Chimeric neurotoxins

Abstract

To provide chimeric neurotoxins with enhanced properties, and the use thereof in therapy.SOLUTION: The invention provides a chimeric neurotoxin comprising a LHN domain from a first neurotoxin covalently linked to a HC domain from a second neurotoxin, where the first and second neurotoxins are different. The C-terminal amino acid residue of the LHN domain corresponds to the first amino acid residue of the 310 helix separating the LHN and HC domains in the first neurotoxin. The N-terminal amino acid residue of the HC domain corresponds to the second amino acid residue of the 310 helix separating the LHN and HC domains in the second neurotoxin.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to chimeric neurotoxins with enhanced properties and their use in therapy. [Background technology]

[0002] Bacteria of the genus Clostridium produce highly potent, specific protein toxins that can poison nerves 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.

[0003] Some of the Clostridial neurotoxins are among the most potent known toxins. As an example, botulinum neurotoxins have a median lethal dose (LD50) value in 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. Botulinum toxins act at the neuromuscular junction and inhibit cholinergic transmission in the peripheral nervous system, whereas tetanus toxin acts in the central nervous system.

[0004] In nature, clostridial neurotoxins are synthesized as single-chain polypeptides, which are post-translationally modified by a proteolytic cleavage event to form two polypeptide chains linked together by a disulfide bond. Cleavage occurs at a specific cleavage site, often referred to as the activation site, located between cysteine ​​residues that provide the interchain disulfide bond. The active form of the toxin is this two-chain form. The two chains are referred to as the heavy chain (H chain), with a molecular weight of approximately 100 kDa, and the light chain (L chain), with a molecular weight of approximately 50 kDa. The H chain is linked to an N-terminal translocation component (H N domain) and the C-terminal targeting component (H CThe cleavage site is located between the L chain and the translocation domain components. After binding of the HC domain to its target neuron and internalization of the bound toxin into the cell by endosomes, the H N The domain translocates the L chain across the endosomal membrane into the cytosol, where the L chain provides the protease function (also known as a non-cytotoxic protease).

[0005] Non-cytotoxic proteases act by proteolytically cleaving intracellular trafficking 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 is derived from Soluble NSF Attachment Receptor, where NSF stands for N-ethylmaleimide-Sensitive Factor. SNARE proteins are essential for intracellular vesicle fusion and thus for the secretion of molecules from cells by vesicular trafficking. The protease function is a zinc-dependent endopeptidase activity and shows high substrate specificity for SNARE proteins. Thus, once delivered to the desired target cell, non-cytotoxic proteases are capable of inhibiting cellular secretion from the target cell. The light chain proteases of clostridial neurotoxins are non-cytotoxic proteases that cleave SNARE proteins.

[0006] Given the ubiquity of SNARE proteins, clostridial neurotoxins, such as botulinum toxins, have been used successfully in a wide range of therapies.

[0007] As an example, the inventors 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 neurotoxin (BoNT), 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, commercially available botulinum toxin products are currently approved for the treatment of indications including focal spasticity, upper limb spasticity, lower limb spasticity, cervical dystonia, blepharospasm, hemifacial spasm, axillary excessive sweating, chronic migraine headaches, neurogenic detrusor hyperactivity, glabellar lines, and severe lateral canthal wrinkles. Additionally, clostridial neurotoxin therapy has been used to treat neuromuscular disorders (see US 6,872,397), uterine disorders (see US 2004 / 0175399), ulcers and gastroesophageal reflux disease (see US 2004 / 0086531), dystonia (see US 6,319,505), eye disorders (see US 2004 / 0234532), blepharospasm (see US 2004 / 0151740), strabismus (see US 2004 / 0126396), pain (see US 6,869,610, US 6,641,821, and US 6,641,821). 0, US 6,464,986 and US 6,113,915), for treating fibromyalgia (see US 6,623,742, US 2004 / 0062776), for treating low back pain (see US 2004 / 0037852), for treating muscle injuries (see US 6,423,319), for treating sinus headaches (see US 6,838,434), for treating tension headaches (see US 6,776,992), for treating headaches (see US 6,458,365), for reducing migraine pain (see US 5,714,469), for treating cardiovascular disease (see US 6,767,544), to treat neurological disorders such as Parkinson's disease (see US 6,620,415, US 6,306,403), to treat neuropsychiatric disorders (see US 2004 / 0180061, US 2003 / 0211121), to treat endocrine disorders (see US 6,827,931), to treat thyroid disorders (see US 6,740,321), and cholinergic influenced sweat gland disorders. for treating chronic myocardial infarction disorders (see US 6,683,049 ), for treating diabetes (see US 6,337,075 , US 6,416,765 ), for treating pancreatic disorders (see US 6,261,572 , US 6,143,306 ), for treating cancer, such as bone tumors (see US 6,565,870 , US 6,368,605 , US 6,139,845 , US 2005 / 0031648 ), for treating ear disorders (see US 6,358,926 , see US 6,265,379 ), to treat autonomic disorders such as gastrointestinal muscular disorders and other smooth muscle dysfunction (see US 5,437,291 ), to treat skin lesions associated with skin cell proliferative disorders (see US 5,670,484 ), for the management of neurogenic inflammatory disorders (see US 6,063,768 ), to reduce hair loss and stimulate hair growth (see US 6,299,893 ), to treat downturned mouth (see US 6,358,917), for reducing appetite (see US2004 / 40253274), for dental treatments and procedures (see US2004 / 0115139), for treating neuromuscular disorders and conditions (see US2002 / 0010138), for treating various disorders and conditions and associated pain (see US2004 / 0013692), for treating conditions resulting from mucus hypersecretion such as asthma and COPD (see WO00 / 10598), and for treating non-neurological conditions such as inflammation, endocrine conditions, exocrine conditions, immunological conditions, cardiovascular conditions, bone conditions, etc. (see WO01 / 21213). All of the above publications are incorporated herein by reference in their entirety.

[0008] The use of non-cytotoxic proteases, such as Clostridial neurotoxins (e.g., BoNT and TeNT), in therapeutic and cosmetic treatments of humans and other mammals is expected to expand the ever-expanding range of diseases and illnesses that could benefit from the properties of these toxins.

[0009] All currently approved drugs / cosmetic preparations containing BoNT contain a naturally occurring neurotoxin purified from a Clostridium strain (BoNT / A in the case of DYSPORT®, BOTOX® or XEOMIN® and BoNT / B in the case of MYOBLOC®).

[0010] Recombinant techniques offer the possibility to modify or optimize the properties of neurotoxins by introducing modifications into their sequence and / or structure. C Domain or H CC The subdomains are derived from different neurotoxins. C Domain or H CC Chimeric neurotoxins have been produced in which the subdomains are substituted.

[0011] “Rummel et al, 2011 (Exchange of the H CCIn "Domain mediating double receptor recognition improves the pharmacodynamic properties of botulinum neurotoxin. FEBS Journal, 278(23), 4506-4515," the authors reported that the AABB, AACC, and BBAA chimeras (the letters represent the four domains L, H, and H) N , H CN , H CC A variety of active full-length hybrid neurotoxins were generated containing the AABB chimera (representing each of the serotype origins of BoNT / A and BoNT / B), which were then used to generate the AABB chimera. The AABB chimera was found to be more potent than BoNT / A in the mouse phrenic nerve hemidiaphragm assay, while the AACC retained only 10% of the potency of BoNT / A. The BBAA chimera retained 85% of the potency of BoNT / A and was comparable to BoNT / B.

[0012] In "Wang et al, 2008 (Novel chimeras of botulinum neurotoxins A and E unveil contributions from the binding, translocation, and protease domains to their functional characteristics. Journal of Biological Chemistry, 283(25), 16993-17002)", AE (BoNT / A-derived LH N and BoNT / E-derived H C ) and EA (BoNT / E-derived LH N and BoNT / A-derived H C ) to create chimeric neurotoxins, and in the case of AE chimeras, LH N and H C A linker was added between the domains. Both were able to induce paralysis in the mouse phrenic nerve hemidiaphragm assay as well as in vivo.

[0013] In "Wang et al., 2012a (Longer-acting and highly potent chimaeric inhibitors of excessive exocytosis created with domains from botulinum neurotoxin A and B. Biochemical Journal, 444(1), 59-67)", AB (BoNT / A-derived LH N and BoNT / B-derived H C、 (LH from BoNT / B) with a linker to improve folding N and BoNT / A-derived H C ) chimeric neurotoxins were created. The AB chimera induced longer neuromuscular paralysis in mice than BoNT / A. The BA chimera was able to reduce exocytosis from non-neuronal cells.

[0014] "Wang et al, 2012b (Novel chimeras of botulinum and tetanus neurotoxins yield insights into their distinct sites of neuroparalysis. The FASEB Journal, 26(12), 5035-5048)" N and TeNT-derived H C ), TxA (LH derived from TeNT N and BoNT / A-derived H C ), ETx (BoNT / E-derived LH N and TeNT-derived H C ) and TxE (TeNT-derived LH N and BoNT / E-derived H C ) chimeras were created. The information provided regarding the protein sequences of these prior art chimeric neurotoxins is summarized in Table 1 below: [Table 1]

[0015] However, there remains a need for optimal design of chimeric neurotoxins that allow for improved therapeutic properties.

[0016] The present invention solves the above problems by providing chimeric neurotoxins, as set forth in the claims. [Prior art documents] [Non-patent literature]

[0017] [Non-Patent Document 1] Rummel et al, 2011, Exchange of the HCC domain mediating double receptor recognition improves the pharmacodynamic properties of botulinum neurotoxin. FEBS Journal, 278(23), 4506-4515 Summary of the Invention

[0018] In one aspect, the present invention provides a method for the production of a neurotoxin comprising administering to a subject a neurotoxin comprising administering to said ... C LH derived from the first neurotoxin covalently linked to the domain N A chimeric neurotoxin comprising a LH domain, wherein the first and second neurotoxins are different, N The C-terminal amino acid residue of the domain is LH N and H C Separate domains 3 10 corresponds to the first amino acid residue of the helix, H C The N-terminal amino acid residue of the domain is LH in the second neurotoxin. N and H C Separate domains 3 10 A chimeric neurotoxin is provided that corresponds to the second amino acid residue of the helix.

[0019] In a second aspect, the present invention provides a nucleotide sequence encoding a chimeric neurotoxin according to the invention.

[0020] In a third aspect, the present invention provides a vector comprising a nucleotide sequence according to the invention.

[0021] In a fourth aspect, the present invention provides a cell comprising a nucleotide sequence or a vector according to the invention.

[0022] In a fifth aspect, the present invention provides a pharmaceutical composition comprising a chimeric neurotoxin according to the present invention.

[0023] In a sixth aspect, the present invention provides a chimeric neurotoxin according to the invention for use in therapy.

