Recombinant Polypeptides of the Members of the Tnf Ligand Family and Use Thereof

• US 20070286843A1
• Filed: 03/24/2005
• Published: 12/13/2007
• Est. Priority Date: 03/26/2004
• Status: Active Grant

• Current Assignee: University of Stuttgart
• Original Assignee: Univeristat Stuttgart
• Inventors: Grunwald, Ingo, Pfizenmaier, Klaus, Scheurich, Peter, Krippner-Heidenreich, Anja
• Granted Patent: US 8,927,205 B2
• Time in Patent Office: Days
• US Class Current: 424/93.100
• 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 45/06 Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• 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 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: C07K 14/525 Tumour necrosis factor [TNF]
• CPC Class Codes: C07K 14/70575 NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154 NGF C07K14/48, TNF C07K14/525
• CPC Class Codes: C07K 16/40 against enzymes
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: G01N 2333/525 Tumor necrosis factor [TNF]
• CPC Class Codes: G01N 33/53 Immunoassay; Biospecific binding assay; Materials therefor
• CPC Class Codes: G01N 33/543 with an insoluble carrier for immobilising immunochemicals
• CPC Class Codes: G01N 33/544 the carrier being organic
• CPC Class Codes: G01N 33/6863 Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
• CPC Class Codes: Y10S 530/811 : Peptides or proteins is immobilized on, or in, an inorganic carrier
• CPC Class Codes: Y10S 530/812 : Peptides or proteins is immobilized on, or in, an organic carrier

Abstract

The invention relates to polypeptides comprising, as constituent A, at least three monomers of a member of the TNF ligand family and, as constituent B, at least two peptide linkers that link the monomers of the member of the TNF ligand family to one another. The invention also relates to the use of these polypeptides for treating diseases and for producing a medicament or a vaccine. The invention also relates to methods for producing and isolating these polypeptides, to nucleic acids that code for these polypeptides, to vectors containing these nucleic acids, to host cells transfected with these vectors, and to pharmaceutical compositions containing these inventive objects. Finally, the invention relates to methods for the extracorporeal manipulation, depletion and/or removal of components contained in body fluids, e.g. by means of apheresis.

Claims

1. Polypeptide comprising at least three components A and at least two components B, wherein each component A is a monomer of a member of the TNF ligand family or a functional fragment, and/or a functional variant thereof, and each component B is a peptide linker.

2. Polypeptide according to claim 1, wherein components A are identical or different.

3. Polypeptide according to any one of the preceding claims, wherein components A stem from the same organism or different organisms.

4. Polypeptide according to any one of the preceding claims, wherein components A are selected from the group, consisting of FasL, TRAIL, TNF, CD30L, CD40L, OX40L, RANKL, TWEAKL, LTalpha, LTheta, LIGHT, CD27L, 41-BB, 41BBL, GITRL, APRIL, EDA, VEGI, and BAFF.

5. Polypeptide according to any one of the preceding claims, wherein components B each link together two of the at least three components A.

6. Polypeptide according to any one of the preceding claims, wherein at least one of components B has the amino acid sequence (GGGS)₃ or (GGGS)₄.

7. Polypeptide according to any one of the preceding claims, wherein components A and components B form a trimeric protein structure.

8. Polypeptide according to claim 7, wherein components A and components B form a homotrimeric protein structure.

9. Polypeptide according to claim 7, wherein components A and components B form a heterotrimeric protein structure.

10. Polypeptide according to any one of the preceding claims, wherein components B are identical or different.

11. Polypeptide according to any one of the preceding claims, wherein components B stem from the same organism or different organisms.

12. Polypeptide according to any one of the preceding claims, wherein the polypeptide has a preferably N-terminal tag sequence, particularly a His tag sequence or a Flag tag sequence.

13. Polypeptide according to any one of the preceding claims, wherein the polypeptide has a preferably N-terminal leader peptide sequence.

14. Polypeptide according to any one of the preceding claims, wherein the polypeptide has at least one other component C, which is an antibody fragment or a different protein or peptide, which selectively recognizes a specific target molecule on the cell surface.

15. Polypeptide according to claim 14, wherein component C is an antibody fragment from a mammal, particularly of murine or human origin, or a humanized antibody fragment.

