Single-chain TNFSF fusion polypeptides

• US 8,450,460 B2
• Filed: 07/18/2009
• Issued: 05/28/2013
• Est. Priority Date: 07/21/2008
• Status: Active Grant

• Current Assignee: Apogenix AG
• Original Assignee: Apogenix Gmbh
• Inventors: Hill, Oliver, Gieffers, Christian, Thiemann, Meinolf
• Primary Examiner(s): LANDSMAN, ROBERT S
• Application Number:
  US13/055,109
• Publication Number:
  US 20110162095A1
• Time in Patent Office: 1,410 Days
• Field of Search: None
• US Class Current: 530/351
• 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: 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 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• 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: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• 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 14/70578 NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95 NGF-receptor C07K14/71, TNF-receptor C07K14/7151
• 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 2317/41 Glycosylation, sialylation, or fucosylation
• CPC Class Codes: C07K 2317/52 Constant or Fc region; Isotype
• CPC Class Codes: C07K 2317/55 Fab or Fab'
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/22 containing a Strep-tag
• 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: C07K 2319/32 : fusions with soluble part of a cell surface receptor, "decoy receptors"
• CPC Class Codes: C07K 2319/35 : containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin
• CPC Class Codes: C07K 2319/74 : containing a fusion for binding to a cell surface receptor
• CPC Class Codes: C12N 15/62 : DNA sequences coding for fusion proteins
• CPC Class Codes: C12N 15/79 : Vectors or expression systems specially adapted for eukaryotic hosts

Abstract

The present invention refers to single-chain fusion proteins comprising three soluble TNF superfamily (TNFSF) cytokine domains and nucleic acid molecules encoding these fusion proteins. The fusion proteins are substantially non-aggregating and suitable for therapeutic, diagnostic and/or research applications.

Claims

1. A single-chain fusion polypeptide comprising:

(i) a first soluble tumor necrosis factor (TNF) superfamily cytokine domain,

(ii) a first peptide linker,

(iii) a second soluble TNF superfamily cytokine domain,

(iv) a second peptide linker, and

(v) a third soluble TNF superfamily cytokine domain,

wherein each of the soluble TNF superfamily cytokine domains lacks a stalk region and the first and the second peptide linkers independently have a length of 3-8 amino acids.

2. The polypeptide of claim 1, wherein the soluble TNF superfamily cytokine domain is selected from soluble CD95 ligand (CD95L) or TNF-related apoptosis-inducing ligand (TRAIL) domains.

3. The polypeptide of claim 1, wherein the second and/or third soluble TNF superfamily cytokine domain is an N-terminally shortened domain which optionally comprises amino acid sequence mutations.

4. The polypeptide of claim 1, wherein at least one of the soluble TNF superfamily cytokine domains is a soluble CD95L domain with an N-terminal sequence which starts at amino acid Arg 144, Lys145, or Val146 of human CD95L, and Arg144 and/or Lys145 is optionally replaced by a neutral amino acid.

5. The polypeptide of claim 4, wherein at least one of the soluble TNF superfamily cytokine domains is a soluble CD95L domain with an N-terminal sequence selected from

(a) Arg144-(Gly/Ser)145-Val146

(b) (Gly/Ser)144-Lys 145-Val146 and

(c) (Gly/Ser)144-(Gly/Ser)145-Val146.

6. The polypeptide of claim 2 wherein the soluble CD95L domain ends with Leu281 of human CD95L and/or optionally comprises a mutation at position Lys177, and/or at position Tyr218.

7. The polypeptide of claim 1 wherein at least one of the soluble TNF superfamily cytokine domains is a soluble TRAIL domain with an N-terminal sequence which starts at amino acid Gln120, Arg121, or Val122 of human TRAIL (SEQ ID NO:

10), and the Arg121 is optionally replaced by a neutral amino acid of serine or glycine.

8. The polypeptide of claim 7, wherein at least one of the soluble TNF superfamily cytokine domains is a soluble TRAIL domain with an N-terminal sequence selected from

(a) Arg121-Val122-Ala123 and

(b) (Gly/Ser)121-Val122-Ala123.

9. The polypeptide of claim 7 wherein the soluble TRAIL domain ends with amino acid G1y281 of human TRAIL and optionally comprises one or more mutations at positions R130, G160, H168, R170, H177, Y189, R191, Q193, E195, N199, K201, Y213, T214, S215, H264, I266, D267 or D269.

10. The polypeptide of claim 1, which additionally comprises an N-terminal signal peptide domain.

11. The polypeptide of claim 1, which additionally comprises a further domain at the N-terminal and/or C-terminal end.

12. An isolated nucleic acid molecule encoding the fusion polypeptide of claim 1.

13. An isolated host cell or a non-human organism transformed or transfected with the nucleic acid molecule of claim 12.

14. A pharmaceutical composition comprising the fusion polypeptide of claim 1 and a pharmaceutically acceptable carrier, diluent and/or adjuvant.

15. The polypeptide of claim 4, wherein the soluble TNF superfamily cytokine domain is soluble TNF superfamily cytokine domain (iii) or (iv).

16. The polypeptide of claim 6, wherein Lys177 mutates to Glu, Asp or Ser, and Tyr218 mutates to Arg, Lys, Ser, or Asp.

17. The polypeptide of claim 1, wherein the first and second peptide linkers are independently glycine/serine linkers.

18. The polypeptide of claim 10, wherein the N-terminal signal peptide domain comprises a protease cleavage site.

19. The polypeptide of claim 11, wherein the further domain is a Fab or Fc fragment domain.

20. The polypeptide of claim 17, wherein the glycine/serine linkers comprises substituted asparagine residues.