Clostridial neurotoxins comprising activated endosomal protease cleavage sites

Abstract

The present invention relates to clostridial neurotoxins engineered to comprise an endosomal protease cleavage site within the activation loop wherein cleavage at said site produces an active double-stranded clostridial neurotoxin. The invention also relates to processes for their manufacture, as well as to related pharmaceutical compositions, nucleotide sequences and therapeutic and cosmetic uses. The invention also relates to a method for processing the single-chain clostridium neurotoxin into the corresponding double-chain clostridium neurotoxin in a proteolysis manner.

Description

Field of the Invention

[0001] The present invention relates to a Clostridial neurotoxin which has been engineered to contain an endosomal protease cleavage site within the activation loop, wherein cleavage at said site produces an active double-stranded Clostridial neurotoxin. The present invention also relates to methods for its manufacture, and related pharmaceutical compositions, nucleotide sequences, and therapeutic and cosmetic uses. The present invention also relates to a method for proteolytically processing said single-stranded Clostridial neurotoxin into the corresponding double-stranded Clostridial neurotoxin. Background of the Invention

[0003] Bacteria of the genus Clostridia are capable of producing highly potent and specific protein toxins that are able to poison neurons and other cells to which they are delivered. Examples of such Clostridial neurotoxins include the neurotoxins produced by Clostridium tetani (TeNT), Clostridium botulinum (BoNT) serotypes A-G and X (see WO 2018 / 009903 A2), as well as the neurotoxins produced by Clostridium baratii and Clostridium butyricum.

[0004] Clostridial neurotoxins are among the most potent toxins known. For example, the median lethal dose (LD 50 ) values for botulinum neurotoxins in mice range from 0.5 to 5 ng / kg, depending on the serotype. Both tetanus and botulinum toxins act by inhibiting the function of affected neurons, particularly the release of neurotransmitters. Botulinum toxins act on the neuromuscular junction and inhibit cholinergic transmission in the peripheral nervous system, while tetanus toxin acts on the central nervous system.

[0005] Clostridial neurotoxins are expressed in Clostridia as single-chain polypeptides. Each Clostridial neurotoxin has a catalytic light chain separated from the heavy chain (which encompasses 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 held together by a disulfide bridge, thereby forming a fully active double-stranded toxin.

[0006] In the standard production process of producing recombinant toxins, this activation process must be reproduced. In traditional production methods, exogenous proteases with defined cleavage motifs (such as trypsin or Lys-C) are used to proteolytically activate single-chain Clostridium neurotoxins. However, for certain Clostridium neurotoxins, incubation with Lys-C or trypsin results in partial or incorrect cleavage of the single-chain polypeptide, thereby generating contaminating single-chain and / or inactive cleavage / degradation products (such as in the case of BoNT / E). For example, for botulinum neurotoxin serotype X (BoNT / X, see WO 2018 / 009903A2), activation is problematic, and cleavage with trypsin or Lys-C completely degrades the polypeptide. Thus, there is currently no general exogenous protease to activate Clostridium neurotoxins. This is particularly problematic when identifying new Clostridium neurotoxins or producing modified (such as chimeric or hybrid) neurotoxins, as multiple proteases need to be screened to determine the correct activation. For retargeted Clostridium neurotoxins, some of the standard proteases used for activation also cleave within the exogenous targeting moiety, resulting in incorrect protein processing and reduced targeting to the desired cell type. To avoid such off-target cleavage, alternative targeting moieties must be identified (which may not always be feasible), or the targeting moiety must be designed to remove the cleavage sites of the standard protease, which may have a negative impact on the structure of the targeting moiety and / or increase the design and production costs.

[0007] In addition, in vitro activation of Clostridium neurotoxins also presents many disadvantages. The use of exogenous proteases (especially GMP-grade proteases) and their removal after activation of Clostridium neurotoxins incurs certain costs. Dependence on a single or limited number of suppliers of GMP-grade proteases also creates a weak supply / production chain. Purifying the activated Clostridium neurotoxin from the activated exogenous protease also affects production efficiency and yield. In addition, producing active double-chain Clostridium neurotoxins according to conventional production methods requires strict safety and control procedures, which also increases production costs and time. Practitioners exposed to active double-chain Clostridium neurotoxins also need to take strict safety precautions.

[0008] The present invention overcomes one or more of the above problems. Summary of the Invention

[0009] Endosomes are small membrane-bound vesicles within eukaryotic cells that are involved in trafficking materials internalized from outside the cell. Some endosomes maintain an acidic pH, which can separate proteins from receptors. Once separated, these different components can then be transported. Some molecules are directed back to the cell surface, while others fuse with lysosomes where the contents are degraded by hydrolytic enzymes. Endosomes also transport materials to and from the Golgi apparatus, or between the apical and basolateral compartments of polarized cells. Endosomes contain a large number of proteases that can degrade internalized proteins.

[0010] The inventors have previously shown that inserting a furin cleavage site into the activation loop of a clostridial neurotoxin allows for in vivo activation of the clostridial neurotoxin (see PCT application No. PCT / GB2022 / 050756; which is incorporated herein by reference in its entirety). This represents a paradigm shift in the production, processing, and activation of clostridial neurotoxins and indeed in their therapeutic use. In particular, to the extent that prior attempts in the art to introduce exogenous cleavage sites into clostridial neurotoxins, the goal has been to facilitate in vitro production and processing of the clostridial neurotoxin and then administer it in a double-stranded form. The inventors have now shown that this potential for in vivo activation of clostridial neurotoxins is not limited to the use of furin, but that other endogenous proteases, in particular endosomal proteases such as cathepsin L and asparaginyl endopeptidase (AEP), are also capable of cleaving an appropriately introduced cleavage site in a clostridial neurotoxin.

[0011] Furthermore, compared to conventionally activated clostridial neurotoxins, the endosomal protease-activated engineered clostridial neurotoxins of the present invention offer a variety of potential benefits such as improved safety for operators (e.g., clinicians or others handling the endosomal protease-activated engineered neurotoxins of the present invention for administration to patients, as well as workers involved in the production of the endosomal protease-activated engineered neurotoxins), and / or reduced manufacturing burden / cost. The endosomal protease-activated engineered neurotoxins of the present invention may also improve the safety profile of patients.

[0012] Thus, the inventors provide for the first time single-chain clostridial neurotoxins with therapeutic potential, such as engineered BoNT / A1 with an endosomal protease cleavage site, without the need to be activated into a double-stranded form prior to administration.

[0013] Accordingly, the present invention provides an engineered Clostridial neurotoxin, the engineered Clostridial neurotoxin comprising an endosomal protease cleavage site, wherein cleavage at the cleavage site results in the production of a double-stranded form of the engineered Clostridial neurotoxin. The endosomal protease cleavage site can be a cleavage site specific for the following enzymes: (a) asparagine endopeptidase (AEP); or (b) cathepsin, optionally cathepsin L1, B, D, K or S. The endosomal protease cleavage site can comprise or consist of the following: (a) an APE core motif selected from SEQ ID NOs: 208-214; (b) a cathepsin L core motif selected from SEQ ID NO: 138 and / or 188-197; (c) a cathepsin B core motif selected from SEQ ID NOs: 20, 181, 198 and / or 199; and / or (d) a cathepsin D core motif selected from SEQ ID NOs: 200-207. The endosomal protease cleavage site can comprise one or more of the following or consist of one or more of the following: SEQ ID NOs: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186 and / or 187. The engineered Clostridial neurotoxin can comprise an exogenous activation loop, the exogenous activation loop comprising any one of the following or consisting of any one of the following: SEQ ID NOs: 35, 36, 37, 38, 127, 128 and / or 129. The endogenous activation loop of the Clostridial neurotoxin or a portion thereof can be replaced with one or more endosomal protease cleavage sites according to the present invention. The endogenous neurotoxin activation loop can be one or more selected from SEQ ID NOs: 89 to 112.

[0014] The Clostridial neurotoxin can be: (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) a chimeric BoNT or a promiscuous BoNT. Preferably, the Clostridial neurotoxin is BoNT / X, BoNT / A (e.g., BoNT / A1) or BoNT / B. In a particularly preferred embodiment, the Clostridial neurotoxin is BoNT / X.

[0015] The engineered Clostridial neurotoxin can be a single-chain Clostridial neurotoxin, the single-chain Clostridial neurotoxin: (a) encoded by a nucleotide sequence having at least 70% sequence identity with SEQ ID NO: 163, wherein SEQ ID NO: 164 within SEQ ID NO: 163 is optionally replaced by a nucleotide sequence encoding at least one endoprotease cleavage site selected from SEQ ID NO: 165, 166, and 167; and / or (b) comprising a polypeptide sequence having at least 70% sequence identity with one or more of SEQ ID NO: 120, wherein SEQ ID NO: 91 within SEQ ID NO: 120 is optionally replaced by at least one endoprotease cleavage site selected from SEQ ID NO: 127-129 or an exogenous activation loop.

[0016] The engineered Clostridial neurotoxin can be a retargeted Clostridial neurotoxin, wherein the endogenous H C or H CC is replaced by an exogenous targeting moiety (TM). Preferably, the engineered Clostridial neurotoxin can be a retargeted BoNT / X or BoNT / A.

[0017] The present invention also provides a retargeted engineered BoNT / X comprising an endoprotease cleavage site, which comprises a polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 160-162, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% sequence identity.

[0018] The present invention also provides a method for proteolytically processing an engineered Clostridial neurotoxin according to the present invention into a corresponding double-chain Clostridial neurotoxin, the method comprising contacting the engineered Clostridial neurotoxin with an endoprotease specific for the endoprotease cleavage site, thereby producing a double-chain Clostridial neurotoxin.

[0019] The present invention also provides a double-chain Clostridial neurotoxin obtainable by the method.

[0020] The present invention also provides a polynucleotide encoding an engineered Clostridial neurotoxin according to the present invention.

[0021] The present invention also provides an expression vector, the expression vector comprising a polynucleotide as defined in claim 15 operably linked to a promoter.

[0022] The polynucleotide or expression vector may comprise or consist of a nucleotide sequence having at least 70% sequence identity with SEQ ID NO: 163, wherein SEQ ID NO: 164 within SEQ ID NO: 163 is optionally replaced with a nucleotide sequence encoding at least one endoprotease cleavage site selected from SEQ ID NO: 165, 166, and 167; and / or (b) encodes a polypeptide sequence having at least 70% sequence identity with one or more of SEQ ID NO: 120, wherein SEQ ID NO: 91 within SEQ ID NO: 120 is optionally replaced with at least one endoprotease cleavage site or an exogenous activation loop selected from SEQ ID NO: 127 - 129.

[0023] The present invention also provides a method for producing the engineered Clostridium neurotoxin of the present invention, the method comprising the steps of: expressing the polynucleotide or expression vector of the present invention in a cell, and recovering the expressed engineered Clostridium neurotoxin. The method may further comprise the step of introducing the polynucleotide or expression vector of the present invention into a cell.

[0024] The present invention also provides a cell that expresses the engineered Clostridium neurotoxin of the present invention. The cell may comprise the polynucleotide or expression vector of the present invention.

[0025] The present invention also provides a pharmaceutical composition comprising the engineered Clostridium neurotoxin of the present invention, or the double-stranded Clostridium neurotoxin of the present invention, and a pharmaceutically acceptable carrier, excipient, diluent, adjuvant, propellant, and / or salt.

[0026] The present invention also provides the engineered Clostridial neurotoxin, double-stranded Clostridial neurotoxin or pharmaceutical composition of the present invention for use in a method of preventing or treating a disease or disorder indicated for treatment with botulinum neurotoxin, wherein optionally, the disease or disorder is selected from disorders associated with unwanted immune secretion, strabismus, blepharospasm, squint, dystonia (such as spastic dystonia, oromandibular dystonia, focal dystonia, tardive dystonia, laryngeal dystonia, limb dystonia, cervical dystonia), torticollis (such as spastic torticollis), cosmetic applications (cosmesis) of beauty therapy benefiting from cell / muscle disablement (via SNARE downregulation or inactivation), neuromuscular disorders or diseases of eye movement (such as concomitant strabismus, vertical strabismus, lateral rectus paralysis, nystagmus, thyroid myopathy), writer's cramp, bruxism, Wilson's disease, tremor, tic, segmental myoclonus, spasm, spasm caused by chronic multiple sclerosis, spasm leading to abnormal bladder control, hostility, back spasm, cramp, levator ani syndrome, spina bifida, tardive dyskinesia, Parkinson's disease, stuttering, hemifacial spasm, eyelid disorders, cerebral palsy, focal spasm, spastic colitis, neurogenic bladder, pelvic floor dyssynergia, limb spasm, tic, tremor, bruxism, anal fissure, achalasia, dysphagia, lacrimation, hyperhidrosis, hypersecretion of saliva, hypersecretion of gastrointestinal tract, muscle pain (such as pain caused by muscle spasm), headache (such as tension headache or migraine), phantom pain (such as phantom limb pain), glabellar wrinkles, skin wrinkles, cancer, uterine disorders, urogenital disorders, urogenital nerve disorders, bladder pain syndrome, interstitial cystitis, chronic neurogenic inflammation and smooth muscle disorders. Preferably, the composition of the present invention can be used to prevent or treat diseases or disorders selected from: limb spasm (upper or lower limb); cervical dystonia; headache disorder (preferably, migraine); blepharospasm; hemifacial spasm; and lower urinary tract disorders (such as bladder pain syndrome (preferably, interstitial cystitis); overactive bladder; and detrusor overactivity (such as neurogenic detrusor overactivity).