16. Polypeptide according to claim 14 or 15, wherein the antibody fragment can be present in different antibody formats, e.g., as scFv, particularly scFv40.

17. Polypeptide according to claim 14, wherein component C is a protein or peptide with specificity for a cell surface molecule, particularly a cytokine receptor, a growth factor receptor, an integrin, or cell adhesion molecule.

18. Polypeptide according to claim 17, wherein the cytokine receptor is selected from the group of the TNFR gene family.

19. Nucleic acid coding for a polypeptide according to any one of the preceding claims 1 through 18.

20. Vector containing the nucleic acid according to claim 19.

21. Host cell containing the nucleic acid according to claim 19 and/or the vector according to claim 20.

22. Method for preparing a host cell according to claim 21, comprising the following steps:

a. Preparation of a nucleic acid according to claim 19 or a vector according to claim 20, and b. Introduction of the nucleic acid and/or vector according to step (a) into a cell.

23. Method for preparing a polypeptide according to any one of claims 1 through 18, comprising the following steps:

a. Culturing of a host cell according to claim 21 under suitable conditions,

b. Expression of the nucleic acid according to claim 19 under suitable conditions, and

c. Isolation of the polypeptide from the host cell and/or the culture supernatant.

24. Use of a polypeptide according to any one of claims 1 through 18, a nucleic acid according to claim 20 [sic], a vector according to claim 20, or a host cell according to claim 21 for the preparation of a medication for the treatment of cancer diseases, particularly solid or lymphatic tumors, infectious diseases, metabolic diseases, inflammatory conditions, hyperproliferative diseases, autoimmune diseases, particularly rheumatoid/arthritic diseases, toxic epidermal necrolysis (TEN), multiple sclerosis, Hashimoto'"'"'s thyroiditis, GVHD, viral hepatitis (HBV, HCV), alcohol-induced hepatitis, rejection reactions in liver transplantation, diseases based on hyperapoptotic reactions, and degenerative diseases, particularly neurodegenerative diseases.

25. Use of a polypeptide according to any one of claims 1 through 18, a nucleic acid according to claim 19, a vector according to claim 20, or a host cell according to claim 21 for the treatment of cancer diseases, particularly solid or lymphatic tumors, infectious diseases, metabolic diseases, inflammatory conditions, hyperproliferative diseases, autoimmune diseases, particularly rheumatoid/arthritic diseases, toxic epidermal necrolysis (TEN), multiple sclerosis, Hashimoto'"'"'s thyroiditis, GVHD, viral hepatitis (HBV, HCV), alcohol-induced hepatitis, rejection reactions in liver transplantation, diseases based on hyperapoptotic reactions, and degenerative diseases, particularly neurodegenerative diseases.

26. Pharmaceutical composition, at least containing a polypeptide according to any one of claims 1 through 18 and/or a nucleic acid according to claim 19 and/or a vector according to claim 20 and/or a host cell according to claim 21, as well as pharmaceutically acceptable aids, additives, and/or carrier substances.

27. Pharmaceutical composition according to claim 26 for the treatment of cancer diseases, particularly solid or lymphatic tumors, infectious diseases, metabolic diseases, inflammatory conditions, hyperproliferative diseases, autoimmune diseases, particularly rheumatoid/arthritic diseases, toxic epidermal necrolysis (TEN), multiple sclerosis, Hashimoto'"'"'s thyroiditis, GVHD, viral hepatitis (HBV, HCV), alcohol-induced hepatitis, rejection reactions in liver transplantation, diseases based on hyperapoptotic reactions, and degenerative diseases, particularly neurodegenerative diseases.

28. Method for extracorporeal manipulation, depletion, and/or removal of soluble, suspended components or cellular blood components comprising the following steps:

a) Optionally separation of the blood into one or more fractions with solid and/or liquid components;

b) Binding of soluble, suspended, or cellular blood components to a surface or particle coupled to a polypeptide according to any one of claims 1 through 18; and

c) Optionally separation of the bound soluble, suspended, or cellular blood components.

29. Method according to claim 28, wherein before step a) or b) blood is taken from a patient.

30. Method according to claim 28, wherein after a step b) or c), the thus treated blood or blood fraction is reinjected into a patient.