[0024] In a seventh aspect, the present invention provides the non-therapeutic use of a chimeric neurotoxin according to the invention for treating an aesthetic or cosmetic condition. [Item 1] H from a second neurotoxin C LH derived from the first neurotoxin covalently linked to the domain N 1. A chimeric neurotoxin comprising a domain, wherein said first and second neurotoxins are different; The above LH N The C-terminal amino acid residue of the domain is LH N and H C Separate domains 3 10 corresponding to the first amino acid residue of the helix, The above H C The N-terminal amino acid residue of the domain is LH N and H C Separate domains 3 10 A chimeric neurotoxin corresponding to the second amino acid residue of the helix. [Item 2] The chimeric neurotoxin according to item 1, wherein the first neurotoxin is botulinum neurotoxin (BoNT) serotype A, serotype B, serotype C, serotype D, serotype E, serotype F or serotype G or tetanus neurotoxin (TeNT), and the second neurotoxin is botulinum neurotoxin (BoNT) serotype A, serotype B, serotype C, serotype D, serotype E, serotype F or serotype G or tetanus neurotoxin (TeNT). [Item 3] LH derived from the first neurotoxin N The domain is - 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 Corresponding to, H from the second neurotoxin C The domain is - 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 3. The chimeric neurotoxin according to item 1 or 2, which corresponds to [Item 4] 4. The chimeric neurotoxin of item 1, 2 or 3, wherein the first neurotoxin is BoNT / A and the second neurotoxin is BoNT / B. [Item 5] 5. The chimeric neurotoxin of item 4, wherein the first neurotoxin is BoNT / A1 and the second neurotoxin is BoNT / B1. [Item 6] LH derived from the first neurotoxin N The domain corresponds to amino acid residues 1 to 872 of BoNT / A1 and is derived from the second neurotoxin. C 6. The chimeric neurotoxin according to item 5, wherein the domain corresponds to amino acid residues 860 to 1291 of BoNT / B1. [Item 7] H derived from the BoNT / B neurotoxin C The H domain has the effect of increasing the binding affinity of the BoNT / B neurotoxin to the human Syt II receptor as compared to the native BoNT / B sequence. CC 7. The chimeric neurotoxin according to item 4, 5 or 6, comprising at least one amino acid residue substitution, addition or deletion in the subdomain. [Item 8] The above H CC 8. The chimeric neurotoxin of item 7, wherein at least one amino acid residue substitution, addition or deletion in the subdomain comprises a substitution mutation selected from the group consisting of V1118M, Y1183M, E1191M, E1191I, E1191Q, E1191T, S1199Y, S1199F, S1199L, S1201V, E1191C, E1191V, E1191L, E1191Y, S1199W, S1199E, S1199H, W1178Y, W1178Q, W1178A, W1178S, Y1183C, Y1183P and combinations thereof. [Item 9] The above H CC8. The chimeric neurotoxin of item 7, wherein at least one amino acid residue substitution, addition or deletion in the 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. [Item 10] 10. The chimeric neurotoxin of item 9, wherein the two substitution mutations are E1191M and S1199Y. [Item 11] The above H CC 8. The chimeric neurotoxin according to item 7, wherein at least one amino acid residue substitution, addition or deletion in the subdomain comprises three substitution mutations: E1191M, S1199W and W1178Q. [Item 12] 4. The chimeric neurotoxin of item 1, 2 or 3, wherein the first neurotoxin is BoNT / B and the second neurotoxin is BoNT / C. [Item 13] 13. The chimeric neurotoxin of item 12, wherein the first neurotoxin is BoNT / B1 and the second neurotoxin is BoNT / C1. [Item 14] LH derived from the first neurotoxin N The domain corresponds to amino acid residues 1 to 859 of BoNT / B1 and is derived from the second neurotoxin. C 14. The chimeric neurotoxin according to item 13, wherein the domain corresponds to amino acid residues 868 to 1291 of BoNT / C1. [Item 15] 15. A nucleotide sequence encoding the chimeric neurotoxin according to any one of items 1 to 14. [Item 16] A vector comprising the nucleotide sequence according to item 15. [Item 17] A cell comprising the nucleotide sequence of item 15 or the vector of item 16. [Item 18] 15. A pharmaceutical composition comprising the chimeric neurotoxin according to any one of items 1 to 14. [Item 19] 20. A kit comprising the pharmaceutical composition of item 18 and instructions for therapeutic or cosmetic administration of said composition to a subject in need thereof. [Item 20] A method for producing the chimeric neurotoxin described in any one of items 1 to 14, comprising culturing the cell described in item 17 under conditions in which the chimeric neurotoxin is produced. [Item 21] 19. The chimeric neurotoxin according to any one of items 1 to 14 or the pharmaceutical composition according to item 18 for use in therapy. [Item 22] 22. The chimeric neurotoxin or pharmaceutical composition according to item 21 for use in the treatment of conditions associated with unwanted neuronal activity, such as conditions selected from the group consisting of spasmodic dysphonia, spasmodic torticollis, laryngeal dystonia, oromandibular dysphonia, lingual dystonia, cervical dystonia, palmar dystonia, blepharospasm, strabismus, hemifacial spasm, eyelid disorders, cerebral palsy, focal spasticity and other dysphonias, spasmodic colitis, irritable bladder, anismus, limb spasticity, tics, tremors, bruxism, anal fissures, achalasia, dysphagia and other muscle tone disorders and other disorders characterized by involuntary movements of muscle groups, lacrimation, excessive sweating, excessive salivation, excessive gastrointestinal secretions, secretory disorders, pain resulting from muscle spasms, headache pain, migraine and dermatological conditions. [Item 23] 19. Non-therapeutic use of a chimeric neurotoxin according to any one of items 1 to 14, or of a pharmaceutical composition according to item 18, for treating an aesthetic or cosmetic condition. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] In one aspect, the present invention provides a method for the production of a neurotoxin comprising administering to a subject a neurotoxin comprising administering to said ... CLH derived from the first neurotoxin covalently linked to the domain N a chimeric neurotoxin comprising a domain, wherein the first and second neurotoxins are different; ·LH N The C-terminal amino acid residue of the domain is LH N and H C Separate domains 3 10 corresponding to the first amino acid residue of the helix, H C The N-terminal amino acid residue of the domain is LH in the second neurotoxin. N and H C Separate domains 3 10 Corresponding to the second amino acid residue of the helix Chimeric neurotoxins are provided.

[0026] As used herein, the terms "a," "an," and "the" can mean one or more.

[0027] The term "neurotoxin" as used herein means any polypeptide that enters neurons and inhibits neurotransmitter release. This process includes binding of the neurotoxin to low or high affinity receptors, internalization of the neurotoxin, translocation of the endopeptidase portion of the neurotoxin to the cytoplasm, and enzymatic modification of the neurotoxin substrate. More specifically, the term "neurotoxin" includes any polypeptide produced by Clostridium bacteria (Clostridial neurotoxins) that enters neurons and inhibits neurotransmitter release, and such polypeptides produced by recombinant or chemical techniques. The active form of the toxin is this two-chain form. The two chains are referred to as heavy chains (H chains) with a molecular weight of approximately 100 kDa and light chains (L chains) with a molecular weight of approximately 50 kDa. Preferably, the first and second neurotoxins are Clostridial neurotoxins.

[0028] An exemplary BoNT / A neurotoxin amino acid sequence is provided as SEQ ID NO: 1 (UniProt Accession No. A5HZZ9). An exemplary BoNT / B neurotoxin amino acid sequence is provided as SEQ ID NO: 2 (UniProt Accession No. B1INP5). An exemplary BoNT / C neurotoxin amino acid sequence is provided as SEQ ID NO: 3 (UniProt Accession No. P18640). An exemplary BoNT / D neurotoxin amino acid sequence is provided as SEQ ID NO: 4 (UniProt Accession No. P19321). An exemplary BoNT / E neurotoxin amino acid sequence is provided as SEQ ID NO: 5 (UniProt Accession No. Q00496). An exemplary BoNT / F neurotoxin amino acid sequence is provided as SEQ ID NO: 6 (UniProt Accession No. Q57236). An exemplary BoNT / G neurotoxin amino acid sequence is provided as SEQ ID NO: 7 (UniProt Accession No. Q60393). An exemplary TeNT neurotoxin amino acid sequence is provided as SEQ ID NO: 8 (UniProt Accession No. P04958). The amino acid sequence of said neurotoxin is shown in the alignment below in FIG. 1 along with the sequences of other neurotoxins (ie, SEQ ID NOs: 58-91).

[0029] The term "chimeric neurotoxin" as used herein refers to a chimeric neurotoxin derived from a first neurotoxin. N H originating from domain and second neurotoxin C By this is meant a neurotoxin comprising or consisting of a domain.

[0030] The term “H C By "domain" herein is meant a functionally distinct region of a neurotoxin heavy chain having a molecular weight of approximately 50 kDa that enables the neurotoxin to bind to a receptor located on the surface of a target cell. C The domain is divided into two structurally distinct subdomains, each of which has a molecular weight of approximately 25 kDa, the "H CN Subdomains (H C N-terminal part of the domain) and "H CC Subdomains (H C It consists of the C-terminal portion of the domain.

[0031] The term “LHN "Domain" as used herein means C The heavy chains lack the endopeptidase domain ("L" or "light chain") and the domain involved in the translocation of the endopeptidase to the cytoplasm (H N It refers to a neurotoxin consisting of the agonist (domain).

[0032] As used herein, "the first neurotoxin is LH N and H C Separate domains 3 10 A reference to "the first amino acid residue of the helix" is N and H C Separate domains 3 10 It refers to the N-terminal residue of the helix.

[0033] As used herein, "the second neurotoxin is LH N and H C Separate domains 3 10 The reference to "the second amino acid residue of the helix" is N and H C Separate domains 3 10 It refers to the amino acid residue following the N-terminal residue of the helix.

[0034] "3 10 A "helix" is a type of secondary structure found in proteins and polypeptides, along with α-helices, β-sheets, and reverse turns. 3 10 The amino acids in the helix are arranged in a right-handed helical structure, where every turn is completed by three residues and ten atoms each, which share intramolecular hydrogen bonds between them. Each amino acid has ten atoms in the ring formed by making hydrogen bonds, corresponding to a 120° turn in the helix (i.e., the helix has three residues per turn) and a translation of 2.0 Å (=0.2 nm) along the helix axis. Most importantly, the NH group of an amino acid forms a hydrogen bond with the C=O group of the amino acid three amino acids before it, and this repeated i+3→i hydrogen bond formation leads to a ring of 10 atoms in the ring. 10 Define the helix. 310 A helix is ​​a standard concept in structural biology with which those of skill in the art are familiar.

[0035] These three 10 The helix corresponds to the four residues that form the actual helix plus two cap (or transition) residues, one at each end of these four residues. N and H C Separate domains 3 10 A "helix" as used herein consists of those six residues.

[0036] By carrying out structural analysis and sequence alignment, the present inventors have found that in tetanus and botulinum neurotoxins, LH N and H C Separate domains 3 10 The three helices were identified. 10 The helix is ​​located at its N-terminus (i.e., LH N at the C-terminal portion of the domain) by an α-helix that C At the N-terminus of the domain, it is surrounded by β-strands. 10 The first (N-terminal) residue of a helix (the cap or transition residue) also corresponds to the C-terminal residue of this α-helix.