[0027] The present invention also provides the use of the engineered Clostridial neurotoxin, double-stranded Clostridial neurotoxin or pharmaceutical composition of the present invention for manufacturing a medicament for preventing or treating a disease or disorder indicated for treatment with a botulinum neurotoxin, wherein optionally, the disease or disorder is selected from disorders associated with unwanted immune secretion, strabismus, blepharospasm, squint, dystonia (such as spastic dystonia, oromandibular dystonia, focal dystonia, tardive dystonia, laryngeal dystonia, limb dystonia, cervical dystonia), torticollis (such as spastic torticollis), cosmetic applications of beauty therapy (cosmesis) benefiting from cell / muscle disability (via SNARE downregulation or inactivation), neuromuscular disorders or diseases of eye movement (such as concomitant strabismus, vertical strabismus, lateral rectus paralysis, nystagmus, dysthyroid myopathy), writer's cramp, bruxism, Wilson's disease, tremor, tic, segmental myoclonus, spasm, spasm caused by chronic multiple sclerosis, spasm leading to abnormal bladder control, hostility, back spasm, cramp, levator ani syndrome, spina bifida, tardive dyskinesia, Parkinson's disease, stuttering, hemifacial spasm, eyelid disorders, cerebral palsy, focal spasm, spastic colitis, neurogenic bladder, pelvic floor dyssynergia, limb spasm, tic, tremor, bruxism, anal fissure, achalasia, dysphagia, lacrimation, hyperhidrosis, hypersalivation, hypersecretion of the gastrointestinal tract, muscle pain (such as pain caused by muscle spasm), headache (such as tension headache or migraine), phantom pain (such as phantom limb pain), glabellar lines, skin wrinkles, cancer, uterine disorders, urogenital disorders, urogenital nerve disorders, bladder pain syndrome, interstitial cystitis, chronic neurogenic inflammation and smooth muscle disorders. Preferably, the composition of the present invention can be used for preventing or treating a disease or disorder selected from the following: limb spasm (upper or lower limb); cervical dystonia; headache disorder (preferably, migraine); blepharospasm; hemifacial spasm; and lower urinary tract disorders (such as bladder pain syndrome (preferably, interstitial cystitis); overactive bladder; and detrusor overactivity (such as neurogenic detrusor overactivity).

[0028] The engineered Clostridial neurotoxin (such as within a pharmaceutical composition) can be administered to a subject in a single-chain form. The Clostridial neurotoxin or pharmaceutical composition can be substantially free of the double-stranded form of the Clostridial neurotoxin. The Clostridial neurotoxin or pharmaceutical composition can contain less than 400 pg of the double-stranded Clostridial neurotoxin per 100 ng of the single-chain Clostridial neurotoxin, or less than 300 pg of the double-stranded Clostridial neurotoxin per 100 ng of the single-chain Clostridial neurotoxin, or less than 200 pg of the double-stranded Clostridial neurotoxin per 100 ng of the single-chain Clostridial neurotoxin, or less than 100 pg of the double-stranded Clostridial neurotoxin per 100 ng of the single-chain Clostridial neurotoxin, or less than 50 pg of the double-stranded Clostridial neurotoxin per 100 ng of the single-chain Clostridial neurotoxin.

[0029] The present invention also provides a cosmetic composition, which comprises the engineered clostridial neurotoxin or double-stranded clostridial neurotoxin of the present invention, as well as a cosmetically acceptable carrier, excipient, diluent, adjuvant, propellant and / or salt.

[0030] The present invention also provides the use of the cosmetic composition of the present invention for preventing or alleviating a cosmetic indication indicative of the application of botulinum neurotoxin. The clostridial neurotoxin can be administered to a subject in single-chain form. The clostridial neurotoxin or the cosmetic composition can be substantially free of the double-stranded form of the clostridial neurotoxin. The clostridial neurotoxin or the cosmetic composition can comprise less than 400 pg of double-stranded clostridial neurotoxin per 100 ng of single-chain clostridial neurotoxin, or less than 300 pg of double-stranded clostridial neurotoxin per 100 ng of single-chain clostridial neurotoxin, or less than 200 pg of double-stranded clostridial neurotoxin per 100 ng of single-chain clostridial neurotoxin, or less than 100 pg of double-stranded clostridial neurotoxin per 100 ng of single-chain clostridial neurotoxin, or less than 50 pg of double-stranded clostridial neurotoxin per 100 ng of single-chain clostridial neurotoxin.

[0031] The present invention also provides a method for proteolytically processing a single-chain clostridial neurotoxin into the corresponding double-stranded clostridial neurotoxin, the method comprising: (a) providing a single-chain clostridial neurotoxin; and (b) contacting the single-chain clostridial neurotoxin with an endosomal protease; wherein the single-chain clostridial neurotoxin has an activation loop comprising or consisting of a polypeptide sequence as defined herein; and wherein the endosomal protease hydrolyzes the peptide bond of the activation loop, thereby producing a double-stranded clostridial neurotoxin. The single-chain clostridial neurotoxin can be: (a) the engineered clostridial neurotoxin of the present invention; (b) encoded by a nucleotide sequence having at least 70% sequence identity with SEQ ID NO: 163, wherein SEQ ID NO: 164 within SEQ ID NO: 163 is optionally replaced by a nucleotide sequence encoding at least one endosomal protease cleavage site selected from SEQ ID NO: 165, 166 and 167; and / or (c) comprising a polypeptide sequence having at least 70% sequence identity with one or more of SEQ ID NO: 120, wherein SEQ ID NO: 91 within SEQ ID NO: 120 is optionally replaced by at least one endosomal protease cleavage site selected from SEQ ID NO: 127-129 or an exogenous activation loop. Description of the Drawings

[0032] Figure 1: (A) Purified and unprocessed CatDReo (BIO4934), Ebo (BIO4935), CatBL (BIO4945), and AEP (BIO4938) were subjected to Coomassie staining at pH 5 and 7.2. (B) Purified and unprocessed CatDReo (BIO4934), Ebo (BIO4935), CatBL (BIO4945), and AEP (BIO4938) were subjected to Western blot analysis with αLC / A antibody at pH 5 and 7.2.

[0033] Figure 2: (A) CatDReo (BIO4934) was subjected to Coomassie staining (left panel), Western blot analysis with αLC / A antibody (middle panel), and Western blot analysis with αHis antibody (right panel), incubated with serial dilutions of cathepsin L1, reduced with DTT, and resolved by SDS PAGE. (B) Ebo (BIO4935) was subjected to Coomassie staining (left panel), Western blot analysis with αLC / A antibody (middle panel), and Western blot analysis with αHis antibody (right panel), incubated with serial dilutions of cathepsin L1, reduced with DTT, and resolved by SDS PAGE.

[0034] Figure 3 : CatBL (BIO4945) was (A) subjected to Coomassie staining, (B) subjected to Western blot analysis with αLC / A antibody, and (C) subjected to Western blot analysis with αHis antibody, incubated with serial dilutions of cathepsin B, reduced with DTT, and resolved by SDS PAGE.

[0035] Figure 4 : AEP (BIO4938) was (A) subjected to Coomassie staining, (B) subjected to Western blot analysis with αLC / A antibody, and (C) subjected to Western blot analysis with αHis antibody, incubated with serial dilutions of AEP, reduced with DTT, and resolved by SDS PAGE. Detailed Description

[0036] Definitions

[0037] 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, 20th ed., John Wiley and Sons, New York (1994), and Hale and Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide one of ordinary skill in the art with a general dictionary of many of the terms used in this disclosure. The meanings and scopes of the terms should be clear; however, in the event of any potential ambiguity, the definitions provided herein shall prevail over any dictionary or extrinsic definition.

[0038] It should be understood that the present invention is not limited to the specific methods, protocols, reagents, etc. described herein, and thus may vary. Specifically, any methods and materials similar or equivalent to those described herein may be used to practice or test the embodiments of this disclosure.

[0039] The description of the embodiments of this disclosure is not intended to be exhaustive or to limit the disclosure to the precise forms disclosed. Although specific embodiments and examples of this disclosure are described herein for illustrative purposes, those skilled in the relevant art will recognize that various equivalent modifications can be made within the scope of this disclosure. For example, although method steps or functions are presented in a given order, alternative embodiments may perform the functions in a different order, or the functions may be performed substantially simultaneously. The teachings of this disclosure provided herein can be applied to other procedures or methods as needed. The various embodiments described herein can be combined to provide additional embodiments. If necessary, aspects of this disclosure can be modified to incorporate the compositions, functions, and concepts of the above references and applications to provide other embodiments of this disclosure. In addition, for considerations of biological functional equivalence, some changes can be made to the protein structure without affecting the biological or chemical actions in kind or quantity. These and other changes can be made to this disclosure in accordance with the detailed description. It is intended that all such modifications fall within the scope of the appended claims.

[0040] Unless otherwise indicated, any nucleic acid sequence is written from left to right in the 5' to 3' direction; amino acid sequences are written from left to right in the amino to carboxyl direction.

[0041] The headings provided herein do not limit the various aspects or embodiments of this disclosure.

[0042] As used herein, the term "capable of", when used in conjunction with a verb, encompasses or means the action of the corresponding verb. For example, "capable of interacting" also means interacting, "capable of cleaving" also means cleaving, "capable of binding" also means binding, and "capable of specifically targeting..." also means specifically targeting.

[0043] Numeric ranges include the numbers defining the range. Where a numerical range is provided, it is understood that each intermediate value between the upper and lower limits of that range is also expressly disclosed, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise. Each smaller range between any stated value or intermediate value in the stated range and any other stated value or intermediate value in that stated range is encompassed within the disclosure. The upper or lower limit of these smaller ranges may independently be included or excluded from the range, and each range that includes either, neither, or both of the limits is also encompassed within the disclosure and is subject to any expressly excluded limit in the stated range. Where the stated range includes one or both of the recited limits, ranges excluding one or both of those included limits are also included in the disclosure.

[0044] Amino acids are referred to herein by their name, three-letter abbreviation, or single-letter abbreviation. The term "protein" as used herein includes proteins, polypeptides, and peptides. As used herein, the term "amino acid sequence" is synonymous with the terms "polypeptide" and / or the term "protein". In some cases, the term "amino acid sequence" is synonymous with the term "peptide". In some cases, the term "amino acid sequence" is synonymous with the term "enzyme". The terms "protein" and "polypeptide" may be used interchangeably herein. In this disclosure and the claims, the conventional one-letter and three-letter codes for amino acid residues may be used. The three-letter codes for amino acids as defined by the Joint Commission on Biochemical Nomenclature (JCBN) of IUPAC / IUB. It should also be understood that due to the degeneracy of the genetic code, a polypeptide may be encoded by more than one nucleotide sequence.

[0045] A "fragment" of a polypeptide generally 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.

[0046] As used herein, the terms "polynucleotide", "nucleic acid", and "nucleic acid sequence" refer to any molecule, preferably a polymeric molecule, having units of ribonucleic acid, deoxyribonucleic acid, or analogs thereof. Nucleic acids can be single-stranded or double-stranded. Single-stranded nucleic acids can be one strand of a denatured double-stranded DNA, or alternatively, they can be single-stranded nucleic acids not derived from any double-stranded DNA. In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA. Suitable nucleic acid molecules are DNA, including genomic DNA or cDNA. Other suitable nucleic acid molecules are RNA, including siRNA, shRNA, and antisense oligonucleotides.

[0047] As defined herein, the term "endosome" refers to a membrane-bounded intracellular organelle. Endosomes are typically part of the endocytic membrane trafficking pathway that originates from the trans-Golgi network. Unless otherwise explicitly stated, the term "endosome" encompasses early endosomes, late endosomes, and recycling endosomes. The term "endosome" can also encompass lysosomes and / or other intracellular vesicles. These "early", "late", "recycling", and "lysosomal" compartments each have unique protein-lipid compositions, morphologies, and luminal pH characteristics.

[0048] Early endosomes typically have a maximum diameter of 1 μm (e.g., 100 - 500 nm) and can be connected by tubules approximately 50 nm in diameter. Markers of early endosomes can include RAB5A and RAB4, RAB11, transferrin, and early endosome antigen 1 (EEA1).

[0049] Late endosomes are typically spherical and not connected by tubules. Late endosomes can contain multiple tightly packed intraluminal vesicles. Markers of late endosomes can include RAB7, RAB9, and the mannose 6-phosphate receptor. The late endosome membrane (and lysosome) can contain a substance called lysobisphosphatidic acid (LBPA). Since endosomes acidify during maturation, the pH of late endosomes (pH ~ 5.0) is typically lower than that of early endosomes (pH ~ 6.5).

[0050] Recycling endosomes are concentrated at the microtubule organizing center and consist mainly of a tubular network. Markers of recycling endosomes include RAB11 and RAB4.

[0051] Lysosomes are small vesicles derived from the Golgi apparatus and contain up to 50 different degradative enzymes. The pH value of lysosomes is usually the lowest in any intracellular vesicular compartment (pH value about 4.5 - 5.0), and the enzymes therein require this acidic pH value to function. Lysosomal markers include highly glycosylated lysosome-associated membrane proteins (LAMP), such as LAMP-1 and LAMP-2, and RAB9.

