# US 10,118,953 B2 · Glucocorticoid-induced tumor necrosis factor receptor ligand (GITRL) single-chain fusion polypeptides

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- Number: US 10,118,953 B2
- Document ID: US10118953B2
- Status: Granted
- Kind: B2
- Application number: 15/643,787
- Filed: 2017-07-07
- Published: 2017-11-02
- Granted: 2018-11-06
- Earliest claimed: 2008-07-21
- Assignees: Apogenix AG
- Applicants: Apogenix AG
- Inventors: Oliver Hill; Christian Gieffers; Meinolf Thiemann
- Web page: https://patentgrep.com/patent/US10118953B2

## CPC

- C07K 14/525: Tumour necrosis factor [TNF]
- A61P 3/00: Drugs for disorders of the metabolism (of the blood or the extracellular fluid A61P7/00)
- A61P 19/02: for joint disorders, e.g. arthritis, arthrosis
- A61P 25/00: Drugs for disorders of the nervous system
- 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
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- A61P 31/00: Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P 35/00: Antineoplastic agents
- A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C07K 14/70575: {NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154 (NGF C07K14/48, TNF C07K14/525)}
- C07K 14/70578: {NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95 (NGF-receptor C07K14/71, TNF-receptor C07K14/7151)}
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C12N 15/62: DNA sequences coding for fusion proteins
- C12N 15/79: Vectors or expression systems specially adapted for eukaryotic hosts
- 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)
- C07K 2317/41: Glycosylation, sialylation, or fucosylation
- C07K 2317/52: Constant or Fc region; Isotype
- C07K 2317/55: Fab or Fab'
- C07K 2319/00: Fusion polypeptide
- C07K 2319/22: containing a Strep-tag
- C07K 2319/30: Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
- C07K 2319/32: fusions with soluble part of a cell surface receptor, "decoy receptors"
- C07K 2319/35: containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin
- C07K 2319/74: containing a fusion for binding to a cell surface receptor
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## 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

### Claim 1

A single-chain fusion polypeptide comprising:
(i) a first soluble glucocorticoid-induced tumor necrosis factor receptor ligand ligand (GITRL) cytokine domain,
(ii) a first peptide linker,
(iii) a second soluble GITRL cytokine domain,
(iv) a second peptide linker, and
(v) a third soluble GITRL cytokine domain,
wherein each of the soluble GITRL cytokine domains lacks a stalk region and the first and the second peptide linkers independently have a length of 3-8 amino acids.

### Claim 2 (depends on 1)

The polypeptide of claim 1, wherein the first, the second, and the third soluble GITRL cytokine domains are independently an N-terminally shortened domain of SEQ ID NO:18, and optionally consists of an amino acid mutation.

### Claim 3 (depends on 1)

The polypeptide of claim 1, wherein the first, the second, and the third soluble GITRL cytokine domains independently have the amino sequence of 52-177 of SEQ ID NO: 18.

### Claim 4 (depends on 1)

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

### Claim 5 (depends on 4)

The polypeptide of claim 4, wherein the glycine/serine linkers comprise substituted asparagine residues.

### Claim 6 (depends on 1)

The polypeptide of claim 1, wherein the first and second peptide linkers are independently selected from the group consisting of SEQ ID NO: 52-53 and SEQ ID NO: 1-26.

### Claim 7 (depends on 1)

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

### Claim 8 (depends on 7)

The polypeptide of claim 7, wherein the N-terminal signal peptide domain comprises a protease cleavage site.

### Claim 9 (depends on 1)

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

### Claim 10 (depends on 1)

The polypeptide of claim 1, which additionally comprises a further domain and a third peptide linker at the C-terminal end.

### Claim 11 (depends on 10)

A dimer comprising two polypeptides of claim 10, wherein the two polypeptides are dimerized via disulfide bridges of the third peptide linkers.

### Claim 12 (depends on 1)

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

### Claim 13 (depends on 12)

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