TNF SUPERFAMILY COLLECTIN FUSION PROTEINS

• US 20100199364A1
• Filed: 07/10/2008
• Published: 08/05/2010
• Est. Priority Date: 07/10/2007
• Status: Active Grant

• Current Assignee: Apogenix AG
• Original Assignee: Apogenix Gmbh
• Inventors: Branschädel, Marcus, Hill, Oliver, Thiemann, Meinolf, Gieffers, Christian
• Granted Patent: US 8,383,774 B2
• Time in Patent Office: Days
• US Class Current: 800/13
• 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 37/02 Immunomodulators
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 14/42 Lectins, e.g. concanavalin, phytohaemagglutinin
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C07K 14/4726 Lectins
• 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 19/00 Hybrid peptides , i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C07K 2319/21 containing a His-tag
• CPC Class Codes: C07K 2319/22 containing a Strep-tag
• CPC Class Codes: C07K 2319/32 fusions with soluble part of a cell surface receptor, "decoy receptors"
• CPC Class Codes: C07K 2319/70 : containing domain for protein-protein interaction
• CPC Class Codes: C12N 15/62 : DNA sequences coding for fusion proteins

Abstract

The present invention refers to a fusion protein comprising a TNF-superfamily (TNFSF) cytokine or a receptor binding domain thereof fused to a collectin trimerization domain, to a nucleic acid molecule encoding the fusion protein, and to a cell comprising the nucleic acid molecule. The fusion protein is present as a trimeric complex or as an oligomer thereof. The fusion protein, the nucleic acid, and the cell is suitable as pharmaceutical composition or for therapeutic, diagnostic and/or research applications.

Claims

1. A fusion protein comprising

(i) a TNF-superfamily cytokine or a receptor binding domain thereof, and

(ii) a collectin trimerization domain.

2. The fusion protein of claim 1 additionally comprising a flexible linker element between (i) and (ii).

3. The fusion of claim 2 wherein the flexible linker element has a length of 3-20 amino acids.

4. The fusion protein of claim 2 wherein the flexible linker element is a glycine/serine linker.

5. The fusion protein of claim 4 wherein the flexible linker element has the amino acid sequence (GSS)_a(SSG)_b(GSG)_c wherein a, b, c is each 0, 1, 2, 3, 4, 5 or 6.

6. The fusion protein of claim 2, wherein the flexible linker element has a length of 9-15 amino acids.

7. The fusion protein of claim 1 wherein (i) is selected from LTA (SEQ ID NO:

1), TNFα

(SEQ ID NO;

2), LTB (SEQ ID NO;

3), OX40L (SEQ ID NO;

4), CD40L (SEQ ID NO;

5), CD95L (SEQ ID NO;

6), CD27L (SEQ ID NO;

7), CD30L (SEQ ID NO;

8), CD137L (SEQ ID NO;

9), TRAIL (SEQ ID NO;

10), RANKL (SEQ ID NO;

11), TWEAK (SEQ ID NO;

12), APRIL 1 (SEQ ID NO;

13), APRIL 2 (SEQ ID NO;

14), BAFF (SEQ ID NO;

15), LIGHT (SEQ ID NO;

16), TL1A (SEQ ID NO;

17), GITRL (SEQ ID NO;

18), EDA-A1 (SEQ ID NO;

19), EDA-A2 (SEQ ID NO;

20), or a receptor binding domain thereof.

8. The fusion protein of claim 1 wherein (i) comprises amino acids 59-205 or 60-205 of LTA (SEQ ID NO:

1), 86-233 of TNFα

(SEQ ID NO;

2), 82-244 or 86-244 of LTB (SEQ ID NO;

3), 52-183 or 55-183 of OX40L (SEQ ID NO;

4), 112-261 or 117-261 of CD40L (SEQ ID NO;

5), 51-193 or 56-193 of CD27L (SEQ ID NO;

7), 97-234, 98-234 or 102-234 of CD30L (SEQ ID NO;

8), 86-254 of CD137L (SEQ ID NO;

9), 161-317 of RANKL (SEQ ID NO;

11), 103-249, 104-249 or 105-249 of TWEAK (SEQ ID NO;

12), 111-247 or 112-247 of APRIL 1 (SEQ ID NO;

13), 111-247 or 112-250 of APRIL 2 (SEQ ID NO;

14), 140-285 of BAFF (SEQ ID NO;

15), 91-240 of LIGHT (SEQ ID NO;

16), 91-251 or 93-251 of TL 1 A (SEQ ID NO;

17), 52-177 of GITRL (SEQ ID NO;

18), 245-391 of EDA-A1 (SEQ ID NO;

19), 245-389 of EDA-A2 (SEQ ID NO;

20).

9. The fusion protein of claim 1 wherein (i) is CD95L or a receptor binding domain thereof, wherein (i) preferably comprises amino acids 142-281 or 144-281 of human CD95L (SEQ ID NO:

6).