[0037] LH N and H C Separate domains 3 10 The helices can be determined, for example, from publicly available crystal structures of botulinum neurotoxins, such as 3BTA (http: / / www.rcsb.org / pdb / explore / explore.do?structureId=3BTA) and 1EPW (http: / / www.rcsb.org / pdb / explore / explore.do?structureId=1EPW) for botulinum neurotoxins A1 and B1, respectively.

[0038] LH in other neurotoxins N and H C Separate domains 310 To determine the position of the helices, publicly available computational modeling and alignment tools can also be used, such as the homology modeling servers LOOPP (Learning, Observing and Outputting Protein Patterns, http: / / loopp.org), PHYRE (Protein Homology / analogY Recognition Engine, http: / / www.sbg.bio.ic.ac.uk / phyre2 / ) and Rosetta (https: / / www.rosettacommons.org / ), the protein superposition server SuperPose (http: / / wishart.biology.ualberta.ca / superpose / ), the alignment program Clustal Omega (http: / / www.clustal.org / omega / ) and several other tools / services listed in Internet Resources for Molecular and Cell Biologists (http: / / molbiol-tools.ca / ). N / H CN " We found that the perijunctional region is highly conserved structurally, making it an ideal region for superimposing different serotypes.

[0039] For example, these three 10 To examine the sequence of the helices, the following methodology was used by the inventors: 1. The structural homology modeling tool LOOP (http: / / loopp.org) was used to obtain predicted structures of all BoNT serotypes and TeNT based on the BoNT / A1 crystal structure (3BTA.pdb). 2. Copy the structure (pdb) file obtained in this way to H CN The N-terminus of the domain and the approximately 80 residues preceding it (H N This results in a highly structurally conserved "H N / H CN " area, edited to preserve To superimpose each serotype onto the 3.3BTA.pdb structure, the protein superposition server SuperPose (http: / / wishart.biology.ualberta.ca / superpose / ) was used. 4. H of BoNT / A1 C 3 at the start of the domain 10 To locate the helices, the overlapping pdb files were examined and the corresponding residues in the other serotypes were then identified. 5. All BoNT serotype sequences were aligned using Clustal Omega to check whether corresponding residues were correct.

[0040] LH determined by this method N , H C and 3 10 Examples of helix domains are shown in Table 2. [Table 2-1] [Table 2-2]

[0041] Using structural analysis and sequence alignment, the inventors have determined that LH N and H C Separate domains 3 10 The beta strand following the helix is ​​a conserved structure in all botulinum and tetanus neurotoxins and is the LH N and H C Separate domains 3 10 It was found that starting from the first residue of the helix, it begins at the eighth residue (eg, at residue 879 of BoNT / A1).

[0042] According to an alternative definition, the first aspect of the invention is a method for producing a neurotoxin-derived H C LH derived from the first neurotoxin covalently linked to the domainN a chimeric neurotoxin comprising a domain, wherein the first and second neurotoxins are different; ·LH N The C-terminal amino acid residue of the domain is H C It corresponds to the 8th amino acid residue from the β-strand located at the beginning (N-terminus) of the domain toward the N-terminus. H C The N-terminal amino acid residue of the domain is H C This corresponds to the seventh amino acid residue from the β-strand located at the beginning (N-terminus) of the domain toward the N-terminus. Chimeric neurotoxins are provided.

[0043] According to yet another definition, the first aspect of the invention relates to a second neurotoxin derived H C LH derived from the first neurotoxin covalently linked to the domain N a chimeric neurotoxin comprising a domain, wherein the first and second neurotoxins are different; ·LH N The C-terminal amino acid residue of the domain is LH N corresponds to the C-terminal amino acid residue of the α-helix located at the end (C-terminus) of the domain, H C The N-terminal amino acid residue of the domain is LH in the second neurotoxin. N It corresponds to the amino acid residue immediately C-terminal to the C-terminal amino acid residue of the α-helix located at the end (C-terminus) of the domain. Chimeric neurotoxins are provided.

[0044] The rationale for the design process of the chimeric neurotoxins according to the present invention was to seek to ensure that the secondary structure was intact, thereby minimizing any alteration of the tertiary structure and function of each domain.

[0045] Some prior art chimeric neurotoxins presumably required the use of LH to ensure acceptable expression and purification. N and H C Linkers are required between the domains (see Table 1).

[0046] Without wishing to be bound by theory, it is hypothesized that structuring the chimeric neurotoxin into a protein form having a tertiary structure that closely mimics that of the native neurotoxin promotes its solubility.

[0047] Without wishing to be bound by theory, three of the chimeric neurotoxins 10 It is further hypothesized that the fact that the four central amino acid residues of the helix are not disrupted ensures an optimal conformation of the chimeric neurotoxin, thereby allowing the chimeric neurotoxin to exert its function to its full potential.

[0048] In fact, the present inventors have surprisingly found that the first neurotoxin 10 The first amino acid residue of the helix and the third of the second neurotoxin 10 It has been found that retaining only the second amino acid residue one step further up the helix not only allows for the production of a soluble and functional chimeric neurotoxin, but further leads to improved properties over other chimeric neurotoxins, in particular increased potency, increased safety factor and / or longer duration of action.

[0049] Undesirable effects of neurotoxins (caused by diffusion of the neurotoxin away from the site of administration) can be experimentally evaluated by measuring the percent weight loss in relevant animal models (e.g., mice where weight loss is detected within 7 days of administration). Conversely, desirable and precise effects of neurotoxins can be experimentally evaluated by the Digital Abduction Score (DAS) assay, a measure of muscle paralysis. The DAS assay may be performed by injection of 20 μL of neurotoxin formulated in gelatin phosphate buffer into the mouse gastrocnemius / soleus muscle complex, followed by evaluation of the digital abduction score using the method of Aoki (Aoki KR, Toxicon 39:1815-1820; 2001). In the DAS assay, mice are briefly suspended by their tails to elicit a characteristic startle response in which the mouse extends its hind limbs and abducts its hind digits. After neurotoxin injection, the fluctuating degree of finger abduction is scored on a 5-point scale (0=normal to 4=maximal reduction in finger abduction and leg extension).

[0050] The safety factor of a neurotoxin can then be expressed as the ratio between the amount of neurotoxin required to achieve a 10% reduction in mouse body weight (measured at peak effect within the first 7 days after dosing in mice) and the amount of neurotoxin required for a DAS score of 2. Thus, a high safety factor score is desirable and indicates a neurotoxin that can efficiently paralyze the target muscle with few undesirable off-target effects.

[0051] A high safety margin is particularly advantageous in therapy, since it represents an increased therapeutic index. In other words, this means that reduced dosages can be used compared to known clostridial toxin therapeutics and / or increased dosages can be used without any additional effects. The possibility of using higher doses of neurotoxin without any additional effects is particularly advantageous, since higher doses usually lead to a longer duration of action of the neurotoxin.

[0052] The efficacy of a neurotoxin is determined by the induction of a given DAS score, e.g., a DAS score of 2 (ED 50The potency of a neurotoxin may also be expressed as the EC50 (EC20) in a cellular assay measuring SNARE cleavage by the neurotoxin. 50 Dose, e.g., EC in a cellular assay measuring SNAP-25 cleavage by chimeric BoNT / AB neurotoxins 50 It may also be expressed as a dose.

[0053] The duration of action of a neurotoxin may also be expressed as the time required to restore a DAS score of 0 following administration of a given dose of neurotoxin, e.g., the minimum dose of neurotoxin that results in a DAS score of 4, to the mouse gastrocnemius / soleus muscle complex.

[0054] In one embodiment, the first neurotoxin is botulinum neurotoxin (BoNT) serotype A, serotype B, serotype C, serotype D, serotype E, serotype F or serotype G or tetanus neurotoxin (TeNT) and the second neurotoxin is botulinum neurotoxin (BoNT) serotype A, serotype B, serotype C, serotype D, serotype E, serotype F or serotype G or tetanus neurotoxin (TeNT). In a preferred embodiment, the first neurotoxin is botulinum neurotoxin (BoNT) serotype A, serotype B or serotype C and the second neurotoxin is botulinum neurotoxin (BoNT) serotype A, serotype B or serotype C.

[0055] Various BoNT serotypes can be distinguished based on inactivation by specific neutralizing antisera, and such classification correlates with the percentage of sequence identity at the amino acid level. BoNT proteins of a given serotype are further divided into different subtypes based on the percentage of amino acid sequence identity.

[0056] Preferably, the first and second neurotoxins are botulinum neurotoxins from different serotypes. In another embodiment, either the first or second neurotoxin is a botulinum neurotoxin and the other neurotoxin is a tetanus neurotoxin.

[0057] X is LH N domain, and Y is HC Using the "XY" designation according to what is the domain, the following chimeric neurotoxins are embodiments of the invention. AB, AC, AD, AE, AF, AG, ATx, BA, BC, BD, BE, BF, BG, BTx, CA, CB, CD, CE, CF, CG, CTx, DA, DB, DC, DE, DF, DG, DTx, EA, EB, EC, ED, EF, EG, ETx, FA, FB, FC, FD, FE, FG, FTx, GA, GB, GC, GD, GE, GF, FTx, TxA, TxB, TxC, TxD, TxE, TxF, TxG, [where A, B, C, D, E, F, G and Tx are botulinum neurotoxin (BoNT) serotype A, serotype B, serotype C, serotype D, serotype E, serotype F, serotype G and tetanus neurotoxin (TeNT), respectively].

[0058] Additionally, using the same "XY" designation as above, the following chimeric neurotoxins are preferred embodiments of the present invention. AB, AC, B.A., B.C., C.A., C.B. [where A, B, and C are botulinum neurotoxin (BoNT) serotypes A, B, and C, respectively].

[0059] In one embodiment, the LH from the first neurotoxin N The domain is - amino acid residues 1 to 872 of SEQ ID NO: 1 or a polypeptide sequence having at least 70% sequence identity thereto; - amino acid residues 1 to 859 of SEQ ID NO: 2 or a polypeptide sequence having at least 70% sequence identity thereto; - amino acid residues 1 to 867 of SEQ ID NO: 3 or a polypeptide sequence having at least 70% sequence identity thereto; - amino acid residues 1 to 863 of SEQ ID NO: 4 or a polypeptide sequence having at least 70% sequence identity thereto; - amino acid residues 1 to 846 of SEQ ID NO: 5 or a polypeptide sequence having at least 70% sequence identity thereto; - amino acid residues 1 to 865 of SEQ ID NO: 6 or a polypeptide sequence having at least 70% sequence identity thereto; - amino acid residues 1 to 864 of SEQ ID NO: 7 or a polypeptide sequence having at least 70% sequence identity thereto; - amino acid residues 1 to 880 of SEQ ID NO: 8 or a polypeptide sequence having at least 70% sequence identity thereto Corresponding to, H from a second neurotoxin C The domain is - amino acid residues 873 to 1296 of SEQ ID NO: 1 or a polypeptide sequence having at least 70% sequence identity thereto; - amino acid residues 860 to 1291 of SEQ ID NO: 2 or a polypeptide sequence having at least 70% sequence identity thereto; - amino acid residues 868 to 1291 of SEQ ID NO: 3 or a polypeptide sequence having at least 70% sequence identity thereto; - amino acid residues 864 to 1276 of SEQ ID NO: 4 or a polypeptide sequence having at least 70% sequence identity thereto; - amino acid residues 847 to 1251 of SEQ ID NO: 5 or a polypeptide sequence having at least 70% sequence identity thereto; - amino acid residues 866 to 1275 of SEQ ID NO: 6 or a polypeptide sequence having at least 70% sequence identity thereto; - amino acid residues 865 to 1297 of SEQ ID NO: 7 or a polypeptide sequence having at least 70% sequence identity thereto; - amino acid residues 881 to 1315 of SEQ ID NO: 8 or a polypeptide sequence having at least 70% sequence identity thereto Corresponds to.