[0052] As used herein, the term "spacer" refers to a flexible peptide used in an exogenous activation loop or a modified BoNT / C activation loop, or a flexible peptide used together with an exogenous protease cleavage site, and generally includes it to maintain the secondary structure of the exogenous activation loop in the engineered Clostridial neurotoxin of the present invention. The spacer for the engineered Clostridial neurotoxin of the present invention may comprise an amino acid sequence of 1 to 30 amino acid residues, such as 5 to 30 amino acid residues, 10 to 25 amino acid residues or about 5 to about 20 amino acid residues. The spacer may comprise small amino acid residues or be composed of small amino acid residues, such as glycine, threonine, arginine, serine, asparagine, glutamine, alanine, aspartic acid, proline, glutamic acid, lysine, leucine and / or valine, especially glycine, serine, alanine, leucine and / or valine. A spacer comprising or consisting of glycine, serine and / or alanine may be preferred, with glycine and serine being particularly preferred. Thus, the most commonly used spacers have sequences mainly composed of Gly and Ser residue segments ("GS" linkers), which include the sequence (Gly-Gly-Gly-Gly-Ser) n (SEQ ID NO:153). Non-limiting examples of GS linkers include GS5 or (GGGGS)1 (SEQ ID NO:154); GS10 or (GGGGS)2 (SEQ ID NO:155); GS15 or (GGGGS)3 (SEQ ID NO:156); GS20 or (GGGGS)4 (SEQ ID NO:157); and GS25 or (GGGGS)5 (SEQ ID NO:158).

[0053] As used herein, the term "core motif" refers to the smallest amino acid sequence that can be cleaved by a given endosomal protease. By way of non-limiting example, the core motif definition of cathepsin L defines the smallest amino acid sequence that can be cleaved by cathepsin L. There may be more than one core motif for any given endosomal protease.

[0054] The terms "increased", "increase", "enhanced", or "activated" are each used herein to mean a statistically significant increase. The terms "increased", "increase", "enhanced", or "activated" can mean an increase of at least 10% compared to a reference level, such as 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% or up to and including 100% increase, or any increase between 10 - 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 of 2-fold to 10-fold or greater compared to a reference level.

[0055] The terms "decrease", "reduced", "reduction", or "inhibited" are each used herein to mean a statistically significant decrease. The terms "reduce", "reduction", or "decrease" or "inhibit" generally mean a decrease of at least 10% compared to a reference level (e.g., in the absence of a given 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, "decrease" or "inhibit" encompasses complete inhibition or decrease compared to a reference level. "Complete inhibition" is 100% inhibition (i.e., elimination) compared to a reference level.

[0056] Other term definitions may occur throughout the specification. Before describing the exemplary embodiments in more detail, it is to be understood that the present disclosure is not limited to the particular embodiments described, and thus may vary. It is also to be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting, as the scope of the present disclosure will be limited only by the appended claims.

[0057] It must be noted that, unless the context clearly dictates otherwise, as used herein and in the appended claims, the singular forms “a / an” and “the” include plural referents. Thus, for example, reference to “a Clostridial neurotoxin” includes a plurality of such agents and reference to “the Clostridial neurotoxin” includes reference to one or more Clostridial neurotoxins and equivalents thereof known to those skilled in the art, and so forth. In addition, the use of the term “including” and other forms such as “includes” and “included” is not limiting.

[0058] “About” generally means an acceptable degree of error in the quantity being measured, taking into account the nature or precision of the measurement. Exemplary degrees of error are within 20 percent (%) of the given value or value range, typically within 10%, more typically within 5%. Preferably, the term “about” herein should be understood as being plus or minus (±) 5% of the numerical value of the number with which it is used, preferably ±4%, ±3%, ±2%, ±1%, ±0.5%, ±0.1%.

[0059] The term “consisting of” refers to the compositions, methods, and their respective components as described herein, excluding any element not recited in the description of the invention.

[0060] As used herein, the term “consisting essentially of” refers to those elements required for a given invention. This term allows for the presence of elements that do not materially affect the basic and novel or functional characteristics of the invention (i.e., inactive or non-immunogenic components).

[0061] Embodiments described herein as “comprising” one or more features may also be considered to disclose corresponding embodiments “consisting of” and / or “consisting essentially of” such features.

[0062] As used herein, the term “deletion” refers to the removal of one or more amino acid residues of a polypeptide without replacement of one or more amino acid residues at the site of deletion. Thus, when one amino acid residue is deleted (for example) from a polypeptide sequence having x number of amino acid residues, the resulting polypeptide has x - 1 amino acid residues.

[0063] As used herein, the term “insertion / deletion” refers to the deletion of one or more amino acid residues of a polypeptide and the insertion at the site of deletion of a number of amino acid residues different from the number of amino acid residues deleted (more or fewer amino acid residues). Thus, for an insertion / deletion in which two amino acid residues are deleted (for example) from a polypeptide sequence having x number of amino acid residues, the resulting polypeptide has x - 1 amino acid residues or x + ≥1 amino acid residues. Insertions and deletions can be made in any order, sequentially or simultaneously.

[0064] As used herein, the term "substitution" refers to the replacement of one or more amino acid residues with the same number of amino acid residues at the same position. Thus, for example, in the case of a substitution of a polypeptide sequence having x number of amino acid residues, the resulting polypeptide also has x number of amino acid residues. Preferably, the substitution is a substitution at a single amino acid position.

[0065] As used herein, the term "insertion" refers to the addition of one or more amino acid residues of a polypeptide at the insertion site without the deletion of one or more amino acid residues of the polypeptide. Thus, when one amino acid residue is inserted into a polypeptide sequence having x number of amino acid residues (for example), the resulting polypeptide has x + 1 amino acid residues.

[0066] Concentrations, amounts, volumes, percentages, and other numerical values may be presented herein in a range format. It should also be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly as including not only the explicitly listed values defining the range boundaries, but also all individual values or sub-ranges encompassed within that range as if each value and sub-range were explicitly listed.

[0067] An individual can be an individual who has been previously diagnosed as or identified as suffering from or having a disorder or one or more complications associated with such a disorder that requires treatment, and optionally, has been previously treated for the disorder or one or more complications associated with the disorder as defined herein. Alternatively, an individual can also be an individual who has not been previously diagnosed as having a disorder or one or more complications associated with the disorder as defined herein. For example, an individual can be an individual who exhibits one or more risk factors for a disorder or one or more complications associated with the disorder, or an individual who does not exhibit risk factors.

[0068] An individual "in need of" treatment for a particular disorder can be an individual who has the disorder, has been diagnosed as having the disorder, or is at risk of developing the disorder.

[0069] The terms "subject", "individual", and "patient" are used interchangeably herein and refer to a mammalian individual. An "individual" can be any mammal. Generally, an individual can be a human; in other words, in one embodiment, the "individual" is a human. An "individual" can be an adult, an adolescent, or an infant. An "individual" can be male or female.

[0070] As used herein, the term "pharmaceutically acceptable" means approved by a federal regulatory agency or a state government, or listed in the United States Pharmacopeia, the European Pharmacopeia, or other generally recognized pharmacopeias.

[0071] The publications discussed herein are provided only for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the appended claims.

[0072] Engineered clostridial neurotoxin

[0073] The present invention provides an engineered clostridial neurotoxin that comprises an endoprotease cleavage site. Typically, cleavage at the endoprotease cleavage site results in the production of a double-stranded form of the engineered clostridial neurotoxin. In other words, cleavage at the endoprotease cleavage site causes activation of the engineered clostridial neurotoxin. The endogenous (native) activation loop of the clostridial neurotoxin may be replaced (or partially replaced) by the endoprotease cleavage site. Thus, the term "endoprotease cleavage site" may be used interchangeably with the term "endoprotease activation site", and an exogenous activation loop as defined herein typically comprises or consists of one or more endoprotease cleavage sites, as described herein. The engineered clostridial neurotoxin of the present invention can be activated in vivo. Thus, the engineered clostridial neurotoxin opens up a new field for the processing and therapeutic applications of clostridial neurotoxins, enabling the toxin to be produced and administered in the form of a single-chain clostridial neurotoxin and then cleaved in vivo to produce the active double-stranded form.

[0074] A typical characteristic of clostridial neurotoxin (before engineering) is that the endogenous activation loop is inefficiently proteolytically processed by one or more endoproteases. Compared to clostridial neurotoxin (before engineering), the engineered clostridial neurotoxin of the present invention is not inefficiently proteolytically processed by one or more endoproteases into which a cleavage site has been introduced and / or peptide bonds located outside the exogenous activation loop of the engineered clostridial neurotoxin are not hydrolyzed by the one or more endoproteases. Thus, clostridial neurotoxin (before engineering) is generally resistant to proteolytic processing by one or more endoproteases. The terms "inefficiently proteolytically processed by one or more endoproteases", "resistant to proteolytic processing by one or more endoproteases", "substantially not hydrolyzed by one or more endoproteases", "inefficiently activated by one or more endoproteases", "resistant to activation by one or more endoproteases", and "substantially not activated by one or more endoproteases" are used interchangeably herein. Typically, clostridial neurotoxin (before engineering) is generally resistant to proteolytic processing by one or more endoproteases into which a cleavage site has been introduced according to the present invention. Clostridial neurotoxin (before engineering) is also resistant to proteolytic processing by one or more endoproteases into which a cleavage site has not been introduced according to the present invention.

[0075] Clostridial neurotoxins (prior to engineering) are generally toxins in which peptide bonds (either within or outside the activation loop) are not or are substantially not hydrolyzed by one or more endosomal proteases. The term "substantially not hydrolyzed" means that less than 10%, 5%, 4%, 3%, 2%, or 1% of the clostridial neurotoxins present in the reaction contain peptide bonds that have been hydrolyzed by one or more of the endosomal proteases in the methods of the present invention.

[0076] Thus, clostridial neurotoxins (prior to engineering) may not contain one or more endosomal protease cleavage sites within their endogenous activation loops (e.g., as defined herein, any of the following: SEQ ID NOs: 1 to 38, 130 - 152, or 171 - 187; 12 to 38, 130 - 152, or 171 - 187; 1 to 34, 130 - 152, or 171 - 187; or 12 to 34, 130 - 152, or 171 - 187). Clostridial neurotoxins (prior to engineering) may not contain one or more endosomal protease core motifs (e.g., as defined herein, any of SEQ ID NOs: 20, 138, 181, and / or 188 - 214).

[0077] Clostridial neurotoxins (prior to engineering) may not contain one or more endosomal protease cleavage sites (e.g., as defined herein, any of the following: SEQ ID NOs: 1 to 38, 130 - 152, or 171 - 187; 12 to 38, 130 - 152, or 171 - 187; 1 to 34, 130 - 152, or 171 - 187; or 12 to 34, 130 - 152, or 171 - 187; or 12 to 34, 130 - 152, or 171 - 187) and / or one or more endosomal protease core motifs (e.g., as defined herein, any of SEQ ID NOs: 20, 138, 181, and / or 188 - 214) that are intended for activation of engineered clostridial neurotoxins by endosomal proteases. By way of a non - limiting example, if an engineered clostridial neurotoxin contains a cathepsin L cleavage site, the corresponding pre - engineered clostridial neurotoxin may not contain a cathepsin L cleavage site and / or core motif within its endogenous activation loop. In some embodiments in which one or more endosomal protease cleavage sites comprise AEP cleavage sites, the clostridial neurotoxin (prior to engineering) can be BoNT / B, BoNT / C, or BoNT / F, particularly BoNT / B or BoNT / C. In a particularly preferred embodiment, the clostridial neurotoxin is BoNT / X or a chimera or hybrid of BoNT / X with another clostridial neurotoxin.

[0078] The present invention may comprise replacing the endogenous activation loop (or a portion thereof) of any Clostridial neurotoxin with one or more endosomal protease (exogenous) cleavage sites as described herein or an exogenous activation loop comprising one or more endosomal protease cleavage sites. The Clostridial neurotoxin may be botulinum neurotoxin (BoNT) or tetanus neurotoxin (TeNT). Preferably, the Clostridial neurotoxin is 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 a particularly preferred embodiment, the Clostridial neurotoxin is BoNT / X or a chimera or hybrid thereof.

[0079] As used herein, the term "endogenous activation loop" refers to the activation loop present in the subject Clostridial neurotoxin (e.g., the subject Clostridial neurotoxin of the indicated serotype). For example, BoNT / A1 comprises a BoNT / A1 heavy chain and a light chain, and thus the endogenous activation loop of BoNT / A1 is the A1 activation loop. For Clostridial neurotoxin chimeras or hybrids, one of ordinary skill in the art can identify the "endogenous activation loop" by, for example, determining the serotype from which the L-chain and H N domains are derived. In some embodiments, a chimeric or hybrid Clostridial neurotoxin may have an endogenous activation loop that is a fusion of activation loops from two different serotypes. By way of example, the chimeric Clostridial neurotoxin BoNT / A1C1 has a BoNT / A1 light chain and translocation domain, and thus the endogenous BoNT / A1C1 activation loop is the A1 activation loop. The endogenous activation loop is typically bound by cysteine residues that form a disulfide bridge and covalently link (prior to engineering) the light and heavy chains of the Clostridial neurotoxin. Thus, endogenous activation loop sequences may be described as including or not including the cysteine residues that bind. One of ordinary skill in the art will understand that these definitions may be used interchangeably and will be able to readily identify endogenous activation loops that include or do not include the cysteine residues that bind.

