Methods for generating monovalent IgG

• US 20070105199A1
• Filed: 10/23/2006
• Published: 05/10/2007
• Est. Priority Date: 10/21/2005
• Status: Active Grant

• Current Assignee: Amgen Inc.
• Original Assignee: Amgen Inc.
• Inventors: Yan, Wei, Wittekind, Michael, Forte, Carla
• Granted Patent: US 8,193,322 B2
• Time in Patent Office: Days
• US Class Current: 435/69.700
• CPC Class Codes: A61P 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 1/04 for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• CPC Class Codes: A61P 15/00 Drugs for genital or sexual disorders for disorders of sex hormones A61P5/24; Contraceptives
• CPC Class Codes: A61P 15/08 for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 17/06 Antipsoriatics
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 21/04 for myasthenia gravis
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 5/00 Drugs for disorders of the endocrine system
• CPC Class Codes: A61P 5/14 of the thyroid hormones, e.g. T3, T4
• CPC Class Codes: A61P 7/06 Antianaemics
• 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 16/2878 against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
• CPC Class Codes: C07K 2317/52 Constant or Fc region; Isotype
• CPC Class Codes: C07K 2317/55 Fab or Fab'
• CPC Class Codes: C07K 2319/00 : Fusion polypeptide
• CPC Class Codes: C07K 2319/30 : Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Abstract

The present invention relates to monovalent antibody, methods of making thereof and therapeutic uses thereof. In particular, the present invention provides a heterodimeric polypeptide comprising an immunoglobulin heavy chain and a fusion protein comprising an immunoglobulin light chain and an Fc molecule.

Claims

1. A heterodimeric polypeptide comprising an immunoglobulin heavy chain which is linked to a fusion protein, in a covalent or non-covalent interaction, said fusion protein comprising an immunoglobulin light chain and a Fc molecule, wherein said heavy chain and said fusion protein are oriented identically with respect to their N- and C-termini, and wherein said heterodimeric polypeptide is capable of specifically binding to a target receptor.

2. The heterodimeric polypeptide according to claim 1, wherein said heavy chain further comprises a tagging moiety.

3. The heterodimeric polypeptide according to claim 2, wherein said tagging moiety is selected from the group consisting of:

hexa-histidine tag, streptavidin-binding peptide, maltose-binding protein, glutathione S-transferase, mic-tag, and FLAG-tag.

4. The heterodimeric polypeptide according to claim 3, wherein said tagging moiety comprises a hexa-histidine tag.

5. The heterodimeric polypeptide according to claim 1, wherein said interaction is via a disulfide bond or via a dimerization domain.

6. The heterodimeric polypeptide according to claim 5, wherein said dimerization domain is located in the CH3 region of said heavy chain and in the CH3 region of said Fc of the fusion protein.

7. The heterodimeric polypeptide according to claim 1, wherein the Fc region of said heavy chain and/or the Fc molecule of said fusion protein is mutated so that the formation of the heterodimeric polypeptide is enhanced.

8. The heterodimeric polypeptide according to claim 1, wherein the hinge region in the Fc portion of said fusion protein is modified so that said heavy chain and said fusion protein are structurally symmetrical to each other.

9. The heterodimeric polypeptide according to claim 8, wherein said hinge region is modified by amino acid insertion, deletion, or substitution.

10. The heterodimeric polypeptide according to claim 1, wherein the sequence of said hinge region is either naturally occurring or non-naturally occurring.

11. The heterodimeric polypeptide according to claim 1, wherein either or both the hinge regions of the heavy chain and the fusion protein are modified.

12. The heterodimeric polypeptide according to claim 1, wherein said heavy chain and said fusion protein contains no cysteine residues that are unpaired.

13. The heterodimeric polypeptide according to claim 1, wherein said immunoglobulin is an IgG.

14. The heterodimeric polypeptide according to claim 13, wherein said IgG is selected from the group consisting of:

IgG1, IgG2, IgG3 and IgG4.

15. The heterodimeric polypeptide according to claim 1, which antagonizes the activity of said target receptor.

16. The heterodimeric polypeptide according to claim 15, wherein said target receptor is a receptor which is activated by the oligomerization of its subunits.

