CD95L or trail fusion proteins
| Title: | CD95L or trail fusion proteins |
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| Patent Number: | 8,147,843 |
| Publication Date: | April 03, 2012 |
| Appl. No: | 12/439486 |
| Application Filed: | August 28, 2007 |
| Abstract: | The present invention refers to fusion proteins comprising a TNF superfamily (TNFSF) cytokine or a receptor binding domain thereof fused to a trimerization domain and a nucleic acid molecule encoding the fusion protein. The fusion protein is present as a trimeric complex or as an oligomer thereof and is suitable for therapeutic, diagnostic and/or research applications. |
| Inventors: | Hill, Oliver (Neckarsteinach, DE); Gieffers, Christian (Dossenheim, DE); Thiemann, Meinolf (Shriesheim, DE) |
| Assignees: | Apogenix GmbH (Heidelberg, DE) |
| Claim: | 1. A fusion protein comprising: (i) a TNF-superfamily cytokine selected from the group consisting of CD95L or TRAIL, or a receptor binding domain thereof; (ii) a fibritin trimerization domain; and (iii) a flexible linker between (i) and (ii), wherein the fibritin trimerization domain is a bacteriophage RB69 trimerization domain, and the linker has 5-20 amino acid residues having the amino acid sequence (GSS) z (SSG) b (GS) c (S) d , a, b, c, d are independently 0, 1, 2, 3, 4, or 5. |
| Claim: | 2. The fusion protein according to claim 1 , wherein the fibritin trimerization domain comprises amino acid residues 455-480 of SEQ ID NO:24, amino acid residues or 456-480 of SEQ ID NO:24. |
| Claim: | 3. The fusion protein according to claim 1 , wherein the TNF-superfamily cytokine or receptor binding domain thereof is fused to the N-terminus of the linker and the linker is fused to the N-terminus of the fibritin trimerization domain. |
| Claim: | 4. The fusion protein according to claim 3 , further comprising: (iv) a N-terminal signal peptide. |
| Claim: | 5. The fusion protein according to claim 4 , further comprising: (v) a protease cleavage site C-terminal to the signal peptide. |
| Claim: | 6. The fusion protein according to claim 3 , further comprising: (iv) a C-terminal flexible element. |
| Claim: | 7. The fusion protein according to claim 6 , further comprising: (v) a recognition or purification domain fused in frame to the C-terminal flexible element. |
| Claim: | 8. The fusion protein according to claim 1 , wherein the fibritin trimerization domain is fused to the N-terminus of the linker and the linker is fused to the N-terminus of the TNF-superfamily cytokine or receptor binding domain thereof. |
| Claim: | 9. The fusion protein according to claim 8 , further comprising: (iv) a N-terminal signal peptide. |
| Claim: | 10. The fusion protein according to claim 9 , further comprising: (v) a protease cleavage site C-terminal to the signal peptide. |
| Claim: | 11. The fusion protein according to claim 8 , further comprising: (iv) a C-terminal flexible element. |
| Claim: | 12. The fusion protein according to claim 11 , further comprising: (v) a recognition or purification domain fused in frame to the C-terminal flexible element. |
| Claim: | 13. The fusion protein according to claim 1 , wherein the fusion protein forms a trimeric complex or an oligomer of a trimeric complex. |
| Claim: | 14. The fusion protein according to claim 13 , wherein the complex consists of three identical fusion proteins. |
| Claim: | 15. The fusion protein according to claim 1 , wherein said TNF-superfamily cytokine is CD95L. |
| Claim: | 16. The fusion protein according to claim 15 , wherein said CD95L has the amino acid sequence of SEQ ID. NO: 30. |
