# US 11,078,472 B2 · Recombinant clostridial neurotoxins with increased duration of effect

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- Number: US 11,078,472 B2
- Document ID: US11078472B2
- Status: Granted
- Kind: B2
- Application number: 15/778,112
- Filed: 2017-01-19
- Published: 2018-11-15
- Granted: 2021-08-03
- Earliest claimed: 2016-01-20
- Assignees: MERZ PHARMA GmbH & CO. KGaA
- Applicants: Merz Pharma GmbH & Co. KGaA
- Inventors: Fred Hofmann; Marcel Jurk; Manuela López De La Paz; Daniel Scheps; Jürgen Frevert
- Web page: https://patentgrep.com/patent/US11078472B2

## CPC

- C12N 9/52: derived from bacteria {or Archaea}
- C07K 14/33: from Clostridium (G)
- C12Y 304/24069: Bontoxilysin (3.4.24.69), i.e. botulinum neurotoxin
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)
- C12Y: ENZYMES
- 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

This invention relates to novel recombinant clostridial neurotoxins exhibiting increased duration of effect and to methods for the manufacture of such recombinant clostridial neurotoxins. These novel recombinant clostridial neurotoxins comprise (i) at least two random coil domains, or (ii) at least two domains having a polyproline II helix conformation, and the methods comprise the steps of inserting at least two nucleic acid sequences each coding for (i) a random coil domain, or (ii) a domain having a polyproline II helix conformation, into a nucleic acid sequence coding for a parental clostridial neurotoxin and expression of the recombinant nucleic acid sequence comprising (i) the random coil domain-coding sequences, or (ii) the sequences encoding the domains having a polyproline II helix conformation in a host cell. The invention further relates to novel recombinant single-chain precursor clostridial neurotoxins used in such methods, nucleic acid sequences encoding such recombinant single-chain precursor clostridial neurotoxins, and pharmaceutical compositions comprising the recombinant clostridial neurotoxin with increased duration of effect.

## Claims

### Claim 1

A recombinant clostridial neurotoxin comprising (i) at least two separate protein segments consisting of alanine, serine, and proline residues, wherein each of said protein segments consists of between 50 and 200 amino acid residues; or (ii) at least two separate domains having a polyproline II helix conformation, wherein each of said domains comprises an amino acid sequence consisting of between 50 and 200 amino acid residues forming said polyproline II helix conformation, and wherein a first of said protein segments or domains is inserted at the N-terminus of the light chain of said recombinant clostridial neurotoxin, and a second of said protein segments or domains is inserted at the C-terminus of the heavy chain of said recombinant clostridial neurotoxin.

### Claim 2 (depends on 1)

The recombinant clostridial neurotoxin of claim 1, wherein at least one of said domains of subpart (ii) consists of alanine, serine and proline residues.

### Claim 3 (depends on 1)

The recombinant clostridial neurotoxin of claim 1, wherein at least one of said protein segments or domains comprises a plurality of amino acid repeats, wherein said repeats consist of Ala, Ser, and Pro residues and wherein no more than six consecutive amino acid residues are identical.

### Claim 4 (depends on 1)

The recombinant clostridial neurotoxin of claim 1, wherein at least one of said protein segments or domains comprises at least one amino acid sequence selected from the group consisting of: (a) ASPAAPAPASPAAPAPSAPA (SEQ ID NO: 1), AAPASPAPAAPSAPAPAAPS (SEQ ID NO: 2), APSSPSPSAPSSPSPASPSS (SEQ ID NO: 3), SAPSSPSPSAPSSPSPASPS (SEQ ID NO: 4), SSPSAPSPSSPASPSPSSPA (SEQ ID NO: 5), AASPAAPSAPPAAASPAAPSAPPA (SEQ ID NO: 6), or ASAAAPAAASAAASAPSAAA (SEQ ID NO: 7); (b) circular permuted versions of ASPAAPAPASPAAPAPSAPA (SEQ ID NO: 1), AAPASPAPAAPSAPAPAAPS (SEQ ID NO: 2), APSSPSPSAPSSPSPASPSS (SEQ ID NO: 3), SAPSSPSPSAPSSPSPASPS (SEQ ID NO: 4), SSPSAPSPSSPASPSPSSPA (SEQ ID NO: 5), AASPAAPSAPPAAASPAAPSAPPA (SEQ ID NO: 6), or ASAAAPAAASAAASAPSAAA (SEQ ID NO: 7); and (c) multimer(s) of ASPAAPAPASPAAPAPSAPA (SEQ ID NO: 1), AAPASPAPAAPSAPAPAAPS (SEQ ID NO: 2), APSSPSPSAPSSPSPASPSS (SEQ ID NO: 3), SAPSSPSPSAPSSPSPASPS (SEQ ID NO: 4), SSPSAPSPSSPASPSPSSPA (SEQ ID NO: 5), AASPAAPSAPPAAASPAAPSAPPA (SEQ ID NO: 6), or ASAAAPAAASAAASAPSAAA (SEQ ID NO: 7) as a whole or parts of these sequences.