[0080] Generally, an "endogenous activation loop" is any activation loop that does not contain one or more endosomal protease cleavage sites as described herein (e.g., SEQ ID NOs: 1 to 38; SEQ ID NOs: 12 to 38; SEQ ID NOs: 1 to 34; or SEQ ID NOs: 12 to 34) or is not composed of one or more endosomal protease cleavage sites as described herein. An "endogenous activation loop" is any activation loop that does not contain one or more endosomal protease cleavage sites selected from the following or is not composed of one or more endosomal protease cleavage sites selected from the following: (i) SEQ ID NOs: 1-10, 12-21 or 27-38; (ii) SEQ ID NOs: 12-21 or 27-38; (iii) SEQ ID NOs: 1-10, 12-21 or 17-34; or (iv) SEQ ID NOs: 12-21 or 27-34).

[0081] In contrast, an "exogenous activation loop" as used herein means an activation loop that is different from the endogenous activation loop present in the subject Clostridial neurotoxin (e.g., the subject Clostridial neurotoxin of the indicated serotype), and wherein the exogenous activation loop contains one or more endosomal protease cleavage sites. For example, the BoNT / C1 activation loop has a polypeptide sequence different from that of the wild-type BoNT / A1 activation loop, and thus the BoNT / C1 activation loop is exogenous to BoNT / A1. For Clostridial neurotoxin chimeras or hybrids, one of ordinary skill in the art can determine whether an activation loop is an "exogenous activation loop" by, for example, determining the serotype from which the L-chain and H N domain are derived. For example, when the L-chain is the BoNT / B L-chain and the H N domain is from BoNT / D, the endogenous activation loop can have a portion of the BoNT / B sequence and a portion of the BoNT / D sequence, and if the activation loop (e.g., the C1 activation loop) is different therefrom and contains one or more endosomal protease cleavage sites, it is considered an "exogenous activation loop".

[0082] One can determine whether an activation loop is an "endogenous activation loop" by aligning the sequence of the subject Clostridial neurotoxin with the activation loop and seeing if the activation loop is present in the subject Clostridial neurotoxin sequence. If it is 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 with an exogenous cleavage site for one or more endosomal proteases, or with an exogenous activation loop that contains one or more endosomal protease cleavage sites.

[0083] Typically, according to the present invention, one or more endoprotease cleavage sites are inserted between the two cysteine residues of the endogenous activation loop that binds to the engineered Clostridium neurotoxin, but the exact position of one or more endoprotease cleavage sites within the endogenous activation loop is not limited, as long as the conformation of the resulting engineered Clostridium neurotoxin is not disrupted and / or the engineered Clostridium neurotoxin does not lose its function.

[0084] The entire endogenous activation loop can be replaced by one or more endoprotease cleavage sites as described herein or an exogenous activation loop containing one or more endoprotease cleavage sites. Alternatively, a part or portion of the endogenous activation loop can be replaced (also referred to herein as partial replacement of the endogenous activation loop), for example, replacing at least 5, 10, 15, 20, 25, 30, 35, or 40 amino acid residues of the endogenous activation loop. Preferably, 5 to 20, more preferably 5 to 15 amino acid residues of the endogenous activation loop are replaced. Typically, partial replacement involves replacement of consecutive amino acids within the endogenous activation loop. Thus, when partially replacing the endogenous activation loop, at least one amino acid residue of the endogenous activation loop is retained. Preferably, about 5 to about 15 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) of about 5 to about 15 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15), for example, about 5 to about 12 (e.g., 5, 6, 7, 8, 9, 10, 11, or 12) are retained. The retained amino acid residues can be located at the N-terminus and / or C-terminus of the endogenous activation loop.

[0085] In some embodiments, the endogenous activation loop is completely replaced in the engineered Clostridium neurotoxin of the present invention. Typically, this means that all amino acid residues of the activation loop (between the cysteine residues that form the disulfide bond in the active double-stranded molecule) are replaced by the exogenous activation loop or exogenous protease cleavage sites of the present invention. Complete replacement of the endogenous activation loop can include introducing an exogenous activation loop consisting entirely of one or more exogenous protease cleavage sites as described herein. Alternatively, complete replacement of the endogenous activation loop can include introducing an exogenous activation loop that contains one or more exogenous protease cleavage sites as described herein and one or more spacer sequences. Each of the one or more spacer sequences is typically a short peptide (e.g., between about 5 and about 25 amino acids, such as between about 5 and about 20 amino acids, between about 5 and about 15 amino acids, or between about 5 and about 10 amino acids). Such spacers can be present when one or more exogenous protease cleavage sites are short motifs (e.g., typically less than 15 amino acids in length, preferably less than 10 or less than 9 amino acids). One or more spacers can be present at the N-terminus and / or C-terminus of each of the said exogenous protease cleavage sites. Preferably, the spacer can be a GS spacer as defined herein.

[0086] The replacement of the endogenous activation loop can be achieved by any method known in the art. For example, the replacement can be achieved by means of amino acid modification. The replacement of the endogenous activation loop can be achieved by deletion of one or more amino acid residues of the endogenous activation loop. The replacement of the endogenous activation loop can be achieved by substituting one or more amino acid residues of the endogenous activation loop with amino acid residues of an exogenous activation loop. The endogenous activation loop (or a portion thereof) can be deleted and one or more endosomal protease cleavage sites or an exogenous activation loop containing one or more endosomal protease cleavage sites can be inserted, preferably at the position formally occupied by the endogenous activation loop. Alternatively, the endogenous activation loop can be retained in the engineered Clostridial neurotoxin of the present invention and is preferably inactivated (e.g., by means of a mutation). Preferably, the endogenous activation loop (a portion or the entire endogenous activation loop) is not present in the engineered Clostridial neurotoxin of the present invention. Preferably, one or more endosomal protease cleavage sites or an exogenous activation loop containing one or more endosomal protease cleavage sites occupy the position formally occupied by the endogenous activation loop in the Clostridial neurotoxin. For the avoidance of doubt, when the endogenous activation loop is modified to contain one or more endosomal protease cleavage sites (e.g., by substituting residues within the endogenous activation loop or by adding one or more amino acids to form one or more endosomal protease cleavage sites within the endogenous activation loop), the modified activation loop is an exogenous activation loop according to the present invention. Thus, potentially, the engineered Clostridial neurotoxin can contain its endogenous activation / cleavage site and one or more endosomal protease cleavage sites and can thus be activated by a native activating protease (or an equivalent used in the production of recombinant BoNT, e.g., trypsin or Lys-C) or by one or more endosomal proteases.

[0087] Methods for modifying proteins by amino acid residue substitution, insertion or deletion are known in the art and can be used in the practice of the present invention. By way of example, amino acid modifications can be introduced by modifying the DNA sequence encoding the Clostridial neurotoxin. This can be achieved using standard molecular cloning techniques, such as site-directed mutagenesis, where a polymerase is used to replace the original coding sequence with a short chain of DNA (oligonucleotide) encoding the desired amino acid, or by inserting / deleting portions of the gene using various enzymes (e.g., ligases and restriction endonucleases). Alternatively, the modified gene sequence can be chemically synthesized. Any other methods known in the art for modifying polypeptides, such as polypeptide synthesis and polypeptide conjugation, can also be used to design the Clostridial neurotoxin according to the present invention.

[0088] Alternative endogenous activation loops according to the invention may comprise or consist of a polypeptide sequence having at least 70% (such as at least 80% or 90%) sequence identity with the following sequences: SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111 or SEQ ID NO:112. Alternative endogenous activation loops according to the invention may comprise or consist of a polypeptide sequence having at least 70% (such as at least 80% or 90%) sequence identity with SEQ ID NO:112. Specifically, the endogenous activation loop may comprise or consist of a polypeptide sequence having at least 95% sequence identity with the following: SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111 or SEQ ID NO:112. Specifically, the endogenous activation loop may comprise or consist of a polypeptide sequence having at least 95% sequence identity with SEQ ID NO:112.Preferably, the endogenous activation loop comprises or consists of a polypeptide sequence shown as follows: SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111 or SEQ ID NO:112. Preferably, the endogenous activation loop comprises or consists of the polypeptide sequence shown as SEQ ID NO:112.

[0089] An alternative endogenous activation loop according to the present invention may comprise or consist of a polypeptide sequence having at least 70% (such as at least 80% or 90%) sequence identity with SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95 or SEQ ID NO:96. The endogenous activation loop may comprise or consist of a polypeptide sequence having at least 95% sequence identity with SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95 or SEQ ID NO:96. Preferably, the endogenous activation loop comprises or consists of a polypeptide sequence shown as SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95 or SEQ ID NO:96.

[0090] Preferably, an alternative endogenous activation loop according to the present invention comprises or consists of a polypeptide sequence having at least 70% (such as at least 80% or 90%) sequence identity with SEQ ID NO:94. The endogenous activation loop may comprise or consist of a polypeptide sequence having at least 95% sequence identity with SEQ ID NO:94. More preferably, the endogenous activation loop comprises or consists of the polypeptide sequence shown as SEQ ID NO:94.

[0091] The present invention encompasses methods and Clostridial neurotoxins as described herein, wherein the endogenous activation loop has been replaced by an exogenous cleavage site or an exogenous activation loop containing one or more endosomal protease cleavage sites, which are one or more endosomal protease cleavage sites.

[0092] The engineered Clostridial neurotoxins of the present invention may comprise an exogenous activation loop that contains one or more of any of the endosomal protease cleavage sites as described herein. The exogenous activation loop can be generated by replacing one or more amino acids of the endogenous activation loop of the Clostridial neurotoxin, as described herein. In some preferred embodiments, the replaced amino acids of the endogenous activation loop are replaced by one or more endosomal protease cleavage sites or an exogenous activation loop having the same number of amino acids. In other words, by way of example, if five amino acids are replaced in the endogenous activation loop, the one or more endosomal protease cleavage sites or the exogenous activation loop containing the one or more endosomal protease cleavage sites that effect the replacement have five amino acids. If ten amino acids are replaced in the endogenous activation loop, the one or more endosomal protease cleavage sites or the exogenous activation loop containing the one or more endosomal protease cleavage sites that effect the replacement have ten amino acids.

[0093] Non-limiting examples of such exogenous activation loops include CQEAANERQQAKKDF FSSHPLREPVNATEDPDLKNVKSGLTNIKTELVTPARDLFGFVGLF RGHHPDC (SEQ ID NO:127), CQLGKNEEGLFGFVGLFRGHHPDELVTPARDFGHFGLSGLTNIKTEC (SEQ ID NO:128), and / or CPGG GNKKIELVTPARDLFGFVGLFRGHHPDLKNVKSKC (SEQ ID NO:129), or corresponding sequences lacking N-terminal and / or C-terminal cysteine residues, since the remaining sequences can be inserted within the endogenous cysteine residues of the pre-engineered Clostridial neurotoxin. All of these are derived from the BoNT / A1 activation loop.

[0094] The present invention provides a method for manufacturing an engineered 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, thereby providing an engineered Clostridial neurotoxin, wherein the exogenous cleavage site is one or more endosomal protease cleavage sites as described herein, or the exogenous activation loop comprises the one or more endosomal protease cleavage sites. Generally, the one or more endosomal protease cleavage sites are selected from sequences comprising or consisting of the following amino acid sequences: SEQ ID NOs: 1 to 38, 130-152 or 171-187; 12 to 38, 130-152 or 171-187; 1 to 34, 130-152 or 171-187; or 12 to 34, 130-152 or 171-187, or the exogenous activation loop comprises the one or more endosomal protease cleavage sites.

[0095] The present invention provides an engineered Clostridial neurotoxin (e.g., obtainable by the method of the present invention), wherein the endogenous activation loop (or a portion thereof) of a Clostridial neurotoxin is replaced with an exogenous activation loop or an exogenous cleavage site, thereby providing an engineered Clostridial neurotoxin, wherein the exogenous cleavage site is one or more endosomal protease cleavage sites as described herein, or the exogenous activation loop comprises the one or more endosomal protease cleavage sites. Generally, the one or more endosomal protease cleavage sites are selected from sequences comprising or consisting of the following amino acid sequences: SEQ ID NOs: 1 to 38, 130-152 or 171-187; 12 to 38, 130-152 or 171-187; 1 to 34, 130-152 or 171-187; or 12 to 34, 130-152 or 171-187, or the exogenous activation loop comprises the one or more endosomal protease cleavage sites, such as any one of SEQ ID NOs: 127-129.

[0096] The Clostridial neurotoxins (e.g., engineered Clostridial neurotoxins) of the present invention can be encoded by a nucleotide sequence having at least 70% sequence identity with SEQ ID NO: 163, and wherein the nucleotide sequence encoding the BoNT / C activation loop (SEQ ID NO: 164) is replaced by a nucleic acid encoding one or more endosomal protease cleavage sites or an exogenous activation loop containing said one or more endosomal cleavage sites. The Clostridial neurotoxins (e.g., engineered Clostridial neurotoxins) of the present invention can be encoded by a nucleotide sequence having at least 80% or 90% sequence identity with SEQ ID NO: 163, wherein the nucleotide sequence encoding the BoNT / C activation loop (SEQ ID NO: 164) is replaced by a nucleic acid encoding one or more endosomal protease cleavage sites or an exogenous activation loop containing said one or more endosomal cleavage sites. Preferably, the Clostridial neurotoxins (e.g., engineered Clostridial neurotoxins) of the present invention are encoded by the nucleotide sequence of SEQ ID NO: 163, wherein the nucleotide encoding the BoNT / C activation loop (SEQ ID NO: 164) is replaced by a nucleotide sequence encoding one or more endosomal protease cleavage sites or an exogenous activation loop containing said one or more endosomal cleavage sites. Non-limiting examples of nucleotide sequences encoding exogenous activation loops that can replace SEQ ID NO: 164 within SEQ ID NO: 163 include SEQ ID NO: 165, 166, and 167. Thus, by way of non-limiting example, the Clostridial neurotoxins (e.g., engineered Clostridial neurotoxins) of the present invention can be encoded by the nucleotide sequences of SEQ ID NO: 168, 169, and 170. The Clostridial neurotoxins (e.g., engineered Clostridial neurotoxins) of the present invention can comprise a polypeptide sequence having at least 70% sequence identity with one or more of SEQ ID NO: 121 or 159 - 162. The Clostridial neurotoxins of the present invention can comprise a polypeptide sequence having at least 80% or 90% sequence identity with one or more of SEQ ID NO: 121 or 159 - 162. Preferably, the Clostridial neurotoxins of the present invention can comprise (more preferably, consist of) the polypeptide sequence shown as any one of SEQ ID NO: 121 or 159 - 162.

