# US 11,441,141 B2 · Methods for the manufacture of proteolytically processed polypeptides

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- Number: US 11,441,141 B2
- Document ID: US11441141B2
- Status: Granted
- Kind: B2
- Application number: 16/928,107
- Filed: 2020-07-14
- Published: 2020-10-29
- Granted: 2022-09-13
- Earliest claimed: 2018-08-20
- Assignees: Ipsen Bioinnovation Limited
- Applicants: Ipsen Bioinnovation Limited
- Inventors: Andreas Rummel
- Web page: https://patentgrep.com/patent/US11441141B2

## CPC

- C12N 9/52: derived from bacteria {or Archaea}
- A61P 1/02: Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
- A61P 1/06: Anti-spasmodics, e.g. drugs for colics, esophagic dyskinesia
- A61P 3/04: Anorexiants; Antiobesity agents
- A61P 11/02: Nasal agents, e.g. decongestants
- A61P 11/04: for throat disorders
- A61P 13/06: Anti-spasmodics
- A61P 13/08: of the prostate
- A61P 13/10: of the bladder
- A61P 15/02: for disorders of the vagina
- A61P 17/00: Drugs for dermatological disorders
- A61P 17/16: Emollients or protectives, e.g. against radiation
- A61P 21/00: Drugs for disorders of the muscular or neuromuscular system
- A61P 21/02: Muscle relaxants, e.g. for tetanus or cramps
- A61P 25/00: Drugs for disorders of the nervous system
- A61P 25/02: for peripheral neuropathies
- A61P 25/08: Antiepileptics; Anticonvulsants
- A61P 25/14: for treating abnormal movements, e.g. chorea, dyskinesia
- A61P 27/02: Ophthalmic agents
- C07K 14/33: from Clostridium (G)
- C12N 9/50: Proteinases {, e.g. Endopeptidases (3.4.21-3.4.25)}
- C12Q 1/37: involving peptidase or proteinase
- G01N 2500/00: Screening for compounds of potential therapeutic value
- 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
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

A proteolytically active polypeptide comprising an acid sequence having at least 50% sequence identity with the sequence of SEQ ID NO: 1 with the proviso that the polypeptide does not comprise the amino acid sequence of SEQ ID NO: 1. A nucleic acid encoding the polypeptide. An antibody specifically binding to the polypeptide. A method for the manufacture of a proteolytically processed polypeptide comprising contacting a first polypeptide having at least 50% sequence identity to SEQ ID NO: 1 with a second polypeptide that is susceptible to proteolysis by said first polypeptide.

## Claims

### Claim 1

A method for producing a proteolytically processed polypeptide, the method comprising contacting a first polypeptide having at least 50% sequence identity with SEQ ID NO: 1 with a second polypeptide that is susceptible to proteolysis by the first polypeptide, resulting in the proteolytic processing of the second polypeptide into at least two cleavage products; wherein: the method occurs in vitro; and/or at least one of the first or second polypeptide is recombinant or purified.

### Claim 2 (depends on 1)

The method of claim 1, wherein the second polypeptide comprises an amino acid sequence having at least 50% sequence identity swill any one of SEQ ID NOs: 3 to 25.

### Claim 3 (depends on 1)

The method of claim 1, wherein the second polypeptide is a clostridial neurotoxin.

### Claim 4 (depends on 1)

The method of claim 1, wherein the second polypeptide is selected from: a clostridial neurotoxin polypeptide that lacks a functional binding domain (Hcc), a clostridial neurotoxin polypeptide that has a modified clostridial neurotoxin binding domain (Hcc) that binds to a native clostridial neurotoxin receptor; or a clostridial neurotoxin polypeptide having a non-clostridial binding domain that binds to a non-native clostridial neurotoxin receptor.

### Claim 5 (depends on 4)

The method of claim 4, wherein the clostridial neurotoxin comprises a light chain from a first clostridial neurotoxin serotype and/or sub-type and a heavy chain from a second and different clostridial neurotoxin serotype and/or subtype.

### Claim 6 (depends on 1)

The method of claim 1, wherein the second polypeptide is a single-chain clostridial neurotoxin prepared by recombinant expression in E. coli.

### Claim 7 (depends on 1)

The method of claim 1, wherein the first polypeptide is purified and has 100% sequence identity to SEQ ID NO: 1.

### Claim 8 (depends on 1)

The method of claim 1, wherein the second polypeptide recombinant.

### Claim 9 (depends on 1)

The method of claim 1, wherein die second polypeptide is recombinant BoNT/A.

### Claim 10

A method for proteolytically processing a polypeptide in a subject wherein a first polypeptide having at least 50% sequence identity with SEQ NO: 1 and a second polypeptide that is susceptible to proteolysis by the first polypeptide are both administered to a subject and allowed to contact each other, resulting in the proteolytic processing of the second polypeptide into at least two cleavage products.

### Claim 11 (depends on 1)

The method of claim 1, wherein the First and second polypeptides are incubated together for at least 30 minutes.

### Claim 12 (depends on 9)

The method of claim 9, wherein the first polypeptide has at least 85% sequence identity with SEQ ID NO: 1.

### Claim 13 (depends on 1)

The method of claim 1, wherein the first polypeptide has at least 90% sequence identity with SEQ ID NO: 1.

### Claim 14 (depends on 1)

The method of claim 1, wherein the first polypeptide has at least 95% sequence identity with SEQ ID NO: 1.

### Claim 15 (depends on 1)

The method of claim 1, wherein the first polypeptide comprises an amino acid sequence that differs from that of SEQ ID NO: 1 by up to 50 conservative amino acid exchanges.

### Claim 16 (depends on 1)

The method of claim 1, wherein the first polypeptide comprises an amino acid sequence that differs from that of SEQ ID NO: 1 by up to 20 conservative amino acid exchanges.

### Claim 17 (depends on 1)

The method of claim 1, wherein the first polypeptide comprises an amino acid sequence that differs from that of SEQ ID NO: 1 by up to 10 conservative amino acid exchanges.

### Claim 18 (depends on 10)

The method of claim 10, wherein the first polypeptide has at least 95% sequence identity with SEQ ID NO: 1.

### Claim 19 (depends on 10)

The method of claim 10, Wherein the first polypeptide comprises an amino acid sequence that differs from that of SEQ ID No: 1 by up to 20 conservative amino acid exchanges.

### Claim 20 (depends on 10)

The method of claim 10, wherein the first polypeptide comprises an amino acid sequence that differs from that of SEQ ID NO: 1 by up to 10 conservative amino acid exchanges.