10. The fusion protein of claim 1 wherein (i) is TRAIL or a receptor binding domain thereof, wherein (i) preferably comprises amino acids 95-281, 116-281, 117-281, 118-281, 119-281 or 120-281 of human TRAIL (SEQ ID NO:

10).

11. The fusion protein of claim 1 wherein (i) comprises a mutant of TRAIL or of a receptor binding domain thereof which binds and/or activates TRAILR1 and/or TRAILR2.

12. The fusion protein of claim 1 wherein (i) comprises at least one amino acid substitution.

13. The fusion protein of claim 11 wherein the amino acid substitution affects at least one of the following amino acid positions of human TRAIL (SEQ ID NO:

10);

R130, G160, Y189, R191, Q193, E195, N199, K201, Y213, T214, 5215, H264, I266, D267, D269, and wherein the amino acid substitution is preferably at least one of the following;

R130E, G160M, Y189A, Y189Q, R191K, Q193S, Q193R, E195R, N199V, N199R, K201R, Y213W, T214R, S215D, H264R, I266L, D267Q, D269H, D269R, or D269K.

14. The fusion protein of claim 1 wherein (ii) is surfactant protein-D, surfactant protein-A, mannan-binding protein-A, mannan-binding-protein-C, collectin liver 1, collectin placenta 1, or collectin-11.

15. The fusion protein of claim 1 wherein (ii) comprises amino acids 217-375, 218-375, 219-375, 220-375, 221-375, 222-375, 223-375, 224-375, 225-375 of human surfactant protein-D of SEQ ID NO:

21.

16. The fusion protein of claim 1 wherein (ii) comprises amino acids 217-257, 218-257, 219-257, 220-257, 221-257, 222-257, 223-257, 224-257, or 225-257 of human surfactant protein-D of SEQ ID NO:

21.

17. The fusion protein of claim 1 wherein (ii) comprises at least one amino acid substitution.

18. The fusion protein of claim 17 wherein the amino acid substitution affects amino acid position F355 of human surfactant protein-D of SEQ ID NO:

21, and wherein the amino acid substitution is one of the following;

F355A, F355S, F355T, F355E, F355D, F355K, or F355R.

19. The fusion protein of claim 1 wherein (ii) comprises a mutant which does not bind to mannose.

20. The fusion protein of claim 1 wherein (ii) comprises amino acids 110-271, 116-271, or 121-271 of human collectin-11 of SEQ ID NO:

22.

21. The fusion protein of claim 1 wherein (ii) comprises amino acids 110-147, 110-148, 110-149, 110-150, 110-151, 116-147, 116-148, 116-149, 116-150, 116-151, 121-147, 121-148, 121-149, 121-150, or 121-151 of human collectin-11 of SEQ ID NO:

22.

22. The fusion protein of claim 1 wherein (ii) is located C-terminally of (i).

23. The fusion protein of claim 1 wherein (ii) is located N-terminally of (i).

24. The fusion protein of claim 1 which additionally comprises an N-terminal signal peptide domain, which may comprise a protease cleavage site, wherein the N-terminal signal peptide domain preferably comprises the sequence SEQ ID NO:

23, SEQ ID NO;

24, or SEQ ID NO;

25.

25. The fusion protein of claim 1 which comprises the sequence of SEQ ID NO:

26 SEQ ID NO;

36, SEQ ID NO;

37, SEQ ID NO;

40-41 or SEQ ID NO;

43-51.

26. The fusion protein of claim 1 wherein the fusion protein further comprises a recognition/purification domain, which is preferably located at the N-terminus or at the C-terminus.

27. The fusion protein of claim 26 wherein the recognition/purification domain is a Strep-tag or a poly His-domain.

28. The fusion protein of claim 1 which additionally comprises a terminal flexible element which may include and/or connect to the recognition/purification domain.

29. The fusion protein of claim 1 which is present as a trimeric complex or as an oligomer of the trimeric complex.

30. The fusion protein of claim 29 wherein the complex is formed by covalent linkage between three fusion proteins, and wherein the covalent linkage preferably consists of disulfide bridges between cysteines of (ii).

31. The fusion protein of claim 1 wherein the complex consists of three identical fusion proteins.

32. A nucleic acid molecule encoding the fusion protein of claim 1.

33. The nucleic acid molecule of claim 32 which is operatively linked to an expression control sequence.

34. The nucleic acid molecule of claim 32 which is located on a vector.

35. A cell transformed or transfected with the nucleic acid molecule of claim 32.

38. A non-human organism transformed or transfected with the nucleic acid molecule of claim 32.

36-37. -37. (canceled)

39-43. -43. (canceled)

44. A polypeptide comprising a collectin trimerization domain form human surfactant protein-D comprising an amino acid substitution selected from F355S, F355T, F355E, F355D, F355K or F355R, and optionally a heterologous polypeptide domain.