Fc VARIANTS AND METHODS FOR THEIR PRODUCTION

• US 20140079689A1
• Filed: 08/02/2013
• Published: 03/20/2014
• Est. Priority Date: 02/04/2011
• Status: Active Grant

• Current Assignee: Genentech, Inc. (Roche Holding AG)
• Original Assignee: Genentech, Inc. (Roche Holding AG)
• Inventors: ELLIOTT, MICHAEL J., SCHEER, JUSTIN
• Granted Patent: US 10,689,447 B2
• Time in Patent Office: Days
• US Class Current: 424/133.1
• CPC Class Codes: C07K 16/244 Interleukins [IL]
• CPC Class Codes: C07K 16/247 IL-4
• CPC Class Codes: C07K 16/2863 against receptors for growth factors, growth regulators
• CPC Class Codes: C07K 16/468 Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies

Abstract

Described herein are Fc variants and methods for the efficient production of antibodies and other multimeric protein complexes (collectively referred to herein as heteromultimeric proteins). Heteromultimeric proteins may be capable of specifically binding to more than one target. The targets may be, for example, different epitopes on a single molecule or located on different molecules. The methods combine efficient, high gene expression level, appropriate assembly, and ease of purification for the heteromultimeric proteins. The invention also provides methods of using these heteromultimeric proteins, and compositions, kits and articles of manufacture comprising these antibodies.

Claims

1-15. -15. (canceled)

16. A variant heteromultimeric protein or modified IgG antibody comprising an Fc variant of a wild-type Fc polypeptide, said Fc variant comprising at least two amino acid modification in the Fc region of said wild-type Fc polypeptide, wherein said variant protein exhibits decreased mispairing, decreased head-to-tail formation or increased overall yield as compared to the wild-type Fc polypeptide.

17. The variant heteromultimeric protein or modified IgG antibody of claim 16 further comprising knob-into-hole modification(s).

32. A host cell that produces the variant heteromultimeric protein or the modified IgG antibody of claim 17.

34. The host cell of claim 32 which is a CHO cell or an E. coli cell.

36. A composition comprising a variant heteromultimeric protein or the modified IgG antibody of claim 17 and a carrier.

18. A variant heteromultimeric protein or modified IgG antibody comprising an Fc variant of a wild-type Fc polypeptide wherein said Fc variant comprises substitutions at residues 241 and 243 (EU numbering) on at least one heavy chain with an amino acid which is different from that present in the wild-type Fc polypeptide thereby causing a decreased mispairing, decreased head-to-tail formation or increased overall yield as compared to the heteromultimer protein or IgG antibody without the substitutions at residues 241 and 243 (EU numbering).

19. The variant heteromultimeric protein or modified IgG antibody of claim 18 comprising mutations on at least one heavy chain selected from F241R/F243S and F241S/F243R.

20. The variant heteromultimeric protein or modified IgG antibody of claim 18 further comprising knob-into-hole modification(s).

21. The variant heteromultimeric protein or modified IgG antibody of claim 20 wherein the knob modification comprises replacing an original amino acid residue from the interface of the Fc polypeptide with an amino acid residue with a larger side chain than the original amino acid residue wherein the substituting amino acid residue is selected from the group consisting of tryptophan, phenylalanine, tyrosine and arginine.

22. The variant heteromultimeric protein or modified IgG antibody of claim 21, wherein the knob modification comprises T366W substitution (EU numbering).

23. The variant heteromultimeric protein or modified IgG antibody of claim 20, wherein the hole modification comprises replacing an original amino acid residue from the interface of the Fc polypeptide with an amino acid residue with a smaller side chain than the original amino acid residue, wherein the substituting amino acid residue is selected from the group consisting of serine, threonine, valine, and alanine.

24. The variant heteromultimeric protein or modified IgG antibody of claim 23, wherein the hole modification comprises two or more amino acid substitutions selected from the group consisting of T366S, L368A and Y407V (EU numbering).

25. The variant heteromultimeric protein or modified IgG antibody of claim 20, wherein the heavy chain comprising the F241R/F243S or F241S/F243R substitution further comprises a knob modification.

26. The variant heteromultimeric protein or modified IgG antibody of claim 20, wherein the heavy chain comprising the F241R/F243S or F241S/F243R substitution further comprises a hole modification.

27. The variant heteromultimeric protein or modified IgG antibody of claim 20, wherein both heavy chains comprise the F241R/F243S or F241S/F243R substitution.

28. The variant heteromultimeric protein or the modified IgG antibody according to claim 20 wherein the protein or antibody is a multi-specific antibody.

29. An isolated nucleic acid encoding the variant heteromultimeric protein or the modified IgG antibody of claim 20.

30. An expression vector comprising the nucleic acid of claim 29.

31. A host cell comprising the expression vector of claim 30.

33. The host cell of claim 31 which is a CHO cell or an E. coli cell.

35. A method of producing a variant heteromultimeric protein or modified IgG antibody comprising culturing the host cell of claim 31 and recovering the variant heteromultimeric protein or the modified IgG antibody from the cell culture.

41. A composition comprising a variant heteromultimeric protein or the modified IgG antibody of claim 20 and a carrier

42. An isolated variant heteromultimeric protein produced by the method of claim 35.

37. A method of preparing a heteromultimeric protein comprising a first Fc-containing polypeptide having a first heterodimerization domain and a second Fc-containing polypeptide having a second heterodimerization domain, wherein the first and/or second Fc-containing polypeptide comprises substitutions at residues 241 and 243 (EU numbering), the method comprising the steps of:

(a) providing a purified first Fc-containing polypeptide having a first heterodimerization domain;

(b) providing a purified second Fc-containing polypeptide having a second heterodimerization domain;

(c) combining the first and second Fc-containing polypeptides; and

(d) refolding the first Fc-containing polypeptide with the second Fc-containing polypeptide to form a heteromultimeric protein.

38. The method of claim 37, further comprising the step of purifying the heteromultimeric protein.

39. The method of claim 37, wherein the first and/or second Fc-containing polypeptide comprises F241R/F243S or F241S/F243R substitutions.

40. The method of claim 39, wherein the first and/or second Fc-containing polypeptide further comprises knob-into-hole modification(s).

43. An isolated heteromultimeric protein produced by the method of claim 37.