[0060] The "percent sequence identity" between two or more nucleic acid or amino acid sequences is a function of the number of identical nucleotides / amino acids at the same positions shared by the aligned sequences. Thus, percent identity can be calculated as the number of identical nucleotides / amino acids at each position in the alignment divided by the total number of nucleotides / amino acids in the aligned sequences, multiplied by 100. The calculation of percent sequence identity can also take into account the number of gaps and the length of each gap that need to be introduced to optimize the alignment of two or more sequences. The sequence comparison and percent identity determination between two or more sequences can be performed using certain mathematical algorithms, in particular global alignment numerical algorithms such as BLAST (Needleman and Wunsch, J. Mol. Biol. vol. 48 (no. 3), pp. 443-453, 1972), which are familiar to those skilled in the art.

[0061] The first or second neurotoxin may be a mosaic neurotoxin. The term "mosaic neurotoxin" as used in this context refers to a naturally occurring Clostridial neurotoxin that contains at least one functional domain derived from another species of Clostridial neurotoxin (e.g., a different serotype of Clostridial neurotoxin), and the Clostridial neurotoxin does not usually contain the at least one functional domain. Examples of mosaic neurotoxins include naturally occurring BoNT / DC and BoNT / CD. BoNT / DC is a neurotoxin that contains an L chain of serotype D and an H chain of serotype D. N Domain and H of serotype C C BoNT / CD contains the L and H chains of serotype C. N Domain and H of serotype D C It consists of a domain.

[0062] The first and second neurotoxins may be modified neurotoxins and derivatives thereof, including but not limited to those described below. The modified neurotoxins or derivatives may contain one or more amino acids that are modified compared to the native (unmodified) form of the neurotoxin, or may contain one or more inserted amino acids that are not present in the native (unmodified) form of the toxin. As an example, the modified Clostridial neurotoxin may have an amino acid sequence in one or more domains that is modified relative to the native (unmodified) Clostridial neurotoxin sequence. Such modifications may modify functional aspects of the neurotoxin, such as biological activity or persistence. Thus, in one embodiment, the first neurotoxin and / or the second neurotoxin is a modified neurotoxin or a modified neurotoxin derivative.

[0063] The modified neurotoxin retains at least one of the functions of a neurotoxin selected from the ability to bind to low or high affinity neurotoxin receptors on a target cell, translocate the endopeptidase portion (light chain) of the neurotoxin into the cytoplasm of the cell, and cleave SNARE proteins. Preferably, the modified neurotoxin retains at least two of these functions. More preferably, the modified neurotoxin retains three of these functions.

[0064] A modified neurotoxin may have one or more modifications in the amino acid sequence of the heavy chain (modified H C domain, the modified heavy chain binds to the target neuronal cell with higher or lower affinity than the native (unmodified) neurotoxin. C Such modifications in the H domain alter binding to ganglioside receptors and / or protein receptors on target neurons. C This may involve modifying residues in the ganglioside binding site of the domain or in the protein (SV2 or synaptotagmin) binding site. Examples of such modified neurotoxins are described in WO2006 / 027207 and WO2006 / 114308, both of which are incorporated herein by reference.

[0065] The modified neurotoxin may have one or more modifications in the amino acid sequence of the light chain, such as modifications in the substrate binding or catalytic domains that may alter or modify the SNARE protein specificity of the modified LC. Examples of such modified neurotoxins are described in WO2010 / 120766 and US2011 / 0318385, both of which are incorporated herein by reference.

[0066] The modified neurotoxin may contain one or more modifications that increase or decrease the biological activity and / or biological persistence of the modified neurotoxin. For example, the modified neurotoxin may contain a leucine- or tyrosine-based motif, which increases or decreases the biological activity and / or biological persistence of the modified neurotoxin. Suitable leucine-based motifs include xDxxxLL, xExxxLL, xExxxIL and xExxxLM (wherein x is any amino acid). Suitable tyrosine-based motifs include Yxx-Hy (wherein Hy is a hydrophobic amino acid). Examples of modified neurotoxins that contain leucine- or tyrosine-based motifs are described in WO2002 / 08268, which is incorporated herein by reference.

[0067] In one embodiment, the first or second neurotoxin is a modified BoNT / A having an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO:1.

[0068] In one embodiment, the first or second neurotoxin is a modified BoNT / B having an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO:2.

[0069] In one embodiment, the first or second neurotoxin is a modified BoNT / C having an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO:3.

[0070] In one embodiment, the first or second neurotoxin is a modified BoNT / D having an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO:4.

[0071] In one embodiment, the first or second neurotoxin is a modified BoNT / E having an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO:5.

[0072] In one embodiment, the first or second neurotoxin is a modified BoNT / F having an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO:6.

[0073] In one embodiment, the first or second neurotoxin is a modified BoNT / G having an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO:7.

[0074] In one embodiment, the first or second neurotoxin is a modified TeNT having an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO:8.

[0075] In one embodiment, the second neurotoxin is BoNT / B. Such chimeric neurotoxins are referred to herein as "BoNT / XB neurotoxins."

[0076] In a preferred embodiment, the first neurotoxin is BoNT / A and the second neurotoxin is BoNT / B. Such chimeric neurotoxins are referred to herein as "BoNT / AB neurotoxins." More preferably, the first neurotoxin is BoNT / A1 and the second neurotoxin is BoNT / B1. More preferably, the first neurotoxin is further enriched in LH from the first neurotoxin. N The domain corresponds to amino acid residues 1-872 of BoNT / A1 and is derived from a second neurotoxin. C The domain corresponds to amino acid residues 860 to 1291 of BoNT / B1. In one preferred embodiment, the LH domain is derived from the first neurotoxin. N The domain corresponds to amino acid residues 1 to 872 of SEQ ID NO: 1 and is H from a second neurotoxin. C The domain corresponds to amino acid residues 860 to 1291 of SEQ ID NO: 2. In other words, a preferred chimeric neurotoxin of the invention comprises or consists of the amino acid sequence SEQ ID NO:13.

[0077] Compared to the BoNT / A serotype, native BoNT / B is considerably less potent, despite the relatively high abundance of its receptor on synaptic vesicles. This is due to a unique amino acid change within the toxin-binding site in human synaptotagmin II (Syt II) as compared to rodent (rat / mouse) Syt II (Peng, L., et al., J Cell Sci, 125(Pt 13):3233-42 (2012); Rummel, A. et al, FEBS J 278:4506-4515 (2011).13,22). As a result of this residue change, human Syt II has greatly reduced binding to native BoNT / B, as well as to native BoNT / DC and / G. These findings provide an explanation for the clinical observation that significantly higher doses of BoNT / B are required than BoNT / A (which binds to a different receptor) to achieve the same level of therapeutic effect in patients. In a preferred embodiment of the BoNT / XB or BoNT / AB neurotoxin of the present invention, H derived from BoNT / B neurotoxin is CThe H domain has the effect of increasing the binding affinity of BoNT / B neurotoxin to human Syt II compared to the native BoNT / B sequence. CC It comprises at least one amino acid residue substitution, addition or deletion in the subdomain.

[0078] BoNT / BH CC Suitable amino acid residue substitutions, additions or deletions in the subdomains are disclosed in WO2013 / 180799, and in the yet to be published PCT / US2016 / 024211 (both incorporated by reference).

[0079] BoNT / BH CC Suitable amino acid residue substitutions, additions or deletions in the subdomains include substitution mutations selected from the group consisting of V1118M, Y1183M, E1191M, E1191I, E1191Q, E1191T, S1199Y, S1199F, S1199L, S1201V, E1191C, E1191V, E1191L, E1191Y, S1199W, S1199E, S1199H, W1178Y, W1178Q, W1178A, W1178S, Y1183C, Y1183P and combinations thereof.

[0080] BoNT / BH CC Suitable amino acid residue substitutions, additions or deletions in the subdomain further include combinations of 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.

[0081] BoNT / BH CCSuitable amino acid residue substitutions, additions or deletions in the subdomains also include the combination of the three substitution mutations: E1191M, S1199W and W1178Q.

[0082] In a preferred embodiment, BoNT / BH CC Suitable amino acid residue substitutions, additions or deletions in the subdomain include a combination of two substitution mutations: E1191M and S1199Y. In other words, a preferred chimeric neurotoxin of the present invention comprises or consists of the amino acid sequence SEQ ID NO:11 or SEQ ID NO:12.

[0083] In another preferred embodiment, the first neurotoxin is BoNT / C and the second neurotoxin is BoNT / B. Such chimeric neurotoxins are referred to herein as "BoNT / CB neurotoxins." More preferably, the first neurotoxin is BoNT / C1 and the second neurotoxin is BoNT / B1. More preferably, the first neurotoxin is further enriched in LH from the first neurotoxin. N The domain corresponds to amino acid residues 1-867 of BoNT / C1 and is derived from a second neurotoxin. C The domain corresponds to amino acid residues 860 to 1291 of BoNT / B1. In one preferred embodiment, the LH domain is derived from the first neurotoxin. N The domain corresponds to amino acid residues 1 to 867 of SEQ ID NO:3 and is derived from a second neurotoxin. C The Hc domain corresponds to amino acid residues 860-1291 of SEQ ID NO: 2. In a preferred embodiment, the Hc domain from a BoNT / B neurotoxin has the effect of increasing the binding affinity of the BoNT / B neurotoxin to human Syt II as compared to the native BoNT / B sequence. CC Contains at least one amino acid residue substitution, addition or deletion in the subdomain. BoNT / BH CC Suitable amino acid residue substitutions, additions or deletions in the subdomains are as described above.

[0084] In a preferred embodiment of the BoNT / XDC neurotoxin of the present invention (a chimeric neurotoxin in which the second neurotoxin is a mosaic BoNT / DC), the H derived from the mosaic BoNT / DC neurotoxin isC The H domain has the effect of increasing the binding affinity of the mosaic BoNT / DC neurotoxin to human Syt II compared to the native mosaic BoNT / DC sequence. CC It comprises at least one amino acid residue substitution, addition or deletion in the subdomain.

[0085] In a preferred embodiment of the BoNT / XG neurotoxin of the present invention, H derived from BoNT / G neurotoxin C The H domain has the effect of increasing the binding affinity of the BoNT / G neurotoxin to human Syt II as compared to the native BoNT / G sequence. CC There is at least one amino acid residue substitution, addition or deletion in the subdomain.

