# US 11,439,710 B2 · Nucleic acids encoding modified relaxin polypeptides

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- Number: US 11,439,710 B2
- Document ID: US11439710B2
- Status: Granted
- Kind: B2
- Application number: 16/885,631
- Filed: 2020-05-28
- Published: 2020-11-19
- Granted: 2022-09-13
- Earliest claimed: 2010-08-17
- Assignees: Ambrx, Inc.
- Applicants: Ambrx, Inc.
- Inventors: Vadim Kraynov; Nick Knudsen; Amha Hewet; Kristine De Dios; Jason Pinkstaff; Lorraine Sullivan
- Web page: https://patentgrep.com/patent/US11439710B2

## CPC

- A61K 47/60: the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
- A61K 38/2221: {Relaxins}
- A61P 3/06: Antihyperlipidemics
- A61P 3/10: for hyperglycaemia, e.g. antidiabetics
- 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 9/08: Vasodilators for multiple indications
- 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 11/00: Drugs for disorders of the respiratory system
- A61P 13/12: of the kidneys
- A61P 15/00: Drugs for genital or sexual disorders (for disorders of sex hormones A61P5/24); Contraceptives
- A61P 15/12: for climacteric disorders
- A61P 17/00: Drugs for dermatological disorders
- A61P 17/02: for treating wounds, ulcers, burns, scars, keloids, or the like
- A61P 19/02: for joint disorders, e.g. arthritis, arthrosis
- A61P 19/04: for non-specific disorders of the connective tissue
- A61P 19/08: for bone diseases, e.g. rachitism, Paget's disease
- 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 31/00: Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P 31/04: Antibacterial agents
- A61P 37/02: Immunomodulators
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C07K 14/64: Relaxins
- 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)
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- 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)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

Modified relaxin polypeptides and their uses thereof are provided.

## Claims

### Claim 1

An isolated nucleic acid encoding a modified relaxin polypeptide chain comprising a non-naturally encoded amino acid, wherein:
(a) said isolated nucleic acid comprises a selector codon that encodes said non-naturally encoded amino acid and said selector codon comprises an amber codon, an ochre codon, an opal codon, or a four base codon;
(b) the encoded modified relaxin polypeptide comprises a relaxin A chain polypeptide having a sequence at least 95% identical to SEQ ID NO: 4; and
(c) said non-naturally encoded amino acid occurs at residue 1 of the A chain of SEQ ID NO:4 or the corresponding position of said modified relaxin polypeptide.

### Claim 2 (depends on 1)

The isolated nucleic acid of claim 1, wherein said nucleic acid encodes a relaxin A chain polypeptide having the sequence of SEQ ID NO: 4 containing said non-naturally occurring amino acid.

### Claim 3 (depends on 2)

The isolated nucleic acid of claim 2, wherein said nucleic acid further encodes a relaxin B chain polypeptide of SEQ ID NO: 5 or SEQ ID NO: 6.

### Claim 4 (depends on 1)

The isolated nucleic acid of claim 1, wherein said nucleic acid further encodes a relaxin B chain polypeptide having a sequence at least 95% identical to SEQ ID NO: 5 or SEQ ID NO: 6.

### Claim 5 (depends on 1)

The isolated nucleic acid of claim 1, wherein said selector codon comprises an amber codon, an ochre codon, or an opal codon.

### Claim 6 (depends on 1)

An isolated host cell comprising the isolated nucleic acid of claim 1.

### Claim 7 (depends on 6)

The isolated host cell of claim 6, wherein said host cell further comprises an orthogonal tRNA that recognizes said selector codon and inserts said non-naturally encoded amino acid during translation of said isolated nucleic acid.

### Claim 8 (depends on 7)

The isolated host cell of claim 7, wherein said host cell further comprises an orthogonal tRNA synthetase that synthesizes said orthogonal tRNA.

### Claim 9 (depends on 6)

The isolated host cell of claim 6, wherein said non-naturally encoded amino acid comprises a functional group selected from a carbonyl group, an aminooxy group, a hydrazide group, a hydrazine group, a semicarbazide group, an azide group, or an alkyne group.

### Claim 10 (depends on 6)

The isolated host cell of claim 6, wherein said non-naturally encoded amino acid comprises a phenylalanine analog or derivative.

### Claim 11 (depends on 6)

The isolated host cell of claim 6, wherein said non-naturally encoded amino acid has the structure:
wherein the R group is any substituent other than the side chain found in alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine.

### Claim 12 (depends on 6)

The isolated host cell of claim 6, wherein said non-naturally encoded amino acid comprises para-acetyl-L-phenylalanine.

### Claim 13 (depends on 6)

The isolated host cell of claim 6, wherein said host cell a bacterial cell.

### Claim 14 (depends on 13)

The isolated host cell of claim 13, wherein said host cell an Escherichia coli cell.

### Claim 15 (depends on 1)

An in vitro translation system comprising the isolated nucleic acid of claim 1 and components that provide for the in vitro translation of said isolated nucleic acid.

### Claim 16 (depends on 1)

The isolated nucleic acid of claim 1, wherein said isolated nucleic acid further comprises a nucleic acid encoding a secretion signal sequence.

### Claim 17

An isolated nucleic acid encoding a modified relaxin polypeptide chain comprising a non-naturally encoded amino acid, wherein:
(a) said isolated nucleic acid comprises a selector codon that encodes said non-naturally encoded amino acid and said selector codon comprises an amber codon, an ochre codon, an opal codon, or a four base codon;
(b) the encoded modified relaxin polypeptide comprises the relaxin A chain polypeptide of SEQ ID NO: 4 containing one non-naturally encoded amino acid substitution; and
(c) said non-naturally encoded amino acid occurs at residue 1 of the A chain of SEQ ID NO:4.

### Claim 18 (depends on 17)

An isolated host cell comprising the isolated nucleic acid of claim 17.

### Claim 19 (depends on 18)

The isolated host cell of claim 18, wherein said host cell an Escherichia coli cell.

### Claim 20 (depends on 1)

A method of making a modified relaxin polypeptide chain comprising a non-naturally encoded amino acid, the method comprising, culturing cells comprising a polynucleotide according to claim 1 and an orthogonal tRNA under conditions to permit expression of the relaxin polypeptide comprising a non-naturally encoded amino acid.