Clostridial neurotoxins containing activated exogenous protease cleavage sites.

Abstract

The present invention relates to clostridial neurotoxins modified to contain an exogenous protease cleavage site within the modified activation loop, where cleavage at said site produces an active di-chain clostridial neurotoxin. The invention also relates to methods for their preparation, as well as related pharmaceutical compositions, nucleotide sequences, and therapeutic and cosmetic uses. The invention further relates to methods for proteolytically cleaving said single-chain clostridial neurotoxins into the corresponding di-chain clostridial neurotoxins.

Description

[Technical Field]

[0001] The present invention relates to clostridial neurotoxins modified to contain an exogenous protease cleavage site within the modified activation loop, where cleavage at said site produces an active di-chain clostridial neurotoxin. The invention also relates to methods for their preparation, as well as related pharmaceutical compositions, nucleotide sequences, and therapeutic and cosmetic uses. The invention further relates to methods for proteolytically cleaving said single-chain clostridial neurotoxins into the corresponding di-chain clostridial neurotoxins. [Background technology]

[0002] Bacteria of the genus Clostridium produce highly potent and specific protein toxins that can harm neurons and other cells to which they are delivered. Examples of such clostridial neurotoxins include those produced by Clostridium tetani (TeNT) and Clostridium botulinum (BoNT) serotypes A to G and X (see WO 2018 / 009903 A2), as well as those produced by Clostridium baratii and Clostridium butyricum.

[0003] Some of the Clostridial neurotoxins are among the most potent known toxins. For example, the median lethal dose (LD) of botulinum neurotoxin in mice is 50 ) values ​​range from 0.5 to 5 ng / kg depending on the serotype. Both tetanus toxin and botulinum toxin act by inhibiting the function of affected neurons, specifically by inhibiting the release of neurotransmitters. Botulinum toxin acts at the neuromuscular junction and inhibits cholinergic transmission in the peripheral nervous system, while tetanus toxin acts in the central nervous system.

[0004] Clostridial neurotoxins are expressed in Clostridium as single-chain polypeptides. Each clostridial neurotoxin has a catalytic light chain that is separated from the heavy chain (which includes an N-terminal translocation domain and a C-terminal receptor-binding domain) by an exposed region called the activation loop. During protein maturation, proteolytic cleavage of the activation loop separates the light and heavy chains of the clostridial neurotoxin (which are linked by a disulfide bridge) to generate the fully activated, di-chain toxin.

[0005] This activation process must be replicated in standard production of recombinant toxins. In traditional production methods, exogenous proteases with well-defined cleavage motifs, such as trypsin or Lys-C, are used to proteolytically activate single-chain clostridial neurotoxins. However, for some clostridial neurotoxins, incubation with Lys-C or trypsin results in partial or inappropriate cleavage of the single-chain polypeptide, resulting in the generation of impurity, single-chain, and / or inactive cleavage / degradation products (e.g., in the case of BoNT / E). For example, botulinum neurotoxin serotype X (BoNT / X, see WO 2018 / 009903 A2) has problems with activation, and cleavage with trypsin or Lys-C results in complete degradation of the polypeptide. Therefore, there is currently no universal exogenous protease for activating clostridial neurotoxins. This is particularly problematic when new clostridial neurotoxins are identified or when engineered (e.g., chimeric or hybrid) neurotoxins are created. In this case, screening of multiple proteases is required to determine correct activation. In retargeted Clostridial neurotoxins, some of the standard proteases used for activation may also cleave within the exogenous targeting moiety, resulting in a mis-cleaved protein with reduced targeting to the desired cell type. To avoid such off-target cleavage, an alternative targeting moiety must be identified (which may not always be possible), or the targeting moiety must be designed to remove the cleavage site for the standard protease, which may adversely affect the structure of the targeting moiety and / or increase design and manufacturing costs.

[0006] Furthermore, in vitro activation of clostridial neurotoxins is associated with many drawbacks. The use of exogenous proteases (especially GMP-grade proteases) and their removal after clostridial neurotoxin activation are costly. Reliance on a single or limited number of suppliers of GMP-grade proteases can also create weaknesses in the supply / production chain. Purification of activated clostridial neurotoxins from activated exogenous proteases can also affect production efficiency and yield. Furthermore, production of active dichain clostridial neurotoxins by conventional manufacturing methods requires strict safety and control procedures, which increases production costs and time. Stringent safety precautions are also required for employees handling active dichain clostridial neurotoxins.

[0007] The present invention solves one or more of the problems set forth above. Summary of the Invention

[0008] The present inventors have previously demonstrated that inserting a furin cleavage site into the activation loop of a clostridial neurotoxin enables in vivo activation of the clostridial neurotoxin (see PCT Application No. PCT / GB2022 / 050756; incorporated herein by reference in its entirety). They have also demonstrated that endosomal proteases can be used to cleave clostridial neurotoxins, opening a new paradigm that enables both in vivo activation of clostridial neurotoxins and provides an alternative means for in vitro activation. The present inventors have also previously shown that the activation loop of BoNT / C1 can be used as a universal activation loop for clostridial neurotoxins (see WO2020 / 065336; incorporated herein by reference in its entirety). However, the use of the BoNT / C1 activation loop may be undesirable in some cases. In particular, the activation loop sequence of native BoNT / C1 is susceptible to cleavage by factor Xa, Lys-C, trypsin, and enterokinase. Some clostridial neurotoxins are susceptible to off-target cleavage by one or more of these enzymes. Furthermore, sourcing these proteases in GMP-compliant grades can be costly and difficult. Therefore, expanding the toolkit of modified clostridial neurotoxins and activating proteases would further benefit this field. In particular, it would be advantageous to provide a means to activate clostridial neurotoxins using proteases that do not suffer from the following problems: (i) sourcing GMP-compliant proteases; (ii) proteases that can function with the desired retargeted clostridial neurotoxin structure; (iii) reducing nonspecific cleavage and, therefore, reducing undesired truncation that requires additional purification and / or impacts yield; and / or (iv) improving utility across clostridial neurotoxin serotypes.

[0009] The present inventors have surprisingly demonstrated that cleavage sites specific for other exogenous proteases, particularly thrombin, tissue plasminogen activator (t-PA), and urokinase (u-PA), can be introduced into the modified BoNT / C1 activation loop, and that this modified BoNT / C1 activation loop can be used to engineer clostridial neurotoxins that can be cleaved and activated by these proteases. Thus, the introduction of one or more exogenous protease cleavage sites into the modified BoNT / C activation loop does not impair the function of the modified BoNT / C activation loop, and the modified BoNT / C activation loop can still be used as a universal activation loop for clostridial neurotoxins. Furthermore, the present inventors have even more surprisingly demonstrated that the modified clostridial neurotoxins can be activated by such proteases with potentially improved cleavage and specificity and reduced off-target protease activity, potentially improving activation rates, reducing the generation of truncated products, and allowing for the use of lower concentrations of the activating protease. Moreover, it may be advantageous to introduce such an exogenous cleavage site while maintaining the clostridial activation loop structure.

[0010] Furthermore, the modified clostridial neurotoxins of the present invention activated by exogenous proteases offer several advantages over clostridial neurotoxins activated by conventional methods, such as improved yields and / or reduced manufacturing burden / costs.

[0011] Thus, the present invention provides a modified clostridial neurotoxin, wherein the endogenous activation loop is replaced with a modified BoNT / C activation loop, and wherein the endogenous activation site of the BoNT / C activation loop, or a portion thereof, is replaced with an exogenous protease cleavage site, wherein cleavage at said cleavage site generates an (active) two-chain form of the modified clostridial neurotoxin.

[0012] The exogenous protease cleavage site is a cleavage site specific for an exogenous protease that may be selected from (a) thrombin; (b) tissue plasminogen activator (t-PA); and / or (c) urokinase (u-PA), where, optionally: (i) the exogenous protease is a human protease; (ii) the exogenous protease is included in the formulation; and / or (iii) the exogenous protease is a recombinant human protease.

[0013] The exogenous protease cleavage site may comprise or consist of (a) the thrombin consensus sequence of SEQ ID NO: 169 or 170; and / or (b) the uPA and / or t-PA consensus sequence of SEQ ID NO: 172 or 173. The exogenous protease cleavage site may comprise or consist of one or more of the following: LTPRGVRL (SEQ ID NO: 15), LVPRGS (SEQ ID NO: 16), ENKSLVPRGS (SEQ ID NO: 17), SGRSA (SEQ ID NO: 25), PPFGRSAG (SEQ ID NO: 33), PGSGRSAG (SEQ ID NO: 26), PGSGRSASGTTGTG (SEQ ID NO: 27), KRV (SEQ ID NO: 24), GRI (SEQ ID NO: 44), or PGRVVGG (SEQ ID NO: 50). The length of the exogenous protease cleavage site may be 3 to 10 amino acids, preferably 3 to 8 amino acids. The exogenous protease cleavage site may consist of: LTPRGVRL (SEQ ID NO: 15), LVPRGS (SEQ ID NO: 16), ENKSLVPRGS (SEQ ID NO: 17), SGRSA (SEQ ID NO: 25), PPFGRSAG (SEQ ID NO: 33), PGSGRSAG (SEQ ID NO: 26), PGSGRSASGTTGTG (SEQ ID NO: 27), KRV (SEQ ID NO: 24), GRI (SEQ ID NO: 44) or PGRVVGG (SEQ ID NO: 50).

[0014] The exogenous protease cleavage site may comprise or consist of LTPRGVRL (SEQ ID NO: 15), LVPRGS (SEQ ID NO: 16) or ENKSLVPRGS (SEQ ID NO: 17), preferably LTPRGVRL (SEQ ID NO: 15), and the exogenous protease cleavage site may be specific for thrombin, optionally wherein the exogenous protease cleavage site consists of LTPRGVRL (SEQ ID NO: 15), LVPRGS (SEQ ID NO: 16) or ENKSLVPRGS (SEQ ID NO: 17), preferably LTPRGVRL (SEQ ID NO: 15).

[0015] The exogenous protease cleavage site may comprise or consist of KRV (SEQ ID NO:24), SGRSA (SEQ ID NO:25), PPFGRSAG (SEQ ID NO:33), PGSGRSAG (SEQ ID NO:26), or PGSGRSASGTTGTG (SEQ ID NO:27), and the exogenous protease cleavage site may be specific for t-PA and / or u-PA, where optionally the exogenous protease cleavage site consists of KRV (SEQ ID NO:24), SGRSA (SEQ ID NO:25), PPFGRSAG (SEQ ID NO:33), PGSGRSAG (SEQ ID NO:26), or PGSGRSASGTTGTG (SEQ ID NO:27).

[0016] The endogenous neurotoxin activation loop may be one or more selected from SEQ ID NOs: 67-90.

[0017] The exogenous protease cleavage site in the modified BoNT / C activation loop may be the only cleavage site for the exogenous protease in the modified clostridial neurotoxin, and contacting the modified clostridial neurotoxin with the exogenous protease may not result in off-site cleavage, and / or contacting the modified clostridial neurotoxin with the exogenous protease may not result in light chain shortening of the modified clostridial neurotoxin.

[0018] The clostridial neurotoxin may be (a) a botulinum neurotoxin (BoNT) serotype A, serotype B, serotype C, serotype D, serotype E, serotype F, serotype G, or serotype X, or a tetanus neurotoxin (TeNT); or (b) a chimeric or hybrid BoNT. The modified clostridial neurotoxin may be a modified clostridial neurotoxin that is a chimeric or hybrid BoNT. C or H CC may be a retargeted Clostridial neurotoxin in which is replaced with an exogenous targeting moiety (TM). In a particularly preferred embodiment, the Clostridial neurotoxin is BoNT / X.

[0019] The modified clostridial neurotoxin may be a single-chain clostridial neurotoxin that satisfies the following conditions: (a) (i) is encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 154, 156, 158, 160, 162, or 164; or (ii) is encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 175, wherein SEQ ID NO: 176 within SEQ ID NO: 175 is encoded by at least one exogenous protease as defined in any one of claims 3 to 9. and / or (b) a polypeptide sequence having at least 70% sequence identity to one or more of SEQ ID NOs: 155, 157, 159, 161, 163, 165, 166, 167, or 168; and / or (c) a polypeptide sequence having: (i) at least 70% sequence identity to SEQ ID NO: 150, wherein SEQ ID NO: 2 within SEQ ID NO: 150 is a nucleotide sequence encoding a modified BoNT / C activation loop; or (ii) at least 70% relative to SEQ ID NO: 152; or (iii) at least 70% relative to SEQ ID NO: 152; or (iv) at least 70% relative to SEQ ID NO: 152; or (v) at least 70% relative to SEQ ID NO: 152; or (vi ... wherein SEQ ID NO: 2 in SEQ ID NO: 152 is replaced by one or more exogenous protease cleavage sites and / or modified BoNT / C activation loops as defined in any one of claims 3 to 9, wherein the one or more exogenous protease sites are preferably selected from one or more of SEQ ID NOs: 15, 33, or 25, and / or the modified BoNT / C activation loop is preferably selected from one or more of SEQ ID NOs: 57, 63, and / or 65.

[0020] The present invention also provides a method for proteolytically cleaving a modified Clostridial neurotoxin of the present invention into a corresponding di-chain Clostridial neurotoxin, the method comprising contacting the modified Clostridial neurotoxin with an exogenous protease specific for the exogenous protease cleavage site, thereby producing a di-chain Clostridial neurotoxin. The present invention further provides a di-chain clostridial neurotoxin obtainable by the method, wherein, optionally, (a) the C-terminus of the clostridial neurotoxin light chain ends with the amino acid sequence LVPR, preferably ALVPR, and the N-terminus of the clostridial neurotoxin heavy chain begins with the amino acid sequence GSK or GSY; (b) the C-terminus of the clostridial neurotoxin light chain ends with the amino acid sequence SLVPR, and the N-terminus of the clostridial neurotoxin heavy chain begins with the amino acid sequence GSY; (c) the C-terminus of the clostridial neurotoxin light chain ends with the amino acid sequence LTPR, preferably ALTPR, and the N-terminus of the clostridial neurotoxin heavy chain begins with the amino acid sequence GVR, preferably GVRL; (d) the C-terminus of the clostridial neurotoxin light chain ends with the amino acid sequence PGR, and the N-terminus of the clostridial neurotoxin heavy chain begins with the amino acid sequence VVG; (e) the C-terminus of the clostridial neurotoxin light chain ends with the amino acid sequence DKR, preferably AIDKR. (f) the C-terminus of the clostridial neurotoxin light chain ends with the amino acid sequence DKR, preferably AIDK, and the N-terminus of the clostridial neurotoxin heavy chain begins with the amino acid sequence RVLY; (g) the C-terminus of the clostridial neurotoxin light chain ends with the amino acid sequence SGR, preferably PGSGR, and the N-terminus of the clostridial neurotoxin heavy chain begins with the amino acid sequence SA, preferably SAY, SAG, or SAS; (h) the C-terminus of the truncated modified clostridial neurotoxin light chain may end with the amino acid sequence SGR, preferably PGSGR, and the N-terminus of the truncated modified clostridial neurotoxin heavy chain may begin with the amino acid sequence TL, preferably TLD or TLDC; or (i) the C-terminus of the clostridial neurotoxin light chain ends with the amino acid sequence FGR, and the N-terminus of the clostridial neurotoxin heavy chain begins with the amino acid sequence SA, preferably SAG.

[0021] The present invention also provides polynucleotides encoding the modified Clostridial neurotoxins of the present invention.

[0022] The present invention further provides an expression vector comprising a polynucleotide of the present invention operably linked to a promoter.

[0023] The polynucleotide or expression vector can: (a) comprise a nucleotide sequence having: (i) at least 70% sequence identity to SEQ ID NO: 154, 156, 158, 160, 162, or 164; or (ii) at least 70% sequence identity to SEQ ID NO: 175, wherein SEQ ID NO: 176 within SEQ ID NO: 175 is replaced by a nucleotide sequence encoding at least one exogenous protease consensus sequence, cleavage site, or modified BoNT / C activation loop as defined in any one of claims 3-9; (b) encodes a polypeptide sequence having at least 70% sequence identity to one or more of SEQ ID NOs: 155, 157, 159, 161, 163, 165, 166, 167, or 168; and / or (c) encodes a polypeptide sequence having: (i) at least 70% sequence identity to SEQ ID NO: 150, wherein SEQ ID NO: 2 within SEQ ID NO: 150 is replaced by a nucleotide sequence encoding at least one exogenous protease consensus sequence, cleavage site, or modified BoNT / C activation loop as defined in any one of claims 3-9. or (ii) at least 70% identity to SEQ ID NO: 152, wherein SEQ ID NO: 2 within SEQ ID NO: 152 is replaced by one or more exogenous protease cleavage sites and / or modified BoNT / C activation loops defined in any one of claims 3-9, wherein the one or more exogenous protease sites are preferably selected from one or more of SEQ ID NOs: 15, 33, or 25, and / or the modified BoNT / C activation loop is preferably selected from one or more of SEQ ID NOs: 57, 63, and / or 65.

[0024] The present invention also provides a method for producing a modified Clostridial neurotoxin of the present invention, the method comprising expressing a polynucleotide of the present invention or an expression vector of the present invention in a cell and recovering the expressed modified Clostridial neurotoxin, and the method may further comprise introducing the polynucleotide of the present invention or the expression vector of the present invention into the cell.

[0025] The present invention further provides a cell expressing a modified Clostridial neurotoxin of the present invention, said cell optionally comprising a polynucleotide of the present invention or an expression vector of the present invention.

[0026] The present invention also provides pharmaceutical compositions comprising a modified clostridial neurotoxin of the present invention or a two-chain clostridial neurotoxin of the present invention and a pharmaceutically acceptable carrier, excipient, diluent, adjuvant, propellant and / or salt.

[0027] The present invention further provides a modified Clostridial neurotoxin of the present invention, a double-chain Clostridial neurotoxin of the present invention, or a pharmaceutical composition of the present invention for use in a method for preventing or treating a disease or disorder for which therapy with a botulinum neurotoxin is indicated, wherein optionally the disease or disorder is selected from the following: conditions associated with unwanted immune secretions, strabismus, blepharospasm, squint, dystonia (e.g., spastic dystonia, oromandibular dystonia, focal dystonia, tardive dystonia, laryngeal dystonia, limb dystonia, cervical dystonia), torticollis (e.g., spasmodic torticollis), cosmetic therapy (cosmetic) applications that would benefit from cell / muscle attenuation (by downregulating or inactivating SNAREs), neuromuscular disorders or conditions of eye movement (e.g., conjugate strabismus, hypertropia, extroversion, ectopia), Rectus paralysis, nystagmus, hypothyroid myopathy), writer's cramp, bruxism, Wilson's disease, tremors, tics, segmental myoclonus, spasms, spasticity due to chronic multiple sclerosis, spasticity resulting in bladder control disorders, animus, back spasms, cramps, levator pelvic syndrome, spina bifida, tardive dyskinesia, Parkinson's disease, stuttering, hemifacial spasm, eyelid disorders, cerebral palsy, focal spasticity, spastic colitis, neurogenic bladder, Anismus, limb spasticity, tics, tremors, teeth grinding, anal fissures, achalasia, dysphagia, lacrimation, hyperhidrosis, excessive salivation, gastrointestinal hypersecretion, muscle pain (e.g., pain due to muscle spasms), headache (e.g., tension headache or migraine), phantom pain (e.g., phantom limb pain), glabellar lines, skin wrinkles, cancer, uterine disorders, urogenital disorders, urogenital neuropathy, bladder pain syndrome, interstitial cystitis, chronic neurogenic inflammation, and smooth muscle disorders. Preferably, the composition of the present invention can be used for the prevention or treatment of a disease or condition selected from the following: limb spasticity (upper or lower limb); cervical dystonia; headache disorders (preferably migraine); blepharospasm; hemifacial spasm; and lower urinary tract disorders (e.g., bladder pain syndrome (preferably interstitial cystitis); overactive bladder; and detrusor overactivity (e.g., neurogenic detrusor overactivity).

[0028] The present invention also provides use of the modified Clostridial neurotoxin of the present invention, the double-chain Clostridial neurotoxin of the present invention, or the pharmaceutical composition of the present invention in the manufacture of a medicament for preventing or treating a disease or disorder for which therapy with a botulinum neurotoxin is indicated, wherein optionally the disease or disorder is selected from the following: conditions associated with unwanted immune secretions, strabismus, blepharospasm, squint, dystonia (e.g., spastic dystonia, oromandibular dystonia, focal dystonia, tardive dystonia, laryngeal dystonia, limb dystonia, cervical dystonia), torticollis (e.g., spasmodic torticollis), cosmetic therapy (cosmetic) applications that would benefit from cell / muscle attenuation (by downregulating or inactivating SNAREs), neuromuscular disorders or conditions of eye movement (e.g., conjugate strabismus, hypertropia, Lateral rectus paralysis, nystagmus, hypothyroid myopathy), writer's cramp, bruxism, Wilson's disease, tremors, tics, segmental myoclonus, spasms, spasticity due to chronic multiple sclerosis, spasticity resulting in bladder control disorders, animus, back spasms, cramps, levator pelvic syndrome, spina bifida, tardive dyskinesia, Parkinson's disease, stuttering, hemifacial spasm, eyelid disorders, cerebral palsy, focal spasticity, spastic colitis, neurogenic bladder , anismus, limb spasticity, tics, tremors, teeth grinding, anal fissures, achalasia, dysphagia, lacrimation, hyperhidrosis, excessive salivation, gastrointestinal hypersecretion, muscle pain (e.g., pain due to muscle spasms), headache (e.g., tension headache or migraine), phantom pain (e.g., phantom limb pain), glabellar lines, skin wrinkles, cancer, uterine disorders, urogenital disorders, urogenital neuropathy disorders, bladder pain syndrome, interstitial cystitis, chronic neurogenic inflammation, and smooth muscle disorders. Preferably, the composition of the present invention can be used for the prevention or treatment of a disease or condition selected from the following: limb spasticity (upper or lower limb); cervical dystonia; headache disorders (preferably migraine); blepharospasm; hemifacial spasm; and lower urinary tract disorders (e.g., bladder pain syndrome (preferably interstitial cystitis); overactive bladder; and detrusor overactivity (e.g., neurogenic detrusor overactivity).

[0029] In such a therapy, the clostridial neurotoxin may be administered to the subject in a single-chain form. The clostridial neurotoxin or pharmaceutical composition may be substantially free of a di-chain form of the clostridial neurotoxin. The clostridial neurotoxin or pharmaceutical composition may comprise less than 400 pg of di-chain clostridial neurotoxin per 100 ng of single-chain clostridial neurotoxin, or less than 300 pg of di-chain clostridial neurotoxin per 100 ng of single-chain clostridial neurotoxin, or less than 200 pg of di-chain clostridial neurotoxin per 100 ng of single-chain clostridial neurotoxin, or less than 100 pg of di-chain clostridial neurotoxin per 100 ng of single-chain clostridial neurotoxin, or less than 50 pg of di-chain clostridial neurotoxin per 100 ng of single-chain clostridial neurotoxin.

[0030] Alternatively, in such therapy, the clostridial neurotoxin may be administered to the subject in a di-chain form. The clostridial neurotoxin or pharmaceutical composition may be substantially free of single-chain forms of the clostridial neurotoxin. The clostridial neurotoxin or pharmaceutical composition may comprise less than 400 pg of single-chain clostridial neurotoxin per 100 ng of double-chain clostridial neurotoxin, or less than 300 pg of single-chain clostridial neurotoxin per 100 ng of double-chain clostridial neurotoxin, or less than 200 pg of single-chain clostridial neurotoxin per 100 ng of double-chain clostridial neurotoxin, or less than 100 pg of single-chain clostridial neurotoxin per 100 ng of double-chain clostridial neurotoxin, or less than 50 pg of single-chain clostridial neurotoxin per 100 ng of double-chain clostridial neurotoxin.

[0031] The present invention also provides a cosmetic composition comprising a modified Clostridial neurotoxin of the present invention or a two-chain Clostridial neurotoxin of the present invention and a cosmetically acceptable carrier, excipient, diluent, adjuvant, propellant and / or salt.

[0032] The present invention further provides the use of the cosmetic composition of the present invention for preventing or alleviating cosmetic indications for which application of a botulinum neurotoxin is indicated.

[0033] For such cosmetic indications, the clostridial neurotoxin may be for administration to a subject in a single-chain form. The clostridial neurotoxin or cosmetic composition may be substantially free of a di-chain form of the clostridial neurotoxin. The clostridial neurotoxin or cosmetic composition may comprise less than 400 pg of a di-chain clostridial neurotoxin per 100 ng of the single-chain clostridial neurotoxin, or less than 300 pg of a di-chain clostridial neurotoxin per 100 ng of the single-chain clostridial neurotoxin, or less than 200 pg of a di-chain clostridial neurotoxin per 100 ng of the single-chain clostridial neurotoxin, or less than 100 pg of a di-chain clostridial neurotoxin per 100 ng of the single-chain clostridial neurotoxin, or less than 50 pg of a di-chain clostridial neurotoxin per 100 ng of the single-chain clostridial neurotoxin.

[0034] Alternatively, for such cosmetic indications, the clostridial neurotoxin may be for administration to a subject in a single-chain form. The clostridial neurotoxin or cosmetic composition may be substantially free of a single-chain form of the clostridial neurotoxin. The clostridial neurotoxin or cosmetic composition may comprise less than 400 pg of single-chain clostridial neurotoxin per 100 ng of double-chain clostridial neurotoxin, or less than 300 pg of single-chain clostridial neurotoxin per 100 ng of double-chain clostridial neurotoxin, or less than 200 pg of single-chain clostridial neurotoxin per 100 ng of double-chain clostridial neurotoxin, or less than 100 pg of single-chain clostridial neurotoxin per 100 ng of double-chain clostridial neurotoxin, or less than 50 pg of single-chain clostridial neurotoxin per 100 ng of double-chain clostridial neurotoxin.

[0035] The present invention also provides a method for proteolytically cleaving a single-chain clostridial neurotoxin into a corresponding two-chain clostridial neurotoxin, the method comprising: (a) providing a single-chain clostridial neurotoxin; and (b) contacting the single-chain clostridial neurotoxin with an exogenous protease; wherein the single-chain clostridial neurotoxin has an exogenous protease cleavage site and / or a modified BoNT / C activation loop as defined herein, and wherein the exogenous protease hydrolyzes a peptide bond of the exogenous protease cleavage site and / or the modified BoNT / C activation loop, thereby generating a two-chain clostridial neurotoxin, wherein optionally the exogenous protease is selected from thrombin, t-PA, u-PA, FIX, and FVIIa.A single-chain Clostridial neurotoxin can: (a) be a modified Clostridial neurotoxin of the present invention; (b) be encoded by a nucleic acid sequence comprising or consisting of a nucleotide sequence having: at least 70% sequence identity to SEQ ID NO: 154, 156, 158, 160, 162, or 164; or (ii) at least 70% sequence identity to SEQ ID NO: 175, wherein SEQ ID NO: 176 within SEQ ID NO: 175 is replaced by a nucleotide sequence encoding at least one exogenous protease consensus sequence, cleavage site, or modified BoNT / C activation loop of the present invention; (c) comprise or consist of a polypeptide sequence having at least 70% sequence identity to one or more of SEQ ID NOs: 155, 157, 159, 161, 163, 165, 166, 167, or 168; and / or (d) comprise or consist of a polypeptide sequence having: (i) at least 70% sequence identity to SEQ ID NO: 150. or (ii) at least 70% identity to SEQ ID NO:152, wherein SEQ ID NO:2 within SEQ ID NO:152 is replaced by one or more exogenous protease cleavage sites and / or modified BoNT / C activation loops of the present invention, wherein the one or more exogenous protease sites are preferably selected from one or more of SEQ ID NOs:15, 33, or 25, and / or the modified BoNT / C activation loop is preferably selected from one or more of SEQ ID NOs:57, 63, and / or 65. [Brief explanation of the drawings]

[0036] [Figure 1]FIG. 1 is an SDS PAGE gel (+ / -DTT) showing the time course of cleavage of A4952 by different concentrations of u-PA. [Figure 2] FIG. 2 is an SDS PAGE gel (+ / -DTT) showing the time course of cleavage of A4953 by different concentrations of FIX. [Figure 3A] FIG. 3A is an SDS PAGE gel (+ / -DTT) showing the time course of cleavage of A4954 by different concentrations of thrombin. [Figure 3B] FIG. 3B is a mass spectrum of A4954 cleaved by thrombin. [Figure 3C] FIG. 3C is an SDS PAGE gel showing that thrombin can cleave the PGR cleavage site without truncation. [Figure 4A] Figure 4A shows an SDS PAGE gel (- / +DTT) showing the cleavage of modified BoNT with an ENKSLVPRGS-modified C-loop treated with thrombin, or modified BoNT with an SGRSA-modified C-loop treated with u-PA or two t-PA variants (full-length (t-PAfl) and functional fragment (t-PAfrag)) after 2 hours. Lane 1 = ENKSLVPRGS loop negative control; Lane 2 = SGRSA loop negative control; Lane 3 = ENKSLVPRGS loop treated with 0.39 μg of thrombin; Lane 4 = ENKSLVPRGS loop treated with 1.95 μg of thrombin; Lane 5 = ENKSLVPRGS loop treated with 3.90 μg of thrombin; Lane 6 = SGRSA loop treated with 0.39 μg of u-PA; Lane 7 = SGRSA loop treated with 1.95 μg of u-PA; Lane 8 = Lane 9 = SGRSA loop treated with 0.39 μg of t-PAfl; Lane 10 = SGRSA loop treated with 1.95 μg of t-PAfl; Lane 11 = SGRSA loop treated with 3.90 μg of t-PAfl; Lane 12 = SGRSA loop treated with 0.39 μg of t-PAfrag; Lane 13 = SGRSA loop treated with 1.95 μg of t-PAfrag; Lane 14 = SGRSA loop treated with 3.90 μg of t-PAfrag. [Figure 4B] FIG. 4B is a mass spectrum of A5045 cleaved by u-PA. [Figure 5A] Figure 5A is an SDS PAGE gel (- / +DTT) showing cleavage of a modified BoNT with an LTPRGVRL-modified C-loop treated with increasing concentrations of thrombin after 2 hours (top) or 20 hours (bottom). Potential truncation products are indicated by black arrows. [Figure 5B] Figure 5B is an SDS PAGE gel (- / +DTT) showing cleavage of modified BoNT with a PGSGRSA-modified C-loop treated with increasing concentrations of u-PA, t-PAfl, or t-PAfrag after 2 hours (left), 4 hours (center), or 20 hours (right). Lanes 1 through 9 are modified BoNT with a PGSGRSA-modified C-loop, and lane 10 is a comparison lane of modified BoNT with an SGRSA-modified C-loop. Potential truncation products are indicated by black arrows. [Figure 5C] Figure 5C is an SDS PAGE gel (- / +DTT) showing cleavage of a modified BoNT with a PGSGRSASGTTGTG-modified C-loop treated with increasing concentrations of u-PA, t-PAfrag, or t-PAfl after 2 hours (left), 4 hours (center), or 20 hours (right). Potential truncation products are indicated by black arrows. [Figure 5D] Figure 5D is an SDS PAGE gel (- / +DTT) showing cleavage of engineered BoNTs with PPFGRSAG-modified C-loops treated with increasing concentrations of u-PA, t-PAfrag, or t-PAfl after 2 hours (left), 4 hours (center), or 20 hours (right). Reteplase is visible in some gels (black arrows). [Figure 5E] Figure 5E is an SDS PAGE gel (- / +DTT) showing cleavage of engineered BoNTs with PPFGRSAG-modified C-loops treated with increasing concentrations of t-PAfrag or t-PAfl after 2 hours (left), 4 hours (center), or 20 hours (right). t-PAfrag (black arrow) and t-PAfl (dotted arrow) are visible in some gels. DETAILED DESCRIPTION OF THE INVENTION

[0037] definition

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994) and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide those of ordinary skill in the art with a general dictionary of many of the terms used in this disclosure. The meaning and scope of the terms should be clear, but in the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions.

[0039] It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein, as these may vary. In particular, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present disclosure.

[0040] The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments and examples of the present disclosure have been described herein, these are for illustrative purposes only, and those skilled in the art will recognize that various equivalent modifications are possible within the scope of the present disclosure. For example, while method steps or functions may be presented in a given order, in alternative embodiments, the functions may be performed in a different order, or functions may be performed substantially simultaneously. The teachings of the present disclosure provided herein can be applied to other procedures or methods, as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the present disclosure can be modified, as appropriate, to employ compositions, functions, and concepts from the above references and applications to provide further embodiments of the present disclosure. Furthermore, given consideration of biological functional equivalence, some changes can be made to protein structure without affecting the type or amount of biological or chemical action. These and other modifications can be made to the present disclosure in light of the detailed description. All such modifications are intended to be within the scope of the appended claims.

[0041] Unless otherwise specified, all nucleic acid sequences are written left to right in 5' to 3' orientation; all amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0042] The headings provided herein do not limit the various aspects or embodiments of the disclosure.

[0043] As used herein, the term "capable of," when used with a verb, encompasses or implies the action of the corresponding verb. For example, "capable of interacting" means to interact, "capable of cleaving" means to cleave, "capable of binding" means to bind, and "capable of specifically targeting" means to specifically target.

[0044] Numerical ranges are inclusive of the numbers defining the range. When a range of values ​​is presented, each intervening value between the upper and lower limit of that range, to the tenth of the unit of the lower limit, is also understood to be specifically disclosed, unless the context clearly dictates otherwise. Each smaller range between any stated value 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 independently be included or excluded in the range, and each range where either limit, neither limit, or both limits are included in the smaller range is also encompassed within the disclosure, subject to the specific exclusion of a limit in the stated range. When a stated range includes either or both limits, ranges excluding either or both of those included limits are also included within the disclosure.

[0045] As used herein, amino acids are represented using the amino acid name, three-letter abbreviation, or single-letter abbreviation. The term "protein," as used herein, includes proteins, polypeptides, and peptides. The term "amino acid sequence," as used herein, is synonymous with the term "polypeptide" and / or the term "protein." In some instances, the term "amino acid sequence" is synonymous with the term "peptide." In some instances, the term "amino acid sequence" is synonymous with the term "enzyme." As used herein, the terms "protein" and "polypeptide" are used interchangeably. Conventional single-letter and three-letter codes for amino acid residues may be used in the present disclosure and claims. The three-letter codes for amino acids are defined in accordance with the Joint Commission on Biochemical Nomenclature (JCBN) of the IUPACIUB. It is also understood that due to the degeneracy of the genetic code, a polypeptide may be encoded by more than one nucleotide sequence.

[0046] A "fragment" of a polypeptide typically comprises at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or more of the original polypeptide.

[0047] As used herein, the terms "polynucleotide," "nucleic acid," and "nucleic acid sequence" refer to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid, or analogs thereof. Nucleic acids may be either single-stranded or double-stranded. A single-stranded nucleic acid may be one nucleic acid strand of a denatured double-stranded DNA. Alternatively, it may be a single-stranded nucleic acid that is not derived from any double-stranded DNA. In one embodiment, a nucleic acid may be DNA. In another embodiment, a nucleic acid may be RNA. A suitable nucleic acid molecule is DNA, including genomic DNA or cDNA. Other suitable nucleic acid molecules are RNA, including siRNA, shRNA, and antisense oligonucleotides.

[0048] As used herein, the term "spacer" refers to a flexible peptide used in an exogenous activation loop or modified BoNT / C activation loop, or in conjunction with an exogenous protease cleavage site, and is typically included to maintain the secondary structure of the exogenous / modified BoNT / C activation loop within the modified clostridial neurotoxins of the present invention. Spacers used in modified clostridial neurotoxins of the present invention may comprise an amino acid sequence of 1 to 30 amino acid residues, e.g., 5 to 30 amino acid residues, 10 to 25 amino acid residues, or about 5 to about 20 amino acid residues. Spacers may comprise or consist of small amino acid residues, such as glycine, threonine, arginine, serine, asparagine, glutamine, alanine, aspartic acid, proline, glutamic acid, lysine, leucine, and / or valine, particularly glycine, serine, alanine, leucine, and / or valine. Spacers comprising or consisting of glycine, serine, and / or alanine may be preferred, with glycine and serine being particularly preferred. Thus, the most commonly used spacer has a sequence consisting primarily of a string of Gly and Ser residues ("GS" linker), (Gly-Gly-Gly-Gly-Ser). n(SEQ ID NO: 137). Non-limiting examples of GS linkers include GS5 or (GGGGS)1 (SEQ ID NO: 138); GS10 or (GGGGS)2 (SEQ ID NO: 139); GS15 or (GGGGS)3 (SEQ ID NO: 140); GS20 or (GGGGS)4 (SEQ ID NO: 141); and GS25 or (GGGGS)5 (SEQ ID NO: 142).

