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US-20260125430-A1 - CHIMERIC NEUROTOXINS

US20260125430A1US 20260125430 A1US20260125430 A1US 20260125430A1US-20260125430-A1

Abstract

Chimeric neurotoxins comprising an LH N domain from a first neurotoxin covalently linked to an H C domain from a different second neurotoxin are disclosed. The C-terminal residue of the LH N domain corresponds to the first amino acid residue of the 3 10 helix separating the LH N and H C domains of the first neurotoxin, and the N-terminal residue of the H C domain corresponds to the second amino acid residue of the corresponding 3 10 helix of the second neurotoxin. Also provided are nucleotide sequences encoding such chimeric neurotoxins, vectors and host cells comprising such nucleotide sequences, methods for producing the chimeric neurotoxins, and pharmaceutical compositions and kits containing the same. The chimeric neurotoxins may be used for aesthetic or cosmetic applications.

Inventors

  • Sai Man LIU

Assignees

  • IPSEN BIOPHARM LIMITED

Dates

Publication Date
20260507
Application Date
20250825
Priority Date
20160505

Claims (20)

  1. 1 . A chimeric neurotoxin comprising a LH N domain from a first neurotoxin covalently linked to a H C domain from a second neurotoxin, wherein said first and second neurotoxins are different, wherein the C-terminal amino acid residue of said LH N domain corresponds to the first amino acid residue of the 3 10 helix separating the LH N and H C domains in said first neurotoxin, and wherein the N-terminal amino acid residue of said H C domain corresponds to the second amino acid residue of the 3 10 helix separating the LH N and H C domains in said second neurotoxin.
  2. 2 . A chimeric neurotoxin according to claim 1 , wherein said first neurotoxin is a Botulinum Neurotoxin (BoNT) serotype A, serotype B, serotype C, serotype D, serotype E, serotype F or serotype G or a Tetanus Neurotoxin (TeNT), and wherein said second neurotoxin is a Botulinum Neurotoxin (BoNT) serotype A, serotype B, serotype C, serotype D, serotype E, serotype F or serotype G or a Tetanus Neurotoxin (TeNT).
  3. 3 . A chimeric neurotoxin according to claim 1 or 2 , wherein said LH N domain from a first neurotoxin corresponds to: amino acid residues 1 to 872 of BoNT/A1, amino acid residues 1 to 859 of BoNT/B1, amino acid residues 1 to 867 of BoNT/C1, amino acid residues 1 to 863 of BoNT/D, amino acid residues 1 to 846 of BoNT/E1, amino acid residues 1 to 865 of BoNT/F1, amino acid residues 1 to 864 of BoNT/G, or amino acid residues 1 to 880 of TeNT. and wherein said H C domain from a second neurotoxin corresponds to: amino acid residues 873 to 1296 of BoNT/A1, amino acid residues 860 to 1291 of BoNT/B1, amino acid residues 868 to 1291 of BoNT/C1, amino acid residues 864 to 1276 of BoNT/D, amino acid residues 847 to 1251 of BoNT/E1, amino acid residues 866 to 1275 of BoNT/F1, amino acid residues 865 to 1297 of BoNT/G, or amino acid residues 881 to 1315 of TeNT.
  4. 4 . A chimeric neurotoxin according to claim 1, 2 or 3 , wherein said first neurotoxin is a BoNT/A and wherein said second neurotoxin is a BoNT/B.
  5. 5 . A chimeric neurotoxin according to claim 4 , wherein said first neurotoxin is a BoNT/A and wherein said second neurotoxin is a BoNT/B.