[0086] Other preferred neurotoxins of the present invention are:

[0087] In a preferred embodiment, the first neurotoxin is BoNT / A and the second neurotoxin is BoNT / C. Such chimeric neurotoxins are referred to herein as "BoNT / AC neurotoxins." More preferably, the first neurotoxin is BoNT / A1 and the second neurotoxin is BoNT / C1. More preferably, the first neurotoxin is further enriched in LH from the first neurotoxin. N The domain corresponds to amino acid residues 1-872 of BoNT / A1 and is derived from a second neurotoxin. C The domain corresponds to amino acid residues 868 to 1291 of BoNT / C1. In one preferred embodiment, the LH domain is derived from the first neurotoxin. N The domain corresponds to amino acid residues 1 to 872 of SEQ ID NO: 1 and is derived from a second neurotoxin. C The domain corresponds to amino acid residues 868 to 1291 of SEQ ID NO:3.

[0088] In another preferred embodiment, the first neurotoxin is BoNT / B and the second neurotoxin is BoNT / A. Such chimeric neurotoxins are referred to herein as "BoNT / BA neurotoxins." More preferably, the first neurotoxin is BoNT / B1 and the second neurotoxin is BoNT / A1. More preferably, the first neurotoxin is further enriched in LH from the first neurotoxin. N The domain corresponds to amino acid residues 1-859 of BoNT / B1 and is derived from a second neurotoxin. C The domain corresponds to amino acid residues 873 to 1296 of BoNT / A1. In one preferred embodiment, the LH domain is derived from the first neurotoxin. N The domain corresponds to amino acid residues 1 to 859 of SEQ ID NO:2 and is derived from a second neurotoxin. C The domain corresponds to amino acid residues 873 to 1293 of SEQ ID NO:1.

[0089] In another preferred embodiment, the first neurotoxin is BoNT / B and the second neurotoxin is BoNT / C. Such chimeric neurotoxins are referred to herein as "BoNT / BC neurotoxins." More preferably, the first neurotoxin is BoNT / B1 and the second neurotoxin is BoNT / C1. More preferably, the first neurotoxin is further enriched in LH from the first neurotoxin. N The domain corresponds to amino acid residues 1-859 of BoNT / B1 and is derived from a second neurotoxin. C The domain corresponds to amino acid residues 868 to 1291 of BoNT / C1. In one preferred embodiment, the LH domain is derived from the first neurotoxin. N The domain corresponds to amino acid residues 1 to 859 of SEQ ID NO:2 and is derived from a second neurotoxin. C The domain corresponds to amino acid residues 868 to 1291 of SEQ ID NO: 3. In other words, a preferred chimeric neurotoxin of the invention comprises or consists of the amino acid sequence SEQ ID NO:56.

[0090] In another preferred embodiment, the first neurotoxin is BoNT / C and the second neurotoxin is BoNT / A. Such chimeric neurotoxins are referred to herein as "BoNT / CA neurotoxins." More preferably, the first neurotoxin is BoNT / C1 and the second neurotoxin is BoNT / A1. More preferably, the first neurotoxin is further enriched in LH from the first neurotoxin. N The domain corresponds to amino acid residues 1-867 of BoNT / C1 and is derived from a second neurotoxin. C The domain corresponds to amino acid residues 873 to 1296 of BoNT / A1. In one preferred embodiment, the LH domain is derived from the first neurotoxin. N The domain corresponds to amino acid residues 1 to 867 of SEQ ID NO:3 and is derived from a second neurotoxin. C The domain corresponds to amino acid residues 873 to 1296 of SEQ ID NO:1.

[0091] The chimeric neurotoxin of the present invention can be produced using recombinant technology.Thus, in one embodiment, the chimeric neurotoxin of the present invention is a recombinant chimeric neurotoxin.It should be easily understood that according to a preferred embodiment, the nucleotide sequence encoding the recombinant chimeric neurotoxin of the present invention, the vector comprising said nucleotide sequence and the cell comprising said vector, as further described below, can be referred to as recombinant, mutatis mutandis.

[0092] In another aspect, the invention provides a nucleotide sequence, e.g., a DNA or RNA sequence, encoding a chimeric neurotoxin of the invention. In a preferred embodiment, the nucleotide sequence is a DNA sequence.

[0093] The nucleic acid molecules of the present invention can be produced using any suitable process known in the art. Thus, the nucleic acid molecules can be produced using chemical synthesis techniques. Alternatively, the nucleic acid molecules of the present invention can be produced using molecular biology techniques.

[0094] The DNA sequences of the present invention are preferably designed on a computer and then synthesized by conventional DNA synthesis techniques.

[0095] The above nucleic acid sequence information is optionally modified to provide codon bias according to the final host cell (eg, E. coli) expression system to be used.

[0096] In another aspect, the present invention provides a vector comprising the nucleotide sequence of the present invention.In one embodiment, the nucleic acid sequence is prepared as a part of a DNA vector comprising a promoter and a terminator.In a preferred embodiment, the vector has a promoter selected from Tac, AraBAD, T7-Lac or T5-Lac.

[0097] The vector may be suitable for in vitro and / or in vivo expression of said nucleic acid sequence. The vector may be a vector for transient and / or stable gene expression. The vector may further comprise regulatory elements and / or selection markers. The vector may be of viral, phage or bacterial origin. For example, the expression vector may be a pET, pJ401, pGEX vector or a derivative thereof.

[0098] In another aspect, the present invention provides a cell comprising the nucleotide sequence or vector of the present invention. The term "cell" may be used herein synonymously with the term "host cell" or "cell line". Suitable cell types include prokaryotic cells, e.g., E. coli, and eukaryotic cells, such as yeast cells, mammalian cells, insect cells, etc. Preferably, the cell is E. coli.

[0099] In another aspect, the present invention provides a method for producing the chimeric neurotoxin of the present invention, said method comprising culturing the cells as described above under conditions in which said chimeric neurotoxin is produced. Said conditions are well known to those skilled in the art and therefore need not be described in further detail herein. Preferably, said method further comprises recovering the chimeric neurotoxin from the culture.

[0100] In another aspect, the present invention provides a pharmaceutical composition comprising the chimeric neurotoxin of the present invention. Preferably, the pharmaceutical composition comprises the chimeric neurotoxin together with at least one component selected from a pharma- ceutically acceptable carrier, excipient, adjuvant, propellant and / or salt.

[0101] In another aspect, the present invention provides the chimeric neurotoxin or pharmaceutical composition of the present invention for use in therapy. More precisely, the present invention relates to the use of the chimeric neurotoxin or pharmaceutical composition as described herein for the manufacture of a medicament. In other words, the present invention relates to a method for treating a subject in need thereof, comprising administering to said subject an effective amount of the chimeric neurotoxin or pharmaceutical composition as described herein. By "effective amount" is meant that the chimeric neurotoxin or pharmaceutical composition is administered in an amount sufficient to provide the indicated effect. As used herein, the term "subject" refers preferably to a human or animal, more preferably to a human.

[0102] The chimeric neurotoxins of the present invention are preferably suitable for use in the treatment of a condition associated with unwanted neuronal activity in a subject in need thereof, such as a condition selected from the group consisting of spasmodic dysphonia, spasmodic torticollis, laryngeal dystonia, oromandibular dysphonia, lingual dystonia, cervical dystonia, palmar dystonia, blepharospasm, strabismus, hemifacial spasm, eyelid disorders, cerebral palsy, focal spasticity and other voice disorders, spasmodic colitis, irritable bladder, anismus, limb spasticity, tics, tremors, bruxism, anal fissures, achalasia, dysphagia and other muscle tone disorders and other disorders characterized by involuntary movement of muscle groups, lacrimation, excessive sweating, excessive salivation, excessive gastrointestinal secretions, secretory disorders, pain resulting from muscle spasms, headache pain, migraine and dermatological conditions. More precisely, the present invention relates to the use of a chimeric neurotoxin or pharmaceutical composition as described herein for the manufacture of a medicament intended to treat a condition associated with an unwanted neuronal activity as described above. In other words, the present invention relates to a method for treating a condition associated with an unwanted neuronal activity as described above in a subject in need thereof, said method comprising administering to said subject an effective amount of a chimeric neurotoxin or pharmaceutical composition as described herein.

[0103] In another aspect, the present invention provides a non-therapeutic use of the chimeric neurotoxin of the present invention for treating an aesthetic or cosmetic condition in a subject in need thereof. In other words, the present invention relates to a method for treating an aesthetic or cosmetic condition in a subject in need thereof, comprising administering to said subject an effective amount of a chimeric neurotoxin or pharmaceutical composition as described herein. According to this aspect of the present invention, the subject to be treated is preferably not suffering from a condition associated with undesired neuronal activity as described above. More preferably, said subject is a healthy subject, i.e. a subject not suffering from any disease.

[0104] In another aspect, the present invention provides a kit for use in a therapeutic or non-therapeutic (cosmetic or aesthetic) method or for therapeutic or non-therapeutic (cosmetic or aesthetic) use as described above, said kit comprising the pharmaceutical composition of the present invention and instructions for carrying out said method or use. More precisely, the present invention relates to a kit comprising the pharmaceutical composition of the present invention and instructions for therapeutic or cosmetic administration of said composition to a subject in need thereof. As used herein, the term "instructions" refers to a publication, record, drawing or any other medium of expression that can be used to convey how to carry out the method or use of the present invention, such as therapeutic or cosmetic administration of said composition to a subject in need thereof. The instructions may, for example, be attached to a container containing said composition or said kit.

[0105] The modified chimeric neurotoxins of the present invention can be formulated for oral, parenteral, continuous infusion, inhalation, or topical application. Compositions suitable for injection can be in the form of a solution, suspension, or emulsion, or a dry powder that is dissolved or suspended in a suitable vehicle prior to use.

[0106] In the case of a chimeric neurotoxin to be delivered locally, the chimeric neurotoxin can be formulated as a cream (eg, for topical application) or for subcutaneous injection.

[0107] Local delivery means can include aerosols or other sprays (e.g., nebulizers). In this regard, aerosol formulations of the chimeric neurotoxins allow for delivery to the lungs and / or other nasal and / or bronchial or airway passages.

[0108] The chimeric neurotoxins of the invention may be administered to a patient by intrathecal injection or by epidural injection in the spinal column at the level of the spinal segment involved in the innervation of the affected organ.

[0109] The preferred routes of administration are laparoscopic and / or by localized, especially intramuscular, injection.

[0110] The dosage ranges for administration of the chimeric neurotoxins of the present invention are those that produce the desired therapeutic effect. It will be appreciated that the required dosage range will vary depending on the exact nature of the chimeric neurotoxin or composition, the route of administration, the nature of the formulation, the age of the patient, the nature, extent or severity of the patient's condition, contraindications, if any, and the judgment of the attending physician. Variations in these dosage levels can be adjusted using standard empirical routines for optimization.

[0111] Fluid dosage forms are usually prepared utilizing the chimeric neurotoxin and a pyrogen-free sterile vehicle. The modified Clostridial toxin can be dissolved or suspended in the vehicle depending on the vehicle and concentration used. In preparing a solution, the chimeric neurotoxin can be dissolved in the vehicle, the solution can be made isotonic by adding sodium chloride as necessary, sterilized by filtration through a sterile filter using aseptic techniques, and then filled into a sterile vial or ampoule and sealed. Alternatively, if the solution stability is appropriate, the solution in the sealed container can be sterilized by autoclaving. Advantageously, additives such as buffers, solubilizers, stabilizers, preservatives or bactericides, suspending or emulsifying agents and / or local anesthetics can be dissolved in the vehicle.