[0049] As used herein, the terms "increased," "increase," "enhance," or "activate" are all used to mean an increase by a statically significant amount. The terms "increased," "increase," "enhance," or "activate" can mean an increase of at least 10% compared to a base level, for example, an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% compared to a base level, or an increase of up to 100%, or any increase between 10 and 100%, or an increase of at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold, or any increase between 2-fold and 10-fold or more compared to a base level.

[0050] The terms "reduce," "reduced," "reduction," or "inhibit" are all used to refer to a statistically significant decrease. The terms "reduce," "reduction," "reduce," or "inhibit" typically refer to a decrease of at least 10% compared to a reference level (e.g., in the absence of a particular treatment), and can include, for example, a decrease of at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more. As used herein, "reduction" or "inhibition" encompasses complete inhibition or reduction compared to a reference level. "Complete inhibition" is 100% inhibition (i.e., abolition) compared to a reference level.

[0051] Other definitions of terms may appear throughout the specification. Before describing exemplary embodiments in further detail, it is to be understood that the present disclosure is not limited to the particular embodiments described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure is defined only by the appended claims.

[0052] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a clostridial neurotoxin" includes a plurality of such candidate substances, and a reference to "the clostridial neurotoxin" includes a reference to one or more clostridial neurotoxins and equivalents thereof known to those skilled in the art, and so forth. Furthermore, the use of the term "including," as well as other forms such as "includes" and "included," is not intended to be limiting.

[0053] "About" can generally refer to an acceptable range of error for the measured quantity, taking into account the nature or precision of the measurement. Exemplary degrees of error are within 20 percent (%), typically within 10%, and more typically within 5% of a given value or range of values. Preferably, the term "about" should be understood herein as plus or minus (±) 5% of the numerical value of the number with which it is used, preferably ±4%, ±3%, ±2%, ±1%, ±0.5%, or ±0.1%.

[0054] The term "consisting of" refers to the compositions, methods, and their respective components described herein, and excludes any element not described in this description of the invention.

[0055] As used herein, the term "consisting essentially of" refers to those elements required for a given invention. The term permits the presence of elements (i.e., inert or non-immunogenic components) that do not materially affect the basic and novel or functional characteristics of the invention.

[0056] Any embodiment described herein as "comprising" one or more features may also be construed as a disclosure of corresponding embodiments "consisting of" and / or "consisting essentially of" such features.

[0057] The term "deletion," as used herein, refers to the removal of one or more amino acid residues of a polypeptide without replacing one or more amino acid residues at the deletion site. Thus, (for example), if one amino acid residue is deleted from a polypeptide sequence having x amino acid residues, the resulting polypeptide will have x-1 amino acid residues.

[0058] The term "indel," as used herein, refers to the deletion of one or more amino acid residues from a polypeptide and the insertion at the site of deletion of a different number of amino acid residues (more or fewer amino acid residues) compared to the number of deleted amino acid residues. Thus, for example, in the case of an indel in which two amino acid residues are deleted from a polypeptide sequence having x amino acid residues, the resulting polypeptide has x-1 amino acid residues, or x+≧1 amino acid residues. Insertions and deletions can be performed sequentially or simultaneously, in any order.

[0059] The term "substitution," as used herein, refers to the replacement of one or more amino acid residues with the same number of amino acid residues at the same positions. Thus, in the case of a substitution of (for example) a polypeptide sequence having x amino acid residues, the resulting polypeptide also has x amino acid residues. Preferably, the substitution is at a single amino acid position.

[0060] The term "insertion," as used herein, refers to the addition of one or more amino acid residues to a polypeptide without deletion of one or more amino acid residues from the polypeptide at the site of insertion. Thus, (for example), if one amino acid residue is inserted into a polypeptide sequence having x amino acid residues, the resulting polypeptide will have x+1 amino acid residues.

[0061] The term "deletion," as used herein, refers to the removal of one or more amino acid residues of a polypeptide without replacing one or more amino acid residues at the deletion site. Thus, (for example), if one amino acid residue is deleted from a polypeptide sequence having x amino acid residues, the resulting polypeptide will have x-1 amino acid residues.

[0062] The term "indel," as used herein, refers to the deletion of one or more amino acid residues from a polypeptide and the insertion at the site of deletion of a different number of amino acid residues (more or fewer amino acid residues) compared to the number of deleted amino acid residues. Thus, for example, in the case of an indel in which two amino acid residues are deleted from a polypeptide sequence having x amino acid residues, the resulting polypeptide has x-1 amino acid residues, or x+≧1 amino acid residues. Insertions and deletions can be performed sequentially or simultaneously, in any order.

[0063] The term "substitution," as used herein, refers to the replacement of one or more amino acid residues with the same number of amino acid residues at the same positions. Thus, in the case of a substitution of (for example) a polypeptide sequence having x amino acid residues, the resulting polypeptide also has x amino acid residues. Preferably, the substitution is at a single amino acid position.

[0064] The term "insertion," as used herein, refers to the addition of one or more amino acid residues to a polypeptide without the deletion of one or more amino acid residues from the polypeptide at the insertion site. Thus, for example, if one amino acid residue is inserted into a polypeptide sequence having x amino acid residues, the resulting polypeptide will have x+1 amino acid residues. Concentrations, amounts, volumes, percentages, and other numerical values ​​may be presented herein in a range format. It should be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values ​​expressly recited as range limits, but also all individual numerical values ​​or subranges subsumed within the range, as if each numerical value and subrange were explicitly recited.

[0065] An individual may be one who has been previously diagnosed with, or identified as suffering from, or having a condition for which treatment is needed or one or more complications associated with such a condition, and optionally may have already been treated for a condition defined herein or one or more complications associated with said condition. Alternatively, an individual may also be one who has not previously been diagnosed with a condition defined herein or one or more complications associated with said condition. For example, an individual may be one who exhibits one or more risk factors for a condition or one or more complications associated with said condition, or a subject who does not exhibit risk factors.

[0066] An "individual in need" of treatment for a particular condition may be an individual who has the condition, who has been diagnosed with the condition, or who is at risk of developing the condition.

[0067] As used herein, the terms "subject," "individual," and "patient" are used interchangeably to refer to a mammalian individual. An "individual" may be any mammal. Generally, an individual may be a human; in other words, in one embodiment, an "individual" is a human. An "individual" may be an adult, juvenile, or infant. An "individual" may be male or female.

[0068] As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government or listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeias.

[0069] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application, and nothing herein should be construed as an admission that such publications constitute prior art to the claims appended hereto.

[0070] Modified Clostridial Neurotoxins

[0071] The present invention provides a modified Clostridial neurotoxin comprising an exogenous protease cleavage site. Typically, cleavage at the exogenous protease cleavage site generates a di-chain form of the modified Clostridial neurotoxin. In other words, cleavage at the exogenous protease cleavage site activates the modified Clostridial neurotoxin.

[0072] Typically, according to the present invention, a modified Clostridial neurotoxin lacks the endogenous activation loop (or a portion thereof) of the original Clostridial neurotoxin from which the modified neurotoxin is derived. The term "endogenous activation loop" is as defined herein. In these modified Clostridial neurotoxins of the present invention, the endogenous activation loop (or a portion thereof) of the original Clostridial neurotoxin is replaced with a modified BoNT / C activation loop, particularly a modified BoNT / C1 activation loop.

[0073] The endogenous BoNT / C1 activation loop contains the consensus sequence Cyst-(Xaa) a -Ile-Asp / Glu-Gly-Arg-(Yaa) b -Cys (SEQ ID NO: 1), where a=1 to 10 and b=4 to 15, typically a=2 to 4 and / or b=6 to 10, preferably a=3 and / or b=8. A specific BoNT / C1 activation loop comprises or consists of CHKAIDGRSLYNKTLDC (SEQ ID NO: 2) or CHKAIEGRSLYNKTLDC (SEQ ID NO: 3). Within the intrinsic BoNT / C1 activation loop, both enterokinase and factor Xa are capable of cleaving immediately C-terminal to the IDGR or IEGR sequence, and the presence of lysine and arginine residues allows cleavage by trypsin and Lys-C.

[0074] In the modified BoNT / C activation loop of the present invention, one or more of these endogenous activation sites within the endogenous activation loop are replaced by one or more exogenous protease cleavage sites. Typically, the modified BoNT / C activation loop of the present invention lacks a factor Xa / enterokinase cleavage site, and thus the modified BoNT / C activation loop is not susceptible to cleavage by factor Xa and / or enterokinase. Therefore, the modified BoNT / C (BoNT / C1) activation loop of the present invention may lack an IDGR or IEGR sequence. Furthermore, the modified BoNT / C (BoNT / C1) activation loop of the present invention typically contains one or more exogenous protease cleavage sites. In other words, the modified BoNT / C (BoNT / C1) activation loop may have the endogenous BoNT / C activation site or a portion thereof replaced by one or more exogenous protease cleavage sites described herein.

[0075] Thus, the present invention provides a modified clostridial neurotoxin, wherein the endogenous activation loop is replaced with a modified BoNT / C (BoNT / C1) activation loop, and wherein the endogenous activation site of the BoNT / C (BoNT / C1) activation loop, or a portion thereof, is replaced with one or more exogenous protease cleavage sites, wherein cleavage at the cleavage sites generates a di-chain form of the modified clostridial neurotoxin.

[0076] A modified BoNT / C (BoNT / C1) activation loop according to the present invention may begin with the N-terminal residue CHKA (SEQ ID NO: 4), e.g., CHKAI (SEQ ID NO: 5) or CHKAID (SEQ ID NO: 6). Typically, such modified BoNT / C (BoNT / C1) activation loops lack the IDGR (SEQ ID NO: 7) or IEGR (SEQ ID NO: 8) sequence, and thus the sequence of the modified BoNT / C (BoNT / C1) activation loop does not include the sequence CHKAIDGR (SEQ ID NO: 9) or CHKAIEGR (SEQ ID NO: 10) at the N-terminus. Alternatively or additionally, a modified BoNT / C (BoNT / C1) activation loop according to the present invention may end with the C-terminal residue TLDC (SEQ ID NO: 11), e.g., KTLDC (SEQ ID NO: 12), YNKTLDC (SEQ ID NO: 13), or LYNKTLDC (SEQ ID NO: 14). A modified BoNT / C (BoNT / C1) activation loop according to the present invention can be about 10 to about 30 (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) amino acids in length, for example, about 15 to about 25 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) amino acids in length, preferably about 17 to about 24 (e.g., 17, 18, 19, 20, 21, 22, 23, or 24) amino acids in length. Particularly preferred is a modified BoNT / C (BoNT / C1) activation loop that is 17 amino acids in length, in which case the modified BoNT / C (BoNT / C1) activation loop is the same length as the endogenous BoNT / C (BoNT / C1) activation loop.

[0077] The modified BoNT / C (BoNT / C1) activation loop may include one or more exogenous protease cleavage sites in the intrinsic activation loop of the clostridial neurotoxin, LTPRGVRL (SEQ ID NO: 15), ENKSLVPRGS (SEQ ID NO: 17), LVPRGS (SEQ ID NO: 16), KRV (SEQ ID NO: 24), SGRSA (SEQ ID NO: 25), PGSGRSAG (SEQ ID NO: 26), PGSGRSASGTTGTG (SEQ ID NO: 27), PPFGRSAG (SEQ ID NO: 33), VVPRVELVA (SEQ ID NO: 32), or GRI (SEQ ID NO: 44), where cleavage at said cleavage site generates a di-chain form of the modified clostridial neurotoxin. In particular, the modified BoNT / C (BoNT / C1) activation loop may include one or more exogenous protease cleavage sites for LTPRGVRL (SEQ ID NO: 15), KRV (SEQ ID NO: 24), SGRSA (SEQ ID NO: 25), VVPRVELVA (SEQ ID NO: 32), PPFGRSAG (SEQ ID NO: 33), or GRI (SEQ ID NO: 44), preferably LTPRGVRL (SEQ ID NO: 15), KRV (SEQ ID NO: 24), SGRSA (SEQ ID NO: 25), or PPFGRSAG (SEQ ID NO: 33). The modified BoNT / C (BoNT / C1) activation loop may include one or more exogenous protease cleavage sites for LTPRGVRL (SEQ ID NO: 15), ENKSLVPRGS (SEQ ID NO: 17), SGRSA (SEQ ID NO: 25), PGSGRSAG (SEQ ID NO: 26), PGSGRSASGTTGTG (SEQ ID NO: 27), or PPFGRSAG (SEQ ID NO: 33).

[0078] The present invention also provides modified clostridial neurotoxins that include an exogenous protease cleavage site formed by introducing one or more amino acid sequences comprising or consisting of LTPRGVRL (SEQ ID NO: 15), ENKSLVPRGS (SEQ ID NO: 17), LVPRGS (SEQ ID NO: 16), KRV (SEQ ID NO: 24), SGRSA (SEQ ID NO: 25), PGSGRSAG (SEQ ID NO: 26), PGSGRSASGTTGTG (SEQ ID NO: 27), PPFGRSAG (SEQ ID NO: 33), VVPRVELVA (SEQ ID NO: 32), or GRI (SEQ ID NO: 44) into the endogenous activation loop of the clostridial neurotoxin, wherein cleavage at the cleavage site generates a di-chain form of the modified clostridial neurotoxin. In particular, the one or more amino acid sequences may comprise or consist of LTPRGVRL (SEQ ID NO: 15), KRV (SEQ ID NO: 24), SGRSA (SEQ ID NO: 25), VVPRVELVA (SEQ ID NO: 32), PPFGRSAG (SEQ ID NO: 33), or GRI (SEQ ID NO: 44), preferably LTPRGVRL (SEQ ID NO: 15), KRV (SEQ ID NO: 24), SGRSA (SEQ ID NO: 25), or PPFGRSAG (SEQ ID NO: 33). The present invention provides modified clostridial neurotoxins that comprise an exogenous protease cleavage site formed by introducing one or more amino acid sequences comprising or consisting of LTPRGVRL (SEQ ID NO: 15), ENKSLVPRGS (SEQ ID NO: 17), SGRSA (SEQ ID NO: 25), PGSGRSAG (SEQ ID NO: 26), PGSGRSASGTTGTG (SEQ ID NO: 27), or PPFGRSAG (SEQ ID NO: 33) into the endogenous activation loop of the clostridial neurotoxin, wherein cleavage at the cleavage site generates a modified clostridial neurotoxin di-chain form. The endogenous (native) activation loop of a clostridial neurotoxin may be replaced (or partially replaced) by said exogenous protease cleavage site. Thus, a modified clostridial neurotoxin of the present invention may comprise an exogenous activation loop.

[0079] The term "exogenous protease cleavage site" can be used interchangeably with the term "exogenous protease activation site." An exogenous activation loop as defined herein will typically comprise or consist of one or more exogenous protease cleavage sites.

[0080] The modified clostridial neurotoxins of the present invention can be activated in vivo whether they contain one or more exogenous protease cleavage sites within the modified BoNT / C (BoNT / C1) activation loop or within the exogenous activation loop. Thus, the modified clostridial neurotoxins open up new areas of clostridial neurotoxin engineering and therapeutic use, allowing the toxins to be produced and administered as single-chain clostridial neurotoxins and then cleaved in vivo to generate the activated two-chain form.

[0081] The modified clostridial neurotoxins of the present invention can be activated in vitro whether they contain one or more exogenous protease cleavage sites within the modified BoNT / C (BoNT / C1) activation loop or within the exogenous activation loop. Introducing an exogenous protease cleavage site into the modified clostridial neurotoxins of the present invention, whether the exogenous protease cleavage site(s) is / are contained within the modified BoNT / C (BoNT / C1) activation loop or within the exogenous activation loop, typically provides advantages in the manufacture and / or excision of the modified clostridial neurotoxins of the present invention, such as: (i) access to a GMP-compatible protease; (ii) a protease that can function with the desired retargeted clostridial neurotoxin structure; (iii) reduction in non-specific cleavage and the resulting undesired truncation that requires additional purification and / or impacts yield; (iv) improved utility across each clostridial neurotoxin serotype described herein, or any combination thereof; and / or (v) improved overall activation rate, which may enable the production of activated two-chain compositions, potentially reducing the amount of protease required and / or reducing the complexity / number of steps during purification.

[0082] A clostridial neurotoxin (before modification) is characterized in that its endogenous activation loop is inefficiently proteolytically excised by one or more exogenous proteases. In contrast to a clostridial neurotoxin (before modification), the modified clostridial neurotoxin of the present invention is not inefficiently proteolytically excised by one or more exogenous proteases into which a cleavage site has been introduced, and / or the peptide bond outside the exogenous activation loop of the modified clostridial neurotoxin is not hydrolyzed by the one or more exogenous proteases. Thus, a clostridial neurotoxin (before modification) is usually resistant to proteolytic excision by one or more exogenous proteases. The terms "inefficiently proteolytically excised by one or more exogenous proteases," "resistant to proteolytic excision by one or more exogenous proteases," "not substantially hydrolyzed by one or more exogenous proteases," "inefficiently activated by one or more exogenous proteases," "resistant to activation by one or more exogenous proteases," and "not substantially activated by one or more exogenous proteases" are used interchangeably herein. Typically, a clostridial neurotoxin (before modification) is typically resistant to proteolytic excision by one or more exogenous proteases into which a cleavage site has been introduced according to the present invention. A clostridial neurotoxin (before modification) may also be resistant to proteolytic excision by one or more exogenous proteases into which a cleavage site has not been introduced according to the present invention.

[0083] Clostridial neurotoxins (before modification) typically have peptide bonds (within or outside the activation loop) that are not hydrolyzed or are not substantially hydrolyzed by one or more exogenous proteases. In the methods of the present invention, the term "not substantially hydrolyzed" means that less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the Clostridial neurotoxins present in a reaction contain peptide bonds that are hydrolyzed by one or more exogenous proteases.

[0084] Thus, a clostridial neurotoxin (before modification) typically does not comprise one or more exogenous protease consensus sequences (e.g., any one or more of SEQ ID NOs: 169, 170, 172, or 173) and / or one or more exogenous protease cleavage sites (e.g., as defined herein, e.g., any one of SEQ ID NOs: 15-22, 24-30, 32-42, 44-46, and / or 50-52, particularly any one of SEQ ID NOs: 15-22, 24-30, and / or 32-42, preferably any one of SEQ ID NOs: 15, 17, 16, 26, 25, 27, 24, 33, and / or 32) within its endogenous activation loop.

[0085] The clostridial neurotoxin (before modification) may contain one or more exogenous protease consensus sequences (e.g., any one or more of SEQ ID NOs: 169, 170, 172, or 173) and / or one or more exogenous protease cleavage sites (e.g., as defined herein, e.g., SEQ ID NOs: 15-22, 24-30, 32-42, 44-46, and and / or any one of SEQ ID NOs: 50-52, particularly any one of SEQ ID NOs: 15-22, 24-30 and / or 32-42, preferably any one of SEQ ID NOs: 15, 17, 16, 25, 26, 27, 24, 33 and / or 32. As a non-limiting example, if a modified clostridial neurotoxin contains a thrombin cleavage site, the corresponding unmodified clostridial neurotoxin may not contain a thrombin cleavage site within its intrinsic activation loop.

[0086] The present invention may involve replacing the intrinsic activation loop (or a portion thereof) of any clostridial neurotoxin with a modified BoNT / C activation loop (or a portion thereof) comprising one or more exogenous protease cleavage sites as described herein. The present invention may involve replacing the intrinsic activation loop (or a portion thereof) of any clostridial neurotoxin with one or more exogenous protease cleavage sites as described herein, or an exogenous activation loop comprising one or more exogenous protease cleavage sites. The clostridial neurotoxin may be a botulinum neurotoxin (BoNT) or a tetanus neurotoxin (TeNT). Preferably, the clostridial neurotoxin is a botulinum neurotoxin, such as BoNT / A, BoNT / B, BoNT / C1, BoNT / D, BoNT / E, BoNT / F, BoNT / G, or BoNT / X, or a chimera or hybrid thereof. In some particularly preferred embodiments, the clostridial neurotoxin is BoNT / X or a chimera or hybrid thereof.

[0087] As used herein, the term "intrinsic activation loop" refers to an activation loop present in a subject Clostridial neurotoxin, e.g., a subject Clostridial neurotoxin of the indicated serotype. For example, BoNT / A1 comprises a BoNT / A1 heavy chain and a light chain, and therefore, the intrinsic activation loop of BoNT / A1 is the A1 activation loop. For chimeric or hybrid Clostridial neurotoxins, those skilled in the art will recognize, for example, the L chain and the H chain. NBy determining the serotype from which the domain is derived, the "endogenous activation loop" can be identified. In some embodiments, a chimeric or hybrid clostridial neurotoxin can have an endogenous activation loop that is a fusion of the activation loops of two different serotypes. As an example, a chimeric clostridial neurotoxin, such as BoNT / A1C1, has the light chain and translocation domain of BoNT / A1, and therefore the endogenous BoNT / A1C1 activation loop is the A1 activation loop. The endogenous activation loop is usually bounded by cysteine ​​residues, which form disulfide bridges and covalently link the light and heavy chains of the clostridial neurotoxin (before modification). Thus, endogenous activation loop sequences can be described with or without the bounding cysteine ​​residues (as described herein). Those skilled in the art will understand that these definitions can be used interchangeably and will be able to easily identify endogenous activation loops with or without the bounding cysteine ​​residues.

[0088] Generally, an "endogenous activation loop" is any activation loop that does not contain or consist of one or more exogenous protease consensus sequences (e.g., any one or more of SEQ ID NOs: 169, 170, 172, or 173) and / or one or more exogenous protease cleavage sites described herein (e.g., SEQ ID NOs: 15-22, 24-30, 32-42, 44-46, and / or 50-52, particularly SEQ ID NOs: 15-22, 24-30, and / or 32-42, preferably SEQ ID NOs: 15, 17, 16, 25, 26, 27, 24, 33, and / or 32, more preferably SEQ ID NOs: 15, 17, 16, 26, 25, and / or 33).

[0089] In contrast, the term "exogenous activation loop" as used herein refers to an activation loop that is different from the endogenous activation loop present in a subject Clostridial neurotoxin, e.g., a subject Clostridial neurotoxin of a designated serotype, where the exogenous activation loop comprises one or more exogenous protease cleavage sites. For example, the BoNT / A activation loop is exogenous to BoNT / X because it has a different polypeptide sequence from the wild-type BoNT / X activation loop. As a further example, the BoNT / C1 activation loop is exogenous to BoNT / A1 because it has a different polypeptide sequence from the wild-type BoNT / A1 activation loop. Thus, the term "exogenous activation loop" as used herein can refer to the modified BoNT / C (BoNT / C1) activation loop of the present invention, unless explicitly stated otherwise. For chimeric or hybrid Clostridial neurotoxins, those skilled in the art will recognize, for example, the L chain and H chain. N By determining the serotype from which the domain is derived, it can be determined whether the activation loop is an "exogenous activation loop." For example, if the L chain is the L chain of BoNT / B and the H N If the domain is derived from BoNT / D, the intrinsic activation loop may have part of the BoNT / B sequence and part of the BoNT / D sequence, and if the activation loop (e.g., the C1 activation loop) is different and contains one or more exogenous protease cleavage sites, it is considered an "exogenous activation loop."

[0090] Determining whether an activation loop is an "endogenous activation loop" can be done by aligning the sequence of a clostridial neurotoxin of interest with the activation loop to see if the activation loop is present in the clostridial neurotoxin sequence of interest. If present, the activation loop can be identified as an endogenous activation loop. As described herein, the endogenous activation loop of a clostridial neurotoxin is replaced by an exogenous cleavage site that is one or more exogenous protease cleavage sites, by an exogenous activation loop that includes one or more exogenous protease cleavage sites, or by a modified BoNT / C (BoNT / C1) activation loop in which the endogenous BoNT / C (BoNT / C1) activation site (or a portion thereof) has been replaced with one or more exogenous protease cleavage sites.

[0091] According to the present invention, one or more exogenous protease cleavage sites can be inserted between the two cysteine ​​residues that bound the endogenous activation loop of the clostridial neurotoxin before modification, but the exact location of the one or more exogenous protease cleavage sites within the endogenous activation loop is not limited, provided that the structure of the resulting modified clostridial neurotoxin is not compromised and / or the function of the modified clostridial neurotoxin is not lost. In the modified BoNT / C (BoNT / C1) activation loop of the present invention, one or more exogenous protease cleavage sites can be inserted between the two cysteine ​​residues that bound the endogenous BoNT / C (BoNT / C1) activation loop.

[0092] The entire endogenous activation loop may be replaced with one or more exogenous protease cleavage sites described herein, or an exogenous activation loop containing one or more exogenous protease cleavage sites. Alternatively, a portion or part of the endogenous activation loop may be replaced, e.g., at least 5, 10, 15, 20, 25, 30, 35, or 40 amino acid residues of the endogenous activation loop (also referred to herein as a partial replacement of the endogenous activation loop). Preferably, 5 to 20 amino acid residues of the endogenous activation loop are replaced, more preferably 5 to 15 amino acid residues of the endogenous activation loop. Typically, a partial replacement involves replacing consecutive amino acids within the endogenous activation loop. Thus, a partial replacement of the endogenous activation loop retains at least one amino acid residue of the endogenous activation loop. Preferably, between about 5 and about 15 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) residues of the endogenous activation loop are retained, for example, between about 5 and about 12 (e.g., 5, 6, 7, 8, 9, 10, 11, or 12) amino acid residues. The retained amino acid residues may be N-terminal and / or C-terminal to the endogenous activation loop.

[0093] As described herein, the entire (unmodified) BoNT / C (BoNT / C1) activation loop may be replaced with one or more exogenous protease cleavage sites. Preferably, a portion or part of the (unmodified) BoNT / C (BoNT / C1) activation loop may be replaced (also referred to herein as a partial replacement of the (unmodified) BoNT / C (BoNT / C1) activation loop), e.g., at least 5, 10, 11, 12, 13, 14, 15, 16, or 17 amino acid residues of the (unmodified) BoNT / C (BoNT / C1) activation loop are replaced. Preferably, 3 to 15 amino acid residues of the (unmodified) BoNT / C (BoNT / C1) activation loop, more preferably 5 to 10 amino acid residues of the (unmodified) BoNT / C (BoNT / C1) activation loop, are replaced. Typically, a partial replacement involves the replacement of consecutive amino acids within the (unmodified) BoNT / C (BoNT / C1) activation loop. Thus, in a partial replacement of the (unmodified) BoNT / C (BoNT / C1) activation loop, at least one amino acid residue of the (unmodified) BoNT / C (BoNT / C1) activation loop is retained. Preferably, between about 5 and about 15 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) amino acids of the (unmodified) BoNT / C (BoNT / C1) activation loop are retained, for example, between about 5 and about 12 (e.g., 5, 6, 7, 8, 9, 10, 11, or 12) amino acid residues of the (unmodified) BoNT / C (BoNT / C1) activation loop. The retained amino acid residues may be at the N-terminus and / or C-terminus of the (unmodified) BoNT / C (BoNT / C1) activation loop.

[0094] In some preferred embodiments, the endogenous activation loop is completely replaced in the modified clostridial neurotoxins of the present invention. Typically, this means that all of the amino acid residues in the activation loop (between the cysteine ​​residues that form disulfide bonds in the active dichain molecule) are replaced with an exogenous activation loop, a modified BoNT / C (BoNT / C1) activation loop, or an exogenous protease cleavage site according to the present invention. A complete replacement of the endogenous activation loop may include the introduction of an exogenous activation loop or a modified BoNT / C (BoNT / C1) activation loop consisting solely of one or more exogenous protease cleavage sites described herein. Alternatively, a complete replacement of the endogenous activation loop may include the introduction of an exogenous activation loop or a modified BoNT / C (BoNT / C1) activation loop comprising one or more spacer sequences along with one or more exogenous protease cleavage sites described herein. Each of the one or more spacer sequences is typically a short peptide (e.g., about 5 to about 25 amino acids, e.g., about 5 to about 20 amino acids, about 5 to about 15 amino acids, or about 5 to about 10 amino acids). Such a spacer may be present when the one or more exogenous protease cleavage sites are short motifs (e.g., typically less than 15 amino acids, preferably less than 10 amino acids, or less than 9 amino acids in length). One or more spacers may be present N-terminally and / or C-terminally to each of the exogenous protease cleavage sites. Preferably, the spacer may be a GS spacer as defined herein.

[0095] Replacement (including partial replacement) of the endogenous activation loop or (unmodified) BoNT / C (BoNT / C1) activation loop with one or more exogenous protease cleavage sites may be achieved by any method known in the art. For example, replacement may be achieved by amino acid modification. The endogenous activation loop may be replaced by deleting one or more amino acid residues in the endogenous activation loop. The endogenous activation loop may be replaced by substituting one or more amino acid residues in the endogenous activation loop with amino acid residues in the exogenous activation loop. The endogenous activation loop (or a portion thereof) may be deleted, and one or more exogenous protease cleavage sites or an exogenous activation loop containing one or more exogenous protease cleavage sites may be inserted, preferably at the position formerly occupied by the endogenous activation loop. Alternatively, the endogenous activation loop may be retained in the modified clostridial neurotoxins of the present invention, preferably inactivated (e.g., by mutation). Preferably, the modified clostridial neurotoxin of the present invention is free of an endogenous activation loop (either a portion thereof or the entire endogenous activation loop). Preferably, one or more exogenous protease cleavage sites, or an exogenous activation loop comprising one or more exogenous protease cleavage sites, occupies the position formally occupied by the endogenous activation loop in the clostridial neurotoxin. For the avoidance of doubt, when an endogenous activation loop is modified to include one or more exogenous protease cleavage sites (e.g., by substituting residues in the endogenous activation loop or by adding one or more amino acids to form one or more exogenous protease cleavage sites in the endogenous activation loop), the modified activation loop is an exogenous activation loop according to the present invention. Thus, a modified Clostridial neurotoxin may contain both its endogenous activation / cleavage site and one or more exogenous protease cleavage sites, and therefore may be activated by the naturally occurring activating protease (or equivalents used in conventional recombinant BoNT production, e.g., trypsin or Lys-C) or by one or more exogenous proteases.

[0096] For example, replacement (including partial replacement) of the unmodified BoNT / C (BoNT / C1) activation loop and / or replacement (including partial replacement) of the endogenous BoNT / C (BoNT / C) activation site can be achieved by amino acid modification. The unmodified BoNT / C (BoNT / C1) activation loop and / or endogenous BoNT / C (BoNT / C) activation site can be replaced by deleting one or more amino acid residues in the unmodified BoNT / C (BoNT / C1) activation loop and / or endogenous BoNT / C (BoNT / C) activation site. The unmodified BoNT / C (BoNT / C1) activation loop and / or endogenous BoNT / C (BoNT / C) activation site can be replaced by substituting one or more amino acid residues in the unmodified BoNT / C (BoNT / C1) activation loop and / or endogenous BoNT / C (BoNT / C) activation site with amino acid residues of an exogenous protease cleavage site. The unmodified BoNT / C (BoNT / C1) activation loop and / or endogenous BoNT / C (BoNT / C) activation site (or a portion thereof) may be deleted, and one or more exogenous protease cleavage sites may be inserted, preferably at the position formerly occupied by the unmodified BoNT / C (BoNT / C1) activation loop and / or endogenous BoNT / C (BoNT / C) activation site. Alternatively, the unmodified BoNT / C (BoNT / C1) activation loop and / or endogenous BoNT / C (BoNT / C) activation site may be retained in the modified clostridial neurotoxin of the present invention, and preferably is inactivated (e.g., by mutation). Preferably, the unmodified BoNT / C (BoNT / C1) activation loop and / or endogenous BoNT / C (BoNT / C) activation site (or a portion thereof) is absent from the modified clostridial neurotoxin of the present invention. Preferably, the one or more exogenous protease cleavage sites occupy the position formally occupied by the unmodified BoNT / C (BoNT / C1) activation loop and / or endogenous BoNT / C (BoNT / C) activation site within the clostridial neurotoxin.For the avoidance of doubt, when an unmodified BoNT / C (BoNT / C1) activation loop and / or an endogenous BoNT / C (BoNT / C) activation site is modified to include one or more exogenous protease cleavage sites (e.g., by substituting residues within the unmodified BoNT / C (BoNT / C1) activation loop and / or the endogenous BoNT / C (BoNT / C) activation site, or by adding one or more amino acids to form one or more exogenous protease cleavage sites within the BoNT / C (BoNT / C1) activation loop and / or the endogenous BoNT / C (BoNT / C) activation site), the modified BoNT / C (BoNT / C1) activation loop and / or modified BoNT / C (BoNT / C) activation site is an exogenous activation loop according to the present invention. Thus, a modified Clostridial neurotoxin may contain both its endogenous activation / cleavage site and one or more exogenous protease cleavage sites, and therefore may be activated by the naturally occurring activating protease (or equivalents used in conventional recombinant BoNT production, e.g., trypsin or Lys-C) or by one or more exogenous proteases.

[0097] In particular, the BoNT / C (BoNT / C1) activation loop and / or the BoNT / C (BoNT / C) activation site can be modified by: (i) substituting one or more amino acids present in the unmodified BoNT / C (BoNT / C1) activation loop and / or the unmodified / endogenous BoNT / C (BoNT / C) activation site; and / or (ii) inserting one or more amino acids into the unmodified BoNT / C (BoNT / C1) activation loop and / or the unmodified / endogenous BoNT / C (BoNT / C) activation site. Preferably, the BoNT / C (BoNT / C1) activation loop and / or BoNT / C (BoNT / C) activation site can be modified by: (i) substituting one or more amino acids present in the unmodified BoNT / C (BoNT / C1) activation loop and / or the unmodified / endogenous BoNT / C (BoNT / C) activation site; or (ii) substituting one or more amino acids present in the unmodified BoNT / C (BoNT / C1) activation loop and / or the unmodified / endogenous BoNT / C (BoNT / C) activation site, and inserting one or more amino acids into the unmodified BoNT / C (BoNT / C1) activation loop and / or the unmodified / endogenous BoNT / C (BoNT / C) activation site. One to ten (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids of the unmodified BoNT / C (BoNT / C1) activation loop and / or the unmodified / endogenous BoNT / C (BoNT / C) activation site may be substituted to form a modified BoNT / C (BoNT / C1) activation loop and / or modified BoNT / C (BoNT / C) activation site, for example, about 3 to 10, 3 to 9, 5 to 9, or 5 to 8 amino acids may be substituted. Alternatively or additionally, 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids may be inserted into the unmodified BoNT / C (BoNT / C1) activation loop and / or the unmodified / endogenous BoNT / C (BoNT / C) activation site to form a modified BoNT / C (BoNT / C1) activation loop and / or modified BoNT / C (BoNT / C) activation site, for example, about 3 to 10, 3 to 9, 4 to 8, or 4 to 7 amino acids may be inserted.

[0098] Methods for modifying proteins by substitution, insertion, or deletion of amino acid residues are known in the art and can be used in the practice of the present invention. For example, amino acid modifications can be introduced by modifying the DNA sequence encoding the clostridial neurotoxin. This can be achieved by standard molecular cloning techniques, such as site-directed mutagenesis, which uses a short strand of DNA (oligonucleotide) encoding the desired amino acid to replace the original coding sequence using a polymerase enzyme, or by inserting / deleting portions of a gene using various enzymes (e.g., ligase and restriction enzymes). Alternatively, modified gene sequences can be chemically synthesized. Any other method known in the art for modifying polypeptides, such as polypeptide synthesis and polypeptide conjugation, can also be used to modify the clostridial neurotoxin according to the present invention.