  6. 6 . A chimeric neurotoxin according to claim 5 , wherein said LH N domain from a first neurotoxin corresponds to amino acid residues 1 to 872 of BoNT/A1 and wherein said H C domain from a second neurotoxin corresponds to amino acid residues 860 to 1291 of BoNT/B1.
  7. 7 . A chimeric neurotoxin according to claim 4, 5 or 6 , wherein said H C domain from a BoNT/B neurotoxin comprises at least one amino acid residue substitution, addition or deletion in the H CC subdomain which has the effect of increasing the binding affinity of the BoNT/B neurotoxin for the human Syt II receptor as compared to the natural BoNT/B sequence.
  8. 8 . A chimeric neurotoxin according to claim 7 , wherein said at least one amino acid residue substitution, addition or deletion in the H CC subdomain comprises a substitution mutation selected from the group consisting of: V1118M; Y1183M; E1191M; E11911; E1191Q; E1191T; S1199Y; S1199F; S1199L; S1201V; E1191C, E1191V, E1191L, E1191Y, S1199W, S1199E, S1199H, W1178Y, W1178Q, W1178A, W1178S, Y1183C, Y1183P and combinations thereof
  9. 9 . A chimeric molecule according to claim 7 , wherein said at least one amino acid residue substitution, addition or deletion in the H CC subdomain comprises two substitution mutations selected from the group consisting of: E1191M and S1199L, E1191M and S1199Y, E1191M and S1199F, E1191Q and S1199L, E1191Q and S1199Y, E1191Q and S1199F, E1191M and S1199W, E1191M and W1178Q, E1191C and S1199W, E1191C and S1199Y, E1191C and W1178Q, E1191Q and S1199W, E1191V and S1199W, E1191V and S1199Y, or E1191V and W1178Q.
  10. 10 . A chimeric neurotoxin according to claim 9 , wherein said two substitution mutations are E1191M and S1199Y.
  11. 11 . A chimeric molecule according to claim 7 , wherein said at least one amino acid residue substitution, addition or deletion in the H CC subdomain comprises three substitution mutations which are E1191M, S1199W and W1178Q.
  12. 12 . A chimeric neurotoxin according to claim 1, 2 or 3 , wherein said first neurotoxin is a BoNT/B and wherein said second neurotoxin is a BoNT/C.
  13. 13 . A chimeric neurotoxin according to claim 12 , wherein said first neurotoxin is a BoNT/B1 and wherein said second neurotoxin is a BoNT/C1.
  14. 14 . A chimeric neurotoxin according to claim 13 , wherein said LH N domain from a first neurotoxin corresponds to amino acid residues 1 to 859 of BoNT/B1 and wherein said H C domain from a second neurotoxin corresponds to amino acid residues 868 to 1291 of BoNT/C1.
  15. 15 . A nucleotide sequence encoding a chimeric neurotoxin according to any one of claim 1 to 14 .
  16. 16 . A vector comprising a nucleotide sequence according to claim 15 .
  17. 17 . A cell comprising a nucleotide sequence according to claim 15 or a vector according to claim 16 .
  18. 18 . A pharmaceutical composition comprising a chimeric neurotoxin according to any one of claims 1 to 14 .
  19. 19 . A kit comprising a pharmaceutical composition of claim 18 and instructions for therapeutic or cosmetic administration of said composition to a subject in need thereof.
  20. 20 . A method for producing a chimeric neurotoxin as defined in any one of claims 1 to 14 , said method comprising the step of culturing a cell of claim 17 , under conditions wherein said chimeric neurotoxin is produced.

