Methods for chemically synthesizing immunoglobulin chimeric proteins

• US 20050027109A1
• Filed: 05/06/2004
• Published: 02/03/2005
• Est. Priority Date: 05/06/2003
• Status: Active Grant

• Current Assignee: Bioverativ Therapeutics Inc. (Sanofi )
• Original Assignee: Syntonix Pharmaceuticals, Inc.
• Inventors: Peters, Robert T., Mezo, Adam R.
• Granted Patent: US 7,381,408 B2
• Time in Patent Office: Days
• US Class Current: 530/391.100
• 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: A61K 47/60 the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• CPC Class Codes: A61K 47/642 the peptide or protein in the drug conjugate being a cytokine, e.g. IL2, chemokine, growth factors or interferons being the inactive part of the conjugate
• CPC Class Codes: A61K 47/68 the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
• CPC Class Codes: A61K 47/6803 Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
• CPC Class Codes: A61K 47/6811 the drug being a protein or peptide, e.g. transferrin or bleomycin
• CPC Class Codes: A61K 47/6813 the drug being a peptidic cytokine, e.g. an interleukin or interferon
• CPC Class Codes: A61K 47/6835 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• 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: C07K 14/475 Growth factors; Growth regulators
• CPC Class Codes: C07K 14/505 Erythropoietin [EPO]
• CPC Class Codes: C07K 14/555 Interferons [IFN]
• CPC Class Codes: C07K 14/56 IFN-alpha
• CPC Class Codes: C07K 14/565 : IFN-beta
• CPC Class Codes: C07K 14/59 : Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
• CPC Class Codes: C07K 14/61 : Growth hormone [GH], i.e. somatotropin
• CPC Class Codes: C07K 14/70503 : Immunoglobulin superfamily
• CPC Class Codes: C07K 14/745 : Blood coagulation or fibrinolysis factors
• CPC Class Codes: C07K 14/755 : Factors VIII , e.g. factor VIII C (AHF), factor VIII Ag (VWF)
• 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 2317/52 : Constant or Fc region; Isotype
• 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
• CPC Class Codes: C12N 9/6437 : Coagulation factor VIIa (3.4.21.21)
• CPC Class Codes: C12N 9/644 : Coagulation factor IXa (3.4.21.22)
• CPC Class Codes: C12N 9/647 : Blood coagulation factors not provided for in a preceding group or according to more than one of the proceeding groups
• CPC Class Codes: C12N 9/96 : Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• CPC Class Codes: C12Y 304/21021 : Coagulation factor VIIa (3.4.21.21)
• CPC Class Codes: C12Y 304/21022 : Coagulation factor IXa (3.4.21.22)

Abstract

The invention provides methods of chemically synthesizing chimeric proteins comprising at least a portion of an immunoglobulin constant region and a biologically active molecule.

Claims

1. A method of producing a chimeric protein comprising combining at least one biologically active molecule and at least a portion of an immunoglobulin constant region, wherein

a) the portion of an immunoglobulin constant region comprises an N terminus cysteine and

b) the biologically active molecule comprises a functional group capable of reacting with an N terminus cysteine to form a bond.

2. The method of claim 1, wherein the chimeric protein is chosen from a dimer and a monomer-dimer hybrid.

3. The method of claim 2, wherein the monomer-dimer hybrid is a dimerically linked monomer-dimer hybrid.

4. The method of claim 1, wherein the at least one biologically active molecule is chosen from a polypeptide, a nucleic acid molecule, a small organic molecule, a small inorganic molecule.

5. The method of claim 1, wherein the biologically active molecule is a polypeptide.

6. The method of claim 1, wherein the biologically active molecule is a nucleic acid molecule.

7. The method of claim 1, wherein the functional group is thioester.

8. The methods of claim 7, wherein the biologically active molecule is a polypeptide comprising an C terminal thioester.

9. The method of claim 1, wherein the functional group is an aldehyde.

10. The method of claim 1, wherein both the biologically active molecule and the portion of an immunoglobulin are produced recombinantly.

11. The method of claim 1, wherein at least one of the biologically active molecule and the portion of an immunoglobulin is produced by chemical synthesis.

12. The method of claim 1, wherein the portion of an immunoglobulin is an FcRn binding partner.

13. The method of claim 12, the FcRn binding partner is an Fc or a fragment thereof.

14. The method of claim 1, wherein the chimeric protein further comprises a linker.

15. The method of claim 1, wherein the reaction is performed in solution.

16. The method of claim 1, wherein the reaction is performed on a solid support.

17. The method of claim 1, wherein the biologically active molecule is chosen from EPO, interferon, a viral fusion inhibitor, and a clotting factor.

18. A method of synthesizing a chimeric protein comprising

a) recombinantly expressing a fusion protein comprising at least a portion of an immunoglobulin constant region and a splicing protein capable of forming a C terminus thioester on the portion of an immunoglobulin constant region;

b) adding a thiol cofactor to the fusion protein of a);

c) adding at least one biologically active molecule having an N terminal cysteine, thereby synthesizing the chimeric protein.

19. The method of claim 18, wherein the splicing protein is intein.

20. The method of claim 18, wherein the thiol cofactor is MESNA.

21. The method of claim 18, wherein the chimeric protein is chosen from a dimer and a monomer-dimer hybrid.

22. The method of claim 21, wherein the monomer-dimer hybrid is a dimerically linked monomer-dimer hybrid.

23. The method of claim 18, wherein the at least one biologically active molecule is chosen from a polypeptide, a nucleic acid molecule, a small organic molecule, a small inorganic molecule.

24. The method of claim 18, wherein the biologically active molecule is a polypeptide.

25. The method of claim 18, wherein the biologically active molecule is a nucleic acid molecule.

26. The method of claim 18, wherein both the biologically active molecule and the portion of an immunoglobulin are produced recombinantly.

27. The method of claim 18, wherein at least one of the biologically active molecule and the portion of an immunoglobulin is produced by chemical synthesis.

28. The method of claim 18, wherein the portion of an immunoglobulin is an FcRn binding partner.

29. The method of claim 28, the FcRn binding partner is an Fc or a fragment thereof.

30. The method of claim 18, wherein the chimeric protein further comprises a linker.

31. The method of claim 18, wherein the reaction is performed in solution.

32. The method of claim 18, wherein the reaction is performed on a solid support.

33. The method of claim 32, wherein the solid support is a chitin bead.

34. The method of claim 18, wherein the biologically active molecule is chosen from EPO, interferon, a viral fusion inhibitor, and a clotting factor.