Modified binding molecules comprising connecting peptides

• US 20050163782A1
• Filed: 06/28/2004
• Published: 07/28/2005
• Est. Priority Date: 06/27/2003
• Status: Abandoned Application

• Current Assignee: Biogen Idec MA Inc. (Biogen, Inc.)
• Original Assignee: Biogen Idec MA Inc. (Biogen, Inc.)
• Inventors: Glaser, Scott, Chinn, Paul, Wu, Xiufeng, Yang, Tzung-Horng, Reff, Mitchell
• Application Number:
  US10/880,028
• Publication Number:
  US 20050163782A1
• Time in Patent Office: Days
• US Class Current: 424/155.100
• CPC Class Codes: A61K 2039/505 comprising antibodies
• 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 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 11/02 Nasal agents, e.g. decongestants
• CPC Class Codes: A61P 11/06 Antiasthmatics
• CPC Class Codes: A61P 13/02 of urine or of the urinary tract, e.g. urine acidifiers
• CPC Class Codes: A61P 13/12 of the kidneys
• 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 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 17/04 Antipruritics
• CPC Class Codes: A61P 17/06 Antipsoriatics
• CPC Class Codes: A61P 17/14 for baldness or alopecia
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• 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 25/08 Antiepileptics; Anticonvulsants
• CPC Class Codes: A61P 27/02 : Ophthalmic agents
• 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 3/00 : Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 3/04 : Anorexiants; Antiobesity agents
• CPC Class Codes: A61P 3/10 : for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 31/04 : Antibacterial agents
• CPC Class Codes: A61P 31/10 : Antimycotics
• CPC Class Codes: A61P 31/12 : Antivirals
• CPC Class Codes: A61P 35/00 : Antineoplastic agents
• CPC Class Codes: A61P 35/02 : specific for leukemia
• CPC Class Codes: A61P 35/04 : specific for metastasis
• CPC Class Codes: A61P 37/00 : Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 37/02 : Immunomodulators
• CPC Class Codes: A61P 37/06 : Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 37/08 : Antiallergic agents antiasthmatic agents A61P11/06; ophthalmic antiallergics A61P27/14
• 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 7/00 : Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 7/02 : Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• CPC Class Codes: A61P 7/04 : Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• CPC Class Codes: A61P 7/06 : Antianaemics
• CPC Class Codes: A61P 9/00 : Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/10 : for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: A61P 9/14 : Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
• 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 16/18 : against material from animals or humans
• CPC Class Codes: C07K 16/2851 : against the lectin superfamily, e.g. CD23, CD72
• CPC Class Codes: C07K 16/2896 : against molecules with a "CD"-designation, not provided for elsewhere
• 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 2317/52 : Constant or Fc region; Isotype
• CPC Class Codes: C07K 2317/53 : Hinge
• CPC Class Codes: C07K 2317/622 : Single chain antibody (scFv)
• CPC Class Codes: C07K 2317/624 : Disulfide-stabilized antibody (dsFv)
• CPC Class Codes: C07K 2319/00 : Fusion polypeptide

Abstract

The instant invention describes methods of separating or preferentially synthesizing dimers which are linked via at least one interchain disulfide linkage from dimers which are not linked via at least one interchain disulfide linkage from a mixture comprising the two types of polypeptide dimers. These forms can be separated from each other using hydrophobic interaction chromatography. In addition, the invention pertains to connecting peptides that result in the preferential biosynthesis of dimers that are linked via at least one interchain disulfide linkage or that are not linked via at least one interchain disulfide linkage. The invention also pertains to compositions in which a majority of the dimers are linked via at least one interchain disulfide linkage or are not linked via at least one interchain disulfide linkage. The invention still further pertains to novel binding molecules, e.g., comprising connecting peptides of the invention.

Claims

1. A composition comprising polypeptide dimers comprising at least four binding sites and at least two polypeptide chains, wherein said at least two polypeptide chains comprise at least one heavy chain portion and a synthetic connecting peptide, and wherein greater than about 50% of the dimers comprise polypeptide chains that are linked via at least one interchain disulfide linkage.

2. The composition of claim 1 wherein greater than about 90% of the dimers are linked via at least one interchain disulfide linkage.

3. The composition of claim 1 wherein at least one of the polypeptide chains comprises a CH3 domain linked to a VL, VH or CH1 domain via the connecting peptide.

4. The composition of claim 1, wherein the polypeptide chains lack all or part of a CH2 domain.

5. The composition of claim 1, wherein the dimers are linked via two or more interchain disulfide linkages.

6. The composition of claim 1, wherein the heavy chain portion is derived from an antibody of an isotype selected from the group consisting of:

IgG1, IgG2, IgG3, and IgG4.

7. The composition of claim 6, wherein the heavy chain portion comprises an amino acid sequence derived from a hinge region selected from the group consisting of:

a γ

1 hinge, a γ

2 hinge a γ

3 hinge, and a γ

4 hinge.

8. The composition of claim 1, wherein the molecules are bispecific.

9. The composition of claim 1, wherein the molecules comprise at least one binding site specific for a soluble ligand.

10. The composition of claim 1, wherein the molecules comprise at least one binding site specific for a cell surface molecule.

11. The composition of claim 1, wherein the molecules comprise two binding sites specific for a tumor cell antigen and two binding sites specific for a prodrug.

12. The composition of claim 11, wherein the binding sites specific for the prodrug are catalytic.

13. The composition of claim 1, wherein the synthetic connecting peptide comprises a proline residue at position 243, Kabat numbering system.

14. The composition of claim 13, wherein the synthetic connecting peptide further comprises an alanine residue at position 244 and a proline residue at position 245, Kabat numbering system.

