NOVEL RECOMBINANT BOTULINUM NEUROTOXINS WITH INCREASED DURATION OF EFFECT

• US 20200131494A1
• Filed: 07/06/2017
• Published: 04/30/2020
• Est. Priority Date: 07/06/2017
• Status: Active Grant

• Current Assignee: Merz Pharma GmbH & Company Kgaa
• Original Assignee: Merz Pharma GmbH & Company Kgaa
• Inventors: FREVERT, Jurgen, HOFMANN, Fred, JURK, Marcel, LOPEZ DE LA PAZ, Manuela, SCHEPS, Daniel
• Granted Patent: US 11,155,802 B2
• Time in Patent Office: Days
• CPC Class Codes: 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
• CPC Class Codes: C07K 14/33 from Clostridium (G)
• CPC Class Codes: C12N 9/52 derived from bacteria or Archaea
• CPC Class Codes: C12Y 304/24069 Bontoxilysin (3.4.24.69), i.e. botulinum neurotoxin

Abstract

This invention relates to novel recombinant botulinum neurotoxins serotype A exhibiting both (i) an increased duration of effect and (ii) a high specific biological activity. These novel recombinant botulinum neurotoxins comprise at least two additional domains consisting of proline, alanine and an additional amino acid residue and at least one amino acid modification which is located at the alpha-exosite or at the beta-exosite of the light chain of the neurotoxin. The invention further relates to novel recombinant single-chain precursor botulinum neurotoxins and compositions comprising the recombinant botulinum neurotoxin with an increased duration of effect and a high specific biological activity.

Claims

1. A recombinant botulinum neurotoxin serotype A comprising at least two domains wherein each domain comprises an amino acid sequence consisting of at least 50 amino acid residues, wherein said amino acid sequence consists of at least one proline, at least one alanine and at least one additional amino acid residue, selected from the group consisting of serine, threonine, tyrosine and glutamine, wherein the neurotoxin further comprises at least one amino acid modification which is located at the alpha-exosite and/or at the beta-exosite of the light chain of the neurotoxin.

2. The recombinant neurotoxin of claim 1, wherein said at least one amino acid modification is located at the alpha-exosite at at least one position selected from D102, T109, K340, I348, N353, K356.

3. The recombinant neurotoxin of claim 1, wherein said at least one amino acid modification is located at the beta-exosite at at least one position selected from G169, T220, P239, S254.

4. The recombinant neurotoxin of claim 2, wherein six amino acid modifications are located at the alpha-exosite at positions D102, T109, K340, I348, N353, K356, wherein these amino acids are substituted as follows D102F, T109R, K340M, I348L, N353M, K356R.

5. The recombinant neurotoxin of claim 3, wherein four amino acid modifications are located at the beta-exosite at positions G169, T220, P239, S254, wherein these amino acids are substituted as follows G169I, T220R, P239M, S254T.

6. The recombinant neurotoxin of claim 4, wherein the neurotoxin comprises two domains wherein each domain comprises an amino acid sequence consisting of between 70 and 260 amino acid residues, particularly 100 amino acid residues, 150 amino acid residues, or 200 amino acid residues, wherein said amino acid sequence consists of proline, alanine and serine residues.

7. The recombinant neurotoxin of claim 1 for the use in the treatment of a disease requiring improved chemodenervation, wherein the recombinant neurotoxin causes both (i) an increased duration of effect relative to a wildtype botulinum neurotoxin serotype A and (ii) an increased specific biological activity relative to a botulinum neurotoxin serotype A comprising two domains consisting of proline, alanine and serine residues without said modifications in the alpha-exosite and/or at the beta-exosite of the light chain of the neurotoxin.

8. A composition comprising the recombinant neurotoxin of claim 1.

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

10. Use of the recombinant neurotoxin of claim 1 for cosmetic treatment.

11. A method for the generation of a recombinant neurotoxin according to claim 1, comprising the step of obtaining a recombinant nucleic acid sequence encoding a recombinant single-chain precursor neurotoxin by the insertion of a nucleic acid sequence encoding said at least two domains consisting of proline, alanine and additional amino acid residues, selected from the group consisting of serine, threonine, tyrosine and glutamine residues into a nucleic acid sequence encoding a parental clostridial neurotoxin and by modifying the nucleic acid sequence encoding a parental botulinum neurotoxin at the alpha-exosite and/or at the beta-exosite of the light chain.

12. A recombinant single-chain neurotoxin, which is a precursor for the recombinant neurotoxin of claim 1.

13. A nucleic acid sequence encoding the recombinant single-chain clostridial neurotoxin of claim 12.

14. A vector comprising the nucleic acid sequence of claim 13.

15. A recombinant host cell comprising the nucleic acid sequence of claim 13.

16. A recombinant host cell comprising the vector of claim 14.

17. The recombinant neurotoxin of claim 5, wherein the neurotoxin comprises two domains wherein each domain comprises an amino acid sequence consisting of between 70 and 260 amino acid residues, particularly 100 amino acid residues, 150 amino acid residues, or 200 amino acid residues, wherein said amino acid sequence consists of proline, alanine and serine residues.