[0099] An endogenous activation loop replaced in accordance with the present invention may comprise or consist of a polypeptide sequence having at least 70% (e.g., at least 80% or 90%) sequence identity to SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, or SEQ ID NO:90. An endogenous activation loop replaced in accordance with the present invention may comprise or consist of a polypeptide sequence having at least 70% (e.g., at least 80% or 90%) sequence identity to SEQ ID NO:90. In particular, the intrinsic activation loop may comprise or consist of a polypeptide sequence having at least 95% sequence identity to SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, or SEQ ID NO:90. The intrinsic activation loop replaced in accordance with the present invention may comprise or consist of a polypeptide sequence having at least 95% sequence identity to SEQ ID NO:90. Preferably, the intrinsic activation loop comprises or consists of the polypeptide sequence set forth in SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, or SEQ ID NO:90. An intrinsic activation loop replaced in accordance with the present invention may comprise or consist of the polypeptide sequence of SEQ ID NO:90.

[0100] An intrinsic activation loop replaced in accordance with the present invention may comprise or consist of a polypeptide sequence having at least 70% (e.g., at least 80% or 90%) sequence identity to SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:90. An intrinsic activation loop may comprise or consist of a polypeptide sequence having at least 95% sequence identity to SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:90. Preferably, the intrinsic activation loop comprises or consists of the polypeptide sequence set forth in SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:90.

[0101] Preferably, the endogenous activation loop replaced in accordance with the present invention comprises or consists of a polypeptide sequence having at least 70% (e.g., at least 80% or 90%) sequence identity to SEQ ID NO: 72 or SEQ ID NO: 90. The endogenous activation loop may also comprise or consist of a polypeptide sequence having at least 95% sequence identity to SEQ ID NO: 72 or SEQ ID NO: 90. More preferably, the endogenous activation loop comprises or consists of the polypeptide sequence set forth in SEQ ID NO: 72 or SEQ ID NO: 90.

[0102] The present invention encompasses methods and modified Clostridial neurotoxins in which the endogenous activation loop is replaced by an exogenous cleavage site that is one or more exogenous protease cleavage sites, or an exogenous activation loop that includes one or more exogenous protease cleavage sites, as described herein.

[0103] The present invention includes methods and modified Clostridial neurotoxins in which the endogenous activation loop is replaced by a modified BoNT / C (BoNT / C1) activation loop, wherein the endogenous activation site of the BoNT / C (BoNT / C1) activation loop (or a portion thereof) is replaced by one or more exogenous protease cleavage sites described herein.

[0104] The modified clostridial neurotoxins of the present invention may include an exogenous activation loop that includes one or more of any of the exogenous protease cleavage sites described herein. The exogenous activation loop may be generated by replacing one or more amino acids in the endogenous activation loop of a clostridial neurotoxin, as described herein. In some preferred embodiments, the replaced amino acids in the endogenous activation loop are replaced by one or more exogenous protease cleavage sites or exogenous activation loops having the same number of amino acids. In other words, for example, if five amino acids in the endogenous activation loop are replaced, the replacing one or more exogenous protease cleavage sites or the exogenous activation loop containing the one or more exogenous protease cleavage sites will have five amino acids. If ten amino acids in the endogenous activation loop are replaced, the replacing one or more exogenous protease cleavage sites or the exogenous activation loop containing the one or more exogenous protease cleavage sites will have ten amino acids.

[0105] Non-limiting examples of such extrinsic activation loops include SEQ ID NOs: 53-66.

[0106] The modified clostridial neurotoxins of the present invention may include a modified BoNT / C (BoNT / C1) activation loop containing one or more of any of the exogenous protease cleavage sites described herein. The modified BoNT / C (BoNT / C1) activation loop can be generated by replacing one or more amino acids in the unmodified BoNT / C (BoNT / C1) activation loop of a clostridial neurotoxin as described herein. In some preferred embodiments, the replaced amino acids in the unmodified BoNT / C (BoNT / C1) activation loop are replaced with one or more exogenous protease cleavage sites having the same number of amino acids. In other words, for example, if five amino acids in the unmodified BoNT / C (BoNT / C1) activation loop are replaced, the replacing one or more exogenous protease cleavage sites will have five amino acids. If 10 amino acids in the unmodified BoNT / C (BoNT / C1) activation loop are replaced, the replacing one or more exogenous protease cleavage sites will have 10 amino acids. Non-limiting examples of such modified BoNT / C (BoNT / C1) activation loops are provided herein.

[0107] The present invention provides a method for producing a modified clostridial neurotoxin according to the present invention, the method comprising replacing the endogenous activation loop (or a portion thereof) of a clostridial neurotoxin with an exogenous activation loop or an exogenous cleavage site or a modified BoNT / C (BoNT / C1) activation loop, thereby providing an exogenous clostridial neurotoxin, wherein the exogenous cleavage site is one or more exogenous protease cleavage sites described herein, or the exogenous activation loop or modified BoNT / C (BoNT / C1) activation loop comprises said one or more exogenous protease cleavage sites. Typically, the one or more exogenous protease cleavage sites are selected from a sequence comprising or consisting of the amino acid sequence of SEQ ID NOs: 15-22, 24-30, 32-42, 44-46, and / or 50-52, particularly SEQ ID NOs: 15-22, 24-30, and / or 32-42, preferably SEQ ID NOs: 15, 17, 16, 26, 25, 27, 24, 33, and / or 32, particularly preferably SEQ ID NOs: 15, 17, 16, 26, 25, and / or 33. Alternatively, the exogenous activation loop or the modified BoNT / C (BoNT / C1) activation loop comprises the one or more exogenous protease cleavage sites.

[0108] The present invention provides modified clostridial neurotoxins (e.g., obtained by the methods of the present invention), in which the endogenous activation loop (or a portion thereof) of the clostridial neurotoxin is replaced with an exogenous activation loop, an exogenous cleavage site, or a modified BoNT / C (BoNT / C1) activation loop, wherein the exogenous cleavage site is one or more exogenous protease cleavage sites described herein, or the exogenous activation loop or modified BoNT / C (BoNT / C1) activation loop comprises said one or more exogenous protease cleavage sites. Typically, the one or more exogenous protease cleavage sites are selected from a sequence comprising or consisting of the amino acid sequence of SEQ ID NOs: 15-22, 24-30, 32-42, 44-46 and / or 50-52, in particular SEQ ID NOs: 15-22, 24-30 and / or 32-42, preferably SEQ ID NOs: 15, 17, 16, 26, 25, 27, 24, 33 and / or 32, particularly preferably SEQ ID NOs: 15, 17, 16, 26, 25 and / or 33. Alternatively, the exogenous activation loop comprises the one or more exogenous protease cleavage sites.

[0109] A clostridial neurotoxin of the present invention (e.g., a modified clostridial neurotoxin) can be encoded by a nucleic acid comprising or consisting of a nucleotide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 154, 156, or 158. A clostridial neurotoxin of the present invention can be encoded by a nucleic acid comprising or consisting of a nucleotide sequence having at least 80% or 90% sequence identity to any one of SEQ ID NOs: 154, 156, or 158. Preferably, a clostridial neurotoxin of the present invention can be encoded by a nucleic acid comprising or consisting of a nucleotide sequence comprising (more preferably consisting of) any one of SEQ ID NOs: 154, 156, or 158.

[0110] A clostridial neurotoxin of the present invention (e.g., a modified clostridial neurotoxin) may comprise or consist of a polypeptide sequence having at least 70% sequence identity to one or more of SEQ ID NOs: 155, 157, 159, 166, 167, or 168. A clostridial neurotoxin of the present invention may comprise or consist of a polypeptide sequence having at least 80% or 90% sequence identity to one or more of SEQ ID NOs: 155, 157, 159, 166, 167, or 168. Preferably, a clostridial neurotoxin of the present invention may comprise or consist of (more preferably, consist of) a polypeptide sequence set forth in any one of SEQ ID NOs: 155, 157, 159, 166, 167, or 168.

[0111] The clostridial neurotoxin (e.g., modified clostridial neurotoxin) of the present invention is preferably BoNT / X, wherein the clostridial neurotoxin is encoded by a nucleic acid comprising or consisting of a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 160, 162, or 164. The clostridial neurotoxin may be encoded by a nucleic acid comprising or consisting of a nucleotide sequence having at least 80% or 90% sequence identity to SEQ ID NO: 160, 162, or 164. Preferably, the clostridial neurotoxin is encoded by a nucleic acid comprising or consisting of a nucleotide sequence comprising (or consisting of) SEQ ID NO: 160, 162, or 164. The clostridial neurotoxin of the present invention is preferably BoNT / X, wherein the clostridial neurotoxin comprises or consists of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 161, 163, 165, 166, 167, or 168. The clostridial neurotoxin may comprise or consist of a polypeptide sequence having at least 80% or 90% sequence identity to SEQ ID NO: 161, 163, 165, 166, 167, or 168. Preferably, the clostridial neurotoxin comprises (or consists of) the polypeptide sequence set forth in SEQ ID NO: 161, 163, 165, 166, 167, or 168.

[0112] The modified clostridial neurotoxin may be a single-chain clostridial neurotoxin encoded by a nucleic acid comprising or consisting of a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 175, wherein SEQ ID NO: 176 within SEQ ID NO: 175 is replaced by a nucleotide sequence encoding at least one exogenous protease cleavage site or a modified BoNT / C activation loop as defined herein, wherein the one or more exogenous protease sites are preferably selected from one or more of SEQ ID NOs: 15, 33, or 25, and / or the modified BoNT / C activation loop is preferably selected from one or more of SEQ ID NOs: 57, 63, and / or 65. The modified clostridial neurotoxin may be a single-chain clostridial neurotoxin encoded by a nucleic acid comprising or consisting of a nucleotide sequence having at least 80% or 90% sequence identity to SEQ ID NO: 175, wherein SEQ ID NO: 176 within SEQ ID NO: 175 is replaced by a nucleotide sequence encoding at least one exogenous protease cleavage site or a modified BoNT / C activation loop as defined herein, wherein the one or more exogenous protease sites are preferably selected from one or more of SEQ ID NOs: 15, 33, or 25, and / or the modified BoNT / C activation loop is preferably selected from one or more of SEQ ID NOs: 57, 63, and / or 65. The modified clostridial neurotoxin may be a single-chain clostridial neurotoxin encoded by a nucleic acid comprising or consisting of the nucleotide sequence of SEQ ID NO: 175, wherein SEQ ID NO: 176 within SEQ ID NO: 175 is replaced by a nucleotide sequence encoding at least one exogenous protease cleavage site or a modified BoNT / C activation loop as defined herein, wherein the one or more exogenous protease sites are preferably selected from one or more of SEQ ID NOs: 15, 33, or 25, and / or the modified BoNT / C activation loop is preferably selected from one or more of SEQ ID NOs: 57, 63, and / or 65.

[0113] Alternatively, or in addition, the modified clostridial neurotoxin may be a single-chain clostridial neurotoxin comprising or consisting of a polypeptide sequence having: (i) at least 70% sequence identity to SEQ ID NO: 150, wherein SEQ ID NO: 2 within SEQ ID NO: 150 is replaced by one or more exogenous protease cleavage sites and / or a modified BoNT / C activation loop as defined herein, wherein the one or more exogenous protease sites are preferably selected from one or more of SEQ ID NOs: 15, 33, or 25, and / or the modified BoNT / C activation loop is preferably is selected from one or more of SEQ ID NOs: 57, 63, and / or 65; or (ii) at least 70% identity to SEQ ID NO: 152, wherein SEQ ID NO: 2 within SEQ ID NO: 152 is replaced by one or more exogenous protease cleavage sites and / or modified BoNT / C activation loops as defined herein, wherein the one or more exogenous protease sites are preferably selected from one or more of SEQ ID NOs: 15, 33, or 25, and / or the modified BoNT / C activation loop is preferably selected from one or more of SEQ ID NOs: 57, 63, and / or 65.The modified clostridial neurotoxin may be a single-chain clostridial neurotoxin comprising or consisting of a polypeptide sequence having: (i) at least 80% or 90% sequence identity to SEQ ID NO: 150, wherein SEQ ID NO: 2 within SEQ ID NO: 150 has been replaced by one or more exogenous protease cleavage sites and / or a modified BoNT / C activation loop as defined herein, wherein the one or more exogenous protease sites are preferably selected from one or more of SEQ ID NOs: 15, 33, or 25, and / or the modified BoNT / C activation loop is preferably selected from SEQ ID NO: 5. or (ii) at least 80% or 90% identical to SEQ ID NO: 152, wherein SEQ ID NO: 2 within SEQ ID NO: 152 is replaced by one or more exogenous protease cleavage sites and / or modified BoNT / C activation loops as defined herein, wherein the one or more exogenous protease sites are preferably selected from one or more of SEQ ID NOs: 15, 33, or 25, and / or the modified BoNT / C activation loop is preferably selected from one or more of SEQ ID NOs: 57, 63, and / or 65.The modified clostridial neurotoxin may be a single-chain clostridial neurotoxin comprising or consisting of: (i) the polypeptide sequence of SEQ ID NO: 150, wherein SEQ ID NO: 2 within SEQ ID NO: 150 is replaced by one or more exogenous protease cleavage sites and / or a modified BoNT / C activation loop as defined herein, wherein the one or more exogenous protease sites are preferably selected from one or more of SEQ ID NOs: 15, 33, or 25, and / or the modified BoNT / C activation loop is preferably selected from one or more of SEQ ID NOs: 57, 63, and and / or 65; or (ii) a polypeptide sequence of SEQ ID NO: 152, wherein SEQ ID NO: 2 within SEQ ID NO: 152 is replaced by one or more exogenous protease cleavage sites and / or modified BoNT / C activation loops described herein, wherein the one or more exogenous protease sites are preferably selected from one or more of SEQ ID NOs: 15, 33, or 25, and / or the modified BoNT / C activation loop is preferably selected from one or more of SEQ ID NOs: 57, 63, and / or 65.

[0114] The polypeptide sequences of the invention (or the nucleotide sequences encoding them) may include a purification tag, such as a His tag. The invention is also intended to encompass polypeptide sequences (and the nucleotide sequences encoding them) from which the purification tag has been removed.

[0115] Exogenous proteases and exogenous protease cleavage sites

[0116] An exogenous protease according to the present invention is a protease that: (i) is not involved in cleavage of a native clostridial holotoxin (i.e., is not a clostridial hydrolase); and (ii) is not commonly used in the conventional production of clostridial neurotoxins (e.g., trypsin and / or Lys-C). The term "exogenous" refers to the relationship of the protease to the endogenous clostridial activation loop. Thus, a protease present in a patient to be treated can be considered an exogenous protease in the context of activating the modified clostridial neurotoxins of the present invention, even though the protease can be described as endogenous to the patient to be treated.

[0117] Thus, non-limiting examples of exogenous proteases according to the present invention may be selected from thrombin; tissue plasminogen activator (t-PA); urokinase (u-PA); factor IX (FIX); factor VIIa (FVIIa) and / or plasminogen. These proteases have a variety of different functions and are typically involved in the formation or degradation of blood clots in vivo. Preferably, the exogenous protease is selected from thrombin, t-PA and / or u-PA, with thrombin being particularly preferred. The exogenous protease is typically a human protease, e.g., a recombinant human protease. Alternatively, or in addition, the exogenous protease may be included in the formulation.

[0118] In other words, the modified clostridial neurotoxins of the present invention typically contain at least one cleavage site for one or more of thrombin, t-PA, u-PA, FIX, FVIIa, and / or plasminogen. Preferably, the modified clostridial neurotoxins of the present invention contain at least one cleavage site for one or more of thrombin, t-PA, and / or u-PA, and particularly preferably contain at least one cleavage site for thrombin.

[0119] Thrombin is a serine protease that hydrolyzes peptide bonds following arginine or lysine residues, including Xaa1-Xaa2-Pro / Ala / Leu / Gly-Arg / / Ser / Ala / Gly-Xaa3-Xaa4-Xaa5 (SEQ ID NO: 18), where Xaa 1乃至5 can each independently be selected from any amino acid, and " / / " indicates the position of the hydrolyzed peptide bond. The term "thrombin" encompasses the thrombins described herein, as well as any protease with structural and / or functional similarity (preferably structural and functional similarity) and the ability to hydrolyze the consensus Xaa1-Xaa2-Pro / Ala / Leu / Gly-Arg / / Ser / Ala / Gly-Xaa3-Xaa4-Xaa5 peptide bond. A preferred thrombin is human thrombin, which has UniProt accession number P00734 (sequence version 2, deposited January 1, 1990, accessed September 4, 2022), and is herein SEQ ID NO: 23. This sequence is the propeptide, which is converted to the human mature form by a process involving cleavage of the N-terminal polypeptide from residues 1 to 271 to form prethrombin 2 (P2), which is then further cleaved at Arg320 to yield thrombin. Human thrombin is commercially available from Merck (#69671).

[0120] Tissue plasminogen activator (t-PA or tPA) is a serine protease that cleaves at various consensus sequences, including: (i) cleavage after the arginine residue in the consensus Arg-Val; (ii) Xaa1-Phe / Tyr-Ser / Gly / Ala-Arg / / Xaa2-Xaa3-Xaa4-Xaa5 (SEQ ID NO: 34), where Xaa 1乃至5 can each independently be selected from any amino acid, and " / / " indicates the position of a hydrolyzed peptide bond; and / or (iii) Gly-Pro-Xaa1-Lys / Arg / / Xaa2-Gly-Gly-Xaa3 (SEQ ID NO: 35), where Xaa 1乃至3are each independently selected from any amino acid, where " / / " indicates the position of the hydrolyzed peptide bond. The term "t-PA" encompasses t-PA as described herein, as well as any protease having structural and / or functional similarity (preferably structural and functional similarity) and capable of hydrolyzing the consensus Arg-Val, Xaa1-Phe / Tyr-Ser / Gly / Ala-Arg / / Xaa1-Xaa2- Xaa3-Xaa4, or Gly-Pro- Xaa1-Lys / Arg / / Xaa2-Gly-Gly- Xaa3 peptide bond. A preferred t-PA is human t-PA, which has UniProt accession number P00750 (sequence version 1, deposited July 21, 1986, accessed September 11, 2022), and is SEQ ID NO: 43 herein. This sequence is the propeptide and is converted to the human mature form by a process that involves cleavage of the N-terminal polypeptide from residues 1 to 35. Human t-PA is commercially available from Merck (#T0831).

[0121] Urokinase (also known as urokinase plasminogen activator, u-PA, or uPA) is a serine protease that cleaves at various consensus sequences, including: (i) cleavage after the arginine residue in the consensus Arg-Val; and / or (ii) Xaa1-Ser-Gly / Ser-Arg / Lys / / Gly-Leu / Arg / Val-Xaa2-Asn / Gly (SEQ ID NO: 28), where Xaa 1乃至2can each independently be selected from any amino acid, and " / / " indicates the position of the hydrolyzed peptide bond. The term "u-PA" encompasses u-PA as described herein, as well as any protease with structural and / or functional similarity (preferably structural and functional similarity) and the ability to hydrolyze the consensus Arg-Val or Xaa1-Ser-Gly / Ser-Arg / Lys / / Gly-Leu / Arg / Val-Xaa2-Asn / Gly peptide bond. A preferred u-PA is human u-PA, which has UniProt Accession Number P29598 (sequence version 1, deposited April 1, 1993, accessed September 11, 2022), and is SEQ ID NO: 31 herein. This sequence is the propeptide and is converted to the human mature form by a process involving cleavage of the N-terminal polypeptide from residues 1 to 158. Human u-PA is commercially available from Merck (#672112).

[0122] Factor IX (FIX) is a serine protease that cleaves at various consensus sequences, including: (i) cleavage after the arginine residue in the consensus Arg-Ile; and / or (ii) Xaa1- Xaa2-Gly-Arg / / Xaa3-Xaa4-Xaa5-Xaa6 (SEQ ID NO: 45), where Xaa 1乃至6 can each independently be selected from any amino acid, and " / / " indicates the position of the hydrolyzed peptide bond. The term "Factor IX" encompasses FIX, as described herein, as well as any protease with structural and / or functional similarity (preferably structural and functional similarity) and the ability to hydrolyze the consensus Arg-Ile or Xaa1-Xaa2-Gly-Arg / / Xaa3-Xaa4-Xaa5-Xaa6 peptide bond. A preferred FIX is human FIX, which has UniProt accession number P00740 (sequence version 2, deposited June 7, 2005, accessed September 11, 2022), and is SEQ ID NO: 47 herein. This sequence is the propeptide and is converted to the human mature form by a process involving cleavage of the N-terminal polypeptide from residues 1 to 46. Human FIX is commercially available from Merck (#233279).

[0123] Factor VIIa (FVIIa) is a serine protease that cleaves at various consensus sequences, including after the arginine residue in the motif Arg-Ile. The term "Factor VIIa" encompasses the FVIIa described herein as well as any protease with structural and / or functional similarity (preferably structural and functional similarity) and the ability to hydrolyze the peptide bond of the consensus Arg-Ile. A preferred FVIIa is human FVIIa, which has UniProt accession number P08709 (sequence version 1, deposited January 1, 1988, accessed September 11, 2022), and is herein SEQ ID NO: 48. This sequence is the propeptide and is converted to the human mature form by a process involving cleavage of the N-terminal polypeptide from residues 1 to 60. Human FVIIa is commercially available from Merck (#P08709).

[0124] Plasminogen is a serine protease that cleaves at various consensus sequences, including: (i) after the lysine residue in the consensus Lys-Xaa, where Xaa is any amino acid; and / or (ii) after the arginine residue in the consensus Arg-Xaa, where Xaa is any amino acid. The term "plasminogen" encompasses plasminogen as described herein, as well as any protease with structural and / or functional similarity (preferably structural and functional similarity) and the ability to hydrolyze the consensus Lys-Xaa or Arg-Xaa peptide bond. A suitable plasminogen is human plasminogen, which has UniProt accession number P00747 (sequence version 1, deposited July 1, 1989, accessed September 11, 2022), and is represented herein by SEQ ID NO: 49. This sequence is the propeptide, which is converted to the human mature form (plasmin) by a process involving cleavage of the peptide bond between Arg580 and Val581. Human AEP is commercially available from Merck (#528185).

[0125] It is within the routine practice of one of ordinary skill in the art to determine the appropriate concentration / unit amount of any exogenous protease to activate the modified Clostridial neurotoxins of the present invention under standard / desired conditions for in vitro and ex vivo use.

[0126] In the context of the present invention, the term "thrombin" encompasses polypeptide sequences having at least 70% sequence identity to SEQ ID NO: 23 or its mature form. Thus, "thrombin" may include polypeptide sequences having at least 80% or 90% sequence identity to SEQ ID NO: 23 or its mature form. Preferably, thrombin comprises (more preferably consists of) SEQ ID NO: 23 or its mature form.

[0127] In the context of the present invention, the term "tissue plasminogen activator" encompasses polypeptide sequences having at least 70% sequence identity to SEQ ID NO: 43 or its mature form. Accordingly, "t-PA" may include polypeptide sequences having at least 80% or 90% sequence identity to SEQ ID NO: 43 or its mature form. Preferably, t-PA comprises (and more preferably consists of) SEQ ID NO: 43 or its mature form.

[0128] In the context of the present invention, the term "urokinase" encompasses polypeptide sequences having at least 70% sequence identity to SEQ ID NO: 31 or its mature form. Accordingly, "u-PA" may include polypeptide sequences having at least 80% or 90% sequence identity to SEQ ID NO: 31 or its mature form. Preferably, u-PA comprises (more preferably consists of) SEQ ID NO: 31 or its mature form.

[0129] In the context of the present invention, the term "Factor IX" encompasses polypeptide sequences having at least 70% sequence identity to SEQ ID NO: 47 or its mature form. Thus, "FIX" can include polypeptide sequences having at least 80% or 90% sequence identity to SEQ ID NO: 47 or its mature form. Preferably, FIX comprises (more preferably consists of) SEQ ID NO: 47 or its mature form.

[0130] In the context of the present invention, the term "Factor VIIa" encompasses polypeptide sequences having at least 70% sequence identity to SEQ ID NO: 48 or its mature form. Thus, "FVIIa" may include polypeptide sequences having at least 80% or 90% sequence identity to SEQ ID NO: 48 or its mature form. Preferably, FVIIa comprises (more preferably consists of) SEQ ID NO: 48 or its mature form.

[0131] In the context of the present invention, the term "plasminogen" encompasses polypeptide sequences having at least 70% sequence identity to SEQ ID NO: 49. Thus, "plasminogen" may include polypeptide sequences having at least 80% or 90% sequence identity to SEQ ID NO: 49 or its mature form. Preferably, plasminogen comprises (more preferably consists of) SEQ ID NO: 49.

[0132] When describing the exogenous protease cleavage sites of the present invention, it is not intended that any one Xaa (e.g., Xaa1, Xaa2, Xaa3, Xaa4, Xaa5, or Xaa6) be limited to only one type of amino acid. Thus, one or more residues present at any Xaa can be independently selected from the following standard amino acids: aspartic acid, glutamic acid, arginine, lysine, histidine, asparagine, glutamine, serine, threonine, tyrosine, methionine, tryptophan, cysteine, alanine, glycine, valine, leucine, isoleucine, proline, and phenylalanine.

[0133] Alternatively / additionally, one or more residues present at any Xaa (e.g., Xaa1, Xaa2, Xaa3, Xaa4, Xaa5, or Xaa6) may be independently selected from non-canonical amino acids (amino acids that are not part of the canonical 20 set described above). For example, non-standard amino acids can include 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline, α-methylserine, trans-3-methylproline, 2,4-methano-proline, cis-4-hydroxyproline, trans-4-hydroxy-proline, N-methylglycine, allo-threonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethylhomo-cysteine, nitro-glutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenyl-alanine, 4-azaphenyl-alanine, L-ornithine, L-2-amino-3-guanidinopropionic acid, or D-isomers of lysine, arginine, and / or ornithine, and 4-fluorophenylalanine. Methods for introducing non-standard amino acids into proteins are known in the art and include recombinant protein synthesis using an auxotrophic expression host such as E. coli.

[0134] The properties of standard amino acids are shown in the table below: [Table 1]

[0135] The following amino acids are considered charged amino acids: aspartic acid (negative), glutamic acid (negative), arginine (positive), and lysine (positive).

[0136] Exemplary (usually consensus) cleavage sites for thrombin, t-PA, u-PA, FIX, FVIIa, and plasminogen are described herein, and the modified clostridial neurotoxins of the invention may contain one or more of these cleavage sites.

[0137] The thrombin cleavage site may comprise or consist of a consensus sequence selected from the following: [ka]

[0138] The amino acid residues shown at each position (P4, P3, P2, P1, P1', P2', P3', and P4') are alternative amino acid residues at each position, with the bold and underlined residues being preferred. " / / " indicates the site of cleavage (i.e., the peptide bond that is hydrolyzed).

[0139] Therefore, the thrombin cleavage site is XX( P ALG)( RK) |(SAG)XXX (SEQ ID NO: 19) and / or XXX( RK) |XXXX (SEQ ID NO: 20), preferably XX PR |(SAG)XXX (SEQ ID NO: 21) and / or XX Penalty kick |(SAG)XXX (SEQ ID NO: 22), more preferably (AL)X( P LAG)( RK )|(SAG)(AVL)(GL)X (SEQ ID NO: 169) and / or (AL)X PR A preferred thrombin cleavage site may comprise or consist of a consensus sequence selected from |G(AVL)(GL)X (SEQ ID NO: 170); where each X can be independently selected from any amino acid, and the amino acid residues shown in brackets are alternative amino acid residues at each position, with bolded and underlined residues being preferred. The "|" indicates the site of cleavage (i.e., the peptide bond that is hydrolyzed). Thus, a preferred thrombin cleavage site may comprise or consist of a PRG motif (SEQ ID NO: 171).

[0140] The t-PA cleavage site may comprise or consist of a consensus sequence selected from the following: [ka]

[0141] The amino acid residues shown at each position (P4, P3, P2, P1, P1', P2', P3', and P4') are alternative amino acid residues at each position, with the bold and underlined residues being preferred. " / / " indicates the site of cleavage (i.e., the peptide bond that is hydrolyzed).

[0142] Therefore, the t-PA cleavage site is XXX R|V XXX (SEQ ID NO: 29), and / or X(FY)(SGA) R |XXXX (SEQ ID NO: 34), and / or S(FY)(SGA) R| XXXX (SEQ ID NO: 38), and / or GPX ( KR) |XGGX (SEQ ID NO: 35), and / or GPY ( KR) |XGGX (SEQ ID NO: 40), and / or GPX ( KR) |KGGX (SEQ ID NO: 41), and / or GPY ( KR )|KGGX (SEQ ID NO: 42); where each X can be independently selected from any amino acid, and the amino acid residues shown in brackets are alternative amino acid residues at each position, with bold and underlined residues being preferred. "|" indicates the site of cleavage (i.e., the peptide bond that is hydrolyzed).

[0143] The u-PA cleavage site may comprise or consist of a consensus sequence selected from the following: [ka]

[0144] The amino acid residues shown at each position (P4, P3, P2, P1, P1', P2', P3', and P4') are alternative amino acid residues at each position, with the bold and underlined residues being preferred. " / / " indicates the site of cleavage (i.e., the peptide bond that is hydrolyzed).

[0145] Therefore, the u-PA cleavage site is XXX R | VXXX (SEQ ID NO: 29), and / or XS( G S)( R K)|G(LRV)X(NG) (SEQ ID NO: 28), preferably XXX R | V XXX (SEQ ID NO: 29), and / or XS GR and may comprise or consist of a consensus sequence selected from |G(LRV)X(NG) (SEQ ID NO: 30); where each X can be independently selected from any amino acid, and the amino acid residues shown in brackets are alternative amino acid residues at each position, with bolded and underlined residues being preferred. "|" indicates the site of cleavage (i.e., the peptide bond that is hydrolyzed).

[0146] The u-PA and / or t-PA cleavage site may preferably comprise or consist of a consensus sequence selected from the following: [ka]

[0147] Therefore, a preferred u-PA and / or t-PA cleavage site is P(GPAL)(SGF)( GS )( R K)|( S GV)( A VLR) (SEQ ID NO: 172), and / or P(GPAL) (SGF) GR | SA (SEQ ID NO: 173), where each X can be independently selected from any amino acid, and the amino acid residues shown in brackets are alternative amino acid residues at each position, with bolded and underlined residues being preferred. The "|" indicates the site of cleavage (i.e., the peptide bond that is hydrolyzed). Thus, a preferred u-PA and / or t-PA cleavage site may comprise or consist of a GRSA motif (SEQ ID NO: 174).

[0148] The FIX cleavage site may comprise or consist of the following consensus sequence: [ka]

[0149] The amino acid residues shown at each position (P4, P3, P2, P1, P1', P2', P3', and P4') are alternative amino acid residues at each position, with the bold and underlined residues being preferred. " / / " indicates the site of cleavage (i.e., the peptide bond that is hydrolyzed).

[0150] Therefore, the FIX cleavage site is XXX R | I XXX (SEQ ID NO: 46), and / or XX GR It may comprise or consist of a consensus sequence of |XXXX (SEQ ID NO: 45), where each X can be independently selected from any amino acid, with bolded and underlined residues being preferred. "|" indicates the site of cleavage (i.e., the peptide bond that is hydrolyzed).

[0151] The FVIIa cleavage site may comprise or consist of a consensus sequence selected from the following: [ka]

[0152] The amino acid residues shown at each position (P4, P3, P2, P1, P1', P2', P3', and P4') are alternative amino acid residues at each position, with the bold and underlined residues being preferred. " / / " indicates the site of cleavage (i.e., the peptide bond that is hydrolyzed).

[0153] Therefore, the FVIIa cleavage site is XXX R | I XXX (SEQ ID NO: 46), where each X can be independently selected from any amino acid, with bolded and underlined residues being preferred. "|" indicates the site of cleavage (i.e., the peptide bond that is hydrolyzed).

[0154] The plasminogen cleavage site may comprise or consist of a consensus sequence selected from the following: [ka]

[0155] The amino acid residues shown at each position (P4, P3, P2, P1, P1', P2', P3', and P4') are alternative amino acid residues at each position, with the bold and underlined residues being preferred. " / / " indicates the site of cleavage (i.e., the peptide bond that is hydrolyzed).

[0156] Therefore, the plasminogen cleavage site is XXX R |XXXX (SEQ ID NO: 52), and / or XXX K XXXX (SEQ ID NO: 51), where each X can be independently selected from any amino acid, with bolded and underlined residues being preferred. The "|" indicates the site of cleavage (i.e., the peptide bond that is hydrolyzed).

[0157] An exogenous protease cleavage site of the present invention can be formed by inserting a peptide less than 10 amino acids in length, for example, from about 3 to about 9 amino acids in length, or from about 3 to about 8 amino acids in length. Thus, an exogenous protease cleavage site of the present invention can typically be formed by inserting a peptide 3, 4, 5, 6, 7, 8, or 9 amino acids in length.

[0158] Exogenous protease cleavage sites of the present invention may comprise or consist of one or more of the following: LTPRGVRL (SEQ ID NO: 15), LVPRGS (SEQ ID NO: 16), ENKSLVPRGS (SEQ ID NO: 17), KRV (SEQ ID NO: 24), SGRSA (SEQ ID NO: 25), VVPRVELVA (SEQ ID NO: 32), PPFGRSAG (SEQ ID NO: 33), PGSGRSAG (SEQ ID NO: 26), PGSGRSASGTTGTG (SEQ ID NO: 27), GRI (SEQ ID NO: 44), or PGRVVGG (SEQ ID NO: 50).

[0159] The exogenous protease cleavage site of the present invention may be a thrombin cleavage site, i.e., the exogenous protease cleavage site may be specific for thrombin. Such a thrombin cleavage site may comprise or consist of LTPRGVRL (SEQ ID NO: 15), ENKSLVPRGS (SEQ ID NO: 17), or LVPRGS (SEQ ID NO: 16), preferably LTPRGVRL (SEQ ID NO: 15) or ENKSLVPRGS (SEQ ID NO: 17). The thrombin cleavage site of LTPRGVRL (SEQ ID NO: 15) is particularly preferred.

[0160] The exogenous protease cleavage site of the present invention may be a t-PA and / or u-PA cleavage site, i.e., the exogenous protease cleavage site may be specific for t-PA and / or u-PA. Such a t-PA and / or u-PA cleavage site may comprise or consist of KRV (SEQ ID NO: 24), SGRSA (SEQ ID NO: 25), VVPRVELVA (SEQ ID NO: 32), or PPFGRSAG (SEQ ID NO: 33), preferably SGRSA (SEQ ID NO: 25), PGSGRSAG (SEQ ID NO: 26), or PGSGRSASGTTGTG (SEQ ID NO: 27), or PPFGRSAG (SEQ ID NO: 33). t-PA and / or u-PA cleavage sites of PPFGRSAG (SEQ ID NO: 33) and / or PGSGRSAG (SEQ ID NO: 26) may be particularly preferred.

[0161] The exogenous protease cleavage site of the present invention may be a u-PA cleavage site, i.e., the exogenous protease cleavage site may be specific for u-PA. Such a u-PA cleavage site may comprise or consist of KRV (SEQ ID NO: 24), SGRSA (SEQ ID NO: 25), PGSGRSAG (SEQ ID NO: 26), PGSGRSASGTTGTG (SEQ ID NO: 27), or PPFGRSAG (SEQ ID NO: 33), preferably SGRSA (SEQ ID NO: 25), PGSGRSAG (SEQ ID NO: 26), or PGSGRSASGTTGTG (SEQ ID NO: 27). The u-PA cleavage site of PGSGRSAG (SEQ ID NO: 26) may be particularly preferred.

[0162] The exogenous protease cleavage site of the present invention may be a t-PA cleavage site, i.e., the exogenous protease cleavage site may be specific for t-PA. Such a t-PA cleavage site may comprise or consist of SGRSA (SEQ ID NO: 25), VVPRVELVA (SEQ ID NO: 32), or PPFGRSAG (SEQ ID NO: 33), preferably PPFGRSAG (SEQ ID NO: 33) or SGRSA (SEQ ID NO: 25). The t-PA cleavage site of PPFGRSAG (SEQ ID NO: 33) may be particularly preferred.

[0163] The exogenous protease cleavage site of the present invention may be a FIX and / or FVIIa cleavage site, i.e., the exogenous protease cleavage site may be specific for FIX and / or FVIIa. Such a FIX and / or FVIIa cleavage site may comprise or consist of GRI (SEQ ID NO: 44).

[0164] The exogenous protease cleavage site of the present invention may be a plasminogen cleavage site, i.e., the exogenous protease cleavage site may be specific for plasminogen. Such a plasminogen cleavage site may comprise or consist of PGRVVGG (SEQ ID NO: 50).

