METHOD FOR MAKING MULTISPECIFIC ANTIBODIES HAVING HETEROMULTIMERIC AND COMMON COMPONENTS

• US 20140322756A1
• Filed: 12/27/2013
• Published: 10/30/2014
• Est. Priority Date: 05/02/1997
• Status: Active Grant

• Current Assignee: Genentech, Inc. (Roche Holding AG)
• Original Assignee: Genentech, Inc. (Roche Holding AG)
• Inventors: PRESTA, Leonard G., ARATHOON, W. Robert, CARTER, Paul J., MERCHANT, Anne M.
• Granted Patent: US 9,409,989 B2
• Time in Patent Office: Days
• US Class Current: 435/69.6
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 1/22 Affinity chromatography or related techniques based upon selective absorption processes
• CPC Class Codes: C07K 16/2809 against the T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 16/2812 against CD4
• CPC Class Codes: C07K 16/2863 against receptors for growth factors, growth regulators
• CPC Class Codes: C07K 16/46 Hybrid immunoglobulins hybrids of an immunoglobulin with a peptide not being an immunoglobulin C07K19/00
• CPC Class Codes: C07K 16/468 Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
• CPC Class Codes: C07K 2317/732 Antibody-dependent cellular cytotoxicity [ADCC]
• CPC Class Codes: Y10S 435/972 Modified antibody, e.g. hybrid, bifunctional

Abstract

The invention relates to a method of preparing heteromultimeric polypeptides such as bispecific antibodies, bispecific immunoadhesins and antibody-immunoadhesin chimeras. The invention also relates to the heteromultimers prepared using the method. Generally, the method provides a multispecific antibody having a common light chain associated with each heteromeric polypeptide having an antibody binding domain. Additionally the method further involves introducing into the multispecific antibody a specific and complementary interaction at the interface of a first polypeptide and the interface of a second polypeptide, so as to promote heteromultimer formation and hinder homomultimer formation; and/or a free thiol-containing residue at the interface of a first polypeptide and a corresponding free thiol-containing residue in the interface of a second polypeptide, such that a non-naturally occurring disulfide bond is formed between the first and second polypeptide. The method allows for the enhanced formation of the desired heteromultimer relative to undesired heteromultimers and homomultimers.

Claims

1-33. -33. (canceled)

34. A method of preparing a multispecific antibody comprising culturing a host cell expressing a multispecific antibody, and recovering the multispecific antibody from the culture, wherein said multispecific antibody comprises four polypeptides, wherein a first and a second of said polypeptides each comprise a heavy chain constant domain and a heavy chain variable domain, and a third and a fourth of said polypeptides are each common light chains that are identical to each other, wherein said first polypeptide and said third polypeptide form a binding domain that binds a first antigen, and wherein said second polypeptide and said fourth polypeptide form a binding domain that binds a different antigen, and wherein said first polypeptide and said second polypeptide dimerize to form a multispecific antibody, wherein the multispecific antibody is an IgG, IgE or IgD antibody.

35. The method of claim 34, wherein said first polypeptide further comprises a first multimerization domain, and said second polypeptide further comprises a second multimerization domain, and wherein the first and second polypeptides dimerize by interaction of the first and second multimerization domains to form said multispecific antibody.

36. The method of claim 35, wherein the multimerization domains of said first and second polypeptide interact at an amino acid side chain protuberance of one of said first and second polypeptides and an amino acid side chain cavity of the other polypeptide.

37. The method of claim 34, wherein the multispecific antibody further comprises a non-naturally occurring disulfide bond between said first and second polypeptide.

38. The method of claim 35, wherein the multimerization domain is at least a part of a C_H3 domain region of an antibody constant domain, and the non-naturally occurring disulfide bond is between the C_H3 multimerization domains of the first and second polypeptides.

39. The method of claim 34, wherein said multispecific antibody following recovery is substantially homogenous.

40. The method of claim 34, wherein said host cell is a mammalian cell.

41. The method of claim 40, wherein said mammalian cell is a monkey cell, human cell, hamster cell, mouse cell or a rat cell.

42. The method of claim 40, wherein said mammalian cell is a rat cell.

43. The method of claim 36, wherein the multispecific antibody further comprises a non-naturally occurring disulfide bond between said first and second polypeptide.

44. The method of claim 34 further comprising the step of purifying the multispecific antibody by chromatography, gel electrophoresis or dialysis.