[0112] Dry powders that are dissolved or suspended in a suitable vehicle prior to use may be prepared by filling pre-sterilized ingredients into a sterile container using aseptic techniques in a sterile area. Alternatively, the ingredients may be dissolved in a suitable container using aseptic techniques in a sterile area. The formulation is then lyophilized and the container is aseptically sealed.

[0113] Parenteral suspensions suitable for intramuscular, subcutaneous, or intradermal injection are prepared in substantially the same manner, except that the sterile components are suspended in a sterile vehicle instead of being dissolved, and sterilization cannot be achieved by filtration. The components may be isolated in a sterile state or may be sterilized after isolation, for example, by gamma irradiation.

[0114] Administration according to the present invention may utilize a variety of delivery techniques, including microparticle encapsulation, viral delivery systems, or high pressure aerosol impingement.

[0115] The present disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in carrying out or testing the embodiments of the present disclosure. Numerical ranges are inclusive of the numbers that define the range. Unless otherwise specified, any nucleic acid sequence is written from left to right in 5' to 3' orientation, and amino acid sequences are written from left to right in amino to carboxy orientation, respectively.

[0116] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range is specifically disclosed to one decimal place of the unit of the lower limit, unless the context clearly indicates otherwise. Each smaller range between any stated or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the disclosure. The upper and lower limits of these smaller ranges may be independently included or excluded in the range, and each range is also encompassed within the disclosure, subject to any specifically excluded limits in the stated range, if either or both of the limits are included in the smaller range, or both are not included, or both are included. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included within the disclosure.

[0117] It must be noted that, herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a clostridial neurotoxin" includes a plurality of such candidate agents, reference to "the clostridial neurotoxin" includes reference to one or more clostridial neurotoxins and equivalents thereof known to those skilled in the art, and so forth.

[0118] The invention will now be described, by way of example only, with reference to the following figures and examples. [Brief description of the drawings]

[0119] [Figure 1] Figure 1 shows a sequence alignment of BoNT / A1-8, / B1-8, / C, / D, / E1-12, / F1-7, / G, / "H" and TeNT using the CLUSTAL Omega (1.2.1) multiple sequence alignment tool. The position of the putative 310 helix separating the LHN and HC domains is in bold, underlined text. [Diagram 2] 2 shows an SDS PAGE of purified recombinant BoNT / AB chimeras 1, 2 and 3A (SEQ ID NOs: 9, 10 and 11, respectively). Lanes are labeled "marker" (molecular weight marker), "-DTT" (oxidized BoNT / AB chimera sample) and "+DTT" (reduced BoNT / AB chimera sample). [Diagram 3]FIG. 3 shows the cleavage of SNAP-25 in rat spinal cord neurons by recombinant BoNT / AB chimeras 1, 2 and 3A (SEQ ID NO: 9, 10 and 11, respectively). Cultured rat primary spinal cord neurons (SCN) were exposed to various concentrations of recombinant BoNT / AB chimeras 1, 2 or 3A for 24 hours at 37° C. in a humidified atmosphere with 10% CO2. Cells were then lysed using 1× NuPAGE buffer supplemented with DTT and benzonase. Samples were transferred to microcentrifuge tubes, heated to 90° C. on a heat block for 5 minutes, stored at −20° C., and then analyzed for SNAP-25 cleavage by Western blot. SNAP-25 was detected using a polyclonal antibody that detects both full-length and cleaved forms of SNAP-25 (Sigma No. S9684). Anti-rabbit HRP (Sigma No. A6154) was used as the secondary antibody. [Figure 4] Figure 4 shows the mouse digit abduction scoring assay. Mice were injected into the gastrocnemius-soleus complex of one hind limb under brief general anesthesia, and muscle weakness was measured using the digit abduction score (DAS) on a 0-4 scale. DAS maximum values ​​for each dose were determined and plotted against dose, and the data were fitted to a four-parameter logistic equation to determine the ED50 and the dose leading to DAS4 (DAS4 dose) values. [Diagram 5] 5 shows an SDS PAGE of purified recombinant BoNT / AB chimeras 3B and 3C (SEQ ID NOs: 12 and 13, respectively). Lanes are labeled "marker" (molecular weight marker), "-DTT" (oxidized BoNT / AB chimera sample) and "+DTT" (reduced BoNT / AB chimera sample). [Figure 6]FIG. 6 shows the cleavage of SNAP-25 by recombinant BoNT / A and BoNT / AB chimeras 3B and 3C (SEQ ID NO: 1, 12 and 13, respectively) in human induced pluripotent stem cell-derived peripheral neurons (PERI.4U-Axiogenesis, Germany). PERI.4U cells were exposed to various concentrations of recombinant BoNT / A or BoNT / AB chimeras 3B or 3C for 24 hours at 37° C. in a humidified CO2 atmosphere containing 5% CO2. Cells were then lysed using 1× NuPAGE buffer supplemented with DTT and benzonase. Samples were transferred to microcentrifuge tubes, heated to 90° C. for 5 minutes on a heat block and stored at −20° C. before analyzing SNAP-25 cleavage by Western blot. SNAP-25 was detected using a polyclonal antibody that detects both full-length and cleaved forms of SNAP-25 (Sigma no. S9684). Anti-rabbit HRP (Sigma no. A6154) was used as secondary antibody. [Figure 7] Figure 7 shows muscle wasting over time in a mouse digit abduction scoring assay. Mice were injected into the gastrocnemius-soleus complex of one hind limb under brief general anesthesia, and muscle wasting was measured using the digit abduction score (DAS) on a 0-4 scale. Animals from the group injected with the lowest dose that induced a DAS of 4 during the first 4 days of injection were monitored until complete recovery of muscle wasting to a DAS of 0 (no muscle wasting observed). [Figure 8] 8 shows an SDS PAGE of purified recombinant BoNT / BC (SEQ ID NO:56). Lanes are labeled "marker" (molecular weight marker), "-DTT" (oxidized BoNT / BC chimera sample), and "+DTT" (reduced BoNT / BC chimera sample). [Figure 9]9 shows the cleavage of VAMP-2 by native BoNT / B, BoNT / BC chimera and inactive recombinant BoNT / B (SEQ ID NO: 2, 56 and 57, respectively) in rat cortical neurons. Cells were exposed to various concentrations of BoNT for 24 hours at 37°C in a humidified CO2 atmosphere containing 5% CO2. Cells were lysed with 1x NuPAGE buffer supplemented with DTT and benzonase, heated to 90°C for 5 minutes and then stored at -20°C. Samples were analyzed for VAMP-2 cleavage by Western blot using polyclonal rabbit anti-VAMP-2 (Abcam ab3347, 1:1000) primary antibody and HRP-conjugated anti-rabbit secondary antibody (Sigma no. A6154). [Figure 10] 10 illustrates cleavage of the VAMP-2 peptide reporter by native BoNT / B and BoNT / BC chimeras (SEQ ID NOs: 2 and 56, respectively) using the BoTest® kit (BioSentinel). Various concentrations of BoNT were incubated with a VAMP-2 peptide bearing a CFP-YFP FRET pair as a reporter for 18 hours at 30° C., and the ratio of uncleaved versus cleaved reporter substrate was measured as the loss of YFP fluorescence intensity at 528 nm and the gain of CFP fluorescence at 485 nm after excitation at 440 nm. EXAMPLES

[0120] The following examples serve to illustrate certain embodiments of the invention and in no way limit the scope of the invention as defined in the claims.

[0121] Example 1 – 3 in Clostridial neurotoxins 10 Mapping the helix

[0122] The amino acid sequences of all BoNT serotypes and TeNT were obtained from public databases (e.g., www.uniprot.org or http: / / www.ncbi.nlm.nih.gov / ) and then modeled onto the known crystal structure of BoNT / A1 (3BTA.pdb) using www.loopp.org. CN The N-terminal part of the domain and the approximately 80 residues preceding it (H N This resulted in a predicted protein structure that was edited to retain only the C-terminal portion of the domain (the “H N / H CN ") is structurally highly conserved, making it the best region to superimpose the various serotypes. Each edited structure was then superimposed onto the 3BTA.pdb using http: / / wishart.biology.ualberta.ca / superpose / , and the H of BoNT / A1 was then compared to the H of BoNT / A1. C The three most prominent 10 Residues corresponding to the helix ( 872 NINTS 876 ) and corresponding residues in other serotypes, which were matched against a sequence alignment of all BoNT serotypes using Clustal Omega (www.ebi.ac.uk / Tools / msa / clustalo / ) (Figure 1).

[0123] By identifying this region that is structurally equivalent among various neurotoxins, it was possible to identify a specific point at which the C-terminal half of one neurotoxin could be transferred to the N-terminal half of another neurotoxin without disturbing the secondary structure of the entire molecule. 10 It was chosen to be the beginning of a helix.

[0124] The results are shown in Table 2 above.

[0125] Example 2 - Cloning, expression and purification of BoNT / AB chimeras

[0126] BoNT / AB chimeric constructs 1, 2, 3A, 3B and 3C (SEQ ID NOs: 9-13) were constructed using standard molecular biology techniques from DNA encoding the parent serotype molecules and appropriate oligonucleotides. These were then enriched with a C-terminal His 10 The A-tags were cloned into the pJ401 expression vector and transformed into BLR(DE3) E. coli cells for overexpression. The cells were grown in 2L baffled Erlenmeyer flasks containing 1L of modified Terrific Broth (mTB) supplemented with the appropriate antibiotic at 37°C and 225 RPM shaking. 600 Once a β-actin ratio of >0.5 was achieved, the incubator temperature was reduced to 16° C., followed one hour later by induction with 1 mM IPTG for 20 hours with 225 RPM shaking to allow expression of the recombinant BoNT / AB construct.

[0127] The harvested cells were lysed by sonication and clarified by centrifugation at 4500 RPM for 1 hour at 4°C. The recombinant BoNT / AB chimera molecule was then extracted with ammonium sulfate and purified by standard fast protein liquid chromatography (FPLC) techniques, which included using a hydrophobic interaction resin for capture and an anion exchange resin for intermediate purification steps. The partially purified molecule was then proteolytically cleaved with endoprotease Lys-C to obtain the active duplex, which was further purified using a second hydrophobic interaction resin to obtain the final BoNT / AB chimera.

[0128] Decahistidine tag (H 10 For BoNT / AB chimera molecules having the following structure (chimeras 1, 2, 3A), the capture step employed the use of immobilized nickel resin instead of a hydrophobic interaction resin.

[0129] The sequences of each chimera are shown in Table 3. [Table 3]

[0130] Example 3 – Comparison of BoNT / AB chimeras 1, 2 and 3A

[0131] C-terminal His 10 BoNT / AB chimeras 1, 2 and 3A carrying the tag and the E1191M / S1199Y double mutation were purified as described in Example 1 (FIG. 2) and tested for functional activity.

