Method For Producing Antibodies

• US 20080187954A1
• Filed: 03/10/2005
• Published: 08/07/2008
• Est. Priority Date: 03/10/2004
• Status: Abandoned Application

• Current Assignee: Gilles Reiss
• Original Assignee: Lonza Biologics PLC (Lonza Group AG), Lonza Limited (Lonza Group AG)
• Inventors: Gay, Robert, Kallmeier, Robert, Kalwy, Stephan, Reiss, Gilles
• Application Number:
  US10/591,812
• Publication Number:
  US 20080187954A1
• Time in Patent Office: Days
• US Class Current: 435/68.1
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/30 from tumour cells
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2319/00 Fusion polypeptide

Abstract

A novel method for expression of immunoglobulines is devised.

Claims

1-57. -57. (canceled)

58. Method for producing an immunoglobulin having Fc receptor activity and/or complement activation activity which immunoglobulin molecule when secreted from a vertebrate host cell comprises at least a first and a second polypeptide chain wherein the first polypeptide is an Ig-Light Chain (L) comprising at least a VL and a CL domain and in that the second polypeptide is an Ig-Heavy Chain (H) comprising at least a VH, CH2 and CH3 domain and a hinge domain, comprising the steps of

a. expressing in a vertebrate host cell having Golgi-only or late-Golgi-only resident furin family endoprotease activity a fusion polypeptide comprising a secretion targeting sequence directing the polypeptide to the secretory pathway and further comprising at least the said first and second polypeptide sequences and at least one cleavage site for the said endoprotease activity and wherein the fusion polypeptide comprises the sequences of said first and second polypeptide separated by a linker and

b. having the fusion polypeptide cleaved in the cells by the furin family endoprotease activity into the first and second polypeptide chains and

c. harvesting the secreted immunoglobulin.

59. Method according to claim 58, characterized in that the Ig molecule comprises a CH1 domain.

60. Method according to claim 58, characterized in that the Light and Heavy Chain are separated by a linker, in that the fusion polypeptide comprises at least two cleavage sites for the furin family endoprotease activity and in that the linker is cleaved off from both Heavy and Light Chain by the furin family endoprotease activity by means of said two cleavage sites.

61. Method according to claim 58, characterized in that the furin family endoprotease activity is an activity naturally present in that host cell line.

62. Method according to claim 58, characterized in that the host cell is devoid of furin family endoprotease activity in the endoplasmic reticulum.

63. Method according to claim 58, characterized in that the furin family endoprotease activity is furin endoprotease or lymphoma proprotein convertase or a functional variant thereof.

64. Method according to claim 58, characterized in that the furin family endoprotease activity is a constitutive endoprotease activity.

65. Method according to claim 58, characterized in that the host cell line is a mammalian cell line.

66. Method according to claim 65, characterized in that the host cell has at least one recombinant furin family endoprotease activity which is a homologously expressed mammalian furin family endoprotease naturally present in that host cell line which further is an constitutive furin family endoprotease or furin family endoprotease belonging to constitutive secretion, in this way achieving an elevated expression level of the natural gene product in its native host cell environment.

67. Method according to claim 65, characterized in that the mammalian host cell line are CHO cells.

68. Method according to claim 58, characterized in that the cleavage sites is a contiguous tetrapeptide sequence comprising at least three basic residues selected from the group consisting of arginine and lysine.

69. Method according to claim 68, characterized in that the tetrapeptide sequence comprises four basic residues selected from the group consisting of arginine and lysine.

70. Method according to claim 58, characterized in that the linker is a non-naturally occurring amino acid sequence.

71. Method according to claim 58, characterized in that the linker comprises at least 20 amino acids.

72. Method according to claim 71, characterized in the linker comprises one or several oligomers consisting of only glycine and either serine, threonine or both.

73. Method according to claim 72, characterized in that the linker consists of one or several oligomers consisting of only glycine and either serine, threonine or both.

74. Method according to claim 72, characterized in that the linker comprises at least >

60% glycine residues.

75. Method for producing an immunoglobulin having Fc receptor activity and/or complement activation activity which immunoglobulin molecule when secreted from a vertebrate host cell comprises at least a first and a second polypeptide chain wherein the first polypeptide is an Ig-Light Chain (L) comprising at least a VL and a CL domain and in that the second polypeptide is an Ig-Heavy Chain (H) comprising at least a a VH, CH2 and CH3 domain and a hinge domain, comprising the steps of

a. expressing in a vertebrate host cell having Golgi-only or late-Golgi-only resident subtilisin/kexin family endoprotease activity a fusion polypeptide comprising a secretion targeting sequence directing the polypeptide to the secretory pathway and further comprising at least the first and second polypeptide sequences and at least one cleavage site for the said endoprotease activity and wherein the fusion polypeptide comprises the sequences of said first and second polypeptide separated by a linker, and

b. having the fusion polypeptide cleaved in the cells by the subtilisin/kexin family endoprotease activity into the first and second polypeptide chains and

c. harvesting the secreted immunoglobulin.