[0165] The modified Clostridial neurotoxins of the present invention may contain one or more exogenous protease cleavage sites as defined herein.

[0166] In some embodiments, the exogenous protease cleavage site of the invention has at least 70% sequence identity to any one of SEQ ID NOs: 15-22, 24-30, 32-42, 44-46 and / or 50-52, particularly SEQ ID NOs: 15-22, 24-30 and / or 32-42, preferably SEQ ID NOs: 15, 17, 16, 26, 25, 27, 24, 33 and / or 32, particularly preferably SEQ ID NOs: 15, 17, 16, 26, 25 and / or 33. The exogenous protease cleavage site may have at least 80%, 85%, or 90% sequence identity to any one of SEQ ID NOs: 15-22, 24-30, 32-42, 44-46 and / or 50-52, in particular SEQ ID NOs: 15-22, 24-30 and / or 32-42, preferably SEQ ID NOs: 15, 17, 16, 26, 25, 27, 24, 33 and / or 32, particularly preferably SEQ ID NOs: 15, 17, 16, 26, 25 and / or 33. Preferably, the exogenous protease cleavage site has at least 95% sequence identity to any one of SEQ ID NOs: 15 to 22, 24 to 30, 32 to 42, 44 to 46 and / or 50 to 52, in particular SEQ ID NOs: 15 to 22, 24 to 30 and / or 32 to 42, preferably SEQ ID NOs: 15, 17, 16, 26, 25, 27, 24, 33 and / or 32, particularly preferably SEQ ID NOs: 15, 17, 16, 26, 25 and / or 33. More preferably, the exogenous protease cleavage site has at least 99% sequence identity to any one of SEQ ID NOs: 15-22, 24-30, 32-42, 44-46 and / or 50-52, in particular SEQ ID NOs: 15-22, 24-30 and / or 32-42, preferably SEQ ID NOs: 15, 17, 16, 26, 25, 27, 24, 33 and / or 32, particularly preferably SEQ ID NOs: 15, 17, 16, 26, 25 and / or 33.Particularly preferred are exogenous protease cleavage sites comprising or consisting of any one of SEQ ID NOs: 15 to 22, 24 to 30, 32 to 42, 44 to 46 and / or 50 to 52, in particular SEQ ID NOs: 15 to 22, 24 to 30 and / or 32 to 42, preferably SEQ ID NOs: 15, 17, 16, 26, 25, 27, 24, 33 and / or 32, particularly preferably SEQ ID NOs: 15, 17, 16, 26, 25 and / or 33.

[0167] Typically, the exogenous protease cleavage site(s) comprises or consists of one or more of the amino acid sequences of any one of SEQ ID NOs: 15-22, 24-30, 32-42, 44-46 and / or 50-52, in particular SEQ ID NOs: 15-22, 24-30 and / or 32-42, preferably SEQ ID NOs: 15, 17, 16, 26, 25, 27, 24, 33 and / or 32, particularly preferably SEQ ID NOs: 15, 17, 16, 26, 25 and / or 33, or the exogenous activation loop comprises the one or more exogenous protease cleavage sites.

[0168] The modified clostridial neurotoxins of the present invention may contain one or more exogenous protease cleavage sites. In other words, the modified clostridial neurotoxins of the present invention may contain one exogenous protease cleavage site described herein, or multiple exogenous protease cleavage sites. The modified clostridial neurotoxins of the present invention may contain 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more exogenous protease cleavage sites. As a non-limiting example, the modified clostridial neurotoxins of the present invention may contain 2 to 7 (2, 3, 4, 5, 6, or 7) exogenous protease cleavage sites. When the modified clostridial neurotoxins of the present invention contain multiple exogenous protease cleavage sites, each of these may be independently selected. Typically, when the modified clostridial neurotoxins of the present invention contain multiple exogenous protease cleavage sites, each of the exogenous protease cleavage sites may be independently selected from the exogenous protease cleavage sites described herein. Thus, the modified clostridial neurotoxins of the present invention may contain multiple exogenous protease cleavage sites that are different from each other, or the modified clostridial neurotoxins of the present invention may contain two or more copies of a specific exogenous protease cleavage site, or any combination thereof.

[0169] Multiple exogenous protease cleavage sites may be introduced into a single location within a clostridial neurotoxin. Alternatively, multiple exogenous protease cleavage sites may be introduced into multiple locations within a clostridial neurotoxin. As a non-limiting example, one exogenous protease cleavage site may be introduced into the activation loop or modified BoNT / C (BoNT / C1) activation loop within a clostridial neurotoxin, and another (the same or different) exogenous protease cleavage site may be introduced into the LH NThe exogenous protease cleavage sites may be introduced into a domain. Typically, when multiple exogenous protease cleavage sites are introduced into a modified clostridial neurotoxin according to the present invention, they are introduced into a single position within the clostridial neurotoxin. Preferably, when multiple exogenous protease cleavage sites are introduced into a modified clostridial neurotoxin according to the present invention, they are each introduced into the activation loop of the clostridial neurotoxin or the modified BoNT / C (BoNT / C1) activation loop. The relative positioning of each exogenous protease cleavage site within the exogenous activation loop or modified BoNT / C (BoNT / C1) activation loop of the present invention can be determined based on the structure-function relationship of the exogenous protease. Based on this structure-function relationship, it is within the ordinary skill of a person skilled in the art to appropriately position each exogenous protease cleavage site within an exogenous activation loop or modified BoNT / C (BoNT / C1) activation loop containing multiple exogenous protease cleavage sites without undue burden.

[0170] An exogenous protease cleavage site can be contained within a modified Clostridial toxin activation loop. The modified Clostridial toxin activation loop can be of the same serotype as the modified Clostridial neurotoxin, or can be derived from a different serotype than the modified Clostridial neurotoxin. Typically, the modified Clostridial toxin activation loop is derived from a different serotype than the modified Clostridial neurotoxin. Preferably, the modified Clostridial activation loop is derived from BoNT / C or BoNT / A, with a modified Clostridial activation loop derived from BoNT / C being particularly preferred.

[0171] The modified clostridial activation loop is one in which the endogenous activation site or a portion thereof has been replaced with an exogenous protease cleavage site as described herein. Preferably, the modified clostridial activation loop may be (i) a BoNT / C activation loop in which the endogenous BoNT / C activation site or a portion thereof has been replaced with an exogenous protease cleavage site as defined herein; or (ii) a BoNT / A activation loop in which the endogenous BoNT / A activation site or a portion thereof has been replaced with an exogenous protease cleavage site as defined herein. Particularly preferred is a modified clostridial activation loop that is a BoNT / C activation loop in which the endogenous BoNT / C activation site or a portion thereof has been replaced with an exogenous protease cleavage site as described herein.

[0172] As described herein, one or more exogenous protease cleavage sites may be included in an exogenous activation loop or a modified BoNT / C activation loop along with one or more spacer sequences as defined herein. A spacer sequence may typically be present when the one or more exogenous protease cleavage sites are short motifs (e.g., typically less than 15 amino acids, preferably less than 10 amino acids, or less than 9 amino acids in length). One or more spacers may be present at the N-terminus and / or C-terminus of each of the exogenous protease cleavage sites. Preferably, the spacer may be a GS spacer as defined herein.

[0173] An exogenous activation loop or modified BoNT / C activation loop can include one or more exogenous protease cleavage sites (e.g., two, three, four, or five exogenous protease cleavage sites, typically two or three exogenous protease cleavage sites). The one or more exogenous protease cleavage sites can be cleavable by different exogenous proteases; as a non-limiting example, the exogenous activation loop or modified BoNT / C activation loop can include a thrombin cleavage site and a t-PA cleavage site. When an exogenous activation loop or modified BoNT / C activation loop includes one or more exogenous protease cleavage sites, and the one or more exogenous protease sites are cleavable by different exogenous proteases, excision of a modified clostridial neurotoxin that includes the exogenous activation loop or modified BoNT / C activation loop, particularly excision of the modified clostridial neurotoxin in vivo, can be increased. Alternatively, multiple copies of the same exogenous protease cleavage site can be included within the exogenous activation loop or modified BoNT / C activation loop. Whether the exogenous activation loop or modified BoNT / C activation loop contains multiple different exogenous protease cleavage sites or multiple copies of the same exogenous protease cleavage site, the cleavage sites can be directly linked or separated by one or more spacers as described herein.

[0174] Non-limiting examples of exogenous activation loops, including modified BoNT / C activation loops according to the present invention, include the following: [Table 2]

[0175] Here, the substituted residues are shown in bold, the inserted residues are shown in italics, the plasminogen residues are double underlined, and the other residues are present in the corresponding unmodified clostridial activation loop.

[0176] An exogenous activation loop or modified BoNT / C activation loop that includes one or more exogenous protease cleavage sites can be of any length, so long as the structure of the exogenous activation loop or modified BoNT / C activation loop, and generally the structure of the clostridial neurotoxin, is maintained and cleavage at the one or more exogenous protease cleavage sites results in the formation of an active two-chain form of the modified clostridial neurotoxin. The exogenous activation loop or modified BoNT / C activation loop can have a length of about 10 to about 80, e.g., about 10 to about 50, about 10 to about 40, or about 10 to about 30 (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 120, 130, 140, 141, The exogenous activation loop or modified BoNT / C activation loop may be 17 amino acids in length, for example, about 15 to about 35 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) amino acids in length, and preferably about 15 to about 30 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) amino acids in length. Particularly preferred is an exogenous activation loop or modified BoNT / C activation loop that is 17 amino acids in length, in which case the exogenous activation loop or modified BoNT / C activation loop is the same length as the endogenous BoNT / C (BoNT / C1) activation loop.

[0177] Clostridial neurotoxins

[0178] As used herein, the term "neurotoxin" refers to any polypeptide that enters neurons and inhibits neurotransmitter release. This process involves 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" encompasses any polypeptide produced by Clostridial bacteria (clostridial neurotoxins) that enters neurons and inhibits neurotransmitter release, as well as such polypeptides produced by recombinant or chemical means. 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.

[0179] The clostridial neurotoxins of the present invention may be catalytically active (also called active) or catalytically inactive. Preferably, the clostridial neurotoxins of the present invention are catalytically active.

[0180] The terms "catalytically active" or "active," as used interchangeably herein, refer to a clostridial neurotoxin L chain (or a clostridial neurotoxin containing such an L chain) that has non-cytotoxic protease activity. Specifically, an active clostridial neurotoxin L chain has endopeptidase activity and is capable of cleaving a protein of the extracellular fusion apparatus within a target cell. The protein of the extracellular fusion apparatus is preferably a SNARE protein, such as SNAP25, synaptobrevin / VAMP, or syntaxin.

[0181] The term "catalytically inactive" as used herein with respect to a clostridial neurotoxin L chain means that the L chain exhibits substantially no cytotoxic protease activity, and preferably, the term "catalytically inactive" as used herein with respect to a clostridial neurotoxin L chain means that the L chain exhibits no cytotoxic protease activity. In one embodiment, a catalytically inactive clostridial neurotoxin L chain does not cleave proteins of the exocellular fusion apparatus in target cells. The term "substantially free of cytotoxic protease activity" means that a clostridial neurotoxin L chain has less than 5% of the cytotoxic protease activity of a catalytically active clostridial neurotoxin L chain, for example, less than 2%, less than 1%, or preferably less than 0.1% of the cytotoxic protease activity of a catalytically active clostridial neurotoxin L chain. Non-cytotoxic protease activity can be measured in vitro by incubating a test Clostridial neurotoxin L chain with a SNARE protein and comparing the amount of SNARE protein cleaved by the test Clostridial neurotoxin L chain with the amount of SNARE protein cleaved by a catalytically active Clostridial neurotoxin L chain under the same conditions. The amount of cleaved SNARE protein can be quantified using conventional techniques such as SDS-polyacrylamide gel electrophoresis and Western blotting. A suitable in vitro assay is described in WO 2019 / 145577 A1, which is incorporated herein by reference.

[0182] The Clostridial neurotoxin (e.g., before modification) may be BoNT / A. Exemplary reference BoNT / A sequences are set forth in SEQ ID NOs: 91-98 and 135, particularly SEQ ID NOs: 91 and 135.

[0183] The clostridial neurotoxin (e.g., before modification) can be BoNT / B. Exemplary reference BoNT / B sequences are set forth in SEQ ID NOs: 99-106, particularly SEQ ID NO: 99.

[0184] The clostridial neurotoxin (e.g., before modification) can be BoNT / C. An exemplary reference BoNT / C1 sequence is set forth in SEQ ID NO:107.

[0185] The clostridial neurotoxin (e.g., before modification) can be BoNT / D. An exemplary reference BoNT / D sequence is set forth in SEQ ID NO:108.

[0186] The clostridial neurotoxin (e.g., before modification) can be BoNT / E. Exemplary reference BoNT / E sequences are set forth in SEQ ID NOs: 111-123, particularly SEQ ID NO: 111.

[0187] The clostridial neurotoxin (e.g., before modification) can be BoNT / F. Exemplary reference BoNT / F sequences are set forth in SEQ ID NOs: 124-130, particularly SEQ ID NO: 124.

[0188] The clostridial neurotoxin (e.g., before modification) can be BoNT / G. An exemplary reference BoNT / G sequence is set forth in SEQ ID NO:131.

[0189] The clostridial neurotoxin (e.g., before modification) can be BoNT / X. An exemplary reference BoNT / X sequence is set forth in SEQ ID NO:133.

[0190] The clostridial neurotoxin (e.g., before modification) can be TeNT. An exemplary reference TeNT sequence is set forth in SEQ ID NO:134.

[0191] In some preferred embodiments, the clostridial neurotoxin (e.g., before modification) is BoNT / A, BoNT / B, or BoNT / X, or a chimera thereof (e.g., BoNT / AB), as described herein.

[0192] As mentioned above, activated clostridial neurotoxins are formed from two polypeptide chains: a heavy chain (H chain) having a molecular weight of approximately 100 kDa, and a light chain (L chain) having a molecular weight of approximately 50 kDa. The H chain contains a C-terminal targeting component (receptor binding domain or H C domain) and N-terminal translocation component (H N domain).

[0193] Examples of light chain reference sequences include: Botulinum type A neurotoxin: amino acid residues 1 to 448 Botulinum type B neurotoxin: amino acid residues 1 to 440 Botulinum type C1 neurotoxin: amino acid residues 1 to 441 Botulinum type D neurotoxin: amino acid residues 1 to 445 Botulinum type E neurotoxin: amino acid residues 1 to 422 Botulinum type F neurotoxin: amino acid residues 1 to 439 Botulinum type G neurotoxin: amino acid residues 1 to 441 Tetanus neurotoxin: amino acid residues 1 to 457

[0194] For the recently identified BoNT / X, the L chain is reported to correspond to amino acids 1 to 439, although the boundaries of the L chain may vary by as much as 25 amino acids (e.g., 1 to 414 or 1 to 464). Preferably, the L chain of the modified clostridial neurotoxin of the present invention is the L chain of BoNT / X.

[0195] The reference sequences provided above should be considered as a guideline and slight variations may occur depending on the serosubtype. For example, US 2007 / 0166332 (hereby incorporated by reference in its entirety) lists slightly different Clostridial sequences. Botulinum type A neurotoxin: amino acid residues M1 to K448 Botulinum type B neurotoxin: amino acid residues M1 to K441 Botulinum type C1 neurotoxin: amino acid residues M1 to K449 Botulinum type D neurotoxin: amino acid residues M1 to R445 Botulinum type E neurotoxin: amino acid residues M1 to R422 Botulinum type F neurotoxin: amino acid residues M1 to K439 Botulinum type G neurotoxin: amino acid residues M1 to K446 Tetanus neurotoxin: amino acid residues M1 to A457

[0196] Alternatively, the L chain of a clostridial neurotoxin can be defined as the amino acid sequence from the first amino acid (with or without the initiating methionine residue) to the first cysteine ​​residue of the intrinsic activation loop. Additionally or alternatively, the L chain of a clostridial neurotoxin can be defined as the amino acid sequence N-terminal to the cleavage site within the intrinsic activation loop.

[0197] The L chain of a clostridial neurotoxin can be defined as a clostridial neurotoxin domain containing a metal-coordinating HExxH motif (SEQ ID NO: 143), which typically functions to cleave SNARE protein substrates.

[0198] The term "light chain" (or "L chain") encompasses variants and fragments thereof, provided that the variants and fragments still exhibit non-cytotoxic protease activity (which can be determined using standard assays known in the art, examples of which are described herein). By way of example, a variant may have at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% or at least 98% amino acid sequence homology with a reference L chain. The term "fragment," when used in reference to an L chain, refers to a peptide having at least 200, preferably at least 250, more preferably at least 300, even more preferably at least 350, and most preferably at least 400 amino acid residues of the reference L chain. In the case of clostridial L chains, the fragment preferably has at least 300, more preferably at least 350, and most preferably at least 400 amino acid residues of the reference L chain. L chain "fragments" of the present invention encompass fragments of variant L chains based on a reference sequence.

[0199] The H chain of a clostridial neurotoxin can be defined as the sequence from the second cysteine ​​to the last amino acid of the intrinsic activation loop. Additionally or alternatively, the H chain of a clostridial neurotoxin can be defined as beginning with the amino acid sequence C-terminal to the cleavage site within the intrinsic activation loop. Additionally or alternatively, the H chain of a clostridial neurotoxin can be defined as beginning with the amino acid C-terminal to the cysteine ​​residue (particularly the second cysteine ​​residue) that forms the disulfide bond between the L chain and the H chain and thus defines the C-terminus of the intrinsic activation loop.

[0200] A translocation domain is a molecule that allows the translocation of a protease to a target cell, thereby functionally expressing protease activity in the cytosol of the target cell. Whether any molecule (e.g., a protein or peptide) has the requisite translocation function of the present invention can be confirmed using any one of several conventional assays.

[0201] For example, Shone C. (1987) describes an in vitro assay using liposomes, in which the liposomes are exposed to a test molecule. The presence of the necessary translocation function is determined by the release of K from the liposomes. + and / or by the release of labeled NAD, which can be easily monitored (see Shone C. (1987) Eur. J. Biochem; vol. 167(1): pp. 175-180).

[0202] A further example is provided by Blaustein R. (1987), who describes a simple in vitro assay utilizing planar phospholipid bilayer membranes: the membrane is exposed to a test molecule, and an increase in conductance across the membrane confirms the required translocation function (see Blaustein (1987) FEBS Letts; vol. 226, no. 1: pp. 115-120).

[0203] Further methodology that allows for the assessment of membrane fusion and thereby the identification of translocation domains suitable for use in the present invention is provided in Methods in Enzymology Vol 220 and 221, Membrane Fusion Techniques, Parts A and B, Academic Press 1993.

[0204] The present invention also encompasses variants and / or fragments of translocation domains, provided that the variant domain still exhibits the requisite translocation activity. By way of example, a variant may have at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% or at least 98% amino acid sequence homology with the reference translocation domain. The term fragment, when used in connection with a translocation domain, refers to a peptide having at least 20, preferably at least 40, more preferably at least 80, and most preferably at least 100 amino acid residues of the reference translocation domain. In the case of a clostridial translocation domain, the fragment preferably has at least 20 amino acid residues of the reference translocation domain (e.g., H N A translocation "fragment" of the present invention includes fragments of variant translocation domains based on a reference sequence.

[0205] Preferably, the translocation domain is capable of forming an ion-permeable pore in a lipid membrane under conditions of low pH. It has been found that preferably only those portions of the protein molecule capable of forming a pore in the endosomal membrane are used.

[0206] The translocation domain may be derived from a microbial protein source, in particular a bacterial or viral protein source. Thus, the translocation domain may be the translocation domain of an enzyme, such as a bacterial toxin or a viral protein.

[0207] The ability of specific domains of bacterial toxin molecules to form such pores has been well documented, and specific translocation domains of membrane fusion proteins expressed by viruses are also known to be capable of forming such pores. Such domains can be used in the present invention.

[0208] The translocation domain may be derived from Clostridium, e.g., H N domain (or a functional component thereof). N means a part or fragment of the H chain of a clostridial neurotoxin approximately equivalent to the amino-terminal half of the H chain, or a domain corresponding to that fragment in an intact H chain. C The function is H C It can be removed by deletion of amino acid sequences (either at the DNA synthesis stage or post-synthesis by treatment with nucleases or proteases). C The function may be inactivated by chemical or biological treatment, and therefore the heavy chain may be unable to bind to the binding site on the target cell to which the native clostridial neurotoxin (i.e., the holotoxin) binds.

[0209] Examples of suitable (reference) translocation domains include: Botulinum type A neurotoxin: amino acid residues (449 to 871) Botulinum type B neurotoxin: amino acid residues (441 to 858) Botulinum type C neurotoxin: amino acid residues (442 to 866) Botulinum type D neurotoxin: amino acid residues (446 to 862) Botulinum type E neurotoxin: amino acid residues (423 to 845) Botulinum type F neurotoxin: amino acid residues (440 to 864) Botulinum type G neurotoxin: amino acid residues (442 to 863) Botulinum type X neurotoxin: amino acid residues (461 to 890) Tetanus neurotoxin: amino acid residues (458 to 879)

[0210] For the recently identified BoNT / X, the translocation domain is reported to correspond to amino acids 460 to 890 of the L chain and H chain. C The boundary can vary by as little as 10 amino acids (eg, 461 to 889 or 454 to 891). Preferably, the translocation domain of a modified clostridial neurotoxin of the present invention is a BoNT / X translocation domain.

[0211] The reference sequences given above should be considered as a guideline and slight variations may occur depending on the serosubtype. For example, US 2007 / 0166332 (hereby incorporated by reference) lists slightly different Clostridial sequences. Botulinum type A neurotoxin: amino acid residues (A449 to K871) Botulinum type B neurotoxin: amino acid residues (A442 to S858) Botulinum type C neurotoxin: amino acid residues (T450 to N866) Botulinum type D neurotoxin: amino acid residues (D446 to N862) Botulinum type E neurotoxin: amino acid residues (K423 to K845) Botulinum type F neurotoxin: amino acid residues (A440 to K864) Botulinum type G neurotoxin: amino acid residues (S447 to S863) Tetanus neurotoxin: amino acid residues (S458 to V879)

[0212] In the context of the present invention, various clostridial neurotoxins H containing a translocation domain N Regions may be useful in embodiments of the present invention, provided that these active fragments promote the release of a non-cytotoxic protease (e.g., a clostridial light chain) from intracellular vesicles into the cytoplasm of a target cell, thereby allowing the clostridial neurotoxin to participate in the overall cellular mechanism of proteolytically cleaving a substrate. N The region is approximately 410 to 430 amino acids long and contains the translocation domain. NIt has been shown that the entire length of the region is not necessary for the translocation activity of the translocation domain. Thus, in the context of the present invention, a translocation domain refers to a Clostridial neurotoxin H containing a translocation domain having a length of, for example, at least 350 amino acids, at least 375 amino acids, at least 400 amino acids, and at least 425 amino acids. N Also included are Clostridial neurotoxin Hs containing translocation domains having lengths of, for example, up to 350 amino acids, up to 375 amino acids, up to 400 amino acids, and up to 425 amino acids. N Areas are also included.

[0213] For further details on the genetic basis of toxin production in Clostridium botulinum and Clostridium tetani, see Henderson et al (1997) The Clostridia: Molecular Biology and Pathogenesis, Academic Press.

[0214] Term H N is a naturally occurring neurotoxin H N modified H having a moiety and a non-naturally occurring amino acid sequence and / or synthetic amino acid residues. N However, modified H N provided that the moiety continues to exhibit the translocation function described above.

[0215] Alternatively, the translocation domain may be of non-clostridial origin. Examples of sources of non-clostridial (reference) translocation domains include the translocation domain of diphtheria toxin (O'Keefe et al., Proc. Natl. Acad. Sci. USA (1992) 89, 6202-6206; Silverman et al., J. Biol. Chem. (1993) 269, 22524-22532; and London, E. (1992) Biochem. Biophys. Acta., 1112, pp.25-51), the translocation domain of Pseudomonas exotoxin type A (Prior et al. Biochemistry (1992) 31, 3555-3559), the translocation domains of anthrax toxin (Blanke et al. Proc. Natl. Acad. Sci. USA (1996) 93, Examples of translocation domains include, but are not limited to, peptides with translocating function (Plank et al. J. Biol. Chem. (1994) 269, 12918-12924; and Wagner et al. (1992) PNAS, 89, pp. 7934-7938), and amphiphilic peptides (Murata et al. (1992) Biochem., 31, pp. 1986-1992). Translocation domains may reflect translocation domains present in naturally occurring proteins, or may contain amino acid variations that do not impair the translocation ability of the translocation domain.

[0216] Specific examples of viral (reference) translocation domains suitable for use in the present invention include specific translocation domains of membrane fusion proteins expressed by viruses. For example, Wagner et al. (1992) and Murata et al. (1992) describe the translocation (i.e., membrane fusion and vesicle formation) functions of several fusogenic and amphipathic peptides derived from the N-terminal region of influenza virus hemagglutinin. Other virally expressed membrane fusion proteins known to have desired translocation activity include the translocation domain of the fusogenic peptide of Semliki Forest virus (SFV), the translocation domain of vesicular stomatitis virus (VSV) glycoprotein G, the translocation domain of the SER virus F protein, and the translocation domain of the foamy virus envelope glycoprotein. Virus-encoded A spike proteins, such as the E1 protein of SFV and the G protein of VSV, are particularly useful in the context of the present invention.

[0217] Use of the translocation domains listed in the table (see below) includes use of sequence variants thereof. Variants may contain one or more conservative nucleic acid substitutions and / or nucleic acid deletions or insertions, provided that the variant retains the required translocation function. Variants may also contain one or more amino acid substitutions and / or amino acid deletions, insertions, or insertion-deletions (indels), so long as the variant retains the required translocation function. [Table 3]

[0218] Clostridial neurotoxin H C Examples of domain reference sequences include: BoNT / A-N872 to L1296 BoNT / B-E859 to E1291 BoNT / C1-N867 to E1291 BoNT / D-S863 to E1276 BoNT / E-R846 to K1252 BoNT / F-K865 to E1274 BoNT / G-N864 to E1297 TeNT-I880 to D1315

[0219] Regarding the recently identified BoNT / X, H C The domain is reported to correspond to amino acids 893 to 1306, and the domain boundary may vary by as much as 25 amino acids (e.g., 868 to 1306 or 918 to 1306). Preferably, the H of the modified clostridial neurotoxin of the present invention C The domain is H of BoNT / X. C It is a domain.

[0220] The clostridial neurotoxins described herein can further comprise a translocation-facilitating domain, which facilitates delivery of the non-cytotoxic protease into the cytosol of a target cell, and is described, for example, in WO 08 / 008803 and WO 08 / 008805, each of which is incorporated herein by reference.

[0221] By way of example, a suitable translocation-facilitating domain includes an enveloped virus fusogenic peptide domain, e.g., suitable fusogenic peptide domains include influenza virus fusogenic peptide domains (e.g., a 23 amino acid influenza A virus fusogenic peptide domain), alphavirus fusogenic peptide domains (e.g., a 26 amino acid Semliki Forest virus fusogenic peptide domain), vesiculovirus fusogenic peptide domains (e.g., a 21 amino acid vesicular stomatitis virus fusogenic peptide domain), respirovirus fusogenic peptide domains (e.g., a 25 amino acid Sendai virus fusogenic peptide domain), morbidity and mortality (e.g., a 25 amino acid ... Examples of such a fusogenic peptide domain include a fusogenic peptide domain of an avian virus (e.g., a 25-amino acid canine distemper virus fusogenic peptide domain), a fusogenic peptide domain of an avian virus (e.g., a 25-amino acid Newcastle disease virus fusogenic peptide domain), a fusogenic peptide domain of a henipavirus (e.g., a 25-amino acid Hendra virus fusogenic peptide domain), a metapneumovirus fusogenic peptide domain (e.g., a 25-amino acid human metapneumovirus fusogenic peptide domain), or a spumavirus fusogenic peptide domain, such as a simian foamy virus fusogenic peptide domain; or a fragment or variant thereof.

[0222] As a further example, the translocation facilitating domain may be a clostridial neurotoxin H CN The neurotoxin may comprise a domain, or a fragment or variant thereof. More particularly, the neurotoxin may comprise a domain, such as Clostridial neurotoxin H CN The translocation facilitating domain may have a length of at least 200 amino acids, at least 225 amino acids, at least 250 amino acids, or at least 275 amino acids. CN The translocation facilitating domain preferably has a length of at most 200 amino acids, at most 225 amino acids, at most 250 amino acids, or at most 275 amino acids. Specific examples (reference examples) include: Botulinum type A neurotoxin: amino acid residues (872 to 1110) Botulinum type B neurotoxin: amino acid residues (859 to 1097) Botulinum type C neurotoxin: amino acid residues (867 to 1111) Botulinum type D neurotoxin: amino acid residues (863 to 1098) Botulinum type E neurotoxin: amino acid residues (846 to 1085) Botulinum type F neurotoxin: amino acid residues (865 to 1105) Botulinum type G neurotoxin: amino acid residues (864 to 1105) Botulinum type X neurotoxin: amino acid residues (890 to 1121) Tetanus neurotoxin: amino acid residues (880 to 1127)

[0223] The above sequence positions may vary slightly depending on the serotype / subtype. CN Further examples of domains include: Botulinum type A neurotoxin: amino acid residues (874 to 1110) Botulinum type B neurotoxin: amino acid residues (861 to 1097) Botulinum type C neurotoxin: amino acid residues (869 to 1111) Botulinum type D neurotoxin: amino acid residues (865 to 1098) Botulinum type E neurotoxin: amino acid residues (848 to 1085) Botulinum type F neurotoxin: amino acid residues (867 to 1105) Botulinum type G neurotoxin: amino acid residues (866 to 1105) Tetanus neurotoxin: amino acid residues (882 to 1127)

[0224] Any of the above-described facilitating domains can be combined with any of the above-described translocation domain peptides suitable for use in the present invention. Thus, by way of example, a non-clostridial facilitating domain can be combined with a non-clostridial translocation domain peptide, or with a clostridial translocation domain peptide. Alternatively, a clostridial neurotoxin H CN The translocation facilitating domain can be combined with a non-clostridial translocation domain peptide.CN The facilitating domain can be combined with a Clostridial translocation domain peptide, such as the following examples: Botulinum type A neurotoxin: amino acid residues (449 to 1110) Botulinum type B neurotoxin: amino acid residues (442 to 1097) Botulinum type C neurotoxin: amino acid residues (450 to 1111) Botulinum type D neurotoxin: amino acid residues (446 to 1098) Botulinum type E neurotoxin: amino acid residues (423 to 1085) Botulinum type F neurotoxin: amino acid residues (440 to 1105) Botulinum type G neurotoxin: amino acid residues (447 to 1105) Tetanus neurotoxin: amino acid residues (458 to 1127)

[0225] In some embodiments, the clostridial neurotoxins of the present invention comprise a functional H C Thus, in the binding assay described by Shone et al. (1985) Eur. J. Biochem. 151, 75-82, the clostridial neurotoxin (Clostridial H C The clostridial neurotoxin is unable to bind to rat synaptosomal membranes (via a component thereof). The clostridial neurotoxin may preferably lack the last 50 C-terminal amino acids of a clostridial neurotoxin holotoxin. The clostridial neurotoxin may preferably lack the last 100, preferably the last 150, more preferably the last 200, particularly preferably the last 250, and most preferably the last 300 C-terminal amino acid residues of a clostridial neurotoxin holotoxin. Alternatively, the clostridial neurotoxin may lack the last 50 C-terminal amino acid residues of a clostridial neurotoxin holotoxin. C Binding activity can be abolished / reduced by mutagenesis. For convenience, referring to BoNT / A, for example, mutation of one or two amino acid residues in the ganglioside-binding pocket (W1266 to L and Y1267 to F) results in the binding of H CThe region loses its receptor binding function. Similar mutations can be made to non-serogroup A clostridial peptide components, for example, constructs based on botulinum B (W1262 to L and Y1263 to F) or botulinum E (W1224 to L and Y1225 to F) with the mutations. Other mutations to the active site can also be made to H. C These mutations eliminate receptor binding activity, for example, Y1267S in botulinum type A toxin and the corresponding highly conserved residue in other clostridial neurotoxins. Details of this and other mutations are described in Rummel et al. (2004) (Molecular Microbiol. 51:631-634), which is incorporated herein by reference.

[0226] Naturally occurring clostridial neurotoxins H C The peptide contains approximately 400 to 440 amino acid residues and is divided into two functionally distinct domains, each of approximately 25 kDa: the N-terminal region (generally H CN peptide or domain) and the C-terminal region (generally H CCThis reality is confirmed by the following publications, each of which is incorporated herein by reference in its entirety: Umland TC (1997) Nat. Struct. Biol. 4: 788-792; Herreros J (2000) Biochem. J. 347: 199-204; Halpern J (1993) J. Biol. 104: 359-364; Lacey DB (1998) Nat. Struct. Biol. 5: 898-902; Knapp (1998) Am. Cryst. Assoc. Abstract Papers 25: 90; Swaminathan and Eswaramoorthy (2000) Nat. Struct. Biol. 7: 1751-1759; and Rummel A (2004) Mol. Microbiol. 51(3), 631-643. Furthermore, the C-terminal region (H CC It has been well established, and the above publications confirm, that the C-terminal 160-200 amino acid residues of the heavy chain H are responsible for the binding of clostridial neurotoxins to their natural cellular receptors, i.e., the nerve endings of the neuromuscular junction. Therefore, throughout this specification, the term "functional heavy chain H" is used to refer to the functional heavy chain H. C When referring to a clostridial heavy chain that lacks a peptide (or domain) that prevents the heavy chain from binding to the cell surface receptor to which a native clostridial neurotoxin binds, it is understood that the clostridial heavy chain is merely a functional H CC This means that the peptide is missing. CC The peptide region can be partially or completely deleted or modified (e.g., by conventional chemical treatment or proteolytic excision) to inactivate its natural binding ability to nerve endings at the neuromuscular junction.

[0227] Therefore, the Clostridial neurotoxin H of the present invention NThe peptide may be C-terminally extended, i.e., Clostridial neurotoxin H C Domain, e.g. H CN , H CC , or H C The clostridial neurotoxin H of the present invention may be associated with all or part of the domain. N Reference to a peptide includes such a C-terminally extended H N peptide, which is Clostridial neurotoxin H C Alternatively, the clostridial neurotoxin H of the present invention may comprise one or more amino acid residues from the clostridial neurotoxin H domain. N The peptide binds to Clostridial neurotoxin H C Domain, e.g. H CN , H CC , or H C It may not be associated with (or lack) all or part of a domain.

[0228] Typically, the clostridial neurotoxin of the present invention or the clostridial neurotoxin H of the present invention N The peptides consist of the C-terminal peptide portion (H CC ) lacking all or part of the H of natural clostridial neurotoxins. C It lacks binding function. For example, the C-terminally extended Clostridium H N The peptide may lack the C-terminal 40 amino acid residues, or the C-terminal 60 amino acid residues, or the C-terminal 80 amino acid residues, or the C-terminal 100 amino acid residues, or the C-terminal 120 amino acid residues, or the C-terminal 140 amino acid residues, or the C-terminal 150 amino acid residues, or the C-terminal 160 amino acid residues of the Clostridial neurotoxin heavy chain. N The peptides consist of the entire C-terminal peptide portion of the clostridial neurotoxin (H CC ) and thus lacking the H of the native clostridial neurotoxin. C It may lack the binding function. For example, Clostridium H NThe peptide may lack the C-terminal 165 amino acid residues, or the C-terminal 170 amino acid residues, or the C-terminal 175 amino acid residues, or the C-terminal 180 amino acid residues, or the C-terminal 185 amino acid residues, or the C-terminal 190 amino acid residues, or the C-terminal 195 amino acid residues of the clostridial neurotoxin heavy chain. N The peptide is a Clostridium H selected from the group consisting of: CC Missing reference sequence: Botulinum type A neurotoxin: amino acid residues (Y1111 to L1296) Botulinum type B neurotoxin: amino acid residues (Y1098 to E1291) Botulinum type C neurotoxin: amino acid residues (Y1112 to E1291) Botulinum type D neurotoxin: amino acid residues (Y1099 to E1276) Botulinum type E neurotoxin: amino acid residues (Y1086 to K1252) Botulinum type F neurotoxin: amino acid residues (Y1106 to E1274) Botulinum type G neurotoxin: amino acid residues (Y1106 to E1297) Botulinum type X neurotoxin: amino acid residues (Y1122 to D1306) Tetanus neurotoxin: amino acid residues (Y1128 to D1315)

[0229] The reference sequences given above should be considered as a guideline and slight variations may occur depending on the serosubtype.