[0132] Rat spinal cord neuron SNAP-25 cleavage assay

[0133] Primary cultures of rat spinal cord neurons (SCN) were prepared and grown for 3 weeks in 96-well tissue culture plates (as described in Masuyer et al., 2011, J. Struct. Biol. Structure and activity of a functional derivative of Clostridium botulinum neurotoxin B, and Chaddock et al., 2002, Protein Expr. Purif. Expression and purification of catalytically active, non-toxic endopeptidase derivatives of Clostridium botulinum toxin type A). Serial dilutions of BoNT / AB were prepared in SCN feeding medium. Growth medium from the wells to be treated was collected and filtered (0.2 μm filter). 125 μL of filtered medium was returned to each test well. 125 μL of diluted toxin was then added to the plate (triplicate wells). Treated cells were incubated at 37° C., 10% CO2 for 24±1 h.

[0134] Analysis of BoNT activity using a SNAP-25 cleavage assay

[0135] After treatment, BoNT was removed and cells were washed once with PBS (Gibco, UK). Cells were lysed with 1x NuPAGE lysis buffer (Life Technologies) supplemented with 0.1M dithiothreitol (DTT) and 250 units / mL benzonase (Sigma). Lysate proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were probed with a primary antibody specific for SNAP-25 (Sigma no. S9684), which recognizes uncleaved SNAP-25 as well as SNAP-25 cleaved by BoNT / A endopeptidase. The secondary antibody used was anti-rabbit IgG conjugated with HRP (Sigma no. A6154). Bands were detected by enhanced chemiluminescence and imaged using pXi6 Access (Synoptics, UK). Band intensities were determined using GeneTools software (Syngene, Cambridge, UK) and the percentage of SNAP-25 cleaved by each concentration of BoNT was calculated. Data were fitted to a four-parameter logistic equation and analyzed for pEC using GraphPad Prism version 6 (GraphPad). 50 was calculated.

[0136] Table 4 below shows the pECs determined for chimeras 1, 2 and 3A in the rat SCN SNAP-25 cleavage assay. 50 These results indicate that the three BoNT / AB chimeras retained the ability to enter rat spinal cord neurons and cleave their target substrates. However, chimera 3A was more potent than chimeras 1 and 2 in this assay (see also FIG. 3). [Table 4]

[0137] Digital Abduction Scoring (DAS) Assay

[0138] The method for measuring the activity of BoNT / AB chimeras 1, 2 and 3A in the DAS assay is based on the startled response toe spreading reflex in mice when briefly suspended by their tails. This reflex is scored as the digit abduction score (DAS) and is inhibited after administration of BoNT into the gastrocnemius-soleus muscle of the hind limb. Mice are briefly suspended by their tails to elicit a characteristic startle response in which the animals extend their hind limbs and then abduct their digits (Aoki et al. 1999, Eur. J. Neurol.; 6 (suppl. 4) S3-S10).

[0139] On the day of injection, mice were anesthetized in an induction chamber receiving 3% isoflurane in oxygen. Each mouse received an intramuscular injection of the BoNT / AB chimera or vehicle (phosphate buffer containing 0.2% gelatin) in the gastrocnemius-soleus muscle of the right hind limb.

[0140] After neurotoxin injection, the variable degree of finger abduction was scored on a scale of zero to four, with 0=normal and 4=maximal reduction in finger abduction and leg extension. The ED50 was determined by nonlinear adjustment analysis using the average of the maximum effect at each dose. The mathematical model used was a four-parameter logistic model.

[0141] The DAS was performed every 2 hours for the first day after dosing, then three times per day for four days.

[0142] Figure 4 shows the fitted curves of chimeras 1, 2 and 3A (SEQ ID NO: 9, 10 and 11, respectively). The chimera 3A curve is shifted to the left, which means that low doses of chimera 3A achieved similar DAS responses compared to chimeras 1 and 2, thus indicating that chimera 3A is more potent than the others in the mouse DAS assay; see also the table below (Table 5) which provides the calculated ED50 and dose values ​​leading to DAS4 (highest score) for each chimera.

[0143] Table 5 below shows the EDs determined for recombinant BoNT / A1 (rBoNT / A1) and chimeras 1, 2, and 3A in the mouse DAS assay. 50 and DAS4. These results indicate that of the three chimeras, chimera 3A has the highest in vivo efficacy in inducing muscle wasting. The studies shown in Figure 4 and Table 5 were performed in mice obtained from Charles River Laboratories. [Table 5]

[0144] Example 4 – Comparison of BoNT / AB chimeras 3B, 3C and BoNT / A1

[0145] The untagged BoNT / AB chimeras 3B and 3C (sequence numbers 12 and 13), respectively, with and without the presence of the E1191M / S1199Y double mutation, were purified as described in Example 1 (Figure 5) and tested for functional activity using recombinant BoNT / A1 (sequence number 1) as a reference.

[0146] Human pluripotent stem cell SNAP-25 cleavage assay

[0147] Cryopreserved PERI.4U-cells were purchased from Axiogenesis (Cologne, Germany). Thawing and plating of cells was performed as recommended by the manufacturer. Briefly, cryovials containing cells were thawed for 2 minutes in a water bath at 37°C. After gentle resuspension, cells were transferred to a 50 mL tube. The cryovial was washed with 1 mL of Peri.4U® Thawing Medium supplied by the manufacturer, and the medium was transferred dropwise to the cell suspension in a 50 mL tube, after which an additional 2 mL of Peri.4U® Thawing Medium dropwise was added to the 50 mL tube. Cells were then counted using a hemocytometer. After this, an additional 6 mL of Peri.4U® Thawing Medium was added to the cell suspension. A cell pellet was obtained by centrifugation at 260xg (e.g., 1,100 RPM) for 6 minutes at room temperature. Cells were then resuspended in complete Peri.4U® culture medium supplied by the manufacturer. Cells were then cultured in cm 2 The cells were plated on cell culture plates coated with poly-L-ornithine and laminin at a density of 50,000–150,000 cells per well. The cells were cultured at 37°C in a humidified CO2 atmosphere, and the medium was changed completely every 2–3 days during culture.

[0148] For toxin treatment, serial dilutions of BoNT were prepared in Peri.4U® culture medium. The medium from the wells to be treated was collected and filtered (0.2 μm filter). 125 μL of filtered medium was returned to each test well. 125 μL of diluted toxin was then added to the plate (triplicate wells). Treated cells were incubated at 37° C., 10% CO2 for 48±1 hours.

[0149] Analysis of BoNT activity using a SNAP-25 cleavage assay

[0150] After treatment, BoNT was removed and cells were washed once with PBS (Gibco, USA). Cells were lysed with 1× NuPAGE lysis buffer (Life Technologies) supplemented with 0.1 M dithiothreitol (DTT) and 250 units / mL benzonase (Sigma). Lysate proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were probed with a primary antibody specific for SNAP-25 (Sigma no. S9684), which recognizes uncleaved SNAP-25 as well as SNAP-25 cleaved by BoNT / A endopeptidase. The secondary antibody used was anti-rabbit IgG conjugated with HRP (Sigma no. A6154). Bands were detected by enhanced chemiluminescence and imaged using pXi6 Access (Synoptics, UK). Band intensities were examined using GeneTools software (Syngene, Cambridge, UK) and the percentage of SNAP-25 cleaved by each concentration of BoNT was calculated. Data were fitted to a four-parameter logistic equation and analyzed for pEC using GraphPad Prism version 6 (GraphPad). 50 was calculated.

[0151] Figure 6 shows that chimeras 3B and 3C were more potent than rBoNT / A1 in cleaving SNAP-25 in induced human pluripotent stem cells, with the former being significantly more potent, which may be explained by a double mutation that increases the affinity of chimera 3B for the human synaptotagmin II protein receptor present in these cells (Figure 6, Table 6). [Table 6]

[0152] Digital Abduction Scoring (DAS) Assay - Safety Factor

[0153] The method for measuring the activity of BoNT in the DAS assay is based on the startle response toe extension reflex of mice when briefly suspended by their tail. This reflex is scored as the digit abduction score (DAS) and is inhibited after administration of BoNT into the gastrocnemius-soleus muscle of the hind limb. Mice are briefly suspended by their tail to elicit a characteristic startle response in which the animal extends its hind limbs followed by abduction of the digits (Aoki et al. 1999, Eur. J. Neurol.; 6 (suppl. 4) S3-S10).

[0154] On the day of injection, mice were anesthetized in an induction chamber receiving 3% isoflurane in oxygen. Each mouse received an intramuscular injection of BoNT or vehicle (phosphate buffer containing 0.2% gelatin) in the gastrocnemius-soleus muscle of the right hind leg.

[0155] After neurotoxin injection, the variable degree of finger abduction was scored on a scale of zero to four, with 0=normal and 4=maximal reduction in finger abduction and leg extension. The ED50 was determined by nonlinear adjustment analysis using the average of the maximum effect at each dose. The mathematical model used was a four-parameter logistic model.

[0156] For all doses, DAS was performed every 2 hours for the first day after dosing, then 3 times per day for 4 days. Animals in the groups injected with vehicle and the lowest dose that induced a DAS of 4 during the first 4 days of injection were then monitored until complete recovery of muscle weakness to a DAS of 0 (no muscle weakness observed).

[0157] To calculate the safety factor, all animals were weighed the day before toxin injection (D0) and once a day thereafter throughout the study period. The mean body weight, its standard deviation and standard error of the mean were calculated daily for each dose group. The safety factor of BoNT (-10%ΔBW / ED 50 To obtain the ED of the BoNT being studied, at any time during the study, the mean body weight of a dose group was 10% lower than the mean body weight of that same dose group at D0. 50A lethal dose was defined as the dose at which one or more animals in that dose group died.

[0158] FIG. 7 shows the duration of muscle weakness over time in a mouse digit abduction scoring assay for rBoNT / A1, chimera 3B and chimera 3C (SEQ ID NOs: 1, 12 and 13), indicating that the chimeras have a longer duration of action.

[0159] Table 7 below shows the EDs determined for rBoNT / A1 and chimeras 3B and 3C in the mouse DAS assay. 50 and DAS4 doses are provided. The table also provides the total duration of action of the DAS4 dose until complete recovery of muscle weakness (no muscle weakness observed) to a DAS of 0. Additionally, the table provides the mouse lethal dose and safety factor (-10%ΔBW / ED 50 ) are shown. Compared to rBoNT / A1, chimeras 3B and 3C have a longer duration of action, a better safety rate and a higher lethal dose. The studies shown in FIG. 7 and Table 7 were performed in mice obtained from the Janvier laboratory. [Table 7]

[0160] Example 5 - Expression and purification of BoNT / BC chimeras and confirmation of functional activity

[0161] BoNT / BC chimera 4 (sequence number 56) was cloned, expressed, and purified as described in Example 2, except for the use of a different expression cell line (BL21) and proteolytic cleavage with trypsin rather than endoprotease Lys-C (Figure 8). [Table 8]

[0162] This chimera was tested for functional activity in a VAMP-2 cleavage assay.