[0097] The Clostridial neurotoxin (e.g., engineered Clostridial neurotoxin) of the present invention is preferably a retargeted BoNT / X, wherein the Clostridial neurotoxin is encoded by a nucleotide sequence having at least 70% sequence identity with SEQ ID NO: 163, and wherein SEQ ID NO: 164 within SEQ ID NO: 163 is optionally replaced by a nucleotide sequence encoding at least one endosomal protease cleavage site selected from SEQ ID NO: 165, 166, and 167. The Clostridial neurotoxin may be encoded by a nucleotide sequence having at least 80% or 90% sequence identity with SEQ ID NO: 163, and wherein SEQ ID NO: 164 within SEQ ID NO: 163 is optionally replaced by a nucleotide sequence encoding at least one endosomal protease cleavage site selected from SEQ ID NO: 165, 166, and 167. Preferably, the Clostridial neurotoxin is encoded by a nucleotide sequence comprising (or consisting of) SEQ ID NO: 163, and wherein SEQ ID NO: 164 within SEQ ID NO: 163 is optionally replaced by a nucleotide sequence encoding at least one endosomal protease cleavage site selected from SEQ ID NO: 165, 166, and 167. The Clostridial neurotoxin of the present invention is preferably a retargeted BoNT / X, wherein the Clostridial neurotoxin comprises a polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 120, and wherein SEQ ID NO: 91 within SEQ ID NO: 120 is optionally replaced by at least one endosomal protease cleavage site or an exogenous activation loop selected from SEQ ID NO: 127 - 129. The Clostridial neurotoxin may comprise a polypeptide sequence having at least 80% sequence identity with SEQ ID NO: 120, and wherein SEQ ID NO: 91 within SEQ ID NO: 120 is optionally replaced by at least one endosomal protease cleavage site or an exogenous activation loop selected from SEQ ID NO: 127 - 129. The Clostridial neurotoxin may comprise a polypeptide sequence having at least 90% sequence identity with SEQ ID NO: 120, and wherein SEQ ID NO: 91 within SEQ ID NO: 120 is optionally replaced by at least one endosomal protease cleavage site or an exogenous activation loop selected from SEQ ID NO: 127 - 129. Preferably, the Clostridial neurotoxin comprises (or consists of) the polypeptide sequence shown as SEQ ID NO: 120, and wherein SEQ ID NO: 91 within SEQ ID NO: 120 is optionally replaced by at least one endosomal protease cleavage site or an exogenous activation loop selected from SEQ ID NO: 127 - 129.

[0098] The polypeptide sequence of the present invention (or the nucleotide sequence encoding the same) may include a purification tag, such as a His tag. It is intended that the present invention also encompasses the polypeptide sequence (and the nucleotide sequence encoding the same) from which the purification tag has been removed.

[0099] Endosomal proteases and endosomal protease cleavage sites

[0100] Endosomes and lysosomes can contain a variety of different proteases, referred to herein as "endosomal proteases" (also interchangeably referred to as endolysosomal proteases). In other words, as used herein, the term "endosomal protease" refers to a protease that may be contained within endosomes, lysosomes, or both. These endosomal proteases have a variety of different functions and are generally involved in the degradation of proteins and peptides taken up extracellularly by endosomes. These enzymes are readily available.

[0101] Non-limiting examples of endosomal proteases according to the present invention include cathepsins and asparagine endopeptidase (AEP, also known as asparaginyl endopeptidase or asparagine endopeptidase (legumain)). The term "cathepsin" describes a family of endosomal proteases, including cathepsin A, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin F, cathepsin G, cathepsin H, cathepsin K, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin W, and cathepsin Z. Preferably, the present invention relates to cathepsin L (e.g., L1), B, D, K, or S, and / or AEP. In other words, the engineered Clostridium neurotoxins of the present invention generally contain at least one cleavage site of one or more of cathepsin L (e.g., L1), B, D, K, or S and / or AEP.

[0102] Cathepsin L1 is a thiol protease with specificity similar to papain and can cleave in a series of consensus sequences, including Xaa1-Xaa2-Leu / Val / Phe / Ile-Xaa3 / / Xaa4-Xaa5-Xaa6-Xaa7, where Xaa 1-7can each independently be selected from any amino acid, and wherein " / / " indicates the position of the hydrolyzed peptide bond. The term "cathepsin L1" encompasses the cathepsin L1 described herein, and any protease having structural and / or functional similarity (preferably structural and functional similarity) that is capable of hydrolyzing the peptide bond of the consensus sequence Xaa1-Xaa2-Leu / Val / Phe / Ile-Xaa3 / / Xaa4-Xaa5-Xaa6-Xaa7. A suitable cathepsin L1 is human cathepsin L1, which has the UniProt accession number P07711 (sequence version 2, deposited on October 1, 1989, accessed on July 30, 2022), herein SEQ ID NO:39. This sequence is a propeptide that can be converted to the mature human form by a process involving cleavage of the N-terminal polypeptide of residues 1-113. Human cathepsin L1 is available from Merck (#SRP6416).

[0103] Cathepsin B is a thiol protease with specificity similar to papain that can cleave in a series of consensus sequences, including (i) after the second arginine residue in the consensus Arg-Arg-Xaa, where X is any amino acid and / or (ii) Xaa1-Xaa2-Xaa3-Gly / / Xaa4-Xaa5-Gly-Xaa6, where Xaa 1-6 can each independently be selected from any amino acid, and wherein " / / " indicates the position of the hydrolyzed peptide bond. The term "cathepsin B" encompasses the cathepsin B described herein, and any protease having structural and / or functional similarity (preferably structural and functional similarity) that is capable of hydrolyzing the peptide bond of the consensus sequence Arg-Arg-Xaa or Xaa1-Xaa2-Xaa3-Gly / / Xaa4-Xaa5-Gly-Xaa6. A suitable cathepsin B is human cathepsin B, which has the UniProt accession number P07858 (sequence version 3, deposited on June 21, 2005, accessed on July 30, 2022), herein SEQ ID NO:40. This sequence is a propeptide that can be converted to the mature human form by a process involving cleavage of the N-terminal polypeptide of residues 1-79. Human cathepsin B is available from Merck (#SRP0289).

[0104] Cathepsin D is a thiol protease with specificity similar to pepsin A that can cleave in a series of consensus sequences, including Xaa1-Xaa2-Xaa3-Leu / Phe / / Xaa4-Xaa5-Xaa6-Xaa7, where Xaa 1-7can each independently be selected from any amino acid, and wherein " / / " indicates the position of the hydrolyzed peptide bond. The term "cathepsin D" encompasses the cathepsin D described herein, and any protease having structural and / or functional similarity (preferably structural and functional similarity) capable of hydrolyzing the peptide bond of the consensus Xaa1-Xaa2-Xaa3-Leu / Phe / / Xaa4-Xaa5-Xaa6-Xaa7. A suitable cathepsin D is human cathepsin D, which has the UniProt accession number P07339 (version 1 of the sequence, deposited on April 1, 1988, accessed on July 30, 2022), herein as SEQ ID NO: 41. This sequence is a propeptide that can be converted to the mature human form by a process involving cleavage of the N-terminal polypeptide of residues 1-64. Human cathepsin D is available from Merck (#SRP6415).

[0105] Cathepsin K is a thiol protease with broad proteolytic activity. The major determinant of specificity is P2 in the standard nomenclature (P4-P3-P2-P1 / / P1’-P2’-P3’-P4’), which can preferably be Leu, Met or Phe rather than Arg. Cathepsin K cleaves in a series of consensus sequences, including Xaa1-Xaa2-Leu / Ile / Val / Pro-Xaa3 / / Xaa4-Xaa5-Xaa6-Xaa7, where Xaa 1-7 can each independently be selected from any amino acid, and wherein " / / " indicates the position of the hydrolyzed peptide bond. The term "cathepsin K" encompasses the cathepsin K described herein, and any protease having structural and / or functional similarity (preferably structural and functional similarity) capable of hydrolyzing the peptide bond of the consensus Xaa1-Xaa2-Leu / Ile / Val / Pro-Xaa3 / / Xaa4-Xaa5-Xaa6-Xaa7. A suitable cathepsin K is human cathepsin K, which has the UniProt accession number P43235 (version 1 of the sequence, deposited on November 1, 1995, accessed on July 30, 2022), herein as SEQ ID NO: 42. This sequence is a propeptide that can be converted to the mature human form by a process involving cleavage of the N-terminal polypeptide of residues 1-114. Human cathepsin K is available from Merck (#SRP6561).

[0106] Cathepsin S is a thiol protease with broad proteolytic activity. Cathepsin S cleaves in a series of consensus sequences, including Xaa1-Xaa2-Leu / Val-Xaa3 / / Xaa4-Xaa5-Xaa6-Xaa7, where Xaa 1-7can each independently be selected from any amino acid, and wherein " / / " indicates the position of the hydrolyzed peptide bond. The term "cathepsin S" encompasses cathepsin S as described herein, and any protease having structural and / or functional similarity (preferably structural and functional similarity) capable of hydrolyzing the peptide bond of the consensus sequence Xaa1-Xaa2-Leu / Val-Xaa3 / / Xaa4-Xaa5-Xaa6-Xaa7. A suitable cathepsin S is human cathepsin S, which has the UniProt accession number P25774 (version 3 of the sequence, deposited on February 21, 2006, accessed on July 30, 2022), herein as SEQ ID NO:43. This sequence is a propeptide that can be converted to the mature human form by a process involving cleavage of the N-terminal polypeptide of residues 1-114. Human cathepsin S is available from Merck (#SRP6297).

[0107] AEP has strict specificity for the hydrolysis of asparaginyl and aspartyl bonds. Thus, AEP cleaves within a series of consensus sequences containing such a bond (Asn / Asp / / Xaa, where Xaa can be any amino acid), which in standard nomenclature can be represented as Xaa1-Xaa2-Xaa3-Asn / Asp / / Xaa4-Xaa5-Xaa6-Xaa7, where Xaa 1-7 can each independently be selected from any amino acid, and wherein " / / " indicates the position of the hydrolyzed peptide bond. The term "AEP" encompasses AEP as described herein, and any protease having structural and / or functional similarity (preferably structural and functional similarity) capable of hydrolyzing asparaginyl and / or aspartyl bonds. A suitable AEP is human AEP, which has the UniProt accession number Q99538 (version 1 of the sequence, deposited on May 1, 1997, accessed on July 30, 2022), herein as SEQ ID NO:44. Human AEP is available from JenaBioscience (#PR-967S or #PR-967L).

[0108] Other exemplary cathepsins include cathepsin A (e.g., UniProt accession number P10619, sequence version 2, deposited on April 16, 2002, accessed on July 30, 2022), cathepsin C (e.g., UniProt accession number P53634, sequence version 2, deposited on January 11, 2011, accessed on July 30, 2022), cathepsin E (e.g., UniProt accession number P14091, sequence version 3, deposited on March 28, 2018, accessed on July 30, 2022), cathepsin F (e.g., UniProt accession number Q9UBX1, sequence version 1, deposited on May 1, 2000, accessed on July 30, 2022), cathepsin G (e.g., UniProt accession number P08311, sequence version 2, deposited on January 1, 1990, accessed on July 30, 2022), cathepsin H (e.g., UniProt accession number P09668, sequence version 4, deposited on February 9, 2010, accessed on July 30, 2022), cathepsin O (e.g., UniProt accession number P43234, sequence version 1, deposited on November 1, 1995, accessed on July 30, 2022), cathepsin V (e.g., UniProt accession number O60911, sequence version 2, deposited on December 1, 2000, accessed on July 30, 2022), cathepsin W (e.g., UniProt accession number P56202, sequence version 2, deposited on September 22, 2009, accessed on July 30, 2022) and cathepsin Z (e.g., UniProt accession number Q9UBR2, sequence version 1, deposited on May 1, 2000, accessed on July 30, 2022).

[0109] For in vitro and ex vivo use, one of ordinary skill in the art can determine the appropriate concentration / unit amount of any endosomal protease that activates the engineered Clostridial neurotoxin of the invention under standard / desired conditions through routine practice.

[0110] In the context of the present invention, the term "cathepsin L (e.g., L1)" encompasses a polypeptide sequence having at least 70% sequence identity with SEQ ID NO:39, or its mature form. Thus, "cathepsin L (e.g., L1)" can include a polypeptide sequence having at least 80% or 90% sequence identity with SEQ ID NO:39, or its mature form. Preferably, cathepsin L (e.g., L1) comprises SEQ ID NO:39 (more preferably, consists of it), or its mature form.