[0230] The present invention is suitable for application to a wide variety of clostridial neurotoxins. Thus, in the context of the present invention, the term "clostridial neurotoxin" includes toxins produced by Clostridium botulinum (botulinum neurotoxin serotypes A, B, C1, D, E, F, G, H, and X), Clostridium tetani (tetanus neurotoxin), Clostridium butyricum (botulinum neurotoxin serotype E), and Clostridium baratii (botulinum neurotoxin serotype F), as well as modified clostridial neurotoxins or derivatives derived from any of the above. The term "clostridial neurotoxin" also includes botulinum neurotoxin serotype H. In some preferred embodiments, the clostridial neurotoxin is BoNT / A, more preferably BoNT / A1. In other preferred embodiments, the clostridial neurotoxin is BoNT / X.

[0231] Botulinum neurotoxins (BoNTs) are produced by Clostridium botulinum in the form of large protein complexes consisting of BoNT itself in complex with several accessory proteins. Currently, nine distinct classes of botulinum neurotoxins exist: botulinum neurotoxin serotypes A, B, C1, D, E, F, G, H, and X, all of which share similar structures and mechanisms of action. Different BoNT serotypes can be distinguished based on inactivation by specific neutralizing antisera, and this serotype classification correlates with the percentage of sequence identity at the amino acid level. BoNT proteins of a given serotype are further divided into distinct subtypes based on the percentage of amino acid sequence identity.

[0232] BoNTs are absorbed in the gastrointestinal tract and enter the systemic circulation, where they bind to the presynaptic membrane of cholinergic nerve terminals and inhibit the release of the neurotransmitter acetylcholine. BoNT / B, BoNT / D, BoNT / F, and BoNT / G cleave synaptobrevin / vesicle-associated membrane protein (VAMP); BoNT / C1, BoNT / A, and BoNT / E cleave 25 kDa synaptosomal-associated protein (SNAP-25); and BoNT / C1 cleaves syntaxin. BoNT / X has been shown to cleave SNAP-25, VAMP1, VAMP2, VAMP3, VAMP4, VAMP5, Ykt6, and syntaxin 1.

[0233] Tetanus toxin is produced by Clostridium tetani in a single serotype: Clostridium butyricum produces BoNT / E, and Clostridium baratii produces BoNT / F.

[0234] The term "clostridial neurotoxin" is also intended to encompass modified clostridial neurotoxins and their derivatives, including, but not limited to, those described below. A modified clostridial neurotoxin or derivative may contain one or more amino acids that are modified compared to the native (unmodified) form of the clostridial neurotoxin, or may contain one or more inserted amino acids that are not present in the native (unmodified) form of the clostridial neurotoxin. By way of example, a modified clostridial neurotoxin may contain a modified amino acid sequence in one or more domains compared to the native (unmodified) clostridial neurotoxin sequence. Such modifications may alter functional aspects of the toxin, such as biological activity or biological persistence. Thus, the clostridial neurotoxin of the present invention may be a modified clostridial neurotoxin, or a modified clostridial neurotoxin derivative, or a clostridial neurotoxin derivative. In particular, the modified clostridial neurotoxin of the present invention may be a modified, modified clostridial neurotoxin, or a modified, modified clostridial neurotoxin derivative, or a modified clostridial neurotoxin derivative.

[0235] A modified Clostridial neurotoxin has one or more modifications in the amino acid sequence of the heavy chain (e.g., modified H C domain), and the modified heavy chain binds to target neurons with higher or lower affinity compared to the native (unmodified) Clostridial neurotoxin. C Such modifications in the 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 Clostridial neurotoxins are described in WO 2006 / 027207 and WO 2006 / 114308, both of which are incorporated by reference in their entireties.

[0236] Most preferably, BoNT / BH C The 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 further comprises a substitution, insertion, indel, or deletion of at least one amino acid residue in the subdomain. CC Suitable amino acid residue substitutions, insertions, indels, or deletions in the subdomains are disclosed in WO 2013 / 180799 and WO 2016 / 154534 (both of which are incorporated herein by reference).

[0237] BoNT / BH CC Suitable amino acid residue substitutions, insertions, indels, or deletions in the subdomains may comprise 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.

[0238] BoNT / BH CC The substitution, insertion, indel, or deletion of a suitable amino acid residue in the subdomain may further comprise a combination 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, E11 91Q 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.

[0239] BoNT / BH CC Suitable amino acid residue substitutions, insertions, indels, or deletions in the subdomains may also include a combination of the three substitution mutations E1191M, S1199W, and W1178Q.

[0240] Preferably, BoNT / BH CC Suitable amino acid residue substitutions, insertions, indels, or deletions in the subdomain may include a combination of two substitution mutations, E1191M and S1199Y. Such modifications are present in the chimeric clostridial neurotoxin of SEQ ID NO: 136. E1191M may correspond to position 1204 and S1199Y to position 1212 of SEQ ID NO: 136. Thus, SEQ ID NO: 136 may include 1204M and 1212Y.

[0241] This modification may be a modification compared to the unmodified BoNT / B set forth as SEQ ID NO:99, where the numbering of the amino acid residues is determined by alignment with SEQ ID NO:99. Because the presence of a methionine residue at position 1 of SEQ ID NO:99 (as well as SEQ ID NOs corresponding to the other clostridial neurotoxin polypeptides described herein, including chimeric clostridial neurotoxin polypeptides) is optional, one skilled in the art would consider the presence / absence of the methionine residue when determining the amino acid residue numbering. For example, if SEQ ID NO:99 contains a methionine, the position numbering would be as defined above (e.g., E1191 would become E1191 in SEQ ID NO:99). Alternatively, if a methionine is absent in SEQ ID NO:99, the amino acid residue numbering must be corrected by −1 (e.g., E1191 would become E1190 in SEQ ID NO:99). Thus, the first methionine amino acid residue in the polypeptide sequence of a chimeric clostridial neurotoxin may be optional or absent. Similar considerations apply for the presence / absence of methionine at position 1 of other polypeptide sequences described herein, and the skilled artisan will readily determine the correct amino acid residue numbering using techniques routine in the art. Alignments may be performed using any of the methods described herein for determining percent sequence homology and / or sequence identity.

[0242] The modified Clostridial neurotoxin may have one or more modifications in the amino acid sequence of the light chain, for example, modifications in the substrate binding or catalytic domain that may alter or modify the SNARE protein specificity of the modified L chain. Examples of such modified Clostridial neurotoxins are described in WO 2010 / 120766 and US 2011 / 0318385, both of which are incorporated by reference in their entireties.

[0243] The modified clostridial neurotoxin may contain one or more modifications that increase or decrease the biological activity and / or biological persistence of the modified clostridial neurotoxin. For example, the modified clostridial neurotoxin may contain a leucine-based or tyrosine-based motif, which increases or decreases the biological activity and / or biological persistence of the modified clostridial neurotoxin. Suitable leucine-based motifs include xDxxxLL (SEQ ID NO: 144), xExxxLL (SEQ ID NO: 145), xExxxIL (SEQ ID NO: 146), and xExxxLM (SEQ ID NO: 147) (where x is any amino acid). Suitable tyrosine-based motifs include Yxx-Hy (where Hy is a hydrophobic amino acid). Examples of modified clostridial neurotoxins containing leucine-based and tyrosine-based motifs are described in WO 2002 / 008268, the entire contents of which are incorporated herein by reference.

[0244] The term "clostridial neurotoxin" is intended to encompass hybrid clostridial neurotoxins and chimeric clostridial neurotoxins. A hybrid clostridial neurotoxin comprises at least a portion of a light chain from one clostridial neurotoxin or subtype thereof and at least a portion of a heavy chain from another clostridial neurotoxin or clostridial neurotoxin subtype. A hybrid clostridial neurotoxin may contain the entire light chain from one clostridial neurotoxin subtype and a heavy chain from another clostridial neurotoxin subtype. A chimeric clostridial neurotoxin may contain a portion of the heavy chain (e.g., the binding domain) from one clostridial neurotoxin subtype, with another portion of the heavy chain being from another clostridial neurotoxin subtype. Chimeric clostridial neurotoxins, particularly chimeric BoNTs, can be defined by the serotype or serosubtype of the four major domains of the neurotoxin: L chain, H chain, and H chain. N , H CN , and H CC (as defined herein). For example, the (before modification) LH of SEQ ID NO: 136 N / A1-H CThe B1 chimera can be described as an AABB chimera. Similarly, or alternatively, a therapeutic element may comprise light chain portions from different clostridial neurotoxins. Such hybrid or chimeric clostridial neurotoxins are useful, for example, as a means of delivering the therapeutic benefits of a particular clostridial neurotoxin to patients who are immunologically resistant to a particular clostridial neurotoxin subtype, to patients who may have lower-than-average receptor concentrations for a particular clostridial neurotoxin heavy chain binding domain, or to patients who may have protease-resistant variants of membrane or vesicular toxin substrates (e.g., SNAP-25, VAMP, and syntaxin). Hybrid and chimeric clostridial neurotoxins are described in U.S. Pat. No. 8,071,110, which is incorporated herein by reference in its entirety. Thus, the clostridial neurotoxins of the present invention may be hybrid or chimeric clostridial neurotoxins. In particular, the modified clostridial neurotoxins of the present invention may be modified hybrid or chimeric clostridial neurotoxins.

[0245] In some preferred embodiments, the clostridial neurotoxin is a BoNT / A that includes at least one domain from a non-BoNT / A clostridial neurotoxin (e.g., a BoNT / A hybrid or chimera). For example, a clostridial neurotoxin of the present invention (including one or more exogenous protease cleavage sites) can include: i. BoNT / A L chain and non-BoNT / A H chain N and H C domain; ii. BoNT / A H N Domains and non-BoNT / A L and H chains C domain iii. BoNT / A H C Domains and non-BoNT / A L and H chains N domain; iv. BoNT / A L chain and H chain N Domain and non-BoNT / A H Cdomain; v. BoNT / A L chain and H chain C Domain and non-BoNT / A H N domain; or vi. BoNT / A H N Domain and H C domains and non-BoNT / A light chains.

[0246] As a non-limiting example, a clostridial neurotoxin (e.g., a modified clostridial neurotoxin) of the present invention may comprise a clostridial neurotoxin comprising the light chain and heavy chain of BoNT / A. N Domain and H of BoNT / B C Domain (e.g., LH N / A1-H C / B1). Unmodified LH, which may be modified to contain one or more exogenous protease cleavage sites in accordance with the present invention. N / A1-H C An example of a B1 chimera is given in SEQ ID NO: 136. N / A1-H C Examples of modified forms of the B1 chimera are given in SEQ ID NOs: 155, 157, and 159. The clostridial neurotoxins (e.g., modified clostridial neurotoxins) of the present invention comprise the light and heavy chains of BoNT / A. N Domain and H of BoNT / C1 C The clostridial neurotoxins of the present invention (e.g., modified clostridial neurotoxins) may comprise the L chain and H chain of BoNT / A. N Domain and H of BoNT / D C The clostridial neurotoxins of the present invention (e.g., modified clostridial neurotoxins) may comprise the L chain and H chain of BoNT / A. N Domain and H of BoNT / E C The clostridial neurotoxins of the present invention (e.g., modified clostridial neurotoxins) may comprise the L chain and H chain of BoNT / A. N Domain and H of BoNT / F C The clostridial neurotoxins of the present invention (e.g., modified clostridial neurotoxins) may comprise the L chain and H chain of BoNT / A. NDomain and H of BoNT / G C The clostridial neurotoxins of the present invention (e.g., modified clostridial neurotoxins) may comprise the L chain and H chain of BoNT / A. N Domain and H of BoNT / X C The clostridial neurotoxins of the present invention (e.g., modified clostridial neurotoxins) may comprise the L chain and H chain of BoNT / A. N Domain and TeNT H C It may include a domain.

[0247] For example, a clostridial neurotoxin of the present invention (e.g., a modified clostridial neurotoxin containing one or more exogenous protease cleavage sites) can include: i. BoNT / B L chain and non-BoNT / B H chain N and H C domain; ii. H of BoNT / B N Domains and non-BoNT / B L and H chains C domain iii. H of BoNT / B C Domains and non-BoNT / B L and H chains N domain; iv. BoNT / B L chain and H chain N Domain and non-BoNT / B H C domain v. BoNT / B L chain and H chain C Domain and non-BoNT / B H N domain; or vi. BoNT / B H N Domain and H C domains and non-BoNT / B light chains.

[0248] For example, a clostridial neurotoxin of the present invention (e.g., a modified clostridial neurotoxin containing one or more exogenous protease cleavage sites) can include: i. BoNT / C1 L chain and non-BoNT / C1 H chain N and H C domain; ii. H of BoNT / C1 N Domains and non-BoNT / C1 L and H chains C domain iii. H of BoNT / C1 C Domains and non-BoNT / C1 L and H chains N domain; iv. BoNT / C1 L chain and H chain N Domain and non-BoNT / C1 H C domain v. BoNT / C1 L chain and H chain C Domain and non-BoNT / C1 H N domain; or vi. BoNT / C1 H N Domain and H C domains and non-BoNT / C1 light chains.

[0249] Non-limiting examples include BoNT / C1 chimeras in which the non-BoNT / C1 element is from BoNT / D (ie, BoNT / CD chimeras).

[0250] For example, a clostridial neurotoxin of the present invention (e.g., a modified clostridial neurotoxin containing one or more exogenous protease cleavage sites) can include: i. BoNT / D L chain and non-BoNT / D H chain N and H C domain; ii. H of BoNT / D N Domains and non-BoNT / D L and H chains C domain iii. H of BoNT / D C Domains and non-BoNT / D L and H chains N domain; iv. BoNT / D L chain and H chain N Domain and non-BoNT / D H C domain v. BoNT / D L chain and H chain C Domain and non-BoNT / D H N domain; or vi. BoNT / D HN Domain and H C domain and non-BoNT / D light chains.

[0251] Non-limiting examples include BoNT / D chimeras in which the non-BoNT / D element is from BoNT / C1 (ie, a BoNT / DC1 chimera).

[0252] For example, a clostridial neurotoxin of the present invention (e.g., a modified clostridial neurotoxin containing one or more exogenous protease cleavage sites) can include: i. BoNT / E L chain and non-BoNT / E H chain N and H C domain; ii. BoNT / E H N Domains and non-BoNT / E L and H chains C domain iii. BoNT / E H C Domains and non-BoNT / E L and H chains N domain; iv. BoNT / E L chain and H chain N Domain and non-BoNT / E H C domain v. BoNT / E L chain and H chain C Domain and non-BoNT / E H N domain; or vi. BoNT / E H N Domain and H C domains and non-BoNT / E light chains.

[0253] For example, a clostridial neurotoxin of the present invention (e.g., a modified clostridial neurotoxin containing one or more exogenous protease cleavage sites) can include: i. BoNT / F L chain and non-BoNT / F H chain N and H C domain; ii. H of BoNT / F N Domains and non-BoNT / F L and H chains C domain iii. H of BoNT / F C Domains and non-BoNT / F L and H chains N domain; iv. BoNT / F L chain and H chain N Domain and non-BoNT / F H C domain v. BoNT / F L chain and H chain C Domain and non-BoNT / F H N domain; or vi. BoNT / F H N Domain and H C domain and non-BoNT / F light chains.

[0254] For example, a clostridial neurotoxin of the present invention (e.g., a modified clostridial neurotoxin containing one or more exogenous protease cleavage sites) can include: i. BoNT / G L chain and non-BoNT / G H chain N and H C domain; ii. H of BoNT / G N Domains and non-BoNT / G L and H chains C domain iii. H of BoNT / G C Domains and non-BoNT / G L and H chains N domain; iv. BoNT / G L chain and H chain N Domain and non-BoNT / G H C domain v. BoNT / G L chain and H chain C Domain and non-BoNT / G H N domain; or vi. BoNT / G H N Domain and H C domains and non-BoNT / G light chains.

[0255] For example, a clostridial neurotoxin of the present invention (e.g., a modified clostridial neurotoxin containing one or more exogenous protease cleavage sites) can include: i. BoNT / X L chain and non-BoNT / X H chain N and H C domain; ii. H of BoNT / X N Domains and non-BoNT / X L and H chains C domain iii. H of BoNT / X C Domains and non-BoNT / X L and H chains N domain; iv. BoNT / X L chain and H chain N Domain and non-BoNT / X H C domain v. BoNT / X L chain and H chain C Domain and non-BoNT / X H N domain; or vi. BoNT / X H N Domain and H C domains and non-BoNT / X light chains.

[0256] For example, a clostridial neurotoxin of the present invention (e.g., a modified clostridial neurotoxin containing one or more exogenous protease cleavage sites) can include: i. TeNT light chain and non-TeNT heavy chain N and H C domain; ii. TeNT H N Domains and non-TeNT L and H chains C domain iii. TeNT H C Domains and non-TeNT L and H chains N domain; iv. TeNT L chain and H chain N Domain and non-TeNT H C domain v. TeNT L chain and H chain C Domain and non-TeNT H N domain; or vi. TeNT H N Domain and H C domains and non-TeNT light chains.

[0257] The term "clostridial neurotoxins" also encompasses newly discovered botulinum neurotoxin and botulinum neurotoxin-like protein family members expressed by non-clostridial microorganisms, such as the toxin encoded by Enterococcus spp. that shares closest sequence identity with BoNT / X, the toxin encoded by Weissella oryzae called BoNT / Wo (NCBI reference sequence: WP_027699549.1) that cleaves VAMP2 at W89-W90, the toxin encoded by Enterococcus faecium (GenBank: OTO22244.1) that cleaves VAMP2 and SNAP25, the toxin encoded by Chryseobacterium pipero (NCBI reference sequence: WP_034687872.1), and the mosquito BoNT-like protein PMP1 (NCBI reference sequence: QEZ70852.1).

[0258] The term "clostridial neurotoxin" is intended to encompass retargeted clostridial neurotoxins. In a retargeted clostridial neurotoxin, the clostridial neurotoxin is modified to include an exogenous ligand (i.e., not derived from the clostridial neurotoxin) known as a targeting moiety (TM). The TM is selected to provide binding specificity for the desired target cell, and as part of the retargeting process, the clostridial neurotoxin's native binding moiety (e.g., H C Domain or H CCdomain) may be removed. Retargeting techniques are described, for example, in: EP-B-0689459; WO 1994 / 021300; EP-B-0939818; US 6,461,617; US 7,192,596; WO 1998 / 007864; EP-B-0826051; US ​​5,989,545; US 6,395,513; US 6,962,703; WO 1996 / 033273; EP-B-0996468; US 7,052,702; WO 1999 / 017806; EP-B-1107794; US 6,632,440; WO 2000 / 010598; WO 2001 / 21213; WO 2006 / 059093; WO 2000 / 62814; WO 2000 / 04926; WO 1993 / 15766; WO 2000 / 61192; and WO 1999 / 58571; all of which are incorporated herein by reference in their entirety. Thus, the clostridial neurotoxin of the present invention may be a retargeted clostridial neurotoxin. In particular, the modified clostridial neurotoxin of the present invention may be a modified, retargeted clostridial neurotoxin. The modified, retargeted clostridial neurotoxin of the present invention may comprise a TM displayed at the N-terminus or C-terminus of the single-chain neurotoxin, or the TM may be displayed centrally within the single-chain neurotoxin. In some preferred embodiments, the modified, retargeted clostridial neurotoxin of the present invention may comprise a TM displayed at the N-terminus or C-terminus of the single-chain neurotoxin.

[0259] Retargeted clostridial neurotoxins may be engineered to utilize TMs that are susceptible to cleavage by proteases traditionally used to activate recombinantly produced retargeted clostridial neurotoxins, such as trypsin, Lys-C, and / or BoNT hydrolase. Thus, retargeted clostridial neurotoxins engineered to contain one or more exogenous protease activation sites in accordance with the present invention may provide improved stability compared to corresponding retargeted clostridial neurotoxins activated by traditional activating proteases, such as Lys-C, trypsin, and / or BoNT hydrolase.

[0260] In some preferred embodiments, the modified, retargeted Clostridial neurotoxin comprises a BoNT / A light chain (LC / A) and / or a BoNT / A translocation domain (H N / A), and particularly preferably LC / A and H N / A, and optionally, in the pre-modification retargeted clostridial neurotoxin, the endogenous BoNT / A activation loop is replaced with the endogenous BoNT / C activation loop. A non-limiting example of a modified retargeted clostridial neurotoxin may include SEQ ID NO: 150, wherein SEQ ID NO: 2 within SEQ ID NO: 150 is replaced by one or more exogenous protease cleavage sites and / or modified BoNT / C activation loops described herein, wherein the one or more exogenous protease sites are preferably selected from one or more of SEQ ID NOs: 15, 33, or 25, and / or the modified BoNT / C activation loop is preferably selected from one or more of SEQ ID NOs: 57, 63, and / or 65.

[0261] In some particularly preferred embodiments, the modified, retargeted Clostridial neurotoxin comprises a BoNT / X light chain (LC / X) and / or a BoNT / X translocation domain (H N / X), and particularly preferably LC / X and H N / X, and optionally, in the pre-modification retargeted clostridial neurotoxin, the endogenous BoNT / X activation loop is replaced with the endogenous BoNT / C activation loop. Such modified retargeted BoNT / X is particularly preferred. A non-limiting example of a modified retargeted clostridial neurotoxin may include SEQ ID NO: 152, in which SEQ ID NO: 2 is replaced with one or more exogenous protease cleavage sites and / or modified BoNT / C activation loops described herein, in which the one or more exogenous protease sites are preferably selected from one or more of SEQ ID NOs: 15, 33, or 25, and / or the modified BoNT / C activation loop is preferably selected from one or more of SEQ ID NOs: 57, 63, and / or 65.

[0262] The clostridial neurotoxins of the present invention (e.g., modified clostridial neurotoxins) contain a functional H C The polypeptide may lack the native binding function of a clostridial neurotoxin, and may also lack any functionally equivalent TM. The polypeptide thus lacks the native binding function of a clostridial neurotoxin, and binds to a clostridial H neurotoxin in the binding assay described by Shone et al. (1985) Eur. J. Biochem. 151, 75-82. C The TM is incapable of binding to rat synaptosomal membranes (through a TM component, or through any functionally equivalent TM). Preferably, the TM is not a wheat germ agglutinin (WGA) peptide. Thus, in some preferred embodiments, the clostridial neurotoxin is incapable of binding to endogenous H of the clostridial neurotoxin. C or H CC Particularly preferred are retargeted clostridial neurotoxins in which the endogenous H of the clostridial neurotoxin is replaced by an exogenous TM. C or H CC is a retargeted Clostridial neurotoxin replaced by exogenous TM.

[0263] The clostridial neurotoxins (e.g., modified clostridial neurotoxins) of the present invention are LH N polypeptides (e.g., modified LH N polypeptides), i.e., clostridial L chains and clostridial H chains as defined herein. N It may include a polypeptide comprising or consisting of a domain.

[0264] Clostridial neurotoxins (e.g., modified clostridial neurotoxins) inhibit LH N polypeptides (e.g., modified LH N polypeptide) and a targeting moiety (TM).

[0265] The present invention also encompasses clostridial neurotoxins with additional, non-naturally occurring protease cleavage sites. Such sites would require an exogenous protease for cleavage, thereby allowing for better control over the timing and location of the cleavage event. Additional non-naturally occurring protease cleavage sites that can be used in clostridial neurotoxins include: TEV (Tobacco Etch Virus) (ENLYFQ↓G) (SEQ ID NO: 148) PreScission (LEVLFQ↓GP) (SEQ ID NO: 149).

[0266] Additional protease cleavage sites include recognition sequences cleaved by non-cytotoxic proteases, such as the light chain of a clostridial neurotoxin. These include SNARE (e.g., SNAP-25, syntaxin, VAMP) protein recognition sequences cleaved by non-cytotoxic proteases, such as the light chain of a clostridial neurotoxin. Clostridial neurotoxins containing non-natural protease cleavage sites are described in US 7,132,259, EP 1206554-B2, and US 2007 / 0166332, all of which are incorporated herein by reference in their entirety. The term protease cleavage site also encompasses inteins, which are self-cleaving sequences. Self-splicing reactions can be controlled, for example, by changing the concentration of reducing agents present.

[0267] The present invention also encompasses clostridial neurotoxins containing a "destructive cleavage site." In the clostridial neurotoxins, a non-natural protease cleavage site is incorporated into the clostridial neurotoxin, and its position is selected so that cleavage at the site reduces the activity of the clostridial neurotoxin or inactivates the clostridial neurotoxin. The destructive protease cleavage site may be susceptible to cleavage by a local protease if the clostridial neurotoxin migrates to a non-target site after administration. Suitable non-natural protease cleavage sites include those described above. Clostridial neurotoxins containing destructive cleavage sites are described in WO 2010 / 094905 and WO 2002 / 044199, both of which are incorporated herein by reference in their entireties.

[0268] The clostridial neurotoxins (e.g., modified clostridial neurotoxins) of the present invention, particularly the light chain component thereof, may be pegylated, which may contribute to improving the stability, e.g., duration of action, of the light chain component. Pegylation is particularly preferred when the light chain comprises BoNT / A, B, or C1 protease. Pegylation preferably involves adding PEG to the N-terminus of the light chain component. For example, the N-terminus of the light chain may be extended with one or more amino acid (e.g., cysteine) residues, which may be the same or different. One or more of the amino acid residues may have their own PEG molecule attached (e.g., covalently attached) thereto. An example of this technology is described in WO2007 / 104567, the entire contents of which are incorporated herein by reference.

[0269] The chimeric Clostridial neurotoxins of the present invention may not include a therapeutic or diagnostic agent (e.g., a nucleic acid, protein, peptide, or small molecule therapeutic, or diagnostic agent) in addition to the light and heavy chains. For example, in one embodiment, the chimeric Clostridial neurotoxin may not include a covalently or non-covalently bound therapeutic or diagnostic agent. Thus, the chimeric Clostridial neurotoxins of the present invention preferably do not function as a delivery vehicle for an additional therapeutic or diagnostic agent.

[0270] In embodiments in which the chimeric Clostridial neurotoxins described herein have a tag for purification (eg, a His tag) and / or a linker, the tag and / or linker are optional.

[0271] The clostridial neurotoxins of the present invention (eg, modified clostridial neurotoxins) may be free of complex proteins present in naturally occurring clostridial neurotoxin complexes.

[0272] The clostridial neurotoxins (e.g., modified clostridial neurotoxins) of the present invention can be produced using recombinant nucleic acid technology. Thus, the modified clostridial neurotoxin (described above) can be a recombinant modified clostridial neurotoxin. The single-chain clostridial neurotoxin (described above) can be a recombinant single-chain neurotoxin.

[0273] Tolerance (i.e., a reduction in the number and / or severity of any side effects) to the modified Clostridial neurotoxins of the present invention may be increased compared to resistance to the corresponding (unmodified) Clostridial neurotoxin. In particular, resistance to the modified Clostridial neurotoxins of the present invention may be increased compared to resistance to the corresponding (unmodified) Clostridial neurotoxin when the unmodified Clostridial neurotoxin is administered (e.g., in a di-chain form).

[0274] The potency of the modified clostridial neurotoxins of the present invention may be equivalent to or increased compared to the potency of the corresponding (unmodified) clostridial neurotoxin. In particular, the potency of the modified clostridial neurotoxins of the present invention may be equivalent to or increased compared to the potency of the corresponding (unmodified) clostridial neurotoxin when the unmodified clostridial neurotoxin is administered to a subject in a di-chain form. As used herein, the term "equivalent potency" means that the modified clostridial neurotoxin has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and up to about 100% of the potency of the corresponding (unmodified) clostridial neurotoxin. Preferably, as used herein, "equivalent potency" means that the modified clostridial neurotoxin has at least about 95%, at least about 99%, at least about 100%, at least about 101%, and up to about 105% of the potency of the corresponding (unmodified) clostridial neurotoxin. As used herein, the term "increased potency" means that the modified clostridial neurotoxin has at least about 10%, at least about 15%, at least about 20%, or at least about 25% greater potency than the potency of the corresponding (unmodified) clostridial neurotoxin. Potency can be measured using any suitable assay, examples of which are described herein.

[0275] The modified clostridial neurotoxins of the present invention generally have an improved safety profile and / or therapeutic range compared to the safety profile and / or therapeutic range of the corresponding (unmodified) clostridial neurotoxin. Without being bound by theory, this may be due to their improved tolerance and / or equivalent or increased efficacy. In particular, the modified clostridial neurotoxins of the present invention may have an improved safety profile and / or therapeutic range compared to the safety profile and / or therapeutic range of the corresponding (unmodified) clostridial neurotoxin when the unmodified clostridial neurotoxin is administered (e.g., in a di-chain form).

[0276] One way in which these advantageous properties (indicating an increase in therapeutic index) can be defined is in terms of the safety factor (for clinical applications) or resistance index (TI, animal model, which can be calculated as described below) of the modified clostridial toxin. In this regard, undesirable effects of a clostridial neurotoxin (e.g., those caused by the neurotoxin diffusing away from the site of administration) can be experimentally evaluated by measuring the percentage of weight loss in a relevant animal model (e.g., mice, where weight loss is detected within 7 days of administration). The desired on-target effect of a clostridial toxin can be experimentally evaluated by any suitable technique, depending on the target cell of interest. Suitable assays are known in the art, and it would be routine for one of skill in the art to select an appropriate assay for a particular target cell type. For clostridial neurotoxins of the present invention that target motor neurons, the digit abduction score (DAS) assay, which measures muscle paralysis, can be used. The DAS assay can be performed by injecting 20 μl of a (modified) clostridial toxin formulated in gelatin phosphate buffer into the mouse gastrocnemius / soleus muscle complex, followed by assessing the digit 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 mice extend their hind limbs and abduct their hind digits. After injection of the clostridial toxin, the varying degrees of digit abduction are scored on a 5-point scale (0 = normal to 4 = maximally reduced digit abduction and leg extension). For clostridial neurotoxins of the present invention that target other subtypes of neurons, any suitable assay known in the art can be used. SNARE cleavage assays can also be used to assess the activity of the modified clostridial neurotoxins of the present invention, examples of which are well described in the art (e.g., Western blot). Assays can also be used to detect and / or quantify the effect of the modified Clostridial neurotoxin on the release of the maker signal molecule. Specific marker signal molecules can be selected depending on the cell type targeted by the modified Clostridial neurotoxin.For example, depending on whether the target is a cell involved in hormone secretion or a pain-sensing neuron, the signal molecule may be a hormone, substance P, CGRP, glutamate, or glycine. In the treatment of pain, animal tests can be used to evaluate whether a subject is more tolerant to noxious stimuli. Typical in vivo assays measure different types of pain (e.g., mechanical, cold, heat), and the readout may include behavior (e.g., licking / biting the treatment site or withdrawal from the noxious stimulus) or the use of the von Frey test. Any suitable nociceptive test can be used, and examples of such tests are well known in the art.

[0277] The safety factor or TI of a clostridial neurotoxin can then be expressed as the ratio of the amount of toxin required to achieve a 10% reduction in body weight (measured at peak effect within the first 7 days after dosing in mice) to the amount of toxin required for a DAS score of 2. A high safety factor score or TI is therefore desirable, indicating a toxin capable of effectively paralyzing target muscles with few undesired off-target effects. The modified toxins of the present invention may have a safety factor and / or TI that is higher than that of a comparable unmodified (pre-modification) single-chain clostridial neurotoxin. The method for calculating the TI may vary depending on the experimental model used.

[0278] For example, in a DAS mouse model, the TI of a modified Clostridial toxin of the invention is at least 8 (e.g., at least 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50), where TI is calculated as: - dose of toxin required for a 10% change in body weight (pg / mouse) ÷ DAS ED 50 (pg / mouse) [ED 50 = dose required to produce a DAS score of 2].

[0279] In clinical use, a safety margin can be calculated.

[0280] Any of the clostridial neurotoxins described herein may be modified in accordance with the present invention to include any exogenous protease cleavage site, an exogenous activation loop comprising one or more exogenous protease cleavage sites, or a modified BoNT / C (BoNT / C1) activation loop described herein.

[0281] The present invention provides nucleic acids (e.g., DNA or RNA) comprising a nucleic acid sequence encoding a clostridial neurotoxin (e.g., a modified clostridial neurotoxin) described herein. The nucleic acid sequence may be prepared as part of an expression vector in which the nucleic acid is operably linked to a promoter. Preferably, the nucleic acid is prepared as part of a DNA expression vector comprising a promoter and a terminator.

[0282] Preferably, the vector has a promoter selected from the following: Promoter Inducer General induction conditions Tac (hybrid) IPTG 0.2mM (0.05 to 2.0mM) AraBAD L-arabinose 0.2% (0.002-0.4%) T7-lac operator IPTG 0.2mM (0.05 to 2.0mM)

[0283] Alternatively, the promoter may preferably be selected from: Promoter Inducer General induction conditions Tac (hybrid) IPTG 0.2mM (0.05 to 2.0mM) AraBAD L-arabinose 0.2% (0.002-0.4%) T7-lac operator IPTG 0.2mM (0.05 to 2.0mM) T5-lac operator IPTG 0.2mM (0.05 to 2.0mM)

[0284] 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.

[0285] The nucleic acid molecules and expression vectors of the present invention can preferably be designed in silico and then synthesized by conventional synthetic techniques, for example, conventional DNA synthesis techniques.

[0286] The above nucleic acid sequence information is optionally corrected for codon bias depending on the ultimate host cell (eg, E. coli) expression system used.

[0287] The present invention provides nucleotide sequences encoding the modified Clostridial neurotoxins of the invention, which encode polypeptides that include one or more exogenous protease cleavage sites as described herein.

[0288] The nucleotide sequence may comprise a sequence having at least 70% sequence identity to SEQ ID NO: 154, 156, 158, 160, 162, or 164. The nucleotide sequence may comprise a sequence having at least 80% or 90% sequence identity to SEQ ID NO: 154, 156, 158, 160, 162, or 164. Preferably, the nucleotide sequence comprises (more preferably consists of) SEQ ID NO: 154, 156, 158, 160, 162, or 164. The nucleotide sequence may encode a modified Clostridial neurotoxin comprising or consisting of an amino acid sequence having at least 70% sequence identity to one or more of SEQ ID NOs: 155, 157, 159, 161, 163, 165, 166, 167, or 168. The nucleotide sequence may encode a modified Clostridial neurotoxin comprising or consisting of an amino acid sequence having at least 80% or 90% sequence identity to one or more of SEQ ID NOs: 155, 157, 159, 161, 163, 165, 166, 167, or 168. Preferably, the nucleotide sequence encodes a modified Clostridial neurotoxin comprising (more preferably consisting of) any one of SEQ ID NOs: 155, 157, 159, 161, 163, 165, 166, 167, or 168.

[0289] The modified Clostridial neurotoxin may be encoded by a nucleic acid comprising or consisting of a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 175, wherein SEQ ID NO: 176 within SEQ ID NO: 175 is replaced by a nucleotide sequence encoding at least one exogenous protease cleavage site or a modified BoNT / C activation loop as defined herein, wherein the one or more exogenous protease sites are preferably selected from one or more of SEQ ID NOs: 15, 33, or 25, and / or the modified BoNT / C activation loop is preferably selected from one or more of SEQ ID NOs: 57, 63, and / or 65. The modified Clostridial neurotoxin may be encoded by a nucleic acid comprising or consisting of a nucleotide sequence having at least 80% or 90% sequence identity to SEQ ID NO: 175, wherein SEQ ID NO: 176 within SEQ ID NO: 175 is replaced by a nucleotide sequence encoding at least one exogenous protease cleavage site or a modified BoNT / C activation loop as defined herein, wherein the one or more exogenous protease sites are preferably selected from one or more of SEQ ID NOs: 15, 33, or 25, and / or the modified BoNT / C activation loop is preferably selected from one or more of SEQ ID NOs: 57, 63, and / or 65. The modified Clostridial neurotoxin may be encoded by a nucleic acid comprising or consisting of the nucleotide sequence of SEQ ID NO: 175, wherein SEQ ID NO: 176 within SEQ ID NO: 175 is replaced by a nucleotide sequence encoding at least one exogenous protease cleavage site or a modified BoNT / C activation loop as defined herein, wherein the one or more exogenous protease sites are preferably selected from one or more of SEQ ID NOs: 15, 33, or 25, and / or the modified BoNT / C activation loop is preferably selected from one or more of SEQ ID NOs: 57, 63, and / or 65.

