# US 9,751,921 B2 · Apelin fusion proteins and uses thereof

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- Number: US 9,751,921 B2
- Document ID: US9751921B2
- Status: Granted
- Kind: B2
- Application number: 15/146,730
- Filed: 2016-05-04
- Published: 2016-08-18
- Granted: 2017-09-05
- Earliest claimed: 2013-03-14
- Assignees: Regeneron Pharmaceuticals, Inc.
- Applicants: Regeneron Pharmaceuticals, Inc.
- Inventors: Panayiotis Stevis; Andrew Murphy
- Web page: https://patentgrep.com/patent/US9751921B2

## CPC

- C07K 14/47: from mammals
- A61P 3/04: Anorexiants; Antiobesity agents
- A61P 3/10: for hyperglycaemia, e.g. antidiabetics
- A61P 3/12: for electrolyte homeostasis
- A61P 9/00: Drugs for disorders of the cardiovascular system
- A61P 9/04: Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
- A61P 9/10: for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
- A61P 9/12: Antihypertensives
- A61P 31/18: for HIV
- A61P 35/00: Antineoplastic agents
- A61P 35/04: specific for metastasis
- C07K 14/435: from animals; from humans
- 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 2319/30: Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
- 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
- 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 invention provides a fusion protein or polypeptide comprising an apelin peptide fused to a multimerizing component. The invention also provides a fusion protein or polypeptide comprising an apelin peptide fused to an Fc domain, a fragment of an Fc domain, or a variant of an Fc domain. Apelin Fc-fusion polypeptides are capable of binding to the apelin receptor (APLNR). Apelin Fc-fusion polypeptides are capable of activating the APLNR and have improved pharmacokinetic properties compared to apelin peptides that are not fused to an Fc or an Fc fragment. Apelin Fc-fusion polypeptides are useful in diseases and conditions related to cardiovascular function, diabetes, cancer, obesity and other apelin-related conditions.

## Claims

### Claim 1

A polypeptide comprising an apelin peptide fused to an Fc domain, a fragment of an Fc domain, or variant of an Fc domain, wherein the N-terminus of the apelin peptide is fused to the C-terminus of the Fc domain or fragment or variant thereof, optionally via a peptide linker, and wherein the apelin peptide is a modified apelin 65-77 (apelin-13) peptide having an additional natural amino acid at its C-terminus.

### Claim 2 (depends on 1)

The polypeptide of claim 1, wherein the modified apelin peptide comprises the amino acid sequence of SEQ ID NO: 42.

### Claim 3 (depends on 1)

The polypeptide of claim 1, wherein the modified apelin peptide comprises the amino acid sequence of SEQ ID NO: 43.

### Claim 4 (depends on 1)

The polypeptide of claim 1, wherein the modified apelin peptide comprises the amino acid sequence of SEQ ID NO: 44.

### Claim 5 (depends on 1)

The polypeptide of claim 1, wherein the apelin peptide is fused to the Fc domain, or fragment thereof, via one or more peptide linkers, optionally wherein the one or more peptide linkers comprise one or more Gly-Ser linkers.

### Claim 6 (depends on 1)

The polypeptide of claim 1, wherein the Fc domain is selected from the group consisting of IgG1 CH2 domain and IgG1 CH3 domain; IgG4 CH2 domain and IgG4 CH3 domain; IgG1 CH2 domain and IgG4 CH3 domain; and IgG4 CH2 domain and an IgG1 CH3 domain, optionally wherein the Fc domain further comprises an IgG hinge domain or a fragment or variant thereof.

### Claim 7 (depends on 6)

The polypeptide of claim 6, wherein the Fc domain comprises an IgG hinge domain comprising SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:21, or SEQ ID NO: 22.

### Claim 8 (depends on 1)

The polypeptide of claim 1, wherein the polypeptide (i) is an APLNR agonist that exhibits an EC50 of less than about 10 nM, or less than about 1 nM when measured in an in vitro APLNR activation assay, and/or (ii) has an in vivo half-life of at least about 1 hour, or at least about 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours.

### Claim 9 (depends on 1)

A composition comprising the polypeptide of claim 1 and at least one pharmaceutically acceptable carrier or diluent.

### Claim 10 (depends on 1)

A nucleic acid molecule encoding the polypeptide of claim 1.

### Claim 11 (depends on 10)

A vector comprising the nucleic acid molecule of claim 10.

### Claim 12 (depends on 10, 11)

A cell comprising the nucleic acid molecule of claim 10 or the vector of claim 11.

### Claim 13 (depends on 12)

The cell of claim 12, wherein the nucleic acid is stably integrated into the genome of the cell.

### Claim 14 (depends on 13)

The cell of claim 13, wherein the cell is a eukaryotic cell, optionally wherein the cell is selected from the group consisting of CHO, COS, retinal cell, Vero, CV1, 293, MDCK, HaK, BHK, HeLa, HepG2, WI38, MRC 5, Colo25, HB 8065, HL-60, Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cell, C127 cell, SP2/0, NS-0, MMT cell, and tumor cell.

### Claim 15 (depends on 13)

The cell of claim 13, wherein the cell is an animal cell, optionally wherein the cell is a mammalian cell.

### Claim 16 (depends on 15)

The cell of claim 15, wherein the cell is a CHO cell.

### Claim 17 (depends on 16)

The cell of claim 16, wherein the cell is a CHO-K1 cell.

### Claim 18

A polypeptide comprising the amino acid sequence of SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41.