Description

FIELD OF THE INVENTION The present invention relates to chimeric neurotoxins with enhanced properties and their use in therapy. SEQUENCE LISTING The Sequence Listing submitted herewith as an XML file named “17811958CONSL.xml” created on Aug. 25, 2025, having a size of 160,329 bytes, is incorporated herein by reference in its entirety. The Sequence Listing provides the sequences disclosed in the present application in accordance with 37 C.F.R. §§ 1.821-1.825 and conforms to the requirements of WIPO Standard ST.26. The information recorded in the Sequence Listing does not include any new matter and is intended to form part of the present disclosure. BACKGROUND Bacteria in the genus clostridia produce highly potent and specific protein toxins, which can poison neurons and other cells to which they are delivered. Examples of such clostridial toxins include the neurotoxins produced by C. tetani (TeNT) and by C. botulinum (BoNT) serotypes A-G, as well as those produced by C. baratii and C. butyricum. Among the clostridial neurotoxins are some of the most potent toxins known. By way of example, botulinum neurotoxins have median lethal dose (LD50) values for mice ranging from 0.5 to 5 ng/kg, depending on the serotype. Both tetanus and botulinum toxins act by inhibiting the function of affected neurons, specifically the release of neurotransmitters. While botulinum toxin acts at the neuromuscular junction and inhibits cholinergic transmission in the peripheral nervous system, tetanus toxin acts in the central nervous system. In nature, clostridial neurotoxins are synthesised as a single-chain polypeptide that is modified post-translationally by a proteolytic cleavage event to form two polypeptide chains joined together by a disulphide bond. Cleavage occurs at a specific cleavage site, often referred to as the activation site, that is located between the cysteine residues that provide the inter-chain disulphide bond. It is this di-chain form that is the active form of the toxin. The two chains are termed the heavy chain (H-chain), which has a molecular mass of approximately 100 kDa, and the light chain (L-chain), which has a molecular mass of approximately 50 kDa. The H-chain comprises an N-terminal translocation component (HN domain) and a C-terminal targeting component (HC domain). The cleavage site is located between the L-chain and the translocation domain components. Following binding of the HC domain to its target neuron and internalisation of the bound toxin into the cell via an endosome, the HN domain translocates the L-chain across the endosomal membrane and into the cytosol, and the L-chain provides a protease function (also known as a non-cytotoxic protease). Non-cytotoxic proteases act by proteolytically cleaving intracellular transport proteins known as SNARE proteins (e.g. SNAP-25, VAMP, or Syntaxin)—see Gerald K (2002) “Cell and Molecular Biology” (4th edition) John Wiley & Sons, Inc. The acronym SNARE derives from the term Soluble NSF Attachment Receptor, where NSF means N-ethylmaleimide-Sensitive Factor. SNARE proteins are integral to intracellular vesicle fusion, and thus to secretion of molecules via vesicle transport from a cell. The protease function is a zinc-dependent endopeptidase activity and exhibits a high substrate specificity for SNARE proteins. Accordingly, once delivered to a desired target cell, the non-cytotoxic protease is capable of inhibiting cellular secretion from the target cell. The L-chain proteases of clostridial neurotoxins are non-cytotoxic proteases that cleave SNARE proteins. In view of the ubiquitous nature of SNARE proteins, clostridial neurotoxins such as botulinum toxin have been successfully employed in a wide range of therapies. By way of example, we refer to William J. Lipham, Cosmetic and Clinical Applications of Botulinum Toxin (Slack, Inc., 2004), which describes the use of clostridial neurotoxins, such as botulinum neurotoxins (BoNTs), BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F and BoNT/G, and tetanus neurotoxin (TeNT), to inhibit neuronal transmission in a number of therapeutic and cosmetic or aesthetic applications—for example, marketed botulinum toxin products are currently approved as therapeutics for indications including focal spasticity, upper limb spasticity, lower limb spasticity, cervical dystonia, blepharospasm, hemifacial spasm, hyperhidrosis of the axillae, chronic migraine, neurogenic detrusor overactivity, glabellar lines, and severe lateral canthal lines. In addition, clostridial neurotoxin therapies are described for treating neuromuscular disorders (see U.S. Pat. No. 6,872,397); for treating uterine disorders (see US 2004/0175399); for treating ulcers and gastroesophageal reflux disease (see US 2004/0086531); for treating dystonia (see U.S. Pat. No. 6,319,505); for treating eye disorders (see US 2004/0234532); for treating blepharospasm (see US 2004/0151740); for treating strabismus (see US 2004/0126396); for treating pain (see U.S. Pat.