[0290] Alternatively, or in addition, the modified clostridial neurotoxin may be a single-chain clostridial neurotoxin comprising or consisting of a polypeptide sequence having: (i) at least 70% sequence identity to SEQ ID NO: 150, wherein SEQ ID NO: 2 within SEQ ID NO: 150 is replaced by one or more exogenous protease cleavage sites and / or a modified BoNT / C activation loop as defined herein, wherein the one or more exogenous protease sites are preferably selected from one or more of SEQ ID NOs: 15, 33, or 25, and / or the modified BoNT / C activation loop is preferably is selected from one or more of SEQ ID NOs: 57, 63, and / or 65; or (ii) at least 70% identity to SEQ ID NO: 152, wherein SEQ ID NO: 2 within SEQ ID NO: 152 is replaced by one or more exogenous protease cleavage sites and / or modified BoNT / C activation loops as defined herein, wherein the one or more exogenous protease sites are preferably selected from one or more of SEQ ID NOs: 15, 33, or 25, and / or the modified BoNT / C activation loop is preferably selected from one or more of SEQ ID NOs: 57, 63, and / or 65.The modified clostridial neurotoxin may be a single-chain clostridial neurotoxin comprising or consisting of a polypeptide sequence having: (i) at least 80% or 90% sequence identity to SEQ ID NO: 150, wherein SEQ ID NO: 2 within SEQ ID NO: 150 has been replaced by one or more exogenous protease cleavage sites and / or a modified BoNT / C activation loop as defined herein, wherein the one or more exogenous protease sites are preferably selected from one or more of SEQ ID NOs: 15, 33, or 25, and / or the modified BoNT / C activation loop is preferably selected from SEQ ID NO: 5. or (ii) at least 80% or 90% identical to SEQ ID NO: 152, wherein SEQ ID NO: 2 within SEQ ID NO: 152 is replaced by one or more exogenous protease cleavage sites and / or modified BoNT / C activation loops as defined herein, wherein the one or more exogenous protease sites are preferably selected from one or more of SEQ ID NOs: 15, 33, or 25, and / or the modified BoNT / C activation loop is preferably selected from one or more of SEQ ID NOs: 57, 63, and / or 65.The modified clostridial neurotoxin may be a single-chain clostridial neurotoxin comprising or consisting of: (i) the polypeptide sequence of SEQ ID NO: 150, wherein SEQ ID NO: 2 within SEQ ID NO: 150 is replaced by one or more exogenous protease cleavage sites and / or a modified BoNT / C activation loop as defined herein, wherein the one or more exogenous protease sites are preferably selected from one or more of SEQ ID NOs: 15, 33, or 25, and / or the modified BoNT / C activation loop is preferably selected from one or more of SEQ ID NOs: 57, 63, and or (ii) a polypeptide sequence of SEQ ID NO: 152, wherein SEQ ID NO: 2 within SEQ ID NO: 152 is replaced by one or more exogenous protease cleavage sites and / or modified BoNT / C activation loops as defined herein, wherein the one or more exogenous protease sites are preferably selected from one or more of SEQ ID NOs: 15, 33, or 25, and / or the modified BoNT / C activation loop is preferably selected from one or more of SEQ ID NOs: 57, 63, and / or 65.

[0291] As used herein, the terms "nucleotide sequence," "nucleic acid," and "polynucleotide" are used synonymously. Preferably, the nucleotide sequence is a DNA sequence.

[0292] The present invention provides a method for producing a single-chain (modified) Clostridial neurotoxin protein having a light chain and a heavy chain, the method comprising expressing a polynucleotide or expression vector described herein in a suitable host cell and recovering the expressed modified Clostridial neurotoxin. Recovering the expressed modified Clostridial neurotoxin may comprise lysing the host cell to obtain a host cell homogenate containing the single-chain (modified) Clostridial neurotoxin protein and / or isolating the single-chain (modified) Clostridial neurotoxin protein. The method may further comprise introducing the polynucleotide or expression vector described herein into the host cell. Suitable host cells include bacterial cell lines used for recombinant production of Clostridial neurotoxins, particularly Escherichia coli cells.

[0293] The present invention provides a method for proteolytically cleaving a (modified) clostridial neurotoxin of the present invention into a corresponding di-chain clostridial neurotoxin, the method comprising contacting the (modified) clostridial neurotoxin with one or more exogenous proteases, thereby producing a di-chain clostridial neurotoxin (e.g., in which the light chain and heavy chain are linked to each other by a disulfide bond).

[0294] Thus, the present invention provides a di-chain clostridial neurotoxin obtainable by the method of the present invention.

[0295] The term "obtained" as used herein also encompasses "obtained." Preferably, the term "obtained" means "obtained."

[0296] Activation of modified clostridial neurotoxins

[0297] The present invention provides a method for proteolytically cleaving a modified Clostridial neurotoxin of the present invention into a corresponding di-chain Clostridial neurotoxin, the method comprising contacting the modified Clostridial neurotoxin with one or more exogenous proteases, thereby producing a di-chain Clostridial neurotoxin. The contacting may be in vitro, ex vivo, or in vivo, preferably in vitro. The therapeutic methods and uses of the present invention may comprise in vivo activation of the modified Clostridial neurotoxin of the present invention by cleavage at one or more exogenous protease activation sites by expression of one or more exogenous proteases in target cells.

[0298] Thus, the methods of the present invention may further comprise contacting the modified Clostridial neurotoxin with one or more exogenous proteases, thereby producing the corresponding double-chain modified Clostridial neurotoxin. Preferably, said contacting is performed in vitro.

[0299] The present invention also provides a method for proteolytically cleaving a single-chain Clostridial neurotoxin into a corresponding two-chain Clostridial neurotoxin, the method comprising: (a) providing a single-chain Clostridial neurotoxin; and (b) contacting the single-chain Clostridial neurotoxin with one or more exogenous proteases; wherein the single-chain Clostridial neurotoxin has one or more exogenous protease consensus sequences (e.g., any one or more of SEQ ID NOs: 169, 170, 172, or 173) or a cleavage site described herein (e.g., SEQ ID NOs: 15-22, 24-26, 25-27, 26-28, 27-30, 28-31, 28-32, 28-33, 28-34, 28-35, 28-36, 28-37, 28-40, 28-41, 28-42, 28-43, 28-44, 28-45, 28-46, 28-47, 28-50, 28-51, 28-52, 28-53, 28-54, 28-55, 28-56, 28-57, 28-60, 28-61, 28-62, 28-63, 28-64, 28-65, 28-70, 28-71, 28-72, 28-73, 28-84, 28-85, 28-90, 28-91, 28-92, 28-93, 28-94, 28-95, 28-96, 28- and an activation loop comprising or consisting of any one or more of SEQ ID NOS: 15-22, 24-30, 32-42, 44-46, and / or 50-52, particularly any one of SEQ ID NOS: 15-22, 24-30, and / or 32-42, preferably any one of SEQ ID NOS: 15, 17, 16, 26, 25, 27, 24, 33, and / or 32, particularly preferably SEQ ID NOS: 15, 17, 16, 26, 25, and / or 33); wherein one or more exogenous proteases hydrolyze the peptide bond of the activation loop, thereby generating the di-chain clostridial neurotoxin. Preferably, the contacting is performed in vitro.

[0300] The present invention includes contacting a single-chain clostridial neurotoxin (e.g., a modified clostridial neurotoxin of the present invention) with one or more exogenous proteases, wherein the one or more exogenous proteases are capable of hydrolyzing a peptide bond within the activation loop of the single-chain clostridial neurotoxin, thereby generating a di-chain clostridial neurotoxin. Preferably, the contacting is performed in vitro.

[0301] The contacting can be carried out under any suitable conditions that result in more than 30%, 40%, 50%, or 60% (preferably more than 70%) of the single-chain clostridial neurotoxins being proteolytically cleaved to produce the corresponding di-chain clostridial neurotoxins without, or substantially without, hydrolysis of peptide bonds outside the activation loop of the clostridial neurotoxins. "Substantially without hydrolysis" can mean that less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the contacted clostridial neurotoxins contain peptide bonds outside the activation loop that are hydrolyzed by one or more exogenous proteases in the methods of the invention.

[0302] Those skilled in the art can select appropriate reaction times, temperatures, buffers, and molar ratios of protease to single-chain clostridial neurotoxin to achieve the above. Optimization of such conditions can be determined empirically using conventional techniques, such as visual analysis of SDS-PAGE (e.g., stained with Coomassie or similarly sensitive dyes) of the reaction products after contact, or spectroscopic techniques (e.g., mass spectrometry).

[0303] The methods of the invention preferably produce only the light and heavy chains of the clostridial neurotoxin, as assessed by SDS-PAGE (eg, stained with Coomassie or similarly sensitive dyes).

[0304] Proteolytic cleavage by one or more exogenous proteases in the methods of the invention typically results in the production of fewer than 5 degradation products of the light or heavy chain of a clostridial neurotoxin, more preferably fewer than 4, fewer than 3, fewer than 2, or fewer than 1 degradation product. Preferably, the light and heavy chains produced by the methods of the invention are full-length light and heavy chains.

[0305] Typically, in the methods of the present invention, cleavage by each of the one or more exogenous proteases hydrolyzes 5 or less, 4 or less, 3 or less, 2 or less, or a single peptide bond in the modified clostridial neurotoxin, preferably 1 or 2 peptide bonds. Preferably, in the methods of the present invention, cleavage by each of the one or more exogenous proteases hydrolyzes 5 or less, 4 or less, 3 or less, 2 or less, or a single peptide bond in the activation loop of the modified clostridial neurotoxin, preferably 1 or 2 peptide bonds. When two or more exogenous protease cleavage sites are present in the modified clostridial neurotoxin of the present invention, the total number of peptide bonds that can be hydrolyzed by the two or more exogenous proteases is typically 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. Preferably, a single peptide bond is hydrolyzed by each of the two or more exogenous proteases. Exemplary exogenous protease cleavage sites are described herein, indicating the position of the peptide bond that is hydrolyzed.

[0306] Any suitable conditions for activation can be used for in vitro activation of modified clostridial neurotoxins with one or more exogenous proteases of the present invention. Determining suitable conditions is within the routine practice of one of ordinary skill in the art. As a non-limiting example, activation can be performed using about 0.5 μg to about 10 μg of thrombin per 1 mg of modified clostridial neurotoxin at room temperature (about 20° C.) and a pH of about 7.2 for 1 hour. As a further non-limiting example, activation can be performed using about 0.5 μg to about 10 μg of u-PA per 1 mg of modified clostridial neurotoxin at room temperature (about 20° C.) and a pH of about 7.2 for 1 hour. As a further non-limiting example, activation can be performed using about 0.5 μg to about 100 μg of t-PA per 1 mg of modified clostridial neurotoxin at room temperature (about 20° C.) and a pH of about 7.2 for 1 hour.

[0307] The step of contacting a clostridial neurotoxin with one or more exogenous proteases according to the present invention may be carried out in a patient treated with the clostridial neurotoxin. In other words, the step of contacting a clostridial neurotoxin with one or more exogenous proteases according to the present invention may involve one or more exogenous proteases that are endogenously present in the patient. Thus, the step of contacting a clostridial neurotoxin with one or more exogenous proteases according to the present invention may occur in vivo after the clostridial neurotoxin is administered to an individual. When the contacting step occurs in vivo, it usually involves one or more exogenous proteases that are endogenously present in the patient treated according to the present invention.

[0308] Typically, the step of contacting a Clostridial neurotoxin with one or more exogenous proteases according to the present invention occurs in vitro, for example during the manufacture of the modified Clostridial neurotoxin.

[0309] The present invention also provides a di-chain clostridial neurotoxin obtainable by the method of the present invention. As described herein, activation to the di-chain form occurs by cleavage at one or more exogenous protease cleavage sites, such that the resulting C-terminal and N-terminal cleaved ends of the di-chain clostridial neurotoxin differ in sequence compared to the corresponding (unmodified) clostridial neurotoxin. Non-limiting examples include: (a) the C-terminal portion of the truncated modified Clostridial neurotoxin light chain may end with the amino acid sequence LVPR, preferably ALVPR, and the N-terminus of the truncated modified Clostridial neurotoxin heavy chain may begin with the amino acid sequence GSK or GSY; (b) the C-terminus of the truncated modified Clostridial neurotoxin light chain may end with the amino acid sequence SLVPR, and the N-terminus of the truncated modified Clostridial neurotoxin heavy chain may begin with the amino acid sequence GSY; (c) the C-terminus of the truncated modified Clostridial neurotoxin light chain may end with the amino acid sequence LTPR, preferably ALTPR, and the N-terminus of the truncated modified Clostridial neurotoxin heavy chain may begin with the amino acid sequence GVR, preferably GVRL; (d) the C-terminus of the truncated modified Clostridial neurotoxin light chain may end with the amino acid sequence PGR. Preferably, the N-terminus of the truncated modified Clostridial neurotoxin heavy chain may begin with the amino acid sequence VVG; (e) the C-terminus of the truncated modified Clostridial neurotoxin light chain may end with the amino acid sequence DKR, preferably AIDKR, and the N-terminus of the truncated modified Clostridial neurotoxin heavy chain may begin with the amino acid sequence VLY; (f) the C-terminus of the truncated modified Clostridial neurotoxin light chain may end with the amino acid sequence DKR, preferably AIDK, and the N-terminus of the truncated modified Clostridial neurotoxin heavy chain may begin with the amino acid sequence RVLY; (g) the C-terminus of the truncated modified Clostridial neurotoxin light chain may end with the amino acid sequence SGR, preferably PGSGR, and the N-terminus of the truncated modified Clostridial neurotoxin heavy chain may begin with the amino acid sequence SA, preferably SAY, SAG, or SAS;(h) The C-terminus of the truncated modified clostridial neurotoxin light chain may end with the amino acid sequence SGR, preferably PGSGR, and the N-terminus of the truncated modified clostridial neurotoxin heavy chain may begin with the amino acid sequence TL, preferably TLD or TLDC; (i) The C-terminus of the truncated modified clostridial neurotoxin light chain may end with the amino acid sequence FGR, and the N-terminus of the truncated modified clostridial neurotoxin heavy chain may begin with the amino acid sequence SA, preferably SAG; or (j) The C-terminus of the truncated modified clostridial neurotoxin light chain may end with the amino acid sequence VVPR, and the N-terminus of the truncated modified clostridial neurotoxin heavy chain may begin with the amino acid sequence VELVA. In contrast, conventional trypsin cleavage of (unmodified) BoNT / A results in a dichain in which the C-terminus of the LC ends with the sequence TSK and the N-terminus of the HC begins with ALNDLC. These dichain clostridial neurotoxins can be used therapeutically as described herein. All disclosures herein regarding therapeutic indications and formulations in the context of the modified or single-chain clostridial neurotoxins of the present invention apply equally and unconditionally to the double-chain clostridial neurotoxins obtained by the methods of the present invention, unless otherwise specified;

[0310] Therapies and Formulations

[0311] The clostridial neurotoxins of the present invention suitably find utility in medicine and / or cosmetics. When used, the modified clostridial neurotoxins of the present invention can be cleaved in vivo by one or more exogenous proteases as described herein, so the clostridial neurotoxins may be in a single-chain form for administration. Preferably, the modified clostridial neurotoxins of the present invention (e.g., obtained by the method of the present invention) are in a two-chain form for administration.

[0312] The (modified) clostridial neurotoxins of the present invention can be used for the prevention or treatment of certain medical or cosmetic diseases and conditions. Accordingly, in a further aspect, the present invention provides the above-mentioned double-chain (modified) clostridial neurotoxins for use in medicine. Furthermore, as described herein, the present invention relates to single-chain clostridial neurotoxins for use in the prevention or treatment of certain medical or cosmetic diseases and conditions, wherein the single-chain clostridial neurotoxins are administered to a subject. Furthermore, as described herein, the present invention relates to double-chain clostridial neurotoxins obtained by the methods of the present invention for use in the prevention or treatment of certain medical or cosmetic diseases and conditions, wherein the double-chain clostridial neurotoxins obtained by the methods of the present invention are administered to a subject. Accordingly, in a further aspect, the present invention provides the above-mentioned (modified) clostridial neurotoxins for use in medicine.

[0313] Accordingly, the present invention provides a Clostridial neurotoxin (e.g., a modified Clostridial neurotoxin) as described above for use in the prevention or treatment of a disease or condition selected from the following: conditions associated with unwanted immune secretion, strabismus, blepharospasm, squint, dystonia (e.g., spastic dystonia, oromandibular dystonia, focal dystonia, tardive dystonia, laryngeal dystonia, limb dystonia, cervical dystonia), torticollis (e.g., spasmodic torticollis), cosmetic therapy (cosmetic) applications that would benefit from cell / muscle attenuation (by downregulating or inactivating SNAREs), neuromuscular disorders or conditions of eye movement (e.g., conjugate strabismus, vertical strabismus, lateral rectus palsy, nystagmus, hypothyroid myopathy), writer's cramp, bruxism. Wilson's disease, tremors, tics, segmental myoclonus, spasms, spasticity due to chronic multiple sclerosis, spasticity resulting in abnormal bladder control, animus, back spasms, cramps, levator pelvic syndrome, spina bifida, tardive dyskinesia, Parkinson's disease, stuttering, hemifacial spasm, eyelid disorders, cerebral palsy, focal spasticity, spastic colitis, neurogenic bladder, animus, limb spasticity, tics , tremors, teeth grinding, anal fissures, achalasia, dysphagia, lacrimation, hyperhidrosis, excessive salivation, gastrointestinal hypersecretion, muscle pain (e.g., pain due to muscle spasms), headache (e.g., tension headache or migraine), phantom pain (e.g., phantom limb pain), glabellar lines, skin wrinkles, cancer, uterine disorders, urogenital disorders, urogenital neuropathy, bladder pain syndrome, interstitial cystitis, chronic neurogenic inflammation, and smooth muscle disorders. In some instances, the condition may be selected from phantom pain (e.g., phantom limb pain) and bladder pain syndrome. Similarly, the present invention also relates to single-chain clostridial neurotoxins and double-chain clostridial neurotoxins obtainable by the methods of the present invention for use in the treatment or prevention of the above-mentioned diseases or conditions. Preferably, the compositions of the present invention may be used for the prevention or treatment of diseases or conditions selected from the following: limb spasticity (upper or lower limbs); cervical dystonia; headache disorders (preferably migraine); blepharospasm; hemifacial spasm; and lower urinary tract disorders (e.g., bladder pain syndrome (preferably interstitial cystitis); overactive bladder; and detrusor overactivity (e.g., neurogenic detrusor overactivity).

[0314] When a clostridial neurotoxin (e.g., a modified clostridial neurotoxin) of the present invention comprises a BoNT / X sequence (or a portion thereof), the clostridial neurotoxin may have the ability to target other types of non-neuronal secretory cells due to its ability to cleave VAMP4, VAMP5, and / or Ykt6. In some embodiments, the targeted secretory cells are secretory immune cells. As used herein, "secretory immune cells" refers to immune cells that secrete cytokines, chemokines, or antibodies. Such secretory immune cells may be innate immune cells, including, but not limited to, natural killer cells, mast cells, eosinophils, basophils, macrophages, neutrophils, and dendritic cells. Antibody-secreting secretory immune cells (e.g., leukocytes) may also be targeted by the clostridial neurotoxins of the present disclosure. Non-limiting examples of antibody-secreting cells include, but are not limited to, plasma B cells, plasmocytes, plasmacytes, and effector B cells. In some embodiments, clostridial neurotoxins can modulate immune responses. Accordingly, therapeutic uses of the clostridial neurotoxins of the present invention for treating conditions associated with unwanted secretions, preferably unwanted immune secretions, are further contemplated herein. Conditions associated with unwanted immune secretions include, but are not limited to, inflammation, psoriasis, allergies, hemophagocytic lymphohistiocytosis, and alcoholic pancreatic disease.

[0315] The present invention also provides the use of the above-described Clostridial neurotoxin (e.g., a modified Clostridial neurotoxin or a two-chain Clostridial neurotoxin obtained by the method of the present invention) in the manufacture of a medicament for use in a method for preventing or treating a disease or disorder described herein.

[0316] The present invention also provides a method for treating a disease or disorder described herein, the method comprising administering a therapeutically effective amount of the above-described Clostridial neurotoxin (e.g., a modified Clostridial neurotoxin or a two-chain Clostridial neurotoxin obtained by the method of the present invention) to a subject in need thereof.

[0317] The present invention also provides non-therapeutic uses of the compositions described herein for treating aesthetic or cosmetic conditions. For cosmetic or aesthetic uses, the individual to be treated preferably does not suffer from a disease or disorder associated with unwanted neural activity, as described above. More preferably, the individual is healthy, i.e., does not suffer from any disease. Preferably, the compositions of the present invention can be used to prevent or treat upper facial wrinkles, i.e., glabellar lines, crow's feet, and / or intrathecal lines.

[0318] The present invention provides a pharmaceutical composition comprising a (modified) clostridial neurotoxin or a two-chain clostridial neurotoxin of the present invention and a pharmaceutically acceptable carrier, excipient, adjuvant, propellant, and / or salt. Preferably, the (modified) clostridial neurotoxin is in a single-chain form (e.g., modified to include one or more exogenous protease cleavage sites). The pharmaceutical composition of the present invention may be a liquid composition (or formulation) or a solid composition (or formulation).

[0319] The present invention also provides a cosmetic composition comprising the (modified) clostridial neurotoxin of the present invention or the two-chain clostridial neurotoxin of the present invention and a cosmetically acceptable carrier, excipient, diluent, adjuvant, propellant, and / or salt. The present invention also provides the use of a cosmetic composition comprising a clostridial neurotoxin (e.g., a modified clostridial neurotoxin or a two-chain clostridial neurotoxin obtained by the method of the present invention) to prevent or alleviate a cosmetic indication for which the application of a botulinum neurotoxin is indicated. The present invention also provides the use of a cosmetic composition comprising a clostridial neurotoxin (e.g., a modified clostridial neurotoxin or a two-chain clostridial neurotoxin obtained by the method of the present invention) to prevent or alleviate a cosmetic indication for which the application of a botulinum neurotoxin is indicated. Preferably, the (modified) clostridial neurotoxin is in a two-chain form (e.g., modified to include one or more exogenous protease cleavage sites and cleaved by one or more exogenous proteases during production). The cosmetic composition of the present invention may be a liquid composition (or preparation) or a solid composition (or preparation).

[0320] The clostridial neurotoxins (e.g., modified clostridial neurotoxins) of the present invention can be formulated for oral, parenteral, continuous infusion, inhalation, or topical application. Compositions suitable for injection may be in the form of a solution, suspension, or emulsion, or a dry powder that is dissolved or suspended in a suitable solvent before use.

[0321] The liquid compositions of the present invention may be (i) pre-lyophilized solutions, (ii) reconstituted solutions, or (iii) solutions not intended for lyophilization and / or not reconstituted after lyophilization. Class (iii) liquid compositions are sometimes referred to as "ready-to-use" compositions or "ready-to-use" solutions because they are manufactured and formulated as liquids and sold for use in liquid form. All disclosures herein regarding liquid formulations apply to all liquid formulations, including pre-lyophilized solutions, reconstituted solutions, and ready-to-use compositions, unless expressly stated to the contrary. As described herein, liquid compositions may be packaged based on the amount (particularly absolute weight) of the chimeric Clostridial neurotoxin of the present invention. Liquid compositions may be packaged to allow for the administration of up to 15 injections from a single container.

[0322] As described herein, the solid composition may be packaged based on the amount (particularly absolute weight) of the modified Clostridial neurotoxin of the present invention.

[0323] In the case of a locally delivered Clostridial neurotoxin (e.g., a modified Clostridial neurotoxin), the Clostridial neurotoxin (e.g., a modified Clostridial neurotoxin) can be formulated as a cream (e.g., for topical application) or for subcutaneous injection.

[0324] Local delivery means can include aerosols or other sprays (e.g., nebulizers). In this regard, aerosol formulations of Clostridial neurotoxins (e.g., modified Clostridial neurotoxins) allow for delivery to the lungs and / or other nasal and / or bronchial or airway passages.

[0325] The clostridial neurotoxins of the present invention (eg, modified clostridial neurotoxins) can be administered to a patient by intrathecal or epidural injection into the spinal column at the level of the spinal segment involved in innervating the affected organ.

[0326] The preferred route of administration is via laparoscopic injection and / or local injection, particularly intramuscular injection.

[0327] The dosage ranges for administration of the compositions of the present invention are those that produce the desired therapeutic effect. A therapeutically effective dose refers to the amount of chimeric neurotoxin used in the compositions of the present invention that prevents, alleviates, or treats symptoms associated with the diseases or conditions described herein. The therapeutic efficacy and toxicity of compounds are generally determined by standard pharmaceutical procedures in cell cultures or experimental animals in the art, e.g., ED 50 (the dose that is therapeutically effective in 50% of the population) and LD 50 (the dose that kills 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, and the LD 50 / ED 50 It will be understood that generally, the dosage range required for any particular pharmaceutical agent will depend on the precise nature of the 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, any contraindications, and the judgment of the attending physician.

[0328] In the context of the modified neurotoxin compositions of the present invention, suitable single unit doses (also referred to as unit doses), i.e., the dose administered per injection site, are described in the art, for example, in WO2021 / 186160, WO2021 / 186167, WO2023 / 047127, WO2023 / 089343, and WO2023 / 041934, each of which is incorporated herein by reference in its entirety. By way of non-limiting example, a single unit dose is 15,000 pg of modified neurotoxin, 25,000 pg of modified neurotoxin, or 36,000 pg of modified neurotoxin.

[0329] Treatment may involve injection at multiple injection sites (usually no more than 20, preferably no more than 15 injection sites), with each injection site receiving a single unit dose.

[0330] Liquid dosage forms are typically prepared using a clostridial neurotoxin (e.g., a modified clostridial neurotoxin) and a sterile, pyrogen-free solvent. The clostridial neurotoxin (e.g., a modified clostridial neurotoxin) may be dissolved or suspended in the solvent, depending on the solvent and concentration used. When preparing a solution, the clostridial neurotoxin (e.g., a modified clostridial neurotoxin) is dissolved in the solvent, sodium chloride is added, if necessary, to make the solution isotonic, and the solution is sterilized by filtration through a sterile filter using aseptic techniques, and then filled into an appropriate sterile vial or ampoule and sealed. Alternatively, if the solution is sufficiently stable, the solution in the sealed container may be sterilized by autoclaving. Advantageously, additives such as buffers, solubilizers, stabilizers, preservatives or bactericides, suspending or emulsifying agents, and / or local anesthetics may be dissolved in the solvent.

[0331] Using aseptic technique in a sterile area, pre-sterilized ingredients may be filled into sterile containers to prepare dry powders that are dissolved or suspended in a suitable solvent before use. Alternatively, the ingredients may be dissolved in a suitable container using aseptic technique in a sterile area. The product is then lyophilized and the container is aseptically sealed.

[0332] Parenteral suspensions suitable for intramuscular, subcutaneous, or intradermal injection are prepared in substantially the same manner, except that the sterile ingredients are suspended in a sterile solvent instead of being dissolved, and sterilization cannot be achieved by filtration. The ingredients may be separated under sterile conditions or may be sterilized after separation, for example, by gamma irradiation.

[0333] Advantageously, the composition contains a suspending agent, such as polyvinylpyrrolidone, to promote uniform distribution of the ingredients.

[0334] Administration according to the present invention may utilize a variety of delivery technologies, including microparticle encapsulation, viral delivery systems, or pressurized aerosol propellants.

[0335] The disclosures relating to the various methods of the present invention are intended to apply equally to other methods, clostridial neurotoxins, e.g., modified clostridial neurotoxins (whether in single-chain or double-chain form), uses or pharmaceutical compositions, and medical uses thereof, and vice versa.

[0336] sequence homology

[0337] Any of a variety of sequence alignment methods may be used to determine percent identity, including, but not limited to, global, local, and hybrid methods, such as segmental approaches. Protocols for determining percent identity are routine procedures within the skill of those in the art. Global methods align the sequence from beginning to end of the molecule, sum the scores of individual residue pairs, and determine optimal alignment by imposing gap penalties. Non-limiting methods include, for example, CLUSTAL W (see, e.g., Julie D. Thompson et al., CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position-Specific Gap Penalties and Weight Matrix Choice, 22(22) Nucleic Acids Research 4673-4680 (1994)); and iterative refinement (see, e.g., Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein. Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments, 264(4) J. Mol. Biol. 823-838 (1996)). Local methods align sequences by identifying one or more conserved motifs shared by all input sequences.Non-limiting methods include, for example, Match-box (see, e.g., Eric Depiereux and Ernest Feytmans, Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8(5) CABIOS 501-509 (1992)); Gibbs sampling (see, e.g., C.E. Lawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131) Science 208-214 (1993)); Align-M (see, e.g., Ivo Van Walle et al., Align-M - A New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bioinformatics:1428-1435 (2004)).

[0338] Thus, percent sequence identity is determined by conventional methods. See, e.g., Altschul et al., Bull. Math. Bio. 48:603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment score using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum 62" scoring matrix of Henikoff and Henikoff (ibid.) as described below (amino acids are represented by standard single-letter codes).

[0339] The "percent sequence identity" between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by the sequences. Thus, percent identity can be calculated by dividing the number of identical nucleotides / amino acids by the total number of nucleotides / amino acids and multiplying by 100. The calculation of percent sequence identity may also take into account the number of gaps that need to be introduced to optimize the alignment of two or more sequences, and the length of each gap. Sequence comparison and determination of percent identity between two or more sequences can be performed using specific mathematical algorithms, such as BLAST, which are well known to skilled artisans.

[0340] Alignment scores for determining sequence identity

number

[0341] The percent identity is then calculated as follows: [total number of identical matches × 100] / [length of the longer sequence + number of gaps introduced in the longer sequence to align the two sequences]

[0342] Substantially homologous polypeptides are characterized by having one or more amino acid substitutions, deletions, or additions. These changes are preferably minor in nature, i.e., conservative amino acid substitutions (see below) and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, usually from 1 to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as, for example, an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag.

[0343] Conservative amino acid substitutions Basicity: Arginine lysine histidine Acidic: glutamic acid Aspartic acid polarity: glutamine Asparagine Hydrophobicity: Leucine Isoleucine Balin Aromatic: phenylalanine Tryptophan Tyrosine Small size: glycine Alanine Serine Threonine methionine

[0344] In addition to the 20 standard amino acids, non-standard amino acids (such as 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline, and α-methylserine) may be substituted for amino acid residues in the polypeptides of the invention. A limited number of non-conservative amino acids, amino acids not encoded by the genetic code, and unnatural amino acids may be substituted for polypeptide amino acid residues. The polypeptides of the invention may also include amino acid residues of non-natural origin.

[0345] Non-naturally occurring amino acids include, but are not limited to, trans-3-methylproline, 2,4-methano-proline, cis-4-hydroxyproline, trans-4-hydroxy-proline, N-methylglycine, allo-threonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethylhomo-cysteine, nitro-glutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenyl-alanine, 3-azaphenyl-alanine, 4-azaphenyl-alanine, and 4-fluorophenylalanine. Several methods for incorporating non-naturally occurring amino acid residues into proteins are known in the art. For example, an in vitro system using chemically aminoacylated suppressor tRNAs to suppress nonsense mutations can be used. Methods for synthesizing amino acids and aminoacylating tRNAs are known in the art. Transcription and translation of plasmids containing nonsense mutations are carried out in a cell-free system containing Escherichia coli S30 extract, commercially available enzymes, and other reagents. Proteins are purified by chromatography. See, e.g., Robertson et al., J. Am. Chem. Soc. 113:2722, 1991; Ellman et al., Methods Enzymol. 202:301, 1991; Chung et al., Science 259:806-9, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA 90:10145-9, 1993). In a second method, translation is carried out in Xenopus oocytes by microinjection of mutant mRNA and chemically aminoacylated suppressor tRNA (Turcatti et al., J. Biol. Chem. 271:1991-8, 1996). In the third method, E. coli cells are cultured in the absence of the natural amino acid to be substituted (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine).Non-naturally occurring amino acids are incorporated into polypeptides in place of their natural counterparts. See Koide et al., Biochem. 33:7470-6, 1994. Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2:395-403, 1993).

[0346] A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for amino acid residues in the polypeptides of the invention.

[0347] Essential amino acids in the polypeptides of the invention can be identified by procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, Science 244:1081-5, 1989). Sites of biological interaction can also be determined by physical analysis of the structure, as determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in combination with mutations of putative contact site amino acids. See, e.g., de Vos et al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; and Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identities of essential amino acids can also be inferred from analysis of homology with related components (e.g., translocation or protease components) of the polypeptides of the invention.

[0348] Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting functional polypeptides, and then sequencing the mutagenized polypeptides to determine the spectrum of permissible substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., U.S. Patent No. 5,223,409; Huse, WIPO Publication WO 92 / 06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

[0349] Sequence information

[0350] Where the first Met amino acid residue or corresponding first codon is shown in any of the following SEQ ID NOs, said residue / codon is optional.

