Recombinant botulinum toxin with increased duration of effect

Patent metadata

Field	Value
Publication number	US-11952601-B2
Application number	US-201716498193-A
Country	US
Kind code	B2
Filing date	Jun 20, 2017
Priority date	Jun 20, 2017
Publication date	Apr 9, 2024
Grant date	Apr 9, 2024

Abstract

This invention relates to novel recombinant clostridial neurotoxins exhibiting an increased duration of effect without a delayed onset of effect and to methods for the manufacture of such recombinant clostridial neurotoxins. These novel recombinant clostridial neurotoxins comprise a domain consisting of proline, alanine, serine, threonine, glycine and glutamate residues, and the methods comprise the steps of inserting a nucleic acid sequence coding for said domain into a nucleic acid sequence coding for a parental clostridial neurotoxin and expression of the recombinant nucleic acid sequence comprising said domain-coding sequence 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 an increased duration of effect without a delayed onset of effect.

First claim

The invention claimed is:

1. A recombinant botulinum neurotoxin comprising: a functionally active botulinum neurotoxin light chain, a functionally active botulinum neurotoxin heavy chain, and at least one domain, wherein the botulinum neurotoxin is selected from Clostridium botulinum neurotoxin serotype A, E, and C; wherein said at least one domain consists of 100-500 amino acid residues consisting of a plurality of amino acid repeats consisting of proline, alanine, serine, threonine, glycine and glutamate residues, wherein no more than six consecutive amino acid residues are identical, and wherein said at least one domain comprises the amino acid sequence of SEQ ID NO: 1, wherein said at least one domain is inserted at a position selected from (i) the N-terminus of the functionally active botulinum neurotoxin light chain, (ii) the C-terminus of the functionally active botulinum neurotoxin light chain, (iii) the N-terminus of the functionally active botulinum neurotoxin heavy chain, or (iv) the C-terminus of the functionally active botulinum neurotoxin heavy chain, and wherein the recombinant botulinum neurotoxin exhibits an increased duration of effect without a delayed onset of effect relative to a wild type botulinum neurotoxin without said at least one domain.

2. The recombinant botulinum neurotoxin of claim 1 , wherein the botulinum neurotoxin is selected from Clostridium botulinum neurotoxin serotype A.

3. The recombinant botulinum neurotoxin of claim 1 , wherein said at least one domain comprises the following amino acid sequence:

(SEQ ID NO: 2)

SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEG

TSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPG

SEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPG

TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPG

TSESATPE.

4. The recombinant botulinum neurotoxin of claim 1 , wherein the botulinum neurotoxin comprises at least one domain comprising the amino acid sequence of SEQ ID NO: 1 and one domain comprising the amino acid sequence of SEQ ID NO: 2.

5. The recombinant botulinum neurotoxin of claim 4 , wherein (i) the domain comprising the amino acid sequence of SEQ ID NO: 1 is inserted at a position of the N-terminus of the functionally active botulinum neurotoxin light chain, and (ii) the domain comprising the amino acid sequence of SEQ ID NO: 2 is inserted at a position of the C-terminus of the functionally active botulinum neurotoxin heavy chain.

6. A composition comprising the recombinant botulinum neurotoxin of claim 1 and a solvent or excipient.

7. A pharmaceutical composition comprising the recombinant botulinum neurotoxin of claim 1 and one or more pharmaceutically acceptable carriers.

8. A method of using a recombinant botulinum neurotoxin for cosmetic treatment, said method comprising the step of administering the recombinant botulinum neurotoxin of claim 1 to a patient.

9. A method for the generation of a recombinant botulinum neurotoxin according to claim 1 , comprising the step of obtaining a recombinant nucleic acid sequence encoding a recombinant single-chain precursor botulinum neurotoxin by the insertion of a nucleic acid sequence encoding said domain into a nucleic acid sequence encoding a parental botulinum neurotoxin, wherein said method further comprises the step of heterologously expressing said recombinant botulinum neurotoxin in a host cell from said nucleic acid sequence.

10. A recombinant single-chain botulinum neurotoxin, which is a precursor comprising the recombinant botulinum neurotoxin of claim 1 .

11. A nucleic acid sequence encoding the recombinant single-chain botulinum neurotoxin of claim 10 .

12. The nucleic acid sequence of claim 11 , wherein the nucleic acid sequence has the nucleotide sequence of SEQ ID NO: 3.

13. The recombinant botulinum neurotoxin of claim 1 , wherein said at least one domain consists of between 100 and 300 amino acid residues.

14. The method of claim 9 , wherein the host cell is a bacterial host cell.

15. The method of claim 9 , wherein the host cell is an E. coli host cell.

16. The recombinant botulinum neurotoxin of claim 1 , wherein the botulinum neurotoxin comprises a first domain inserted at a position of the N-terminus of the functionally active botulinum neurotoxin light chain, and a second domain inserted at a position of the C-terminus of the functionally active botulinum neurotoxin heavy chain,
wherein both the first and second domain consist of 100-500 amino acid residues consisting of a plurality of amino acid repeats consisting of proline, alanine, serine, threonine, glycine and glutamate residues, wherein no more than six consecutive amino acid residues are identical, and wherein the first and second domain comprise the amino acid sequence of SEQ ID NO: 1.

Assignees

• Merz Pharma Gmbh & Co Kgaa

Inventors

• Hofmann Fred
• Jurk Marcel
• López De La Paz Manuela
• Scheps Daniel
• FREVERT Jürgen

Classifications

• C12N9/52 Primary

  derived from bacteria {or Archaea} · CPC title

• A61K8/66

  Enzymes · CPC title

• C12Y304/24069

  Bontoxilysin (3.4.24.69), i.e. botulinum neurotoxin · CPC title

• A61K38/00

  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) · CPC title

• C07K2319/31

  fusions, other than Fc, for prolonged plasma life, e.g. albumin · CPC title