[0111] In the context of the present invention, the term "cathepsin B" encompasses polypeptide sequences having at least 70% sequence identity with SEQ ID NO: 40, or mature forms thereof. Thus, "cathepsin B" may include polypeptide sequences having at least 80% or 90% sequence identity with SEQ ID NO: 40, or mature forms thereof. Preferably, cathepsin B comprises (more preferably, consists of) SEQ ID NO: 40, or a mature form thereof.

[0112] In the context of the present invention, the term "cathepsin D" encompasses polypeptide sequences having at least 70% sequence identity with SEQ ID NO: 41, or mature forms thereof. Thus, "cathepsin D" may include polypeptide sequences having at least 80% or 90% sequence identity with SEQ ID NO: 41, or mature forms thereof. Preferably, cathepsin D comprises (more preferably, consists of) SEQ ID NO: 41, or a mature form thereof.

[0113] In the context of the present invention, the term "cathepsin K" encompasses polypeptide sequences having at least 70% sequence identity with SEQ ID NO: 42, or mature forms thereof. Thus, "cathepsin K" may include polypeptide sequences having at least 80% or 90% sequence identity with SEQ ID NO: 42, or mature forms thereof. Preferably, cathepsin K comprises (more preferably, consists of) SEQ ID NO: 42, or a mature form thereof.

[0114] In the context of the present invention, the term "cathepsin S" encompasses polypeptide sequences having at least 70% sequence identity with SEQ ID NO: 43, or mature forms thereof. Thus, "cathepsin S" may include polypeptide sequences having at least 80% or 90% sequence identity with SEQ ID NO: 43, or mature forms thereof. Preferably, cathepsin S comprises (more preferably, consists of) SEQ ID NO: 43, or a mature form thereof.

[0115] In the context of the present invention, the term "AEP" encompasses polypeptide sequences having at least 70% sequence identity with SEQ ID NO: 44. Thus, "AEP" may include polypeptide sequences having at least 80% or 90% sequence identity with SEQ ID NO: 44. Preferably, AEP comprises (more preferably, consists of) SEQ ID NO: 44.

[0116] In the context of the present invention, the term "cathepsin A" encompasses polypeptide sequences having at least 70% sequence identity with UniProt accession number P10619 as described herein, or mature forms thereof. Thus, "cathepsin A" can include polypeptide sequences having at least 80% or 90% sequence identity with UniProt accession number P10619 as described herein, or mature forms thereof. Preferably, cathepsin A comprises (more preferably, consists of) UniProt accession number P10619 as described herein, or mature forms thereof.

[0117] In the context of the present invention, the term "cathepsin C" encompasses polypeptide sequences having at least 70% sequence identity with UniProt accession number P53634 as described herein, or mature forms thereof. Thus, "cathepsin C" can include polypeptide sequences having at least 80% or 90% sequence identity with UniProt accession number P53634 as described herein, or mature forms thereof. Preferably, cathepsin C comprises (more preferably, consists of) UniProt accession number P53634 as described herein, or mature forms thereof.

[0118] In the context of the present invention, the term "cathepsin E" encompasses polypeptide sequences having at least 70% sequence identity with UniProt accession number P14091 as described herein, or mature forms thereof. Thus, "cathepsin E" can include polypeptide sequences having at least 80% or 90% sequence identity with UniProt accession number P14091 as described herein, or mature forms thereof. Preferably, cathepsin E comprises (more preferably, consists of) UniProt accession number P14091 as described herein, or mature forms thereof.

[0119] In the context of the present invention, the term "cathepsin F" encompasses polypeptide sequences having at least 70% sequence identity with UniProt accession number Q9UBX1 as described herein, or mature forms thereof. Thus, "cathepsin F" can include polypeptide sequences having at least 80% or 90% sequence identity with UniProt accession number Q9UBX1 as described herein, or mature forms thereof. Preferably, cathepsin F comprises (more preferably, consists of) UniProt accession number Q9UBX1 as described herein, or mature forms thereof.

[0120] In the context of the present invention, the term "cathepsin G" encompasses polypeptide sequences having at least 70% sequence identity with UniProt accession number P08311 as described herein, or mature forms thereof. Thus, "cathepsin G" can include polypeptide sequences having at least 80% or 90% sequence identity with UniProt accession number P08311 as described herein, or mature forms thereof. Preferably, cathepsin G comprises (more preferably, consists of) UniProt accession number P08311 as described herein, or mature forms thereof.

[0121] In the context of the present invention, the term "cathepsin H" encompasses polypeptide sequences having at least 70% sequence identity with UniProt accession number P09668 as described herein, or mature forms thereof. Thus, "cathepsin H" can include polypeptide sequences having at least 80% or 90% sequence identity with UniProt accession number P09668 as described herein, or mature forms thereof. Preferably, cathepsin H comprises (more preferably, consists of) UniProt accession number P09668 as described herein, or mature forms thereof.

[0122] In the context of the present invention, the term "cathepsin O" encompasses polypeptide sequences having at least 70% sequence identity with UniProt accession number P43234 as described herein, or mature forms thereof. Thus, "cathepsin O" can include polypeptide sequences having at least 80% or 90% sequence identity with UniProt accession number P43234 as described herein, or mature forms thereof. Preferably, cathepsin O comprises (more preferably, consists of) UniProt accession number P43234 as described herein, or mature forms thereof.

[0123] In the context of the present invention, the term "cathepsin V" encompasses polypeptide sequences having at least 70% sequence identity with UniProt accession number O60911 as described herein, or mature forms thereof. Thus, "cathepsin V" can include polypeptide sequences having at least 80% or 90% sequence identity with UniProt accession number O60911 as described herein, or mature forms thereof. Preferably, cathepsin V comprises (more preferably, consists of) UniProt accession number O60911 as described herein, or mature forms thereof.

[0124] In the context of the present invention, the term "cathepsin W" encompasses polypeptide sequences having at least 70% sequence identity with UniProt accession number P56202 as described herein, or mature forms thereof. Thus, "cathepsin W" can include polypeptide sequences having at least 80% or 90% sequence identity with UniProt accession number P56202 as described herein, or mature forms thereof. Preferably, cathepsin W comprises (more preferably, consists of) UniProt accession number P56202 as described herein, or mature forms thereof.

[0125] In the context of the present invention, the term "cathepsin Z" encompasses polypeptide sequences having at least 70% sequence identity with UniProt accession number Q9UBR2 as described herein, or mature forms thereof. Thus, "cathepsin Z" can include polypeptide sequences having at least 80% or 90% sequence identity with UniProt accession number Q9UBR2 as described herein, or mature forms thereof. Preferably, cathepsin Z comprises (more preferably, consists of) UniProt accession number Q9UBR2 as described herein, or mature forms thereof.

[0126] When describing the endosomal cleavage site of the present invention, it is not meant that any Xaa (e.g., Xaa1, Xaa2, Xaa3, Xaa4, Xaa5, Xaa6 or Xaa7) is limited to one type of amino acid. Thus, one or more residues present at any Xaa can independently be selected from the 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.

[0127] Alternatively / Additionally, one or more residues present at any Xaa (e.g., Xaa1, Xaa2, Xaa3, Xaa4, Xaa5, Xaa6 or Xaa7) can be independently selected from non-standard amino acids (amino acids that do not belong to the above 20 standard groups). For example, non-standard amino acids can include 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline, α-methylserine, trans-3-methylproline, 2,4-methylene-proline, cis-4-hydroxyproline, trans-4-hydroxy-proline, N-methylglycine, allothreonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethyl-homocysteine, nitro-glutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, 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 Escherichia coli auxotrophic expression hosts.

[0128] The properties of the standard amino acids are shown in the following table:

[0129] Amino acid Side chain Aspartic acid Asp D Charged (acidic) Glutamic acid Glu E Charged (acidic) Arginine Arg R Charged (basic) Lysine Lys K Charged (basic) Histidine His H Uncharged (polar) Asparagine Asn N Uncharged (polar) Glutamine Gln Q Uncharged (polar) Serine Ser S Uncharged (polar) Threonine Thr T Uncharged (polar) Tyrosine Tyr Y Uncharged (polar) Methionine Met M Uncharged (polar) Tryptophan Trp W Uncharged (polar) Cysteine Cys C Uncharged (polar) Alanine Ala A Uncharged (hydrophobic) Glycine Gly G Uncharged (hydrophobic) Valine Val V Uncharged (hydrophobic) Leucine Leu L Uncharged (hydrophobic) Isoleucine Ile I Uncharged (hydrophobic) Proline Pro P Uncharged (hydrophobic) Phenylalanine Phe F Uncharged (hydrophobic)

[0130] The following amino acids are considered charged amino acids: aspartic acid (negative), glutamic acid (negative), arginine (positive) and lysine (positive).

[0131] Exemplary (and generally common) cleavage sites for cathepsin L (e.g., L1), B, D, K or S and AEP are described herein. The engineered clostridial neurotoxins of the invention can comprise one or more of these cleavage sites.

[0132] The cathepsin L (e.g., L1) cleavage site can comprise or consist of a consensus sequence selected from:

[0133]

[0134] The amino acid residues shown at each position (P4, P3, P2, P1, P1', P2', P3' and P4') are the possible amino acid residues for substitution at each position, where the bold and underlined residues are preferred. “ / / ” indicates the cleavage site (i.e., the hydrolyzed peptide bond).

[0135] Thus, the cathepsin L cleavage site can comprise or consist of a consensus sequence selected from: and / or Preferably, L and / or The amino acid residues shown in parentheses are the possible amino acid residues for substitution at each position, and the residues in bold and underlined are preferred. "|" indicates the cleavage site (i.e., the hydrolyzed peptide bond).

[0136] The cathepsin B cleavage site may comprise or consist of a consensus sequence selected from the following:

[0137]

[0138] The amino acid residues shown at each position (P4, P3, P2, P1, P1', P2', P3' and P4') are the possible amino acid residues for substitution at each position, and the residues in bold and underlined are preferred. " / / " indicates the cleavage site (i.e., the hydrolyzed peptide bond).

[0139] Thus, the cathepsin B cleavage site may comprise or consist of a consensus sequence selected from the following: and / or Preferably, and / or The amino acid residues shown in parentheses are the possible amino acid residues for substitution at each position, and the residues in bold and underlined are preferred. "|" indicates the cleavage site (i.e., the hydrolyzed peptide bond).

[0140] The cathepsin D cleavage site may comprise or consist of a consensus sequence selected from the following:

[0141]

[0142] The amino acid residues shown at each position (P4, P3, P2, P1, P1', P2', P3' and P4') are the possible amino acid residues for substitution at each position, and the residues in bold and underlined are preferred. " / / " indicates the cleavage site (i.e., the hydrolyzed peptide bond).

[0143] Thus, the cathepsin D cleavage site may comprise or consist of a consensus sequence selected from the following: Preferably, The amino acid residues shown in parentheses are the possible amino acid residues for substitution at each position, and the residues in bold and underlined are preferred. "|" indicates the cleavage site (i.e., the hydrolyzed peptide bond).

[0144] The AEP cleavage site may comprise or consist of the following consensus sequence:

[0145]

[0146] The amino acid residues shown at each position (P4, P3, P2, P1, P1', P2', P3' and P4') are the possible amino acid residues that can be substituted at each position, where the residues in bold and underlined are preferred. " / / " indicates the cleavage site (i.e., the hydrolyzed peptide bond).

[0147] Thus, the AEP cleavage site can comprise or consist of the following consensus sequence: where the amino acid residues shown in parentheses are the possible amino acid residues that can be substituted at each position, where the residues in bold and underlined are preferred. "|" indicates the cleavage site (i.e., the hydrolyzed peptide bond).