[0351] SEQ ID NO: 1 C1 activation loop consensus C-(X) a -ID / EGR-(Y) b -C where a=1 to 10 and b=4 to 15

[0352] SEQ ID NO: 2 C1 activation loop CHKAIDGRSLYNKTLDC

[0353] SEQ ID NO: 3 C1 activation loop CHKAIEGRSLYNKTLDC

[0354] SEQ ID NO: 4 N-terminal residue of modified C1 activation loop CHKA

[0355] SEQ ID NO: 5 N-terminal residue of modified C1 activation loop CHKAI

[0356] SEQ ID NO: 6 N-terminal residue of modified C1 activation loop CHKAID

[0357] SEQ ID NO: 7 Residues missing from modified C1 activation loop IDGR

[0358] SEQ ID NO: 8 Residues missing from modified C1 activation loop IEGR

[0359] SEQ ID NO: 9 Residues missing from modified C1 activation loop CHKAIDGR

[0360] SEQ ID NO: 10 Residues missing from modified C1 activation loop CHKAIEGR

[0361] SEQ ID NO: 11 C-terminal residue of modified C1 activation loop TLDC

[0362] SEQ ID NO: 12 C-terminal residue of modified C1 activation loop KTLDC

[0363] SEQ ID NO: 13 C-terminal residue of modified C1 activation loop YNKTLDC

[0364] SEQ ID NO: 14 C-terminal residue of modified C1 activation loop LYNKTLDC

[0365] SEQ ID NO: 15 Thrombin cleavage site LTPRGVRL

[0366] SEQ ID NO: 16 Thrombin cleavage site LVPRGS

[0367] SEQ ID NO: 17 Thrombin cleavage site ENKSLVPRGS

[0368] SEQ ID NO: 18 Thrombin cleavage site consensus X1-X2-X3-R-X4-X5-X6-X7 In the formula, X3 is P, A, L or G X4 is S, A or G

[0369] SEQ ID NO: 19 Thrombin cleavage site consensus X1-X2-X3-X4-X5-X6-X7-X8 In the formula, X3 is P, A, L or G X4 is R or K X5 is S, A or G

[0370] SEQ ID NO: 20 Thrombin cleavage site consensus X1-X2- X3-X4-X5-X6-X7-X8 In the formula, X4 is R or K

[0371] SEQ ID NO: 21 Thrombin cleavage site consensus X1-X2-PR-X3-X4-X5-X6 In the formula, X3 is S, A or G

[0372] SEQ ID NO: 22 Thrombin cleavage site consensus X1-X2- PK-X3-X4-X5-X6 In the formula, X3 is S, A or G

[0373] SEQ ID NO: 23 Thrombin MAHVRGLQLPGCLALAALCSLVHSQHVFLAPQQARSLLQRVRRANTFLEEVRKGNLERECVEETCSYEEAFEALESSTATDVFWAKYTACETARTPRDKLAACLEGNCAEGLGTNYRGHVNITRSGIECQLWRSRYPHKPEINSTTHPGADLQEN FCRNPDSSTTGPWCYTTDPTVRRQECSIPVCGQDQVTVAMTPRSEGSSVNLSPPLEQCVPDRGQQYQGRLAVTTHGLPCLAWASAQAKALSKHQDFNSAVQLVENFCRNPDGDEEGVWCYVAGKPGDFGYCDLNYCEEAVEEETGDGLDEDSDRAI EGRTATSEYQTFFNPRTFGSGEADCGLRPLFEKKSLEDKTERELLESYIDGRIVEGSDAEIGMSPWQVMLFRKSPQELLCGASLISDRWVLTAAHCLLYPPWDKNFTENDLLVRIGKHSRTRYERNIEKISMLEKIYIHPRYNWRENLDRDIALM KLKKPVAFSDYIHPVCLPDRETAASLLQAGYKGRVTGWGNLKETWTANVGKGQPSVLQVVNLPIVERPVCKDSTRIRITDNMFCAGYKPDEGKRGDACEGDSGGPFVMKSPFNNRWYQMGIVSWGEGCDRDGKYGFYTHVFRLKKWIQKVIDQFGE

[0374] SEQ ID NO: 24 Urokinase (u-PA) cleavage site KRV

[0375] SEQ ID NO: 25 Urokinase (u-PA) cleavage site SGRSA

[0376] SEQ ID NO: 26 Urokinase (u-PA) cleavage site PGSGRSAG

[0377] SEQ ID NO: 27 Urokinase (u-PA) cleavage site PGSGRSASGTTGTG

[0378] SEQ ID NO: 28 u-PA cleavage site consensus X1-S-X2-X3-G-X4-X5-X6 In the formula, X2 is G or S X3 is R or K X4 is L, R or V X6 is N or G

[0379] SEQ ID NO: 29 u-PA cleavage site consensus X1-X2-X3-RV-X4-X5-X6

[0380] SEQ ID NO: 30 u-PA cleavage site consensus X1-SGRG- X1-X2-X3 In the formula, X1 is L, R or V X3 is N or G

[0381] SEQ ID NO: 31 u-PA MRVWLASLFLCALVANSEGGSELEASDESNCGCQNGGVCVSYKYFSSIRRCSCPKKFKGEHCEIDTSKTCYHGNGQSYRGKANTDTKGRPCLAWNSPAVLQQTYNAHR SDALSLGLGKHNYCRNPDNQRRPWCYVQIGLKQFVQECMVQDCSLSKKPSSTVDQQGFQCGQKALRPRFKIVGGEFTVVENQPWFAAIYLKNKGGSPPSFKCGGSLIS PCWVASATHCFVNQPKKEEYVVYLGQSKRNSYNPGEMKFEVEQLILHEDFSDETLAFHNDIALLKIRTSTGQCAQPSRTIQTICLPPRFGDAPFGSDCEITGFGQESA TDYFYPKDLKMSVVKIISHEQCKQPHYYGSEINYKMLCAADPEWKTDSCSGDSGGPLICNIDGRPTLSGIVSWGSGCAEKNKPGVYTRVSYFLNWIQSHIGEENGLAF

[0382] SEQ ID NO: 32 Tissue plasminogen activator (t-PA) cleavage site VVPRVELVA

[0383] SEQ ID NO: 33 Tissue plasminogen activator (t-PA) cleavage site PPFGRSAG

[0384] SEQ ID NO: 34 t-PA cleavage site consensus X1-X2-X3-R-X4-X5 In the formula, X2 is F or Y X3 is S, G or A

[0385] SEQ ID NO: 35 t-PA cleavage site consensus GP-X1-X2-X3-GG-X4 In the formula, X2 is K or R

[0386] SEQ ID NO: 36 t-PA cleavage site consensus X1-X2-X3-RV-X4-X5-X6

[0387] SEQ ID NO: 37 t-PA cleavage site consensus X1-X2-X3-R-X4-X5-X6-X7 In the formula, X2 is F or Y X3 is S, G or A

[0388] SEQ ID NO: 38 t-PA cleavage site consensus S-X1-X2-R-X3-X4-X5-X6 In the formula, X1 is F or Y X2 is S, G or A

[0389] SEQ ID NO: 39 t-PA cleavage site consensus GP-X1-X2-X3-GG-X4 In the formula, X2 is K or R

[0390] SEQ ID NO: 40 t-PA cleavage site consensus GPY-X1-X2-GG-X2 In the formula, X1 is F or Y

[0391] SEQ ID NO: 41 t-PA cleavage site consensus GP-X1- X2-KGG-X3 In the formula, X2 is K or R

[0392] SEQ ID NO: 42 t-PA cleavage site consensus GPY-X1-KGG-X2 In the formula, X1 is K or R

[0393] SEQ ID NO: 43 t-PA MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARSYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRCFNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTRATCYEDQGISYRGTWS TAESGAECTNWNSSALAQKPYSGRRPDAIRLGLGNHNYCRNPDRDSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVL KNRRLTWEYCDVPSCSTCGLRQYSQPQFRIKGGLFADIASHPWQAAIFAKHRRSPGERFLCGGILISSCWILSAAHCFQERFPPHHLTVILGRTYRVVPGEEEQKFEVEKYIVHKEFDDDTYDNDIALLQLKSDSSRCAQ ESSVVRTVCLPADLQLPDWTECELSGYGKHEALSPFYSERLKEAHVRLYPSSRCTSQHLLNRTVTDNMLCAGDTRSGGPQANLHDACQGDSGGPLVCLNDGRMTLVGIISWGLGCGQKDVPGVYTKVTNYLDWIRDNMRP

[0394] SEQ ID NO: 44 FVII / FIX cleavage site GRI

[0395] SEQ ID NO: 45 FIX cleavage site consensus X1- X2-GR-X3-X4-X5-X6

[0396] SEQ ID NO: 46 FVII / FIX cleavage site consensus X1 - X2 - X3 - R - I - X4 - X5 - X6

[0397] Array number 47 FIX MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT

[0398] Array number 48 FVII MVSQALRLLCLLLGLQGCLAAGGVAKASGGETRDMPWKPGPHRVFVTQEEAHGVLHRRRRANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQ NGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKPQGRIVGGKVCPKGECPWQVLLLVN GAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLD RGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFP

[0399] SEQ ID NO: 49 Plasminogen MEHKEVVLLLLLFLKSGQGEPLDDYVNTQGASLFSVTKKQLGAGSIEECAAKCEEDEEFTCRAFQYHSKEQQCVIMAENRKSSIIIRMRDVLFEKKVYLSECKTGNGKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILECEEECMHCSGENYDGKISKTMS GLECQAWDSQSPHAHGYIPSKFPNKNLKKNYCRNPDRELRPWCFTTDPNKRWELCDIPRCTTPPPSSGPTYQCLKGTGENYRGNVAVTVSGHTCQHWSAQTPHTHNRTPENFPCKNLDENYCRNPDGKRAPWCHTTSQVRWEYCKIPSCDSSPVSTEQLAPTAPPELTPVVQDCYHGDGQSYRGTSSTTTTGKCCQSWSSMT PHRHQKTPENYPNAGLTMNYCRNPDADKGPWCFTTDPSVRWEYCNLKKCSGTEASVVAPPPVVLLPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFC GGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN

[0400] SEQ ID NO: 50 Plasminogen cleavage site PGRVVGG

[0401] SEQ ID NO: 51 Plasminogen cleavage site consensus X1- X2- X3-K-X4-X5-X6-X7

[0402] SEQ ID NO: 52 Plasminogen cleavage site consensus X1- X2- X3-R-X4-X5-X6-X7

[0403] SEQ ID NO: 53 Modified C-loop with minimal uPA / tPA cleavage site [ka]

[0404] SEQ ID NO: 54 Modified C-loop with minimal thrombin cleavage site [ka]

[0405] SEQ ID NO: 55 Modified C-loop with minimal FIX / FVII cleavage site [ka]

[0406] SEQ ID NO: 56 Modified C-loop with an intact thrombin cleavage site [ka]

[0407] SEQ ID NO: 57 Modified C-loop with alternative thrombin cleavage site [ka]

[0408] SEQ ID NO: 58 Modified C-loop with native plasminogen cleavage site [ka]

[0409] SEQ ID NO: 59 Modified C-loop with native plasminogen cleavage site with spacer [ka]

[0410] SEQ ID NO: 60 Optimal u-PA cleavage site within the plasminogen loop [ka]

[0411] SEQ ID NO: 61 Optimal u-PA cleavage site with spacer [ka]

[0412] SEQ ID NO: 62 Modified C-loop with alternative t-PA cleavage site [ka]

[0413] SEQ ID NO: 63 Modified C-loop with alternative t-PA cleavage site [ka]

[0414] SEQ ID NO: 64 Modified C-loop with alternative t-PA cleavage site [ka]

[0415] SEQ ID NO: 65 Modified C-loop with alternative u-PA cleavage site [ka]

[0416] SEQ ID NO: 66 Modified C-loop with alternative t-PA cleavage site [ka]

[0417] SEQ ID NO: 67 Endogenous BoNT / D activation loop CLRLTKNSRDDSTC

[0418] SEQ ID NO: 68 Endogenous BoNT / DC activation loop CLRLTRNSRDDSTC

[0419] SEQ ID NO: 69 Endogenous BoNT / C1 and CD activation loop CHKAIDGRSLYNKTLDC

[0420] SEQ ID NO: 70 Endogenous BoNT / A4 activation loop CVRGIITSKTKSLDEGYNKALNELC

[0421] SEQ ID NO: 71 Endogenous BoNT / A5 and A7 activation loop CVRGIITSKTKSLDEGYNKALNDLC

[0422] SEQ ID NO: 72 Endogenous BoNT / A1 and A6 activation loop CVRGIITSKTKSLDKGYNKALNDLC

[0423] SEQ ID NO: 73 Endogenous BoNT / A3 activation loop CVRGIIPFKTKSLDEGYNKALNYLC

[0424] SEQ ID NO: 74 Endogenous BoNT / A2 and A8 activation loop CVRGIIPFKTKSLDEGYNKALNDLC

[0425] SEQ ID NO: 75 Endogenous BoNT / H activation loop CSNSNTKNSLC

[0426] SEQ ID NO: 76 Endogenous BoNT / E1 to E5, E9 and E12 activation loop CKNIVSVKGIRKSIC

[0427] SEQ ID NO: 77 Endogenous BoNT / E11 activation loop CTNIFSPKGIRKSIC

[0428] SEQ ID NO: 78 Endogenous BoNT / E7, E8 and E10 activation loop CKNIVFSKGITKSIC

[0429] SEQ ID NO: 79 Endogenous BoNT / E6 activation loop CKNIVFSKGIRKSIC

[0430] SEQ ID NO: 80 Endogenous BoNT / F7 activation loop CKSIVSKKGTKNSLC

[0431] SEQ ID NO: 81 endogenous BoNT / F5 activation loop CLNSSFKKNTKKPLC

[0432] SEQ ID NO: 82 Endogenous BoNT / F1 and F6 activation loop CKSVIPRKGTKAPPRLC

[0433] SEQ ID NO: 83 Endogenous BoNT / F4 activation loop CKSIIPRKGTKAPPRLC

[0434] SEQ ID NO: 84 Endogenous BoNT / F2 and F3 activation loop CKSIIPRKGTKQSPSLC

[0435] SEQ ID NO: 85 endogenous TeNT activation loop CKKIIPPTNIRENLYNRTASLTDLGGELC

[0436] SEQ ID NO: 86 Endogenous BoNT / G activation loop CKPVMYKNTGKSEQC

[0437] SEQ ID NO: 87 Endogenous BoNT / B4 activation loop CKSVKVPGIC

[0438] SEQ ID NO: 88 Endogenous BoNT / B2, B3, B6 and B8 activation loop CKSVRAPGIC

[0439] SEQ ID NO: 89 Endogenous BoNT / B1, B5 and B7 activation loop CKSVKAPGIC

[0440] SEQ ID NO: 90 Endogenous BoNT / X activation loop CPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTNVSYPCSLLNGC

[0441] SEQ ID NO: 91 (BoNT / A1-UniProt P10845) [ka] In some embodiments, valine 27 may be substituted with alanine, as shown in SEQ ID NO:135. The intrinsic activation loop is indicated by a dashed underline.

[0442] SEQ ID NO: 92 (BoNT / A2 - GenBank accession number X73423.1) [ka] The intrinsic activation loop is indicated by a dashed underline.

[0443] SEQ ID NO: 93 (BoNT / A3 - GenBank accession number DQ185900.1) [ka] The intrinsic activation loop is indicated by a dashed underline.

[0444] SEQ ID NO: 94 (BoNT / A4 - GenBank accession number EU341307.1) [ka] The intrinsic activation loop is indicated by a dashed underline.

[0445] SEQ ID NO: 95 (BoNT / A5 - GenBank accession number EU679004.1) [ka] The intrinsic activation loop is indicated by a dashed underline.

[0446] SEQ ID NO: 96 (BoNT / A6 - GenBank accession number FJ981696.1) [ka] The intrinsic activation loop is indicated by a dashed underline.

[0447] SEQ ID NO: 97 (BoNT / A7 - GenBank accession number JQ954969.1) [ka] The intrinsic activation loop is indicated by a dashed underline.

[0448] SEQ ID NO: 98 (BoNT / A8 - GenBank accession number KM233166.1) [ka] The intrinsic activation loop is indicated by a dashed underline.

[0449] SEQ ID NO: 99 (BoNT / B1-UniProt P10844) [ka] The intrinsic activation loop is indicated by a dashed underline.

[0450] SEQ ID NO: 100 (BoNT / B2 - GenBank accession number AB084152.1) [ka] The intrinsic activation loop is indicated by a dashed underline.

[0451] SEQ ID NO: 101 (BoNT / B3 - GenBank accession number EF028400.1) [ka] The intrinsic activation loop is indicated by a dashed underline.

[0452] SEQ ID NO: 102 (BoNT / B4 - GenBank accession number EF051570.1) [ka] The intrinsic activation loop is indicated by a dashed underline.

[0453] SEQ ID NO: 103 (BoNT / B5 - GenBank accession number EF033130.1) [ka] The intrinsic activation loop is indicated by a dashed underline.

[0454] SEQ ID NO: 104 (BoNT / B6 - GenBank accession number AB302852.1) [ka] The intrinsic activation loop is indicated by a dashed underline.

[0455] SEQ ID NO: 105 (BoNT / B7 - GenBank accession number JQ354985.1) [ka] The intrinsic activation loop is indicated by a dashed underline.

[0456] SEQ ID NO: 106 (BoNT / B8 - GenBank accession number JQ964806.1) [ka] The intrinsic activation loop is indicated by a dashed underline.

[0457] SEQ ID NO: 107 (BoNT / C1-UniProt P18640) [ka] The intrinsic activation loop is indicated by a dashed underline.

[0458] SEQ ID NO: 108 (BoNT / D-UniProt P19321) [ka] The intrinsic activation loop is indicated by a dashed underline.

[0459] SEQ ID NO: 109 (BoNT / CD-GenBank accession number AB200360.1) [ka] The intrinsic activation loop is indicated by a dashed underline.

[0460] SEQ ID NO: 110 (BoNT / DC - GenBank accession number AB745660.1) [ka] The intrinsic activation loop is indicated by a dashed underline.

[0461] SEQ ID NO: 111 (BoNT / E-UniProt Q00496) [ka] The intrinsic activation loop is indicated by a dashed underline.

[0462] SEQ ID NO: 112 (BoNT / E1 - GenBank accession number GQ244314.1) [ka] The intrinsic activation loop is indicated by a dashed underline.

[0463] SEQ ID NO: 113 (BoNT / E2 - GenBank accession number EF028404.1) [ka] The intrinsic activation loop is indicated by a dashed underline.

[0464] SEQ ID NO: 114 (BoNT / E3 - GenBank accession number EF028403.1) [ka] The intrinsic activation loop is indicated by a dashed underline.

[0465] SEQ ID NO: 115 (BoNT / E4 - GenBank accession number AB088207.1) [ka] The intrinsic activation loop is indicated by a dashed underline.

[0466] SEQ ID NO: 116 (BoNT / E5 - GenBank accession number AB037711.1) [ka] The intrinsic activation loop is indicated by a dashed underline.

[0467] SEQ ID NO: 117 (BoNT / E6 - GenBank accession number AM695759.1) [ka] The intrinsic activation loop is indicated by a dashed underline.

[0468] SEQ ID NO: 118 (BoNT / E7 - GenBank accession number JN695729.1) [ka] The intrinsic activation loop is indicated by a dashed underline.

[0469] SEQ ID NO: 119 (BoNT / E8 - GenBank accession number JN695730.1) [ka] The intrinsic activation loop is indicated by a dashed underline.

[0470] SEQ ID NO: 120 (BoNT / E9 - GenBank accession number JX424534.1) [ka] The intrinsic activation loop is indicated by a dashed underline.

[0471] SEQ ID NO: 121 (BoNT / E10 - GenBank accession number KF861917.1) [ka] The intrinsic activation loop is indicated by a dashed underline.

[0472] SEQ ID NO: 122 (BoNT / E11 - GenBank accession number KF861875.1) [ka] The intrinsic activation loop is indicated by a dashed underline.

[0473] SEQ ID NO: 123 (BoNT / E12 - GenBank accession number KM370319.1) [ka] The intrinsic activation loop is indicated by a dashed underline.

[0474] SEQ ID NO: 124 (BoNT / F1-UniProt A7GBG3) [ka] The intrinsic activation loop is indicated by a dashed underline.

[0475] SEQ ID NO: 125 (BoNT / F2 - GenBank accession number GU213209.1) [ka] The intrinsic activation loop is indicated by a dashed underline.

[0476] SEQ ID NO: 126 (BoNT / F3 - GenBank accession number GU213227.1) [ka] The intrinsic activation loop is indicated by a dashed underline.

[0477] SEQ ID NO: 127 (BoNT / F4 - GenBank accession number GU213214.1) [ka] The intrinsic activation loop is indicated by a dashed underline.

[0478] SEQ ID NO: 128 (BoNT / F5 - GenBank accession number GU213211.1) [ka] The intrinsic activation loop is indicated by a dashed underline.

[0479] SEQ ID NO: 129 (BoNT / F6 - GenBank accession number M92906.1) [ka] The intrinsic activation loop is indicated by a dashed underline.

[0480] SEQ ID NO: 130 (BoNT / F7 - GenBank accession number GU213233.1) [ka] The intrinsic activation loop is indicated by a dashed underline.

[0481] SEQ ID NO: 131 (BoNT / G-UniProt Q60393) [ka] The intrinsic activation loop is indicated by a dashed underline.

[0482] SEQ ID NO: 132 (BoNT / FA - GenBank accession number KGO15617.1) [ka] The intrinsic activation loop is indicated by a dashed underline.

[0483] SEQ ID NO: 133 (Polypeptide sequence of BoNT / X) [ka] The intrinsic activation loop is indicated by a dashed underline.

[0484] SEQ ID NO: 134 (TeNT-UniProt P04958) [ka] The intrinsic activation loop is indicated by a dashed underline.

[0485] SEQ ID NO: 135 (BoNT / A GenBank accession number AF488749.1) [ka] The intrinsic activation loop is indicated by a dashed underline.

[0486] SEQ ID NO: 136 Unmodified BoNT / AB chimera [ka] Syt II binding mutations E1191M and S1199Y are shown in bold and underlined. The intrinsic activation loop is indicated by a dashed underline.

[0487] SEQ ID NO: 137 GS linker consensus (GGGGS) n

[0488] SEQ ID NO: 138 GS5 linker GGGGS

[0489] SEQ ID NO: 139 GS10 linker GGGGSGGGGS

[0490] SEQ ID NO: 140 GS15 linker GGGGSGGGGSGGGGS

[0491] SEQ ID NO: 141 GS20 linker GGGGSGGGGSGGGGSGGGGS

[0492] SEQ ID NO: 142 GS25 linker GGGGSGGGGSGGGGSGGGGSGGGGS

[0493] SEQ ID NO: 143 Metal-coordinated SNARE cleavage motif HEXXH

[0494] SEQ ID NO: 144 Leucine base motif xDxxxLL

[0495] SEQ ID NO: 145 Leucine base motif xExxxLL

[0496] SEQ ID NO: 146 Leucine base motif xExxxIL

[0497] SEQ ID NO: 147 Leucine base motif xExxxLM

[0498] SEQ ID NO: 148 TEV (Tobacco Etch Virus) cleavage motif ENLYFQG

[0499] SEQ ID NO: 149 PreScission cleavage motif LEVLFQGP

[0500] SEQ ID NO: 150 LC / A1-C loop-H N / A1 [ka] LC / A1 is shown in italics. The BoNT / C activation loop is shown in bold and underlined. H N A1 is C-terminal to the C activation loop and is neither underlined nor italicized.

[0501] SEQ ID NO: 151 LC / A1-Extrinsic Activation Loop-H N / A1 [ka] LC / A1 is shown in italics. The extrinsic activation loop is shown in bold and underlined. H N A1 is C-terminal to the C activation loop and is neither underlined nor italicized.

[0502] SEQ ID NO: 152: LC / XC loop-H N / X [ka] LC / X is shown in italics. The BoNT / C activation loop is shown in bold and underlined. H N X is C-terminal to the C activation loop and is neither underlined nor italicized.

[0503] SEQ ID NO: 153: LC / X-Extrinsic Activation Loop-H N / X [ka] LC / X is shown in italics. The extrinsic activation loop is shown in bold and underlined. H N X is C-terminal to the C activation loop and is neither underlined nor italicized.

[0504] SEQ ID NO: 154: Nucleic acid encoding an exemplary modified BoNT / A1B1 chimera with modified C-loop / PPFGRSAG of SEQ ID NO: 63

[0505] SEQ ID NO: 155: Exemplary modified BoNT / A1B1 chimera with modified C-loop / PPFGRSAG of SEQ ID NO: 63 [ka] LC / A1 is shown in italics. The exogenous activation loop of SEQ ID NO: 63 is shown in bold and underlined. H N B1 is C-terminal to the activation loop and is neither underlined nor italicized.

[0506] SEQ ID NO: 156: Nucleic acid encoding an exemplary modified BoNT / A1B1 chimera with modified C-loop / SGRSA of SEQ ID NO: 65

[0507] SEQ ID NO: 157: Exemplary modified BoNT / A1B1 chimera with modified C-loop / SGRSA of SEQ ID NO: 65 [ka] LC / A1 is shown in italics. The extrinsic activation loop of SEQ ID NO: 65 is shown in bold and underlined. H N B1 is C-terminal to the activation loop and is neither underlined nor italicized.

[0508] SEQ ID NO: 158: Nucleic acid encoding an exemplary modified BoNT / A1B1 chimera with modified C-loop / LTPRGVRL of SEQ ID NO: 57

[0509] SEQ ID NO: 159: Exemplary modified BoNT / A1B1 chimera with modified C-loop / LTPRGVRL of SEQ ID NO: 57 [ka] LC / A1 is shown in italics. The extrinsic activation loop of SEQ ID NO:57 is shown in bold and underlined. H N B1 is C-terminal to the activation loop and is neither underlined nor italicized.

[0510] SEQ ID NO: 160: Exemplary modified LC / X-modified C-loop (LTPRGVRL)-H of SEQ ID NO: 57 N / X-encoding nucleic acid

[0511] SEQ ID NO: 161: Exemplary modified LC / X-modified C-loop (LTPRGVRL)-H of SEQ ID NO: 57 N / X [ka] LC / X is shown in italics. The extrinsic activation loop of SEQ ID NO:57 is shown in bold and underlined. H N X is C-terminal to the activation loop and is neither underlined nor italicized.

[0512] SEQ ID NO: 162: Exemplary modified LC / X-modified C-loop (SGRSA)-H of SEQ ID NO: 65 N / X-encoding nucleic acid

[0513] SEQ ID NO: 163: Exemplary engineered LC / X-SEQ ID NO: 65 modified C-loop (SGRSA)-H N / X [ka] LC / X is shown in italics. The extrinsic activation loop of SEQ ID NO: 65 is shown in bold and underlined. H N X is C-terminal to the activation loop and is neither underlined nor italicized.

[0514] SEQ ID NO: 164: Exemplary modified LC / X-modified C-loop (PPFGRSAG)-H of SEQ ID NO: 63 N / X-encoding nucleic acid

[0515] SEQ ID NO: 165: Exemplary modified LC / X-modified C-loop (PPFGRSAG)-H of SEQ ID NO: 63 N / X [ka] LC / X is shown in italics. The exogenous activation loop of SEQ ID NO: 63 is shown in bold and underlined. H N X is C-terminal to the activation loop and is neither underlined nor italicized.

[0516] SEQ ID NO: 166: Exemplary modified LC / A1 - modified C-loop (LTPRGVRL)-H of SEQ ID NO: 57 N / A1 [ka] LC / A1 is shown in italics. The extrinsic activation loop of SEQ ID NO:57 is shown in bold and underlined. H NA1 is C-terminal to the activation loop and is neither underlined nor italicized.

[0517] SEQ ID NO: 167: Exemplary modified LC / A1 - modified C-loop (PPFGRSAG)-H of SEQ ID NO: 63 N / A1 [ka] LC / A1 is shown in italics. The exogenous activation loop of SEQ ID NO: 63 is shown in bold and underlined. H NA1 is C-terminal to the activation loop and is neither underlined nor italicized.

[0518] SEQ ID NO: 168: Exemplary modified LC / A1 - modified C-loop (SGRSA)-H of SEQ ID NO: 65 N / A1 [ka] LC / A1 is shown in italics. The extrinsic activation loop of SEQ ID NO: 65 is shown in bold and underlined. H NA1 is C-terminal to the activation loop and is neither underlined nor italicized.

[0519] SEQ ID NO: 169: Preferred thrombin consensus sequence [ka] where each X can be independently selected from any amino acid, the amino acid residues shown in parentheses are alternative amino acid residues at each position, with bold and underlined residues being preferred, and "|" indicates the site of cleavage (i.e., the peptide bond that is hydrolyzed).

[0520] SEQ ID NO: 170: Preferred thrombin consensus sequence [ka] where each X can be independently selected from any amino acid, the amino acid residues shown in parentheses are alternative amino acid residues at each position, with bold and underlined residues being preferred, and "|" indicates the site of cleavage (i.e., the peptide bond that is hydrolyzed).

[0521] SEQ ID NO: 171: Preferred motif within the thrombin cleavage site PRG

[0522] SEQ ID NO: 172: Preferred uPA and / or t-PA consensus sequence [ka] where each X can be independently selected from any amino acid, the amino acid residues shown in parentheses are alternative amino acid residues at each position, with bold and underlined residues being preferred, and "|" indicates the site of cleavage (i.e., the peptide bond that is hydrolyzed).

[0523] SEQ ID NO: 173: Preferred uPA and / or t-PA consensus sequence [ka] where each X can be independently selected from any amino acid, the amino acid residues shown in parentheses are alternative amino acid residues at each position, with bold and underlined residues being preferred, and "|" indicates the site of cleavage (i.e., the peptide bond that is hydrolyzed).

[0524] SEQ ID NO: 174: Preferred motifs within the uPA and / or t-PA cleavage site GRSA

[0525] SEQ ID NO: 175 LC / XC loop-H N Exemplary Nucleic Acids Encoding / X [ka] The nucleic acid encoding the C-loop is shown in bold and underlined. The nucleic acid encoding the 2xAP linker is shown with a dashed underline.

[0526] SEQ ID NO: 176: Exemplary nucleic acid C-loop tgtcataaagccattgatggtcgcagcctgtataacaaaaccctggat [Example]

[0527] The present invention is further described by the following examples, which are intended to be merely illustrative of the present invention and in no way limiting. In addition to the data presented below, the contents of PCT / GB2022 / 050756, particularly the Examples section thereof, are incorporated herein by reference. Although the examples relate to modified clostridial neurotoxins that contain a furin cleavage site rather than an exogenous protease cleavage site, data obtained from modified clostridial neurotoxins that share features with those of the present invention, as well as related synthetic methods, are included.

[0528] Example 1 - Design and generation of modified BoNTs containing exogenous protease cleavage sites

[0529] The modified BoNTs were generated by determining the desired amino acid sequence, back-translating it into the corresponding nucleic acid sequence, and then codon-optimizing it for recombinant expression in bacteria.

[0530] The resulting gene sequence was verified to ensure that commonly used restriction sites (NdeI, XhoI, BamHI, HindIII, NcoI, and EcoRI) were not present within the sequence. A start codon was added to the 5' end, a stop codon to the 3' end, and appropriate terminal restriction sites (e.g., an NdeI restriction site at the 5' end and a BamHI restriction site at the 3' end) were added to allow subcloning into an expression vector.

[0531] The modified BoNT gene sequences were then subcloned into the pK8 vector (containing a kanamycin resistance gene, a T7 promoter, a T7 terminator, a pBR322 origin of replication, and a multiple cloning site). Plasmids for each modified BoNT were then amplified in the Escherichia coli strain DH5α under kanamycin selection and extracted by miniprep using standard molecular biology techniques.

[0532] Next, the λDE3-harboring E. coli expression strain BL21 was transformed with the plasmid DNA, spread onto agar plates supplemented with kanamycin, and incubated overnight at 37°C. Colonies were then collected and used to prepare glycerol stocks. Next, 100 mL of modified TB medium supplemented with kanamycin was inoculated using a stub and then incubated overnight at 37°C with shaking at 225 RPM for aeration. Ten mL of this preculture was used to inoculate multiple baffled Erlenmeyer flasks, each containing up to 1 L of the same nutrient medium and antibiotics. The cultures were grown under the same conditions for several hours until the optical density (A600) reached 0.6 or higher, after which the incubator temperature was set to 16°C. The cultures were then induced to express the modified BoNT by adding IPTG one hour later. After 20 hours, the cells were harvested by centrifugation and stored at -80°C until use.

[0533] Cells were thawed in 0.25 M NaCl in 50 mM Tris pH 7.4 (5 mL per gram of cells) and lysed at 4°C by two passes through a cell homogenizer at 20 k PSI or by sonication (10 30-second on / off cycles). Cell debris was removed by centrifugation, and the clarified supernatant was loaded onto a nickel affinity column pre-equilibrated with 0.5 M NaCl in 50 mM Tris pH 7.4 ("Buffer NA") using an FPLC system (GE). The column was washed with Buffer NA until a stable baseline was obtained at A280. The wash and eluted protein were collected from the column with a linear gradient of 0 to 0.5 M imidazole in Buffer NA over 25 column volumes (CV), collecting 3 mL fractions.

[0534] All collected material was stored at 4°C during analysis of the samples by stained SDS PAGE (Invitrogen). Fractions that showed a strong protein band with the MW of the target molecule calculated based on the protein marker were pooled, and the total protein concentration was measured using a Nanodrop (Thermo Fisher).

[0535] Pooled fractions were desalted into 50 mM Tris pH 8 ("Buffer QA") for further purification by anion exchange chromatography (e.g., Q HP). After washing the column with Buffer QA and achieving a stable baseline, the protein was eluted with a linear gradient of 0 to 0.5 M NaCl in Buffer QA over 25 CV. Samples were analyzed by SDS-PAGE, and fractions containing pure target were desalted to 150 mM NaCl in 50 mM HEPES pH 7.2 before being aliquoted and stored at -80°C. Samples of the final product were analyzed by SDS-PAGE in the presence and absence of DTT, revealing a single band.

[0536] Using the above method, structure LC / A1-Exo Loop-H N X modified BoNTs were generated with the same targeting moiety (TM), BoNT / A1 light chain (LC / A1), and BoNT / A1 translocation domain (H). The Exo loops are a modified C-loop containing a KRV cleavage site (A4952), a modified C-loop containing a GRI cleavage site (A49523), and a modified C-loop containing a PRG cleavage site (A4954). All three modified BoNTs share the same targeting moiety (TM), BoNT / A1 light chain (LC / A1), and BoNT / A1 translocation domain (H). N The modified BoNTs each contain a modified LC / A1-C loop H, but have different modified C loops containing different exogenous protease cleavage sites. N The / A1 sequence is SEQ ID NO: 150, in which the C-loop (SEQ ID NO: 2) is replaced by a modified C-loop. In particular, A4952 contains a modified C-loop of SEQ ID NO: 53, which itself contains the cleavage site KRV specific for u-PA and t-PA. A49523 contains a modified C-loop of SEQ ID NO: 55, which itself contains the cleavage site GRI specific for FIX and FVII. A49524 contains a modified C-loop of SEQ ID NO: 54, which itself contains the cleavage site PRG specific for thrombin.

[0537] Example 2 - BoNT modified to contain a KRV-modified C-loop is efficiently cleaved by u-PA

[0538] A4952 from Example 1 was incubated with increasing concentrations of urokinase ((i) 1 μl, final u-PA concentration 0.85 μg / mL; (ii) 5 μl, final u-PA concentration 4.22 μg / mL; and (iii) 10 μl, final u-PA concentration 8.41 μg / mL). Samples taken at 3, 4, 5, and 19 hours after the start of the reaction were analyzed by SDS-PAGE under reducing and non-reducing conditions. As can be seen in Figure 1A, partial cleavage of A4952 was observed as early as 3 hours at higher concentrations of uPA (4.22 μg / mL and 8.41 μg / mL). By 19 hours, cleavage was observed at all concentrations of u-PA tested. The following table describes the lanes of the gel in Figure 1A. [Table 4]

[0539] Example 3 - BoNT modified to contain a GRI-modified C-loop was not efficiently cleaved by FIX

[0540] A4953 from Example 1 was incubated with increasing concentrations of FIX ((i) 1 μl, final FIX concentration 1 μg / mL; (ii) 5 μl, final FIX concentration 5 μg / mL; and (iii) 10 μl, final FIX concentration 10 μg / mL). Samples taken at 1, 2, 3, and 19 hours after the start of the reaction were analyzed by SDS-PAGE under reducing and non-reducing conditions. As can be seen in Figure 2, no cleavage of A4953 was observed, even with the higher FIX concentrations and 19-hour reaction time. The following table describes the lanes of the gel in Figure 2. [Table 5]

[0541] Example 4 - BoNT modified to contain a PRG-modified C-loop is efficiently cleaved by thrombin

[0542] A4954 from Example 1 was incubated with increasing concentrations of thrombin ((i) 1 μl, final thrombin concentration 0.46 μg / mL; (ii) 5 μl, final thrombin concentration 2.3 μg / mL; and (iii) 10 μl, final thrombin concentration 4.6 μg / mL). Samples taken at 1, 2, 3, 4, and 19 hours after the start of the reaction were analyzed by SDS-PAGE under reducing and non-reducing conditions. As can be seen in Figure 3A, cleavage was observed after 1 hour even with the lowest concentration of thrombin (0.46 μg / mL), and complete cleavage of A4954 was observed by 19 hours with higher concentrations of thrombin (2.3 μg / mL and 4.6 μg / mL). The following table describes the lanes of the gel in Figure 3A. [Table 6]

[0543] The resulting cleaved A4954 product was analyzed by mass spectrometry. Samples were desalted using a thermoset ZEBA 0.5m 7K MWCO spin column. Before thawing the sample, the column was prepared by washing three times with 300 μl of 100 mM Tris. After desalting, protein concentration was measured using a nanodrop filter, corrected to 0.1 AU, and 1 μl of DTT was added per 99 μl of sample and incubated at 37°C for 30 minutes. The sample was then analyzed by LC / MS. The HPLC column used was BioResolve, RP mAb Polyphenyl 450Å 2.7 μm 2.1×150 mm, S / N 01153907216925. One microliter of sample was injected. Samples were analyzed using the Bioresolve sensitivity 6 min segmented gradient method SXNA004472 (NBK88-86). As shown in Figure 3B, a single cleavage site was identified at residue R437 within the activation loop. Furthermore, no off-site cleavage of A4954 by thrombin was observed.

[0544] Furthermore, LC / A1-Exo loop-H NIn three additional retargeted BoNTs with the / A1-TM structure but different targeting moieties (A00005361, A00005362, and A00005363), a modified C-loop containing the same PRG cleavage site present in A4954 was used to replace the BoNT / A activation loop. As shown in Figure 3C, in each case, thrombin was able to cleave the PRG cleavage site in the modified C-loop, and no truncation products were observed. The following table describes the lanes of the gel in Figure 3C. [Table 7]

[0545] Example 5 - Design of additional exogenous activation loops and modified BoNT / C1 activation loops

[0546] Using the methodology of Example 1, the same LC / A1-Exo Loop-H as in Example 1 was N Modified BoNTs were generated that have the / A1-TM structure but with exogenous activation loops and modified BoNT / C1 activation loops as shown in the table below. [Table 8]

[0547] Example 6 - BoNTs modified to contain an ENKSLVPRGS- or SGRSA-modified C-loop are efficiently cleaved by thrombin and urokinase, respectively

[0548] 200 μg of modified BoNT with an ENKSLVPRGS-modified C-loop was treated with increasing amounts of (i) thrombin (0.39 μg, 1.95 μg, and 3.90 μg) at 20°C, and samples were taken at 2, 4, and 20 h after activation.