17. The heterodimeric polypeptide according to claim 15, wherein said oligomerization forms at least a dimer, a trimer, or a tetramer.

18. The heterodimeric polypeptide according to claim 15, wherein said oligomerization forms at least an oligomer comprising at least four subunits.

19. The heterodimeric polypeptide according to claim 15, wherein said receptor is a member selected from the group consisting of:

TNF/TNF receptor superfamily, cytokine receptors, receptor tyrosine kinases, G-protein coupled receptors (GPCRs), Fc receptors (FcRs), AT1 receptors, tissue factors, and integrins.

20. The heterodimeric polypeptide according to claim 19, wherein said receptor is a member of the TNF/TNF receptor superfamily.

21. The heterodimeric polypeptide according to claim 20, wherein said member is selected from the group consisting of:

TNFR1, TNFR2, NGFR, Troy, EDAR, XEDAR, CD40, DcR3, Fas, OX40, AITR, CD30, HveA, 4-1BB, DR3, CD27, LTβ

R, RANK, TWEK receptor, TACI, BCMA, DR6, OPG, DR4, DR5, DcR1 and DcR2.

22. The heterodimeric polypeptide according to claim 19, wherein said receptor is a receptor tyrosine kinase.

23. The heterodimeric polypeptide according to claim 22, wherein said receptor tyrosine kinase is selected from the group consisting of:

growth hormone receptor, erythropoietin receptor, VEGF receptor, FGF receptor, PFGF receptor and EGF receptor.

24. The heterodimeric polypeptide according to claim 23, wherein said EGF receptor is selected from the group consisting of:

ErbB1, ErbB2, ErbB3 and ErbB4.

25. The heterodimeric polypeptide according to claim 19, wherein said receptor is an interleukin receptor.

26. The heterodimeric polypeptide according to claim 25, wherein said interleukin receptor is a receptor of an interleukin selected from the group consisting of:

IL-1, IL-2, IL-4, IL-15, IL7, TSLP, LIF, IL-13, IL-23 and IL-31.

27. The heterodimeric polypeptide according to claim 1, wherein the amino acid sequences of said heavy chain and said fusion protein are fully human, humanized, or chimeric.

28. The heterodimeric polypeptide according to claim 15, which does not agonize the activity of said target receptor.

29. A composition comprising the heterodimeric polypeptide according to claim 1 and a pharmaceutically acceptable carrier.

48. A method of treating a subject having a condition mediated by a target receptor which is activated by the oligomerization of its subunits, comprising administering to said subject an effective amount of the heterodimeric polypeptide according to claim 1, wherein said heterodimeric polypeptide is capable of specifically binding to at least one subunit of said target receptor thereby antagonizing said oligomerization.

49. A method according to claim 48, wherein said oligomerization forms a dimer, a trimer, or a tetramer.

50. A method according to claim 48, wherein said target receptor is selected from the group consisting of:

TNF/TNF receptor superfamily, cytokine receptors, receptor tyrosine kinases, G-protein coupled receptors (GPCRs), Fc receptors (FcRs), AT1 receptors, tissue factors, and integrins.

51. A method according to claim 48, wherein said target receptor is a member of the TNF/TNF receptor superfamily.

52. A method according to claim 51, wherein said target receptor is selected from the group consisting of:

TNFR1, TNFR2, NGFR, Troy, EDAR, XEDAR, CD40, DcR3, Fas, OX40, AITR, CD30, HveA, 4-1BB, DR3, CD27, LTβ

R, RANK, TWEK receptor, TACI, BCMA, DR6, OPG, DR4, DR5, DcR1 and DcR2.

53. A method according to claim 48, wherein said target receptor is a receptor tyrosine kinase.

54. A method according to claim 53, wherein said receptor tyrosine kinase is selected from the group consisting of:

growth hormone receptor, erythropoietin receptor, VEGF receptor, FGF receptor, PFGF receptor and EGF receptor.

55. A method according to claim 54, wherein said EGF receptor is selected from the group consisting of:

ErbB1, ErbB2, ErbB3 and ErbB4.