15. The composition of claim 1, wherein the heavy chain portion comprises a chimeric hinge.

16. The composition of claim 1, wherein the synthetic connecting peptide comprises at least a portion of an IgG1 hinge domain, at least a portion of an IgG3 hinge domain.

17. The composition of claim 1, wherein the connecting peptide comprises an amino acid sequence selected from the group consisting of:

SEQ ID NOs;

8-15 and 48.

18. A method of treating a subject that would benefit from treatment with an antigen binding molecule comprising administering a composition of claim 1 to the subject such that treatment occurs.

19. The method of claim 18, wherein the subject is suffering from cancer.

20. The method of claim 18, wherein the subject is suffering from lymphoma.

21. The method of claim 18, wherein the subject is suffering from an autoimmune disease or disorder.

22. The method of claim 18, wherein the subject is suffering from an inflammatory disease or disorder.

23. A nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide chain of claim 1.

24. The composition of claim 1, wherein the polypeptide dimers comprise four polypeptide chains and wherein two of the polypeptide chains comprise at least one heavy chain portion and a synthetic connecting peptide.

32. The nucleic acid molecule of any one of claims 24-30 which is in a vector.

33. A host cell comprising the vector of claim 32.

41. The composition of claim 1, wherein said binding sites are individually selected from the group consisting of:

an antigen binding site, a ligand binding portion of a receptor, and a receptor binding portion of a ligand.

42. The method of claim 41, wherein the polypeptide chains have at least one binding site derived from an antibody selected from the group consisting of:

2B8, Lym 1, Lym 2, LL2, Her2, B1, MB1, BH3, B4, B72.3, CC49, 5E8, B3F6, and 5E10.

43. The composition of claim 1, wherein the polypeptide dimers are tetravalent minibody molecules.

44. The composition of claim 1, wherein the polypeptide dimers are tetravalent domain deleted antibody molecules.

45. The composition of claim 1, wherein the polypeptide dimers are diabodies.

58. A method of treating a subject that would benefit from treatment with an antigen binding molecule comprising administering a composition of claim 45 to the subject such that treatment occurs.

25. A nucleic acid molecule comprising a nucleotide sequence shown in FIG. 8B (SEQ ID NO:

17)

26. A nucleic acid molecule comprising a nucleotide sequence shown in FIG. 8C (SEQ ID NO:

18)

27. A nucleic acid molecule comprising a nucleotide sequence shown in FIG. 10B (SEQ ID NO:

23)

28. A nucleic acid molecule comprising a nucleotide sequence shown in FIG. 12A (SEQ ID NO:

26).

29. A nucleic acid molecule comprising a nucleotide sequence shown in FIG. 12B (SEQ ID NO:

27).

30. A nucleic acid molecule comprising a nucleotide sequence shown in FIG. 14 (SEQ ID NO:

30).

31. A nucleic acid molecule comprising a nucleotide sequence shown in FIG. 15 (SEQ ID NO:

31).

34. A binding molecule comprising the amino acid sequence of FIG. 9B (SEQ ID NO:

20).

35. A binding molecule comprising the amino acid sequence of FIG. 9C (SEQ ID NO:

21).

36. A binding molecule comprising the amino acid sequence of FIG. 11B (SEQ ID NO:

25).

37. A binding molecule comprising the amino acid sequence of FIG. 13A (SEQ ID NO:

28).

38. A binding molecule comprising the amino acid sequence of FIG. 13B (SEQ ID NO:

29).

39. An binding molecule comprising the amino acid sequence of FIG. 16 (SEQ ID NO:

32).

40. A binding molecule comprising the amino acid sequence of FIG. 17 (SEQ ID NO:

33).

46. A composition comprising minibody molecules comprising two polypeptide chains, wherein the polypeptide chains comprise a heavy chain portion and a synthetic connecting peptide, wherein the polypeptide chains lack all or part of a CH2 domain, and wherein greater than about 50% of the molecules are present in a form in which one of the polypeptide chains are linked via at least one interchain disulfide linkage.

47. The composition of claim 46, wherein greater than about 90% of the dimers are linked via at least one interchain disulfide linkage.

48. The composition of claim 46 wherein at least one of the polypeptide chains comprises a CH3 domain genetically fused to a VL, VH or CH1 domain via the connecting peptide.

49. The composition of claim 46, wherein the polypeptide chains lack the entire CH2 domain.

50. The composition of claim 46, wherein the dimers are linked via two or more interchain disulfide linkages.

51. The composition of claim 46, wherein a heavy chain portion is derived from an antibody of an isotype selected from the group consisting of:

IgG1, IgG2, IgG3, and IgG4.

52. The composition of claim 46, wherein the heavy chain portion comprises an amino acid sequence is derived from a hinge region selected from the group consisting of:

a γ

1 hinge, a γ

2 hinge a γ

3 hinge, and a γ

4 hinge.

53. The composition of claim 46, wherein said binding sites are individually selected from the group consisting of:

an antigen binding site, a ligand binding portion of a receptor, and a receptor binding portion of a ligand.

54. The composition of claim 46, wherein the molecules are bispecific.

55. The composition of claim 46, wherein the connecting peptide comprises a proline residue at position 243, Kabat numbering system.

56. The composition of claim 46, wherein the synthetic connecting peptide comprises a chimeric hinge.

57. The composition of claim 46, wherein the synthetic connecting peptide comprises at least a portion of an IgG1 hinge domain, at least a portion of an IgG3 hinge domain.

59. A nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide chain of claim 46.

60. A composition comprising polypeptide dimers having at least four binding sites and at least two polypeptide chains, wherein said at least two polypeptide chains comprise at least one heavy chain portion and lack all or part of a CH2 domain, wherein greater than 50% of the polypeptide dimers comprise polypeptide chains that are linked via at least one interchain disulfide linkage.