[0549] 200 μg of modified BoNT with an SGRSA-modified C-loop was administered in a 200 μg / mL PBS containing increasing amounts of (i) urokinase; (ii) a functional t-PA fragment (t-PA frag or (iii) full-length t-PA (t-PAfl ) at increasing concentrations (0.39 μg, 1.95 μg, and 3.90 μg) at 20°C, and samples were taken at 2, 4, and 20 hours after activation.

[0550] As shown in Figure 4A, thrombin cleavage of the modified BoNT with the ENKSLVPRGS-modified C-loop was nearly complete at 2 hours, even at the lowest thrombin concentration tested. The heavy and light chains are visible on the non-reducing gel (black arrows). Cleavage of the modified BoNT with the SGRSA-modified C-loop began at 2 hours with 0.39 μg of urokinase, revealing traces of what appeared to be truncated products (dotted arrows). However, neither t-PA nor the modified BoNT with the SGRSA-modified C-loop showed any appreciable cleavage. The following table provides a description of the lanes in the gel in Figure 4A. [Table 9]

[0551] Cleavage progressed over time, and by 4 hours, thrombin cleavage of the modified BoNT with the ENKSLVPRGS-modified C-loop was nearly complete at all concentrations of thrombin tested (data not shown). Cleavage of the modified BoNT with the SGRSA-modified C-loop was nearly complete at 0.39 μg of urokinase, with trace amounts of what appeared to be truncated products (data not shown).

[0552] By 20 hours, thrombin cleavage of the modified BoNT with the ENKSLVPRGS-modified C-loop was nearly complete at all concentrations of thrombin tested. Cleavage of the modified BoNT with the SGRSA-modified C-loop was nearly complete at 0.39 μg of urokinase. However, the modified BoNT with the SGRSA-modified C-loop was only minimally cleaved by either t-PA.

[0553] The results of urokinase cleavage of the modified BoNT with an SGRSA-modified C-loop were compared with urokinase cleavage of A4952 from Example 2. Compared with the KVR cleavage site, inclusion of the SGRSA cleavage site in the modified C-loop improved cleavage and specificity and reduced off-target activity. Thus, these results suggest that an SGRSA-modified C-loop may be advantageous compared to a KVR-modified C-loop when modifying BoNT for urokinase activation.

[0554] The results of thrombin cleavage of the modified BoNT with an ENKSLVPRGS-modified C-loop were compared to the thrombin cleavage of A4954 in Example 4. Compared to the PRG cleavage site, inclusion of the ENKSLVPRGS cleavage site in the modified C-loop improved the cleavage rate, and no off-target activity was observed. Thus, these results suggest that the ENKSLVPRGS-modified C-loop may be advantageous compared to the PRG-modified C-loop when modifying BoNT for thrombin activation.

[0555] Furthermore, even 72 hours after thrombin activation (20°C, pH 7.2, 150 mM NaCl), there was no observable shortening of the modified BoNT with the ENKSLVPRGS-modified C-loop, even when the thrombin concentration was increased 10-fold (19.5 μg) (data not shown), suggesting that the ENKSLVPRGS-modified C-loop may advantageously promote the stability of the modified BoNT, for example, during delays in production.

[0556] The products obtained from cleavage of a modified BoNT (A5045) with an SGRSA-modified C-loop were analyzed by mass spectrometry. Samples were desalted using a thermo ZEBA 0.5m 7K MWCO spin column. Before thawing the sample, the column was washed three times with 300 μl of 100 mM Tris. After desalting, the protein concentration was measured using a nanodrop filter, corrected to 0.1 AU, and 1 μl of DTT was added per 99 μl of sample and incubated at 37°C for 30 minutes. The samples were then analyzed by LC / MS. The HPLC column used was BioResolve, RP mAb Polyphenyl 450Å 2.7 μm 2.1x150 mm, S / N 01153907216925. One microliter of sample was injected. Samples were analyzed using the SXNA004472 Bioresolve sensitivity 6 min segmented gradient assay described in NBK88-86. As shown in Figure 4B, a single cleavage site was identified at residue R437 within the activation loop. Two off-site cleavages of A5045 were observed at residues F167 and K683, both of which could potentially be removed by genetic engineering.

[0557] Example 7 - Design of additional exogenous activation loops and modified BoNT / C1 activation loops

[0558] Based on the experimental results observed in Example 6, the same LC / A1-Exo Loop-H as in Example 1 was prepared using the methodology of Example 1. N Further modified BoNTs were generated that had the / A1-TM structure but additionally introduced exogenous protease cleavage sites into the C-loop, as shown in the table below. [Table 10]

[0559] Example 8 - BoNTs modified to contain an LTPRGVRL-, PGSGRSAG-, or PPFGRSAG-modified C-loop are efficiently cleaved by thrombin, urokinase, and / or t-PA, respectively

[0560] 200 μg of modified BoNT with an LTPRGVRL-modified C-loop was treated with increasing amounts of (i) thrombin (0.39 μg, 1.95 μg, and 3.90 μg) at 20°C, pH 7.2, and samples were taken at 2, 4, and 20 hours after activation.

[0561] 200 μg of modified BoNT with a PGSGRSAG-modified C-loop or an extended PGSGRSAG-modified C-loop (including PGSGRSASGTTGTG) was administered to a subject in a 24-well plate containing increasing amounts of: (i) urokinase; (ii) a functional fragment of t-PA (t-PA frag or (iii) full-length t-PA (t-PA fl ) at increasing concentrations (0.39 μg, 1.95 μg, and 3.90 μg) at 20°C and pH 7.2, and samples were taken at 2, 4, and 20 hours after activation.

[0562] 200 μg of modified BoNT with a PPFGRSAG-modified C-loop was administered in a 24-well plate containing increasing amounts of (i) urokinase; (ii) t-PA frag or (iii) t-PA fl were treated with increasing concentrations (0.39 μg, 1.95 μg, and 3.90 μg) at 20°C and pH 7.2, and samples were taken at 2, 4, and 20 hours after activation.

[0563] As shown in Figure 5A, thrombin cleavage of the modified BoNT with the LTPRGVRL-modified C-loop was nearly complete at 2 hours, even at the lowest thrombin concentration tested. Cleavage was essentially complete by 4 hours (data not shown). With extended incubation after activation was complete, some shortening was observed by 20 hours. The following table describes the lanes of the gel in Figure 5A. [Table 11]

[0564] As shown in Figure 5B, at 2 hours, urokinase-mediated cleavage of the modified BoNT with a PGSQRSA-modified C-loop was ongoing even at the lowest urokinase concentration tested, with significant activation already achieved when 1.95 μg or 3.90 μg of urokinase was used. Compared with the modified BoNT with an SGRSA-modified C-loop, the rate of urokinase-mediated activation of the modified BoNT with a PGSQRSA-modified C-loop was enhanced. Cleavage of the modified BoNT with a PGSQRSA-modified C-loop was essentially complete by 4 hours when 1.95 μg or 3.90 μg of urokinase was used. Trace amounts of truncated products (black arrows), likely generated by urokinase, were observed at all time points. However, the modified BoNT with a PGSQRSA-modified C-loop showed no significant activation of t-PA. frag or t-PA fl The following table describes the lanes of the gel in Figure 5B. [Table 12]

[0565] As shown in Figure 5C, at 2 hours, urokinase cleavage of the modified BoNT with the PGSGRSASGTTGTG-modified C-loop was ongoing even at the lowest urokinase concentration tested, with significant activation already achieved when 1.95 μg or 3.90 μg of urokinase was used. Cleavage of the modified BoNT with the PGSGRSASGTTGTG-modified C-loop was essentially complete by 4 hours when 3.90 μg of urokinase was used. Trace amounts of truncated products (black arrows), likely generated by urokinase, were observed at all time points. However, the modified BoNT with the PGSGRSASGTTGTG-modified C-loop was significantly reduced by t-PA. frag or t-PA fl The following table describes the lanes of the gel in Figure 5C. [Table 13]

[0566] As shown in Figure 5D, at 2 hours, cleavage of the modified BoNT with the PPFGRSAG-modified C-loop was significantly enhanced by urokinase, t-PA, and ATP. frag , and t-PA fl In the case of urokinase, some activation was observed even at the lowest concentration of urokinase tested. frag and t-PA fl Then, 1.95 μg or 3.90 μg of t-PA frag or t-PA fl Partial activation was achieved when the modified BoNT with the PPFGRSAG-modified C-loop was used. Cleavage of the modified BoNT with the PPFGRSAG-modified C-loop progressed over time, and by 20 hours, activation was significantly enhanced by urokinase, t-PA, and t-PA at each concentration tested. frag , and t-PA fl Significant cleavage was observed for all of the t-PA frag or t-PA fl No truncated products generated using ATP were observed at any time point. The table below describes the lanes of the gel in Figure 5D. [Table 14]

[0567] Using a modified BoNT with a PPFGRSAG-modified C-loop, t-PA frag and t-PA fl The cleavage experiment was repeated using increasing concentrations of t-PA. frag or t-PA fl Increasing concentrations (3.9 μg, 7.8 μg, 15.6 μg, and 31.2 μg) of t-PA were treated at 20°C and pH 7.2, and samples were taken at 2, 4, and 20 hours after activation. frag and t-PA flIncreasing the concentration of t-PA increased the cleavage of the modified BoNT with the PPFGRSAG-modified C-loop at each time point, and at higher concentrations, nearly complete activation was observed. frag and t-PA fl Even with t-PA frag and t-PA fl No truncated products generated using ATP were observed at any time point. The table below describes the lanes of the gel in Figure 5E. [Table 15]

[0568] Consideration

[0569] The highly effective ENKSLVPRGS and LTPRGVRL modified C-loops suggest that they may be used to enhance activation and / or reduce the amount of protease required for activation. Furthermore, minimal shortening was observed for the range of modified BoNTs tested, with activation complete by 4 hours and only slight shortening observed after 20 hours. Furthermore, thrombin appears to be compatible with BoNT / X-based modified molecules (data not shown). Therefore, the use of thrombin, particularly with the ENKSLVPRGS and LTPRGVRL modified C-loops, most preferably the LTPRGVRL modified C-loop, is a commercially preferred option.

[0570] For activation by urokinase, the SGRSA- and PGSGRSAG-modified C-loops show improved cleavage by urokinase. For urokinase, the PGSGRSASGTTGTG-modified C-loop also shows improvement over the KRV-modified C-loop. Although some truncated products were observed, these may be manageable during the production and purification process, and / or sensitive sites in the BoNT backbone may be removed by further engineering.

[0571] Regarding activation by t-PA, the VVPRVELVA-modified C-loop showed no evidence of cleavage by any of the proteases tested. The SGRSA-modified C-loop and the extended PGSGRSASGTTGTG-modified C-loop were significantly different from the t-PA variant (t-PA frag and t-PA fl ), and the PPFGRSAG-modified C-loop exhibits modest activation at higher concentrations of t-PA variants. Importantly, no shortening of the SGRSA-modified C-loop, the extended PGSGRSASGTTGTG-modified C-loop, or the PPFGRSAG-modified C-loop was observed even at high concentrations of t-PA variants.

[0572] All publications mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the described methods and systems of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the following claims. [Explanation of symbols]

[0573] [Figure 3B] TIC [counts]: TIC [counts] Retention time [min]: Retention time [minutes] Intensity [counts]: Intensity [counts] Mass [Da]: Mass [Da] Identity: Identification information A00004954 LC - Cleaved at R 437: A00004954 LC - Cleaved at R 437 Observed m / z: Observed m / z Theoretical m / z: Theoretical m / z [Figure 3C] - non-reducing: - non-reducing state + reducing: + reduced state [Figure 4A] 2 hrs: 2 hours [Figure 4B] BIO100005045 HC Cleaved at R 437 + ABESF (protease inhibitor): BIO100005045 HC - Cleaved at R 437 + ABESF (protease inhibitor) [Figure 5A] 2 hrs: 2 hours 20 hours: 20 hours [Figure 5B] 2 hrs: 2 hours 4 hrs: 4 hours 20 hours: 20 hours

Claims

1. a modified Clostridial neurotoxin, wherein the endogenous activation loop is replaced with a modified BoNT / C activation loop, and wherein the endogenous activation site of the BoNT / C activation loop, or a portion thereof, is replaced with an exogenous protease cleavage site; Cleavage at the cleavage site now generates a two-chain form of the modified Clostridial neurotoxin.

2. 2. The modified Clostridial neurotoxin of claim 1, wherein the exogenous protease cleavage site is a cleavage site specific for an exogenous protease selected from: (a) Thrombin; (b) tissue plasminogen activator (t-PA); and / or (c) urokinase (u-PA); where, optionally: (i) the exogenous protease is a human protease; (ii) the exogenous protease is included in the formulation; and / or (iii) the exogenous protease is a recombinant human protease.

3. 3. The modified Clostridial neurotoxin of claim 1 or 2, wherein the exogenous protease cleavage site comprises or consists of: (a) the thrombin consensus sequence of SEQ ID NO: 169 or 170; and / or (b) uPA and / or t-PA consensus sequence of SEQ ID NO: 172 or 173.

4. 10. The modified Clostridial neurotoxin of any one of the preceding claims, wherein the exogenous protease cleavage site comprises one or more of, or consists of, one or more of: LTPRGVRL (SEQ ID NO: 15), LVPRGS (SEQ ID NO: 16), ENKSLVPRGS (SEQ ID NO: 17), SGRSA (SEQ ID NO: 25), PPFGRSAG (SEQ ID NO: 33), PGSGRSAG (SEQ ID NO: 26), PGSGRSASGTTGTG (SEQ ID NO: 27), KRV (SEQ ID NO: 24), GRI (SEQ ID NO: 44) or PGRVVGG (SEQ ID NO: 50).

5. A modified Clostridial neurotoxin according to any one of the preceding claims, wherein the exogenous protease cleavage site is 3 to 10 amino acids in length, preferably 3 to 8 amino acids in length.

6. 10. A modified Clostridial neurotoxin according to any one of the preceding claims, wherein the exogenous protease cleavage site consists of one or more of the following: LTPRGVRL (SEQ ID NO: 15), LVPRGS (SEQ ID NO: 16), ENKSLVPRGS (SEQ ID NO: 17), SGRSA (SEQ ID NO: 25), PPFGRSAG (SEQ ID NO: 33), PGSGRSAG (SEQ ID NO: 26), PGSGRSASGTTGTG (SEQ ID NO: 27), KRV (SEQ ID NO: 24), GRI (SEQ ID NO: 44) or PGRVVGG (SEQ ID NO: 50).

7. 10. A modified Clostridial neurotoxin according to any one of the preceding claims, wherein said exogenous protease cleavage site comprises or consists of LTPRGVRL (SEQ ID NO: 15), LVPRGS (SEQ ID NO: 16) or ENKSLVPRGS (SEQ ID NO: 17), preferably LTPRGVRL (SEQ ID NO: 15), and said exogenous protease cleavage site is specific for thrombin, optionally wherein said exogenous protease cleavage site consists of LTPRGVRL (SEQ ID NO: 15), LVPRGS (SEQ ID NO: 16) or ENKSLVPRGS (SEQ ID NO: 17), preferably LTPRGVRL (SEQ ID NO: 15).

8. 7. The modified Clostridial neurotoxin of any one of claims 1 to 6, wherein the exogenous protease cleavage site comprises or consists of KRV (SEQ ID NO: 24), SGRSA (SEQ ID NO: 25), PPFGRSAG (SEQ ID NO: 33), PGSGRSAG (SEQ ID NO: 26) or PGSGRSASGTTGTG (SEQ ID NO: 27), and the exogenous protease cleavage site is specific for t-PA and / or u-PA, and optionally the exogenous protease cleavage site consists of KRV (SEQ ID NO: 24), SGRSA (SEQ ID NO: 25), PPFGRSAG (SEQ ID NO: 33), PGSGRSAG (SEQ ID NO: 26) or PGSGRSASGTTGTG (SEQ ID NO: 27).

9. 10. A modified Clostridial neurotoxin according to any one of the preceding claims, wherein the endogenous neurotoxin activation loop is one or more selected from SEQ ID NOs: 67-90.

10. A modified Clostridial neurotoxin according to any one of the preceding claims, wherein: (a) the exogenous protease cleavage site within the modified BoNT / C activation loop is the only cleavage site for the exogenous protease within the engineered clostridial neurotoxin; (b) contacting the modified Clostridial neurotoxin with the exogenous protease does not result in off-site cleavage; and / or (c) contacting the modified Clostridial neurotoxin with the exogenous protease does not result in light chain truncation of the modified Clostridial neurotoxin.

11. 10. A modified Clostridial neurotoxin according to any one of the preceding claims, wherein said Clostridial neurotoxin is: (a) botulinum neurotoxin (BoNT) serotype A, serotype B, serotype C, serotype D, serotype E, serotype F, serotype G, or serotype X, or tetanus neurotoxin (TeNT); or (b) Chimeric or hybrid BoNTs.

12. 9. The modified Clostridial neurotoxin of claim 8, wherein the modified Clostridial neurotoxin is: (a) BoNT / A, optionally BoNT / A1; or (b) BoNT / X.

13. Endogenous H of clostridial neurotoxins C or H CC 10. The modified Clostridial neurotoxin according to any one of the preceding claims, which is a retargeted Clostridial neurotoxin in which is replaced by an exogenous targeting moiety (TM).

14. 10. The modified clostridial neurotoxin according to any one of the preceding claims, which is a single-chain clostridial neurotoxin that satisfies the following conditions: (a)(i) encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 154, 156, 158, 160, 162, or 164; or (ii) encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 175, wherein SEQ ID NO: 176 within SEQ ID NO: 175 is replaced by a nucleotide sequence encoding at least one exogenous protease consensus sequence, cleavage site, or modified BoNT / C activation loop as defined in any one of claims 3 to 9; and / or (b) comprises a polypeptide sequence having at least 70% sequence identity to one or more of SEQ ID NOs: 155, 157, 159, 161, 163, 165, 166, 167, or 168; and / or (c) comprises a polypeptide sequence having: (i) at least 70% sequence identity to SEQ ID NO: 150, wherein SEQ ID NO: 2 within SEQ ID NO: 150 is replaced by one or more exogenous protease cleavage sites and / or modified BoNT / C activation loops as defined in any one of claims 3-9, wherein the one or more exogenous protease sites are preferably selected from one or more of SEQ ID NOs: 15, 33, or 25, and / or the modified BoNT / C activation loop is preferably selected from one or more of SEQ ID NOs: 57, 63, and / or 65; or (ii) at least 70% identity to SEQ ID NO: 152, wherein SEQ ID NO: 2 within SEQ ID NO: 152 is replaced by one or more exogenous protease cleavage sites and / or modified BoNT / C activation loops as defined in any one of claims 3-9, wherein the one or more exogenous protease sites are preferably selected from one or more of SEQ ID NOs: 15, 33, or 25, and / or the modified BoNT / C activation loop is preferably selected from one or more of SEQ ID NOs: 57, 63, and / or 65.

15. A method for proteolytically cleaving a modified clostridial neurotoxin described in any one of claims 1 to 14 into a corresponding two-chain clostridial neurotoxin, comprising contacting the modified clostridial neurotoxin with an exogenous protease specific to the exogenous protease cleavage site, thereby producing a two-chain clostridial neurotoxin.

16. 16. A di-chain Clostridial neurotoxin obtainable by the method of claim 15, wherein optionally: (a) the C-terminus of the light chain of the clostridial neurotoxin ends with the amino acid sequence LVPR, preferably ALVPR, and the N-terminus of the heavy chain of the clostridial neurotoxin begins with the amino acid sequence GSK or GSY; (b) the C-terminus of the light chain of said clostridial neurotoxin ends with the amino acid sequence SLVPR and the N-terminus of the heavy chain of said clostridial neurotoxin begins with the amino acid sequence GSY; (c) the C-terminus of the light chain of said clostridial neurotoxin ends with said amino acid sequence LTPR, preferably ALTPR, and the N-terminus of the heavy chain of said clostridial neurotoxin begins with the amino acid sequence GVR, preferably GVRL; (d) the C-terminus of the light chain of said clostridial neurotoxin ends with the amino acid sequence PGR and the N-terminus of the heavy chain of said clostridial neurotoxin begins with the amino acid sequence VVG; (e) the C-terminus of the light chain of the clostridial neurotoxin ends with the amino acid sequence DKR, preferably AIDKR, and the N-terminus of the heavy chain of the clostridial neurotoxin begins with the amino acid sequence VLY; (f) the C-terminus of the light chain of said clostridial neurotoxin ends with said amino acid sequence DKR, preferably AIDK, and the N-terminus of the heavy chain of said clostridial neurotoxin begins with the amino acid sequence RVLY; (g) the C-terminus of the light chain of the clostridial neurotoxin ends with the amino acid sequence SGR, preferably PGSGR, and the N-terminus of the heavy chain of the clostridial neurotoxin begins with the amino acid sequence SA, preferably SAY, SAG, or SAS; (h) the C-terminus of the truncated modified Clostridial neurotoxin light chain may end with the amino acid sequence SGR, preferably PGSGR, and the N-terminus of the truncated modified Clostridial neurotoxin heavy chain may begin with the amino acid sequence TL, preferably TLD or TLDC; or (i) The C-terminus of the light chain of said clostridial neurotoxin ends with the amino acid sequence FGR, and the N-terminus of the heavy chain of said clostridial neurotoxin begins with the amino acid sequence SA, preferably SAG.

17. 15. A polynucleotide encoding a modified Clostridial neurotoxin as defined in any one of claims 1 to 14.

18. 18. An expression vector comprising the polynucleotide defined in claim 17 operably linked to a promoter.

19. 19. The polynucleotide of claim 16, or the expression vector of claim 18, wherein the polynucleotide or expression vector comprises: (a) comprising a nucleotide sequence having: (i) at least 70% sequence identity to SEQ ID NO: 154, 156, 158, 160, 162, or 164; or (ii) at least 70% sequence identity to SEQ ID NO: 175, wherein SEQ ID NO: 176 within SEQ ID NO: 175 is replaced by a nucleotide sequence encoding at least one exogenous protease consensus sequence, cleavage site, or modified BoNT / C activation loop as defined in any one of claims 3-9; (b) encoding a polypeptide sequence having at least 70% sequence identity to one or more of SEQ ID NOs: 155, 157, 159, 161, 163, 165, 166, 167, or 168; and / or (c) encoding a polypeptide sequence having: (i) at least 70% sequence identity to SEQ ID NO: 150, wherein SEQ ID NO: 2 within SEQ ID NO: 150 is replaced by one or more exogenous protease cleavage sites and / or modified BoNT / C activation loops as defined in any one of claims 3-9, wherein the one or more exogenous protease sites are preferably selected from one or more of SEQ ID NOs: 15, 33, or 25, and / or the modified BoNT / C activation loop is preferably selected from one or more of SEQ ID NOs: 57, 63, and / or 65; or (ii) at least 70% identity to SEQ ID NO: 152, wherein SEQ ID NO: 2 within SEQ ID NO: 152 is replaced by one or more exogenous protease cleavage sites and / or modified BoNT / C activation loops as defined in any one of claims 3-9, wherein the one or more exogenous protease sites are preferably selected from one or more of SEQ ID NOs: 15, 33, or 25, and / or the modified BoNT / C activation loop is preferably selected from one or more of SEQ ID NOs: 57, 63, and / or 65.

20. A method for producing a modified Clostridial neurotoxin as defined in any one of claims 1 to 14, comprising expressing a polynucleotide as defined in claim 17 or 19 or an expression vector as defined in claim 18 or 19 in a cell, and recovering the expressed modified Clostridial neurotoxin.

21. 21. The method of claim 20, further comprising introducing into said cell a polynucleotide as defined in claim 17 or 19 or an expression vector as defined in claim 18 or 19.

22. 15. A cell expressing a modified Clostridial neurotoxin as defined in any one of claims 1 to 14.

23. 23. A cell according to claim 22, comprising a polynucleotide as defined in claim 17 or 19 or an expression vector as defined in claim 18 or 19.

24. 17. A pharmaceutical composition comprising a modified clostridial neurotoxin as defined in any one of claims 1 to 14 or a two-chain clostridial neurotoxin as defined in claim 16, and a pharmaceutically acceptable carrier, excipient, diluent, adjuvant, propellant and / or salt.

25. 26. A modified Clostridial neurotoxin as defined in any one of claims 1 to 14, a double-chain Clostridial neurotoxin as defined in claim 16, or a pharmaceutical composition as defined in claim 24 for use in a method for preventing or treating a disease or disorder in which therapy with a botulinum neurotoxin is indicated, wherein optionally the disease or disorder is a condition associated with unwanted immune secretion, strabismus, blepharospasm, squint, dystonia (e.g., spastic dystonia, oromandibular dystonia, focal dystonia, tardive dystonia, laryngeal dystonia, limb dystonia, cervical dystonia), torticollis (e.g., spasmodic torticollis), cosmetic therapy (cosmetic) applications that would benefit from cell / muscle attenuation (by downregulation or inactivation of SNAREs), neuromuscular disorders or conditions of eye movement (e.g., conjugate strabismus, hypertropia, Lateral rectus paralysis, nystagmus, hypothyroid myopathy), writer's cramp, bruxism, Wilson's disease, tremors, tics, segmental myoclonus, spasms, spasticity due to chronic multiple sclerosis, spasticity resulting in bladder dysfunction, animus, back spasms, cramps, levator pelvic syndrome, spina bifida, tardive dyskinesia, Parkinson's disease, stuttering, hemifacial spasm, eyelid disorders, cerebral palsy, focal spasticity, spastic colitis, neurogenic bladder, anis The symptom is selected from: muscular aches, limb spasticity, tics, tremors, teeth grinding, anal fissures, achalasia, dysphagia, lacrimation, hyperhidrosis, excessive salivation, gastrointestinal hypersecretion, muscle pain (e.g., pain due to muscle spasms), headache (e.g., tension headache or migraine), phantom pain (e.g., phantom limb pain), glabellar lines, skin wrinkles, cancer, uterine disorders, genitourinary disorders, genitourinary neurological disorders, bladder pain syndrome, interstitial cystitis, chronic neurogenic inflammation, and smooth muscle disorders.

26. 24. Use of a modified Clostridial neurotoxin as defined in any one of claims 1 to 14, a double-chain Clostridial neurotoxin as defined in claim 16, or a pharmaceutical composition as defined in claim 24, in the manufacture of a medicament for preventing or treating a disease or disorder in which therapy with a botulinum neurotoxin is indicated, wherein optionally said disease or disorder is a condition associated with unwanted immune secretion, strabismus, blepharospasm, squint, dystonia (e.g., spastic dystonia, oromandibular dystonia, focal dystonia, tardive dystonia, laryngeal dystonia, limb dystonia, cervical dystonia), torticollis (e.g., spasmodic torticollis), cosmetic therapy (cosmetic) that would benefit from cell / muscle attenuation (by downregulation or inactivation of SNAREs), neuromuscular disorders or conditions of eye movement (e.g., conjunct strabismus, vertical strabismus, , lateral rectus paralysis, nystagmus, hypothyroid myopathy), writer's cramp, bruxism, Wilson's disease, tremors, tics, segmental myoclonus, spasms, spasticity due to chronic multiple sclerosis, spasticity resulting in bladder dysfunction, animus, back spasms, cramps, levator pelvic syndrome, spina bifida, tardive dyskinesia, Parkinson's disease, stuttering, hemifacial spasm, eyelid disorders, cerebral palsy, focal spasticity, spastic colitis, neurogenic bladder, animus The condition is selected from: slurred speech, limb spasticity, tics, tremors, teeth grinding, anal fissures, achalasia, dysphagia, lacrimation, hyperhidrosis, excessive salivation, gastrointestinal hypersecretion, muscle pain (e.g., pain due to muscle spasms), headache (e.g., tension headache or migraine), phantom pain (e.g., phantom limb pain), glabellar lines, skin wrinkles, cancer, uterine disorders, genitourinary disorders, genitourinary neurological disorders, bladder pain syndrome, interstitial cystitis, chronic neurogenic inflammation, and smooth muscle disorders.

27. 27. The modified Clostridial neurotoxin or pharmaceutical composition for use according to claim 25, or the use of the modified Clostridial neurotoxin or pharmaceutical composition according to claim 26, wherein the Clostridial neurotoxin is administered to the subject in a single-chain form.

28. 28. A modified Clostridial neurotoxin or pharmaceutical composition for use according to claim 27, or use of the modified Clostridial neurotoxin or pharmaceutical composition according to claim 27, wherein the Clostridial neurotoxin or pharmaceutical composition is substantially free of a two-chain form of the Clostridial neurotoxin.

29. 29. A modified Clostridial neurotoxin or pharmaceutical composition for use according to claim 27 or 28, or use of the modified Clostridial neurotoxin or pharmaceutical composition according to claim 27 or 28, wherein said Clostridial neurotoxin or pharmaceutical composition comprises less than 400 pg of double-chain Clostridial neurotoxin per 100 ng of single-chain Clostridial neurotoxin, or less than 300 pg of double-chain Clostridial neurotoxin per 100 ng of single-chain Clostridial neurotoxin, or less than 200 pg of double-chain Clostridial neurotoxin per 100 ng of single-chain Clostridial neurotoxin, or less than 100 pg of double-chain Clostridial neurotoxin per 100 ng of single-chain Clostridial neurotoxin, or less than 50 pg of double-chain Clostridial neurotoxin per 100 ng of single-chain Clostridial neurotoxin.

30. 27. The modified Clostridial neurotoxin or pharmaceutical composition for use according to claim 25, or the use of the modified Clostridial neurotoxin or pharmaceutical composition according to claim 26, wherein the Clostridial neurotoxin is administered to the subject in a di-chain form.

31. 31. A modified Clostridial neurotoxin or pharmaceutical composition for use, or use of a modified Clostridial neurotoxin or pharmaceutical composition, according to claim 30, wherein said Clostridial neurotoxin or pharmaceutical composition is substantially free of single-chain forms of said Clostridial neurotoxin.

32. 32. The modified Clostridial neurotoxin or pharmaceutical composition for use, or the use of the modified Clostridial neurotoxin or pharmaceutical composition according to claim 30 or 31, wherein the Clostridial neurotoxin or pharmaceutical composition comprises less than 400 pg of single-chain Clostridial neurotoxin per 100 ng of double-chain Clostridial neurotoxin, or less than 300 pg of single-chain Clostridial neurotoxin per 100 ng of double-chain Clostridial neurotoxin, or less than 200 pg of single-chain Clostridial neurotoxin per 100 ng of double-chain Clostridial neurotoxin, or less than 100 pg of single-chain Clostridial neurotoxin per 100 ng of double-chain Clostridial neurotoxin, or less than 50 pg of single-chain Clostridial neurotoxin per 100 ng of double-chain Clostridial neurotoxin.

33. 17. A cosmetic composition comprising a modified clostridial neurotoxin as defined in any one of claims 1 to 14 or a two-chain clostridial neurotoxin as defined in claim 16, and a cosmetically acceptable carrier, excipient, diluent, adjuvant, propellant and / or salt.

34. 34. Use of a cosmetic composition as defined in claim 33 for preventing or alleviating a cosmetic indication for which said application of a botulinum neurotoxin is indicated.

35. 35. Use of the cosmetic composition of claim 33 or the cosmetic composition of claim 34, wherein the Clostridial neurotoxin is for administration to a subject in a single-chain form.

36. 36. The cosmetic composition or use of a cosmetic composition according to claim 35, wherein the clostridial neurotoxin or cosmetic composition is substantially free of a di-chain form of the clostridial neurotoxin.

37. 37. The cosmetic composition of claim 35 or 36, or use of the cosmetic composition of claim 34 or 35, wherein the clostridial neurotoxin or cosmetic composition comprises less than 400 pg of double-chain clostridial neurotoxin per 100 ng of single-chain clostridial neurotoxin, or less than 300 pg of double-chain clostridial neurotoxin per 100 ng of single-chain clostridial neurotoxin, or less than 200 pg of double-chain clostridial neurotoxin per 100 ng of single-chain clostridial neurotoxin, or less than 100 pg of double-chain clostridial neurotoxin per 100 ng of single-chain clostridial neurotoxin, or less than 50 pg of double-chain clostridial neurotoxin per 100 ng of single-chain clostridial neurotoxin.

38. 34. The cosmetic composition of claim 33, or use of the cosmetic composition of claim 33, wherein the Clostridial neurotoxin is for administration to a subject in a single-chain form.

39. 39. The cosmetic composition or use of a cosmetic composition according to claim 38, wherein the clostridial neurotoxin or cosmetic composition is substantially free of single-chain forms of the clostridial neurotoxin.

40. 40. The cosmetic composition or use of a cosmetic composition according to claim 38 or 39, wherein the clostridial neurotoxin or cosmetic composition comprises less than 400 pg of single-chain clostridial neurotoxin per 100 ng of double-chain clostridial neurotoxin, or less than 300 pg of single-chain clostridial neurotoxin per 100 ng of double-chain clostridial neurotoxin, or less than 200 pg of single-chain clostridial neurotoxin per 100 ng of double-chain clostridial neurotoxin, or less than 100 pg of single-chain clostridial neurotoxin per 100 ng of double-chain clostridial neurotoxin, or less than 50 pg of single-chain clostridial neurotoxin per 100 ng of double-chain clostridial neurotoxin.

41. 1. A method for proteolytically cleaving a single-chain clostridial neurotoxin into the corresponding two-chain clostridial neurotoxin, comprising: (a) providing a single-chain clostridial neurotoxin; and (b) contacting said single-chain Clostridial neurotoxin with an exogenous protease; wherein the single-chain clostridial neurotoxin has an exogenous protease cleavage site and / or a modified BoNT / C activation loop as defined in any one of claims 3 to 9; The exogenous protease hydrolyzes the peptide bond of the exogenous protease cleavage site and / or the modified BoNT / C activation loop, thereby generating a di-chain clostridial neurotoxin, and optionally the exogenous protease is selected from thrombin, t-PA, u-PA, FIX, and FVIIa.

42. 42. The method of claim 41, wherein the single-chain clostridial neurotoxin is (a) a modified Clostridial neurotoxin as defined in any one of claims 1 to 14; (b) encoded by a nucleic acid sequence comprising or consisting of a nucleotide sequence having: At least 70% sequence identity to SEQ ID NO: 154, 156, 158, 160, 162, or 164; or (ii) at least 70% sequence identity to SEQ ID NO: 175, wherein SEQ ID NO: 176 within SEQ ID NO: 175 is replaced by a nucleotide sequence encoding at least one exogenous protease consensus sequence, cleavage site, or modified BoNT / C activation loop as defined in any one of claims 3-9; (c) comprising or consisting of a polypeptide sequence having at least 70% sequence identity to one or more of SEQ ID NOs: 155, 157, 159, 161, 163, 165, 166, 167, or 168; and / or (d) comprising or consisting of a polypeptide sequence having: (i) at least 70% sequence identity to SEQ ID NO: 150, wherein SEQ ID NO: 2 within SEQ ID NO: 150 is replaced by one or more exogenous protease cleavage sites and / or modified BoNT / C activation loops as defined in any one of claims 3-9, wherein the one or more exogenous protease sites are preferably selected from one or more of SEQ ID NOs: 15, 33, or 25, and / or the modified BoNT / C activation loops are preferably selected from one or more of SEQ ID NOs: 57, 63, and / or 65; or (ii) at least 70% identity to SEQ ID NO: 152, wherein SEQ ID NO: 2 within SEQ ID NO: 152 is replaced by one or more exogenous protease cleavage sites and / or modified BoNT / C activation loops as defined in any one of claims 3-9, wherein the one or more exogenous protease sites are preferably selected from one or more of SEQ ID NOs: 15, 33, or 25, and / or the modified BoNT / C activation loop is preferably selected from one or more of SEQ ID NOs: 57, 63, and / or 65.

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