| Claim: | 17. The fusion protein according to claim 15 , wherein the CD95L comprises amino acid residues 142-281 or amino acid residues 144-281 of SEQ ID NO:30. |
| Claim: | 18. The fusion protein according to claim 1 , wherein the TNF-superfamily cytokine is TRAIL, which comprises amino acid residues 116-281, amino acid residues 118-281, or amino acid residues 120-281 of SEQ ID NO:34. |
| Claim: | 19. The fusion protein according to claim 1 , wherein the linker has a length of 6, 9, 12, 15 or 18 amino acid residues. |
| Claim: | 20. The fusion protein according to claim 1 , wherein the linker has the amino acid sequence of GSS GSS GSS GS (SEQ ID NO: 46). |
| Claim: | 21. A pharmaceutical composition comprising: the fusion protein according to claim 1 , and a pharmaceutically acceptable carrier, diluent or adjuvant. |
| Claim: | 22. A nucleic acid encoding the fusion protein according to claim 1 . |
| Claim: | 23. An expression vector comprising the nucleic acid according to claim 22 . |
| Claim: | 24. A cell transformed or transfected with the expression vector according to claim 23 . |
| Current U.S. Class: | 4241/921 |
| Patent References Cited: | 1365027 November 2003 |
| Other References: | Belousova, Natalya et al.; “Genetically Targeted Adenovirus Vector Directed to CD40-Expressing Cells”; 2003, Journal of Virology, vol. 77, No. 21, pp. 11367-11377. cited by other Efimov, Vladimir P. et al.; “Bacteriophage T4 as a Surface Display Vector”; 1995, Virus Genes, vol. 10, No. 2, pp. 173-177. cited by other Frank, Sabine et al.; “Stabilization of Short Collagen-like Triple Helices by Protein Engineering”; 2001, J. Mol. Biol., vol. 308, pp. 1081-1089. cited by other Krasnykh, Victor et al.; “Genetic Targeting of an Adenovirus Vector via Replacement of the Fiber Protein with the Phage T4 Fibritin”; 2001, Journal of Virology, vol. 75, No. 9, pp. 4176-4183. cited by other Meier, Sebastian et al.; “Foldon, The Natural Trimerization Domain of T4 Fibritin, Dissociates into a Monomeric A-state Form containing a Stable β-Hairpin: Atomic Details of Trimer Dissociation and Local β-Hairpin Stability from Residual Dipolar Couplings”; 2004, J. Mol. Biol., vol. 344, pp. 1051-1069. cited by other Miroshnikov, Konstantin A. et al.; Engineering trimeric fibrous proteins based on bacteriophage T4 adhesins; 1998, Protein Engineering, vol. 11, No. 4, pp. 329-332. cited by other Sissoëff, Ludmilla et al.; “Stable trimerization of recombinant rabies virus glycoprotein ectodomain is required for interaction with the p75NTR receptor”; 2005, Journal of General Virology, vol. 86, pp. 2543-2552. cited by other Shiraishi, Tetsuya et al.; “Increased cytotoxicity of soluble Fas Ligand by fusing isoleucine zipper motif”; 2004, Biochemical and Biophysical Research Communications, vol. 322, pp. 197-202. cited by other Sun, Kuang-Hui et al.; “Expression, purification, refolding, and characterization of recombinant human soluble-Fas ligand from Escherichia coli”; 2005, Enzyme and Microbial Technology, vol. 36, No. 4, pp. 527-534. cited by other Yang, Xinzhen et al.; “Highly Stable Trimers Formed by Human Immunodeficiency Virus Type 1 Envelope Glycoproteins Fused with the Trimeric Motif of T4 Bacteriophage Fibritin”; 2002, Journal of Virology, vol. 76, No. 9, pp. 4634-4642. cited by other “Wac fibritin neck whiskers”; 2003, retrived from EBI accession No. UNIPROT: Q7Y4X5, 1 page. cited by other |
| Primary Examiner: | Stoica, Elly-Gerald |
| Attorney, Agent or Firm: | Perkins Coie LLP Kung, Viola T. |
| Accession Number: | edspgr.08147843 |
| Database: | USPTO Patent Grants |
| Language: | English |
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