[0148] The endosomal protease cleavage site of the present invention may comprise or consist of one or more of the following: STSQKSIVAYTMSLGADSS (SEQ ID NO:12); LFRGGHHPD (SEQ ID NO:13); ELVTPARDFGHFGLS (SEQ ID NO:14); QAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQA (SEQ ID NO:15); STSQKSIVAYTMSLGADSSTGFGTNE (SEQ ID NO:16); LFRGGHHPDTGFGTNE (SEQ ID NO:17); ELVTPARDFGHFGLSTGFGTNE (SEQ ID NO:18); QAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNE (SEQ ID NO:19); LFGFVG (SEQ ID NO:20); ALVEKLLELKKK (SEQ ID NO:21); QEAANERQQ (SEQ ID NO:22); SGLTNIKTE (SEQ ID NO:23); PDLKNVKSK (SEQ ID NO:24); PGGGNKKIE (SEQ ID NO:25); QLGKNEEGA (SEQ ID NO:26); QKVGKAMYAP (SEQ ID NO:27); GFLG (SEQ ID NO:28); TVIVITLVMLKKKQ (SEQ ID NO:29); PVETDSEEQPYLEMDL (SEQ ID NO:30); LEGMELIVSQVHPETKENEIYPVWSGLP (SEQ ID NO:31); QKEYALLYKLDIEP (SEQ ID NO:32); SLAEEEVVIRSED (SEQ ID NO:33); ERNSNLVGAA (SEQ ID NO:34); PDLKNVKS (SEQ ID NO:130); DLFGFVGL (SEQ ID NO:131); GFVGLFRG (SEQ ID NO:132); GSGLFGFVGGSG (SEQ ID NO:133); LFGFVGLFGFVG (SEQ ID NO:134); LFGFVGLFGFVGLFGFVG (SEQ ID NO:135); GLFGFVGL (SEQ ID NO:136); QAKKDFFSSHPLREPVNATED (SEQ ID NO:137); ELVTPARD (SEQ ID NO:138); RDFGHFGL (SEQ ID NO:139);GLFRGHHP (SEQ ID NO:140); GSGLFRGHHPDGSG (SEQ ID NO:141); LFRGHHPDLFRGHHPD (SEQ ID NO:142); ELVTPARDFGHFGLS (SEQ ID NO:143); QAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFLGTNE (SEQ ID NO:144); LFRGHHPDSTSQKSIVAYTMSLGADSS (SEQ ID NO:145); STSQKSIVAYTMSLGADSSLFRGHHPD (SEQ ID NO:146); STSQKSIVAYTMSLGADSSSTSQKSIVAYTMSLGADSS (SEQ ID NO:147); LFRGHHPDLFRGHHPDLFRGHHPD (SEQ ID NO:148); ELVTPARDFGHFGLSELVTPARDFGHFGLS (SEQ ID NO:149); STSQKSIVAYTMSLGADSSELVTPARDFGHFGLSLFRGHHPD (SEQ ID NO:150); QLGKNEEG (SEQ ID NO:151); GERGFFYTPKT (SEQ ID NO:152); GYYSTTIRYQATGFGTNE (SEQ ID NO:171); GYYSTTIRYQATGFGTNE (SEQ ID NO:171); LFRGHHPD (SEQ ID NO:172); GLFRGHHPD (SEQ ID NO:173); QAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNEP (SEQ ID NO:174); QAKKDFFSSHPL (SEQ ID NO:175); REPVNATEDPSSGYYS (SEQ ID NO:176); TTIRYQATGFGTNE (SEQ ID NO:177); TTIRYQATGFGTNEP (SEQ ID NO:178); EVDLLIGSS (SEQ ID NO:179); EVDLLIGSSGE (SEQ ID NO:180); GLAGFLGG (SEQ ID NO:181); GLFGFVGG (SEQ ID NO:182); TVGSFGFE (SEQ ID NO:183); TVGSFGFEGG (SEQ ID NO:184); LASLLELPEFLLFLQ (SEQ ID NO:185);GLTTELFSPVD (SEQ ID NO:186); and / or LERNSNLVGAA (SEQ ID NO:187).;

[0149] The endosomal protease cleavage site of the present invention may comprise or consist of one or more core cathepsin L cleavage motifs selected from the following: MSLGADSS (SEQ ID NO:188); LFRGHHP (SEQ ID NO:189); GLFRGHHP (SEQ ID NO:190); ELVTPARD (SEQ ID NO:138); KDFFSSHP (SEQ ID NO:191); EPVNATED (SEQ ID NO:192); TGFGTNE (SEQ ID NO:193); TGFGTNEP (SEQ ID NO:194); QKVGKAMY (SEQ ID NO:195); LLIGSS (SEQ ID NO:196); and / or LLIGSSGE (SEQ ID NO:197).

[0150] The endosomal protease cleavage site of the present invention may comprise or consist of one or more cathepsin L cleavage sites selected from the following: STSQKSIVAYTMSLGADSS (SEQ ID NO:12); LFRGGHHPD (SEQ ID NO:13); ELVTPARDFGHFGLS (SEQ ID NO:14); QAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQA (SEQ ID NO:15); STSQKSIVAYTMSLGADSS (SEQ ID NO:16); LFRGGHHPD (SEQ ID NO:17); ELVTPARDFGHFGLS (SEQ ID NO:18); QAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNE (SEQ ID NO:19); QKVGKAMYAP (SEQ ID NO:27); QAKKDFFSSHPLREPVNATED (SEQ ID NO:137); ELVTPARD (SEQ ID NO:138); RDFGHFGL (SEQ ID NO:139); GLFRGHHP (SEQ ID NO:140); GSGLFRGHHPDGSG (SEQ ID NO:141); LFRGHHPDLFRGHHPD (SEQ ID NO:142); ELVTPARDFGHFGLS (SEQ ID NO:143); QAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFLGTNE (SEQ ID NO:144); LFRGHHPDSTSQKSIVAYTMSLGADSS (SEQ ID NO:145); STSQKSIVAYTMSLGADSSLFRGHHPD (SEQ ID NO:146); STSQKSIVAYTMSLGADSSSTSQKSIVAYTMSLGADSS (SEQ ID NO:147); LFRGHHPDLFRGHHPDLFRGHHPD (SEQ ID NO:148); ELVTPARDFGHFGLSELVTPARDFGHFGLS (SEQ ID NO:149); STSQKSIVAYTMSLGADSSELVTPARDFGHFGLSLFRGHHPD (SEQ ID NO:150); GYYSTTIRYQATGFGTNE (SEQ ID NO:171); LFRGHHPD (SEQ ID NO:172); GLFRGHHPD (SEQ ID NO:173);QAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNEP (SEQ ID NO:174); QAKKDFFSSHPL (SEQ ID NO:175); REPVNATEDPSSGYYS (SEQ ID NO:176); TTIRYQATGFGTNE (SEQ ID NO:177); TTIRYQATGFGTNEP (SEQ ID NO:178); EVDLLIGSS (SEQ ID NO:179); and / or EVDLLIGSSGE (SEQ ID NO:180).;

[0151] The endosomal protease cleavage site of the present invention may comprise or consist of one or more core cathepsin B cleavage motifs selected from the following: LFGFVF (SEQ ID NO:20); GLAGFLGG (SEQ ID NO:181); GLFGFVGG (SEQ ID NO:182); GSFGFE (SEQ ID NO:198); and / or GSFGFEGG (SEQ ID NO:199).

[0152] The endosomal protease cleavage site of the present invention may comprise or consist of one or more cathepsin B cleavage sites selected from the following: LFGFVG (SEQ ID NO:20); GFLG (SEQ ID NO:28); DLFGFVGL (SEQ ID NO:131); GFVGLFRG (SEQ ID NO:132); GSGLFGFVGGSG (SEQ ID NO:133); LFGFVGLFGFVG (SEQ ID NO:134); LFGFVGLFGFVGLFGFVG (SEQ ID NO:135); GLFGFVGL (SEQ ID NO:136); GLAGFLGG (SEQ ID NO:181); GLFGFVGG (SEQ ID NO:182); TVGSFGFE (SEQ ID NO:183); and / or TVGSFGFEGG (SEQ ID NO:184).

[0153] The endosomal protease cleavage site of the present invention may comprise or consist of one or more core cathepsin D cleavage motifs selected from the following: VEKLLELK (SEQ ID NO:200); VITLVMLK (SEQ ID NO:201); GMELIVSQ (SEQ ID NO:202); QPYLEMDL (SEQ ID NO:203); EYALLYKL (SEQ ID NO:204); LAEEEVVI (SEQ ID NO:205); LASLLELP (SEQ ID NO:206); and / or TTELFSPV (SEQ ID NO:207).

[0154] The endosomal protease cleavage site of the present invention may comprise or consist of one or more cathepsin D cleavage sites selected from the following: ALVEKLLELKKK (SEQ ID NO:21); TVIVITLVMLKKKQ (SEQ ID NO:29); PVETDSEEQPYLEMDL (SEQ ID NO:30); LEGMELIVSQVHPETKENEIYPVWSGLP (SEQ ID NO:31); QKEYALLYKLDIEP (SEQ ID NO:32); SLAEEEVVIRSED (SEQ ID NO:33); GERGFFYTPKT (SEQ ID NO:152); LASLLELPEFLLFLQ (SEQ ID NO:185); and / or GLTTELFSPVD (SEQ ID NO:186).

[0155] The endosomal protease cleavage site of the present invention may comprise or consist of one or more core AEP cleavage motifs selected from the following: EAANERQQ (SEQ ID NO:208); GLTNIKTE (SEQ ID NO:209); DLKNVKSK (SEQ ID NO:210); GGGNKKIE (SEQ ID NO:211); LGKNEEGA (SEQ ID NO:212); ERNSNLV (SEQ ID NO:213); and / or LERNSNLV (SEQ ID NO:214).

[0156] The endosomal protease cleavage site of the present invention may comprise or consist of one or more AEP cleavage sites selected from the following: QEAANERQQ (SEQ ID NO:22); SGLTNIKTE (SEQ ID NO:23); PDLKNVKSK (SEQ ID NO:24); PGGGNKKIE (SEQ ID NO:25); QLGKNEEGA (SEQ ID NO:26); ERNSNLVGAA (SEQ ID NO:34); PDLKNVKS (SEQ ID NO:130); QLGKNEEG (SEQ ID NO:151); and / or LERNSNLVGAA (SEQ ID NO:187).

[0157] The engineered Clostridial neurotoxin of the present invention may comprise one or more endosomal protease cleavage sites as defined herein.

[0158] In some embodiments, the endosomal protease cleavage site has at least 70% sequence identity to any of the following: SEQ ID NO: 1 to 38, 130 - 152 or 171 - 187; 12 to 38, 130 - 152 or 171 - 187; 1 to 34, 130 - 152 or 171 - 187; or 12 to 34, 130 - 152 or 171 - 187. The endosomal protease cleavage site can have at least 80%, 85% or 90% sequence identity to any of the following: SEQ ID NO: 1 to 38, 130 - 152 or 171 - 187; 12 to 38, 130 - 152 or 171 - 187; 1 to 34, 130 - 152 or 171 - 187; or 12 to 34, 130 - 152 or 171 - 187. Preferably, the endosomal protease cleavage site has at least 95% sequence identity to any of the following: SEQ ID NO: 1 to 38, 130 - 152 or 171 - 187; 12 to 38, 130 - 152 or 171 - 187; 1 to 34, 130 - 152 or 171 - 187; or 12 to 34, 130 - 152 or 171 - 187. More preferably, the endosomal protease cleavage site has at least 99% sequence identity to any of the following: SEQ ID NO: 1 to 38, 130 - 152 or 171 - 187; 12 to 38, 130 - 152 or 171 - 187; 1 to 34, 130 - 152 or 171 - 187; or 12 to 34, 130 - 152 or 171 - 187. Particularly preferred is an endosomal protease cleavage site comprising any of or consisting of the following: SEQ ID NO: 1 to 38, 130 - 152 or 171 - 187; 12 to 38, 130 - 152 or 171 - 187; 1 to 34, 130 - 152 or 171 - 187; or 12 to 34, 130 - 152 or 171 - 187.

[0159] Generally, the endosomal protease cleavage site comprises one or more or consists of one or more of the following amino acid sequences: SEQ ID NO: 1 to 38, 130 - 152 or 171 - 187; 12 to 38, 130 - 152 or 171 - 187; 1 to 34, 130 - 152 or 171 - 187; or 12 to 34, 130 - 152 or 171 - 187, or the exogenous activation loop contains one or more endosomal protease cleavage sites.

[0160] As described herein, one or more endoprotease cleavage sites may be included in an exogenous activation loop together with one or more spacer sequences as defined herein. When one or more endoprotease cleavage sites are short motifs (e.g., typically less than 15 amino acids in length, preferably less than 10 or less than 9 amino acids), spacer sequences may generally be present. One or more spacers may be present at the N-terminus and / or C-terminus of each said endoprotease cleavage site. Preferably, the spacer may be a GS spacer as defined herein.

[0161] The engineered Clostridial neurotoxin of the invention may comprise one or more endoprotease cleavage sites. In other words, the engineered Clostridial neurotoxin of the invention may comprise one endoprotease cleavage site as described herein, or multiple endoprotease cleavage sites. The engineered Clostridial neurotoxin of the invention may comprise two, three, four, five, six, seven, eight, nine, ten or more endoprotease cleavage sites. By way of non-limiting example, the engineered Clostridial neurotoxin of the invention may comprise from 2 to 7 (2, 3, 4, 5, 6 or 7) endoprotease cleavage sites. When the engineered Clostridial neurotoxin of the invention comprises multiple endoprotease cleavage sites, these sites may be selected independently of each other. Generally, when the engineered Clostridial neurotoxin of the invention comprises multiple endoprotease cleavage sites, each endoprotease cleavage site may be independently selected from the endoprotease cleavage sites described herein. Thus, the engineered Clostridial neurotoxin of the invention may comprise multiple endoprotease cleavage sites that are different from each other, or the engineered Clostridial neurotoxin of the invention may comprise two or more copies of a particular endoprotease cleavage site, or any combination thereof. Whether the exogenous activation loop comprises multiple different endoprotease cleavage sites or multiple copies of the same endoprotease cleavage site, the cleavage sites may be directly linked, or may be separated by one or more spacers as described herein.

[0162] Multiple endoprotease cleavage sites may be introduced at a single position within the Clostridial neurotoxin. Alternatively, multiple endoprotease cleavage sites may be introduced at multiple positions within the Clostridial neurotoxin. By way of non-limiting example, one endoprotease cleavage site may be introduced within the activation loop of the Clostridial neurotoxin, and one may be introduced at LH NAnother (identical or different) endoprotease cleavage site is introduced within the domain. Generally, when multiple endoprotease cleavage sites are introduced into the engineered Clostridial neurotoxin according to the present invention, they are introduced at a single position within the Clostridial neurotoxin. Preferably, when multiple endoprotease cleavage sites are introduced into the engineered Clostridial neurotoxin according to the present invention, they are each introduced within the activation loop of the Clostridial neurotoxin. The relative positioning of each endoprotease cleavage site within the exogenous activation loop of the present invention can be determined by the structure - function relationship of the endoprotease in question. It is routine practice for those of ordinary skill in the art to appropriately position each endoprotease cleavage site within the exogenous activation loop containing multiple endoprotease cleavage sites based on this structure - function relationship without undue burden.