56. A method according to claim 48, wherein said target receptor is an interleukin receptor.

57. A method according to claim 48, wherein said target receptor, wherein said interleukin receptor is a receptor of an interleukin selected from the group consisting of:

IL-1, IL-2, IL-4, IL-15, IL-7, TSLP, LIF, IL-13, IL-23 and IL-31.

30. A heterodimeric polypeptide comprising a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:

9 and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO;

10.

31. A heterodimeric polypeptide comprising

(a) an immunoglobulin heavy chain comprising a CDR of at least 90% identical to an amino acid sequence selected from the group consisting of:

SEQ ID NOS;

11, 12 and 13, and

(b) a fusion protein comprising an immunoglobulin light chain, a hinge region and a Fc, wherein said light chain comprises a CDR of at least 90% identical to an amino acid sequence selected from the group consisting of;

SEQ ID NOS;

18, 19 and 20,

wherein said heterodimeric polypeptide is capable of binding to and inhibiting the activity of TNFR1.

32. A polypeptide comprising an immunoglobulin light chain which is fused to an Fc molecule.

33. A polypeptide according to claim 32, wherein said light chain is fused to said Fc molecule via a hinge region.

34. A heterodimeric polypeptide comprising the polypeptide according to claim 32 which is linked to an immunoglobulin heavy chain in a covalent or non-covalent interaction.

35. A vector comprising a first nucleic acid encoding an immunoglobulin heavy chain and a second nucleic acid encoding a fusion protein, said fusion protein comprising an immunoglobulin light chain, a hinge region, and an Fc molecule, wherein said first nucleic acid and said second nucleic acid are inserted in said vector at the same or different sites.

36. The vector according to claim 35, wherein said first nucleic acid encodes an immunoglobulin heavy chain which is fused to a tagging moiety.

37. A host cell transformed with the vector according to claim 35 or 36.

38. A transformed or transfected host cell comprising at least two vectors, at least one of said vectors comprising a nucleic acid sequence encoding at least an immunoglobulin heavy chain and at least another one of said vectors comprising a nucleic acid sequence encoding at least a fusion protein comprising an immunoglobulin light chain, a hinge region and an Fc molecule.

39. A process for producing in a host cell a heterodimeric polypeptide, said heterodimeric polypeptide comprising an immunoglobulin heavy chain which is optionally fused to a tagging moiety, said heavy chain is linked to a fusion protein comprising an immunoglobulin light chain, a hinge region, and an Fc molecule, wherein said heavy chain and said fusion protein are oriented identically with respect to their N- and C-termini, and wherein said heterodimeric polypeptide is capable of specifically binding to a target receptor, said process comprising the steps of:

(a) transforming or transfecting said host cell with a first nucleic acid encoding said immunoglobulin heavy chain and a second nucleic acid encoding said fusion protein, and

(b) expressing said first nucleic acid and said second nucleic acid so that said immunoglobulin heavy chain and said fusion protein are produced separately in said transformed host cell.

40. A process according to claim 39, wherein said heavy chain and said fusion protein are expressed in said host cell and secreted therefrom as a heterodimeric polypeptide which is capable of binding to a target receptor.

41. The process according to claim 39, wherein said first and second nucleic acids are present in a single vector.

42. The process according to claim 39, wherein said first and second nucleic acids are present in different vectors.

43. The process according to claim 39, wherein said host cell is prokaryotic or eukaryotic.

44. The process according to claim 39, wherein said host cell is a mammalian cell.

45. The process according to claim 44, wherein said mammalian cell is selected from the group consisting of:

CHO, VERO, BHK, HeLa, Cos, MDCK, 293, 3T3, a myeloma cell line, and WI38 cells.

46. A process according to claim 39, wherein said heavy chain and said fusion protein are produced in insoluble form and are solubilized and allowed to refold in solution to form a heterodimeric polypeptide which is capable of specifically binding to a target receptor.

47. A nucleic acid encoding a fusion protein comprising an immunoglobulin light chain which is fused to an Fc molecule.