[0163] Rat cortical neuron VAMP-2 cleavage assay

[0164] Rat cortical neurons were prepared and maintained on poly-L-ornithine (PLO)-coated 96-well plates at a density of 20,000 cells / well in 125 μL of Neurobasal medium containing 2% B27 nutrient supplement, 0.5 mM GlutaMAX, 1% fetal bovine serum (FBS) and 100 U / mL penicillin / streptomycin at 37 °C in a humidified atmosphere containing 5% CO2. On day 4 of in vitro culture, an additional 125 μL of Neurobasal medium containing 2% B27, 0.5 mM GlutaMAX was added. Cells were maintained by replacing half the medium every 3-4 days. On day 11 of in vitro culture, 1.5 μM cytosine β-D-arabinofuranoside (AraC) was added to the medium to prevent the proliferation of non-neuronal cells. On days 19–21 of in vitro culture, cortical neurons were treated with a range of concentrations of BoNT (30 fM–3 nM) for 24 h at 37°C.

[0165] Analysis of BoNT activity using a VAMP-2 cleavage assay

[0166] Cells were washed briefly with assay medium (Neurobasal w / o phenol red, 2% B27, 0.5 mM GlutaMAX, 10 μM TFB-TBOA ((3S)-3-[[3-[[4-(trifluoromethyl)benzoyl]amino]phenyl]methoxy]-L-aspartic acid) and then lysed in 100 μL of lysis buffer (NuPage LDS sample buffer, 1 mM DTT and 1:500 benzonase) and heated at 90 °C for 5 min. 15 μL of lysate was run on a 12% Bis-Tris gel with MES buffer at 200 V for 50 min. Proteins were transferred onto nitrocellulose membranes by Transblot Turbo (Biorad) using a low MW program. Membranes were blocked with 5% low fat milk in PBST and then probed with rabbit anti-VAMP-2 (Abcam ab3347, 1:1000) primary antibody, followed by HRP-conjugated anti-rabbit secondary antibody (Sigma #A6154). Membranes were developed with SuperSignal West Dura chemiluminescent substrate and visualized using a Syngene PXi system. Band densitometry was analyzed using GeneTools software (Syngene) to determine the percentage of VAMP-2 cleavage at each concentration of BoNT relative to control wells. Data were fitted to a four-parameter logistic equation and pEC 50 was calculated.

[0167] Figure 9 shows that chimera 4 can bind to rat spinal cord neurons, translocate into the cytoplasm, and specifically cleave its substrate VAMP-2. As a reference point, this chimera is clearly functional compared to the inactive recombinant BoNT / B1 molecule (also referred to herein as BoNT / B1(0) with double mutations at E231Q and H234Y) (SEQ ID NO: 57), and is almost as active as the natural BoNT / B1 molecule (SEQ ID NO: 2) (Table 9). This may be explained by the high affinity binding of BoNT / B to synaptotagmin and various gangliosides present on the rat cell surface, while the binding domain of C in chimera 4 is known to bind gangliosides only with lower affinity. This is supported by data obtained from light chain protease activity assays as further shown below. [Table 9]

[0168] Light chain protease activity assay

[0169] Serotype B light chain activity was assessed using the BoTest® (BioSentinel A1009) cell-free assay according to the manufacturer's instructions. For example, BoNT was diluted to 1.39 nM in BoTest reaction buffer (50 mM HEPES-NaOH, 5 mM NaCl, 10 μM ZnCl2, 0.1% Tween-20, 0.1 mg / ml BSA, pH 7.1) and reduced with 5 mM DTT for 30 min at room temperature. A final concentration of 200 nM of VAMP-2 peptide reporter (CFP-VAMP-2(33-94)-YFP in 50 mM HEPES-NaOH, 10 mM NaCl, 15% glycerol) was combined with a concentration range of BoNT (500 fM to 1.25 nM, final) in a black Maxisorp plate (Nunc) in a final assay volume of 100 μL / well. Plates were sealed and incubated away from light at 30°C for 18 hours. The loss of CFP to YFP FRET fluorescence at 528 nm and the gain of GFP fluorescence at 485 nm after excitation at 440 nm were measured using a BioTek Synergy HT plate reader. The fluorescence emission ratio of uncleaved to cleaved reporter substrate at each BoNT concentration was fitted to a four-parameter logistic equation and calculated as pEC 50 was calculated.

[0170] Light chain protease activity assays confirm that the light chain of chimera 4 is as active as the one of native BoNT / B1 (see FIG. 10 and Table 10), and therefore, as explained above, the results of the VAMP-2 cleavage assay can be explained by the fact that BoNT / B has a higher affinity for synaptotagmin and various gangliosides present on the rat cell surface compared to the binding domain of C in chimera 4, which only binds to gangliosides. [Table 10] [Explanation of symbols]

[0171] [Figures 2, 5, 8] Marker: Marker SEQ ID No.: Sequence number [Figure 3] %SNAP-25 cleavage (in rat spinal cord neurons): % of SNAP-25 cleavage (in rat spinal cord neurons) Un-Tr: Untreated SEQ ID NO: Sequence number [Figure 4] Mean DAS: Average DAS Dose (pg / mouse): Dose (pg / mouse) SEQ ID NO: Sequence number [Figure 6] %SNAP-25 cleavage (in human pluripotent stem cells): % of SNAP-25 cleavage (in human pluripotent stem cells) Un-Tr: Untreated SEQ ID NO: Sequence number [Figure 7] Time (day): Time (day) SEQ ID NO: Sequence number [Figure 9] %VAMP2 cleavage (in rat spinal cord neurons): % of VAMP2 cleavage (in rat spinal cord neurons) Blank: Blank SEQ ID NO: Sequence number [Figure 10] RFU ratio: RFU ratio Blank: Blank SEQ ID NO: Sequence number

Claims

1. H from a second neurotoxin C LH derived from the first neurotoxin covalently linked to the domain N 1. A chimeric neurotoxin comprising a domain, wherein said first and second neurotoxins are different; The above LH N The C-terminal amino acid residue of the domain is LH N and H C Separate domains 3 10 corresponding to the first amino acid residue of the helix, The above H C The N-terminal amino acid residue of the domain is LH N and H C Separate domains 3 10 A chimeric neurotoxin corresponding to the second amino acid residue of the helix.

2. The chimeric neurotoxin of claim 1, wherein the first neurotoxin is botulinum neurotoxin (BoNT) serotype A, serotype B, serotype C, serotype D, serotype E, serotype F or serotype G or tetanus neurotoxin (TeNT), and the second neurotoxin is botulinum neurotoxin (BoNT) serotype A, serotype B, serotype C, serotype D, serotype E, serotype F or serotype G or tetanus neurotoxin (TeNT).

3. LH derived from the first neurotoxin N The domain is - 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 Corresponding to, H from the second neurotoxin C The domain is - 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 3. The chimeric neurotoxin of claim 1 or 2, which corresponds to:

4. 4. The chimeric neurotoxin of claim 1, 2 or 3, wherein the first neurotoxin is BoNT / A and the second neurotoxin is BoNT / B.

5. 5. The chimeric neurotoxin of claim 4, wherein the first neurotoxin is BoNT / A1 and the second neurotoxin is BoNT / B1.

6. LH derived from the first neurotoxin N The domain corresponds to amino acid residues 1-872 of BoNT / A1 and is derived from the second neurotoxin. C The chimeric neurotoxin of claim 5, wherein the domain corresponds to amino acid residues 860 to 1291 of BoNT / B1.

7. H derived from the BoNT / B neurotoxin C The H domain has the effect of increasing the binding affinity of the BoNT / B neurotoxin to the human Syt II receptor as compared to the native BoNT / B sequence. CC 7. The chimeric neurotoxin of claim 4, 5 or 6, comprising at least one amino acid residue substitution, addition or deletion in the subdomain.

8. The above H CC 8. The chimeric neurotoxin of claim 7, wherein at least one amino acid residue substitution, addition or deletion in the subdomain comprises a substitution mutation selected from the group consisting of V1118M, Y1183M, E1191M, E1191I, E1191Q, E1191T, S1199Y, S1199F, S1199L, S1201V, E1191C, E1191V, E1191L, E1191Y, S1199W, S1199E, S1199H, W1178Y, W1178Q, W1178A, W1178S, Y1183C, Y1183P and combinations thereof.

9. The above H CC 8. The chimeric neurotoxin of claim 7, wherein at least one amino acid residue substitution, addition or deletion in the 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. The chimeric neurotoxin of claim 9, wherein the two substitution mutations are E1191M and S1199Y.

11. The above H CC 8. The chimeric neurotoxin of claim 7, wherein at least one amino acid residue substitution, addition or deletion in the subdomain comprises three substitution mutations: E1191M, S1199W and W1178Q.

12. 4. The chimeric neurotoxin of claim 1, 2 or 3, wherein the first neurotoxin is BoNT / B and the second neurotoxin is BoNT / C.

13. 13. The chimeric neurotoxin of claim 12, wherein the first neurotoxin is BoNT / B1 and the second neurotoxin is BoNT / C1.

14. LH derived from the first neurotoxin N The domain corresponds to amino acid residues 1-859 of BoNT / B1 and is derived from the second neurotoxin. C The chimeric neurotoxin of claim 13, wherein the domain corresponds to amino acid residues 868 to 1291 of BoNT / C1.

15. A nucleotide sequence encoding the chimeric neurotoxin of any one of claims 1 to 14.

16. A vector comprising the nucleotide sequence of claim 15.

17. 17. A cell comprising the nucleotide sequence of claim 15 or the vector of claim 16.

18. 15. A pharmaceutical composition comprising the chimeric neurotoxin of any one of claims 1 to 14.

19. 20. A kit comprising the pharmaceutical composition of claim 18 and instructions for therapeutic or cosmetic administration of the composition to a subject in need thereof.

20. A method for producing a chimeric neurotoxin described in any one of claims 1 to 14, comprising culturing a cell described in claim 17 under conditions in which the chimeric neurotoxin is produced.

21. 19. A chimeric neurotoxin according to any one of claims 1 to 14 or a pharmaceutical composition according to claim 18 for use in therapy.

22. 22. The chimeric neurotoxin or pharmaceutical composition of claim 21 for use in the treatment of a condition associated with unwanted neuronal activity, such as a condition selected from the group consisting of spasmodic dysphonia, spasmodic torticollis, laryngeal dystonia, oromandibular dysphonia, lingual dystonia, cervical dystonia, palmar dystonia, blepharospasm, strabismus, hemifacial spasm, eyelid disorders, cerebral palsy, focal spasticity and other voice disorders, spasmodic colitis, irritable bladder, anismus, limb spasticity, tics, tremors, bruxism, anal fissures, achalasia, dysphagia and other muscle tone disorders and other disorders characterized by involuntary movement of muscle groups, lacrimation, excessive sweating, excessive salivation, excessive gastrointestinal secretions, secretory disorders, pain resulting from muscle spasms, headache pain, migraine and dermatological conditions.

23. 19. Non-therapeutic use of a chimeric neurotoxin according to any one of claims 1 to 14, or of a pharmaceutical composition according to claim 18, for treating an aesthetic or cosmetic condition.

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