claim 13 , wherein the mammalian cell is a CHO cell.

15. The method of claim 1 , wherein the first or second half-antibody is an IgG half-antibody.

16. The method of claim 15 , wherein the IgG half-antibody is of the IgG1, IgG2 or IgG4 isotype.

17. The method of claim 15 , wherein the first or second half-antibody comprises an Fc component.

18. The method of claim 15 , wherein the first or second half-antibody comprises a V L domain, a V H domain, a hinge domain, a CH 2 domain and a CH 3 domain.

19. The method of claim 18 , wherein the first or second half-antibody comprises a single chain polypeptide that further comprises a tether, and wherein said single chain polypeptide comprises domains positioned relative to each other in an N-terminal to C-terminal direction as follows: V L -tether-V H -hinge-CH 2 —CH 3 .

20. The method of claim 18 , wherein the first or second half-antibody further comprises a C L domain and a CH 1 domain.

21. The method of claim 20 , wherein the first or second half-antibody comprises a single chain polypeptide that further comprises a tether, and wherein said single chain polypeptide comprises domains positioned relative to each other in an N-terminal to C-terminal direction as follows: V L —C L -tether-V H —CH 1 -hinge-CH 2 —CH 3 .

22. The method of claim 1 , wherein one or more of steps a-d are heated at a temperature of between 25° C. and 42° C.

23. The method of claim 22 , wherein one or more of steps a-d are heated at a temperature of between 32° C. and 37° C.

24. The method of claim 22 , wherein the first half-antibody of step a and the second half-antibody of step b are heated.

25. The method of claim 22 , wherein the assembly mixture of step d is heated.

26. The method of claim 22 , wherein all of steps a-d are heated.

27. The method of claim 26 , wherein all of steps a-d are heated at a temperature between 32° C. and 37° C.

28. The method of claim 1 , wherein the reducing condition has an oxidation potential of between −200 to −600 mV.

29. The method of claim 1 , wherein the GSH is added in 100-300× molar excess to the assembly mixture.

30. The method of claim 29 , wherein the GSH is added in 200× molar excess to the assembly mixture.

31. The method of claim 1 wherein the assembly mixture is incubated at pH 7 to pH 9.

32. The method of claim 1 , wherein the heteromultimerization domain of the first or second half antibody 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.

33. The method of claim 32 , wherein the heteromultimerization domain of the first half-antibody comprises a knob and the heteromultimerization second domain of the half-antibody comprises a hole.

34. The method of claim 1 , further comprising adding a stabilizer in one or more of steps a-d.

35. The method of claim 34 , wherein the stabilizer is added to step c or step d.

36. The method of claim 35 , wherein the stabilizer is arginine or polyvinylpyrrolidone (PVP).

37. The method of claim 1 , further comprising the step of recovering the heteromultimeric protein.

38. The method of claim 37 , wherein the step of recovering the heteromultimeric protein comprises purifying the heteromultimeric protein.

39. A method of producing a multispecific antibody comprising the steps of
a. providing a first half-antibody at pH 5-9 in the presence of arginine or histidine, wherein the first half-antibody comprises a heteromultimerization domain;
b. providing a second half-antibody at pH 5-9 in the presence of arginine or histidine, wherein the second half-antibody comprises a heteromultimerization domain;
c. mixing the first and second half-antibodies to form an assembly mixture in a reducing condition comprising 50-400× molar excess of glutathione (GSH) with respect to the total amount of the half-antibodies; and
d. incubating the assembly mixture to form a multispecific antibody comprising the first and second half-antibodies, wherein the first half-antibody interacts with the second half-antibody at the heteromultimerization domain.

40. The method of claim 39 , wherein the arginine or histidine is present at a concentration of between 20 mM and 400 mM.

41. The method of claim 39 , wherein the first half-antibody and the second half-antibody are purified before mixing.

42. The method of claim 39 , wherein the first and second half-antibodies are produced by a bacterial cell, a yeast cell, a baculovirus, or a mammalian cell.

43. The method of claim 42 , wherein the first and second half-antibodies are produced by a mammalian cell.

Assignees

• Genentech Inc

Inventors

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

Classifications

• C07K2317/622

  Single chain antibody (scFv) · CPC title

• C07K2317/526

  CH3 domain · CPC title

• C07K2317/524

  CH2 domain · CPC title

• C07K2317/53

  Hinge · CPC title

• C07K2317/522

  CH1 domain · CPC title