45. The method of claim 44, wherein said chromatography is affinity chromatography, hydroxylapatite chromatography, ion-exchange chromatography, reverse phase HPLC or chromatography on silica.

46. The method of claim 35, wherein said multispecific antibody comprises an insertion or substitution of a residue within the amino acid sequence of said first or second polypeptides forming said multispecific antibody, wherein said multispecific antibody maintains its binding to said antigens.

47. The method of claim 46, wherein said insertion or substitution is not within the multimerization domains of said first or second polypeptides.

48. A method of preparing a multispecific antibody comprising culturing a host cell expressing a multispecific antibody, and recovering said multispecific antibody from the culture, wherein said multispecific antibody comprises two heteromeric polypeptides and two light chains, wherein:

(a) the first heteromeric polypeptide comprises a first heavy chain variable domain and a first multimerization domain; and

(b) the second heteromeric polypeptide comprises a second heavy chain variable domain and a second multimerization domain;

wherein the two light chains have an amino acid sequence identity of 100%, wherein the first and second heteromeric polypeptides dimerize by interaction of the first and second multimerization domains to form a multispecific antibody, and wherein the multispecific antibody is an IgG, IgE or IgD antibody.

49. The method of claim 48, wherein the first multimerization domain has a protuberance modification and the second multimerization domain has a cavity modification, and wherein the protuberance is positionable in the cavity.

50. The method of claim 49, wherein the multispecific antibody further comprises a non-naturally occurring disulfide bond between the first and second heteromeric polypeptides.

51. The method of claim 48, wherein the multispecific antibody further comprises a non-naturally occurring disulfide bond between the first and second heteromeric polypeptides.

52. The method of claim 51, wherein the multimerization domains comprise a part of a C_H3 domain of an antibody constant domain, and the non-naturally occurring disulfide bond is between the C_H3 multimerization domains of the first and second heteromeric polypeptides.

53. The method of claim 48, wherein said multispecific antibody following recovery is substantially homogenous.

54. The method of claim 48, wherein said host cell is a mammalian host cell.

55. The method of claim 54, wherein said mammalian host cell is a monkey cell, human cell, hamster cell, mouse cell or a rat cell.

56. The method of claim 54, wherein said mammalian host cell is a human or a rat cell.

57. The method of claim 48 further comprising the step of purifying the multispecific antibody by chromatography, gel electrophoresis or dialysis.

58. The method of claim 57, wherein said chromatography of said multispecific antibody is affinity chromatography, hydroxylapatite chromatography, ion-exchange chromatography, reverse phase HPLC or chromatography on silica.

59. The method of claim 48, wherein said multispecific antibody comprises an insertion or substitution of a residue within the amino acid sequence of said first or second heteromeric polypeptides forming said multispecific antibody, wherein said multispecific antibody maintains its multispecificity.

60. The method of claim 59, wherein said insertion or substitution is not within the multimerization domains of said first or second heteromeric polypeptides.

61. A vector comprising nucleic acid sequences capable of encoding a first heteromeric polypeptide, a second heteromeric polypeptide and a common light chain polypeptide that form a multispecific antibody, wherein:

(a) the first heteromeric polypeptide comprises a first heavy chain variable domain and a first multimerization domain; and

(b) the second heteromeric polypeptide comprises a second heavy chain variable domain and a second multimerization domain;

wherein the first and second heteromeric polypeptides dimerize by interaction of the first and second multimerization domains to form the multispecific antibody, and wherein the multispecific antibody is an IgG, IgE or IgD antibody.

62. The vector of claim 61, wherein said vector is an expression vector.

63. The vector of claim 61, wherein said vector comprises one or more of:

a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter or a transcription termination sequence.

64. The vector of claim 61, wherein said vector can express said first heteromeric polypeptide, said second heteromeric polypeptide and said common light chain polypeptide in a mammalian cell.

65. An isolated host cell comprising one or more vectors, wherein said one or more vectors comprise nucleic acid sequences capable of encoding a first heteromeric polypeptide, a second heteromeric polypeptide and a common light chain polypeptide that form a multispecific antibody, wherein:

(a) the first heteromeric polypeptide comprises a first heavy chain variable domain and a first multimerization domain; and

(b) the second heteromeric polypeptide comprises a second heavy chain variable domain and a second multimerization domain;

wherein the first and second heteromeric polypeptides dimerize by interaction of the first and second multimerization domains to form the multispecific antibody.