# US 10,246,504 B2 · Engineered receptor and their use

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- Number: US 10,246,504 B2
- Document ID: US10246504B2
- Status: Granted
- Kind: B2
- Application number: 15/455,047
- Filed: 2017-03-09
- Published: 2017-07-06
- Granted: 2019-04-02
- Earliest claimed: 2012-12-10
- Assignees: University of Pittsburgh—Of the Commonwealth System of Higher Education
- Applicants: University of Pittsburgh—Of the Commonwealth System of Higher Education
- Inventors: Yan Xu; Pei Tang; Tommy S. Tillman
- Web page: https://patentgrep.com/patent/US10246504B2

## CPC

- C07K 14/705: Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)}
- A61K 38/164: {from bacteria}
- A61K 38/177: {Receptors; Cell surface antigens; Cell surface determinants}
- A61P 11/00: Drugs for disorders of the respiratory system
- A61P 25/04: Centrally acting analgesics, e.g. opioids
- C07K 14/195: from bacteria
- C07K 14/27: Erwinia (G)
- 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)
- B82Y 5/00: Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
- C07K 2319/00: Fusion polypeptide
- C07K 2319/03: containing a transmembrane segment
- 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)
- 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
- B82Y: SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES

## Abstract

Engineered chloride channel receptors, nucleic acids encoding these receptors, expression vectors including these nucleic acids are disclosed herein. Nanoparticles and pharmaceutical compositions including these engineered chloride channel receptors, nucleic acids, and expression vectors are disclosed. The use of these compositions and nanoparticles, such as for the treatment of pain, cystic fibrosis and asthma, is also disclosed.

## Claims

### Claim 1

A chloride channel receptor, comprising:
a) a transmembrane domain of a mammalian glycine receptor in the absence of the extracellular and intracellular domains of the mammalian glycine receptor, wherein the transmembrane domain comprises domains transmembrane (TM) 1, TM2, and TM3, wherein the transmembrane domain comprises a mutation at S16′ of the TM2; and
b) an extracellular domain of a pH-gated pentameric ligand-gated ion channel from Gloeobacter violaceus (GLIC); and
c) interfacial sequences between the extracellular domain and transmembrane domain, wherein the interfacial sequences comprise, in N-terminal to C-terminal order, loop 2, loop 7, loop 9, pre-TM1 linker, TM2-TM3 linker, and C-terminus, wherein the interfacial sequences and C-terminus are from Gloeobacter violaceus or a human;
and wherein opening of the chloride channel receptor is induced by pH.

### Claim 2 (depends on 1)

The chloride channel receptor of claim 1, further comprising domain TM4 of the transmembrane domain of the glycine receptor.

### Claim 3 (depends on 1)

The chloride channel receptor of claim 1, further comprising a C-terminal segment of GLIC.

### Claim 4 (depends on 2)

The chloride channel receptor of claim 2, further comprising a linker between the TM3 domain and the TM4 domain.

### Claim 5 (depends on 4)

The chloride channel of claim 4, wherein the linker comprises the amino acid sequence GGGGG (SEQ ID NO: 49), SQPARAA (SEQ ID NO: 17), or HHRQANGVEDD (SEQ ID NO: 48).

### Claim 6 (depends on 1)

The chloride channel receptor of claim 1, wherein the TM2 domain comprises at least one additional amino acid substitution in wherein the wild-type TM2 amino acid sequence is SEQ ID NO: 50 or SEQ ID NO: 51.

### Claim 7 (depends on 1)

The chloride channel receptor of claim 1, comprising a mutation at T13′ of TM2 of the transmembrane domain.

### Claim 8 (depends on 7)

The chloride channel receptor of claim 7, comprising a T13'S mutation.

### Claim 9 (depends on 1)

The chloride channel receptor of claim 1, wherein the channel is not permeable to chloride ions at a pH of 7.4.

### Claim 10 (depends on 9)

The chloride channel receptor of claim 9, wherein the channel is permeable to chloride ions at a pH of about 6.8 to about 7.2.

### Claim 11 (depends on 1)

The chloride channel receptor of claim 1, wherein the channel is permeable to chloride ions at a pH of about 7.0.

### Claim 12 (depends on 2)

The chloride channel receptor of claim 2, comprising the amino acid sequence at least 90% identical to the amino acid sequence set forth as one of SEQ ID NOs: 1-7.

### Claim 13 (depends on 12)

The chloride channel receptor of claim 12, comprising the amino acid sequence set forth as one of SEQ ID NOs: 1-7.

### Claim 14 (depends on 2)

The chloride channel receptor of claim 2, comprising the amino acid sequence of SEQ ID NO: 3.

### Claim 15 (depends on 2)

The chloride channel receptor of claim 2, comprising the amino acid sequence of SEQ ID NO: 2.

### Claim 16 (depends on 1)

A pharmaceutical composition comprising the chloride channel receptor of claim 1 and a pharmaceutically acceptable carrier.

### Claim 17 (depends on 1)

A nanoparticle comprising the chloride channel receptor of claim 1.

### Claim 18 (depends on 17)

A pharmaceutical composition comprising the nanoparticle of claim 17 and a pharmaceutically acceptable carrier.

### Claim 19 (depends on 1)

A method of reducing inflammatory pain in a subject, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 1, thereby reducing inflammatory pain in the subject.