### Claim 5 (depends on 1)

The recombinant clostridial neurotoxin of claim 1, wherein the sequence of said clostridial neurotoxin is selected from the group consisting of the sequences of: Clostridium botulinum neurotoxin serotype A, B, C, D, E, F, G or H; the sequence of a functional variant of a Clostridium botulinum neurotoxin of serotype A, B, C, D, E, F, G or H; the sequence of a chimeric Clostridium botulinum neurotoxin, wherein the clostridial neurotoxin light chain and heavy chain are from different clostridial neurotoxin serotypes; and the sequence of a chimeric Clostridium botulinum neurotoxin, wherein the clostridial neurotoxin light chain and heavy chain are from different clostridial neurotoxin serotypes and wherein said recombinant clostridial neurotoxin has a light chain and a heavy chain comprised in the amino acid sequence as found in SEQ ID NO: 8.

### Claim 6 (depends on 1)

The recombinant clostridial neurotoxin of claim 1 for treatment of a disease, wherein the recombinant clostridial neurotoxin causes longer lasting denervation relative to an identical clostridial neurotoxin with either one of or no said protein segments or domains.

### Claim 7 (depends on 1)

A pharmaceutical composition comprising the recombinant clostridial neurotoxin of claim 1.

### Claim 8 (depends on 1)

A method of cosmetic treatment of a patient comprising administering the recombinant clostridial neurotoxin of claim 1 to the patient.

### Claim 9 (depends on 1)

A method for the generation of a recombinant clostridial neurotoxin according to claim 1, comprising the step of obtaining a recombinant nucleic acid sequence encoding a recombinant single-chain precursor clostridial neurotoxin by the insertion of one or more nucleic acid sequences each encoding one of said protein segments or domains at at least two positions into a nucleic acid sequence encoding a parental clostridial neurotoxin.

### Claim 10 (depends on 9)

The method of claim 9, further comprising the step of heterologously expressing said recombinant nucleic acid sequence in a host cell wherein the host cell is a bacterial host cell or an E. coli host cell.

### Claim 11 (depends on 1)

A recombinant single-chain clostridial neurotoxin comprising (i) at least two separate protein segments consisting of alanine, serine, and proline residues, wherein each of said protein segments consists of between 50 and 200 amino acid residues; or (ii) at least two separate domains having a polyproline II helix conformation, wherein each of said domains comprises an amino acid sequence consisting of between 50 and 200 amino acid residues forming said polyproline II helix conformation, which is a precursor for the recombinant clostridial neurotoxin of claim 1, optionally wherein the sequence of said clostridial neurotoxin is selected from the group consisting of the sequences of: Clostridium botulinum neurotoxin serotype A, B, C, D, E, F, G or H; the sequence of a functional variant of a Clostridium botulinum neurotoxin of serotype A, B, C, D, E, F, G or H; and the sequence of a chimeric Clostridium botulinum neurotoxin, wherein the clostridial neurotoxin light chain and heavy chain are from different clostridial neurotoxin serotypes.

### Claim 12 (depends on 11)

A nucleic acid sequence encoding the recombinant single-chain clostridial neurotoxin of claim 11, optionally wherein the nucleic acid sequence is as found in SEQ ID NO:9.

### Claim 13 (depends on 12)

A vector comprising the nucleic acid sequence of claim 12.

### Claim 14 (depends on 12)

A recombinant host cell comprising the nucleic acid sequence of claim 12.

### Claim 15 (depends on 11)

A method for producing the recombinant single-chain precursor clostridial neurotoxin of claim 11, comprising the step of expressing a nucleic acid sequence encoding the recombinant single-chain clostridial neurotoxin of claim 11, optionally wherein the nucleic acid sequence is as found in SEQ ID NO:9, in a recombinant host cell.

### Claim 16 (depends on 3)

The recombinant clostridial neurotoxin of claim 3, wherein the proline residues comprised in said protein segment or domain constitute more than 4% and less than 40% of the amino acids of said protein segment or domain.

### Claim 17 (depends on 4)

The recombinant clostridial neurotoxin of claim 4 wherein said protein segment or domain comprises (ASPAAPAPASPAAPAPSAPA)n, with n being:
a. (i) an integer selected from 3 to 25 (SEQ ID NO: 10),
(ii) an integer selected from 4 to 8 (SEQ ID NO: 11),
(iii) an integer selected from 5 to 10 (SEQ ID NO: 12),
(iv) wherein n is 5 (SEQ ID NO: 13) or
(v) wherein n is 10 (SEQ ID NO: 14);
or
b. (i) wherein n is an integer selected from 5 to 150 (SEQ ID NO: 15),
(ii) wherein n is an integer selected from 6 to 20 (SEQ ID NO: 16),
(iii) wherein n is an integer selected from 7 to 13 (SEQ ID NO: 17),
(iv) wherein n is an integer selected from 8 to 12 (SEQ ID NO: 18), or
(v) wherein n is an integer selected from 9 to 11 (SEQ ID NO: 19).