[0163] Examples of polypeptide sequences containing multiple endoprotease cleavage sites are:

[0164] or

[0165]

[0166] The bold and underlined residues identify the P1 residue within the endoprotease cleavage site, i.e., the site where peptide bond hydrolysis occurs. It can be seen that contains three endoprotease cleavage sites (two for cathepsin L and one for cathepsin D); contains two cathepsin L cleavage sites; contains three endoprotease cleavage sites (two for cathepsin L and one for cathepsin B); and contains five different AEP cleavage sites.

[0167] Thus, the engineered Clostridial neurotoxin of the present invention can comprise one or more of SEQ ID NO: 35 to 38.

[0168] An exogenous activation loop comprising one or more endopeptidase cleavage sites can be of any length, provided that the structure of the exogenous activation loop and generally of the clostridial neurotoxin is retained and cleavage at one or more endopeptidase activation loops results in the formation of an active double-stranded form of the engineered clostridial neurotoxin. The length of the exogenous activation loop can be from about 10 to about 80 amino acids, such as from about 10 to about 50 amino acids, from about 10 to about 40 amino acids or from about 10 to about 30 amino acids (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 or 50 amino acids), such as a length of from about 15 to about 35 amino acids (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), preferably a length between from about 15 to about 30 amino acids (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids). Particularly preferred is an exogenous activation loop of 17 amino acids in length as this length of exogenous activation loop is the same as the endogenous BoNT / C (BoNT / C1) activation loop.

[0169] Clostridial neurotoxin

[0170] As used herein, the term "neurotoxin" means any polypeptide that enters a neuron and inhibits neurotransmitter release. The process encompasses binding of the neurotoxin to a low or high affinity receptor, internalization of the neurotoxin, translocation of the endopeptidase portion of the neurotoxin into the cytoplasm, and enzymatic modification of the neurotoxin substrate. More specifically, the term "neurotoxin" encompasses any polypeptide produced by Clostridium bacteria that enters a neuron and inhibits neurotransmitter release (clostridial neurotoxin), as well as such polypeptides produced by recombinant or chemical techniques. The double-stranded form is the active form of the toxin. The two chains are referred to as the heavy chain (H chain), which has a molecular weight of about 100 kDa; and the light chain (L chain), which has a molecular weight of about 50 kDa.

[0171] The clostridial neurotoxin of the invention can be catalytically active (also referred to as active) or non-catalytically active. Preferably, the clostridial neurotoxin of the invention is catalytically active.

[0172] As used interchangeably herein, the term "catalytic activity" or "activity" refers to a Clostridial neurotoxin L chain (or a Clostridial neurotoxin comprising such an L chain) having non-cytotoxic protease activity. Specifically, an active Clostridial neurotoxin L chain has endopeptidase activity and is capable of cleaving proteins of the exocytic fusion apparatus in target cells. The proteins of the exocytic fusion apparatus are preferably SNARE proteins such as SNAP25, synaptobrevin / VAMP, or syntaxin.

[0173] As used herein, the term "non-catalytic activity" with respect to a Clostridial neurotoxin L chain means that the L chain essentially does not exhibit non-cytotoxic protease activity. Preferably, as used herein, the term "non-catalytic activity" with respect to a Clostridial neurotoxin L chain means that the L chain does not exhibit non-cytotoxic protease activity. In one embodiment, a non-catalytically active Clostridial neurotoxin L chain is a chain that does not cleave proteins of the exocytic fusion apparatus in target cells. The term "substantially free of non-cytotoxic protease activity" means that a Clostridial neurotoxin L chain has less than 5% of the non-cytotoxic protease activity of a catalytically active Clostridial neurotoxin L chain, such as less than 2%, 1%, or preferably less than 0.1% of the non-cytotoxic protease activity of a catalytically active Clostridial neurotoxin L chain. The non-cytotoxic protease activity can be determined 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. Conventional techniques (such as SDS-PAGE and Western blotting) can be used to quantify the amount of cleaved SNARE protein. Suitable in vitro assays are described in WO 2019 / 145577 A1, which is incorporated herein by reference.

[0174] The Clostridial neurotoxin (e.g., before engineering) can be BoNT / A. Exemplary reference BoNT / A sequences are the BoNT / A1 sequences shown as SEQ ID NO:45 or 117. Other non-limiting examples of BoNT / A sequences include the sequences of SEQ ID NO:46 to 52.

[0175] The Clostridial neurotoxin (e.g., before engineering) can be BoNT / B. An exemplary reference BoNT / B sequence is the BoNT / B1 sequence shown as SEQ ID NO:53. Other non-limiting examples of BoNT / B sequences include the sequences of SEQ ID NO:54 to 60.

[0176] The Clostridial neurotoxin (e.g., before engineering) can be BoNT / C. An exemplary reference BoNT / C1 sequence is shown as SEQ ID NO:61.

[0177] The Clostridial neurotoxin (e.g., before engineering) can be BoNT / D. An exemplary reference BoNT / D sequence is shown as SEQ ID NO:62.

[0178] The Clostridial neurotoxin (e.g., before engineering) can be a BoNT / CD chimera. An exemplary reference BoNT / CD sequence is shown as SEQ ID NO:63.

[0179] The Clostridial neurotoxin (e.g., before engineering) can be a BoNT / DC chimera. An exemplary reference BoNT / DC sequence is shown as SEQ ID NO:64.

[0180] The Clostridial neurotoxin (e.g., before engineering) can be BoNT / E. An exemplary reference BoNT / E sequence is shown as SEQ ID NO:65. Other non-limiting examples of BoNT / E sequences include the sequences of SEQ ID NOs:66 to 77.

[0181] The Clostridial neurotoxin (e.g., before engineering) can be BoNT / F. The exemplary reference BoNT / F sequence is the BoNT / F1 sequence shown as SEQ ID NO:78. Other non-limiting examples of BoNT / F sequences include the sequences of SEQ ID NOs:79 to 84.

[0182] The Clostridial neurotoxin (e.g., before engineering) can be BoNT / G. An exemplary reference BoNT / G sequence is shown as SEQ ID NO:85.

[0183] The Clostridial neurotoxin (e.g., before engineering) can be a BoNT / FA chimera. An exemplary reference BoNT / FA sequence is shown as SEQ ID NO:86.

[0184] The Clostridial neurotoxin (e.g., before engineering) can be BoNT / X. An exemplary reference BoNT / X sequence is shown as SEQ ID NO:87.

[0185] The Clostridial neurotoxin (e.g., before engineering) can be TeNT. An exemplary reference TeNT sequence is shown as SEQ ID NO:88.

[0186] In some preferred embodiments, the Clostridial neurotoxin (e.g., before engineering) is BoNT / A, BoNT / B or BoNT / X as described herein, or a chimera thereof (e.g., BoNT / AB).

[0187] As described above, the activated Clostridial neurotoxin is formed by two polypeptide chains: a heavy chain (H chain), which has a molecular weight of approximately 100 kDa; and a light chain (L chain), which has a molecular weight of approximately 50 kDa. The H chain contains a C-terminal targeting component (receptor binding domain or H C domain) and an N-terminal translocation component (H N domain).

[0188] Examples of light chain reference sequences include:

[0189] Botulinum neurotoxin type A: amino acid residues 1 - 448

[0190] Botulinum neurotoxin type B: amino acid residues 1 - 440

[0191] Botulinum neurotoxin type C1: amino acid residues 1 - 441

[0192] Botulinum neurotoxin type D: amino acid residues 1 - 445

[0193] Botulinum neurotoxin type E: amino acid residues 1 - 422

[0194] Botulinum neurotoxin type F: amino acid residues 1 - 439

[0195] Botulinum neurotoxin type G: amino acid residues 1 - 441

[0196] Tetanus neurotoxin: amino acid residues 1 - 457

[0197] For the most recently identified BoNT / X, the L chain has been reported to correspond to its amino acids 1 - 439, where the L chain boundaries may vary by approximately 25 amino acids (e.g., 1 - 414 or 1 - 464). Preferably, the L chain in the engineered Clostridial neurotoxin of the present invention is the BoNT / X L chain.

[0198] The reference sequences identified above should be considered as a guide, as minor variations may occur depending on the sub-serotype. For example, US2007 / 0166332 (incorporated herein by reference in its entirety) cites slightly different Clostridial sequences:

[0199] Botulinum neurotoxin type A: amino acid residues M1 - K448

[0200] Botulinum neurotoxin type B: amino acid residues M1 - K441

[0201] Botulinum neurotoxin type C1: amino acid residues M1 - K449

[0202] Botulinum neurotoxin type D: amino acid residues M1 - R445

[0203] Clostridial neurotoxin type E: Amino acid residues M1 - R422

[0204] Clostridial neurotoxin type F: Amino acid residues M1 - K439

[0205] Clostridial neurotoxin type G: Amino acid residues M1 - K446

[0206] Tetanus neurotoxin: Amino acid residues M1 - A457

[0207] Alternatively, the L chain of a clostridial neurotoxin can be defined as the first amino acid (including or excluding the initial methionine residue) to the first cysteine residue of the endogenous 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 endogenous activation loop.

[0208] The L chain of a clostridial neurotoxin can be defined as a clostridial neurotoxin domain that contains a metal - coordinating HExxH motif (SEQ ID NO:113), which typically functions to cleave SNARE protein substrates.

[0209] The term "light chain" (or "L chain") encompasses its variants and fragments, 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). For example, a variant can 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. When used in relation to an L chain, the term fragment means 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 a clostridial L chain, 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. The 'fragment' of the L chain of the present invention includes fragments of variant L chains based on the reference sequence.

[0210] The H chain of a clostridial neurotoxin can be defined as the second cysteine of the endogenous activation loop to the last amino acid. Additionally or alternatively, the L chain of a clostridial neurotoxin can be defined as starting from the amino acid sequence C - terminal to the cleavage site within the endogenous activation loop. Additionally or alternatively, the H chain of a clostridial neurotoxin can be defined as starting from the amino acid C - terminal to the cysteine residue (usually the second cysteine residue) that forms a disulfide bond between the L chain and the H chain, thereby delimiting the C - terminal of the endogenous activation loop.

[0211] A translocation domain is a molecule that can translocate a protease into a target cell, such that the functional expression of the protease activity occurs within the cytosol of the target cell. Whether any molecule (such as a protein or peptide) has the translocation function required by the present invention can be confirmed by any of a variety of conventional assays.

[0212] For example, Shone C. (1987) describes an in vitro assay using liposomes, which are excited with the test molecule. The presence of the required translocation function is confirmed by the release of K + and / or labeled NAD from the liposomes, which can be easily monitored (see Shone C. (1987) Eur. J. Biochem; Vol. 167(1): pp. 175 - 180).

[0213] Blaustein R. (1987) provides another example, which describes a simple in vitro assay using planar phospholipid bilayer membranes. The membrane is excited with the test molecule, and the required translocation function is confirmed by an increase in the conductivity across the membrane (see Blaustein (1987) FEBS Letts; Vol. 226, No. 1: pp. 115 - 120).

[0214] Methods in Enzymology Volumes 220 and 221, Membrane Fusion Techniques, Parts A and B, Academic Press 1993 provide additional methods that can evaluate membrane fusion and thus identify translocation domains suitable for the present invention.

[0215] The present invention also encompasses variants and / or fragments of the translocation domain, provided that the variant domain still exhibits the required translocation activity. For example, the variant can 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 translocation domain. When used in relation to a translocation domain, the term fragment means 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 the Clostridium translocation domain, the fragment preferably has at least 100, preferably at least 150, more preferably at least 200 and most preferably at least 250 amino acid residues of the reference translocation domain (such as the H N domain). The translocation 'fragments' of the present invention include fragments of variant translocation domains based on the reference sequence.

[0216] The translocation domain preferably is capable of forming an ion - permeable pore in a lipid membrane under low pH conditions. Preferably, it has been found that only a protein molecular moiety that can form a pore within the endosomal membrane is used.

[0217] The translocation domain can be obtained from a microbial protein source, particularly from 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.

[0218] There is sufficient evidence to show that certain domains of bacterial toxin molecules can form such pores. It is also known that certain translocation domains of membrane fusion proteins expressed by viruses can form such pores. Such domains can be used in the present invention.

[0219] The translocation domain can be from Clostridium, such as the H N domain (or its functional component). The H N refers to the portion or fragment of the Clostridium neurotoxin H chain that is approximately equivalent to the first half of the amino terminus of the H chain, or the domain in the intact H chain corresponding to this fragment. The H C function can be removed from the H chain by deleting the H C amino acid sequence (at the DNA synthesis level, or at the post-synthesis level by nuclease or protease treatment). Alternatively, the H C function can be inactivated by chemical or biological treatment. Thus, the H chain may be unable to bind to the binding site on the target cell to which the native Clostridium neurotoxin (i.e., the holotoxin) binds.

[0220] Examples of suitable (reference) translocation domains include:

[0221] Botulinum neurotoxin type A - amino acid residues (449 - 871)

[0222] Botulinum neurotoxin type B - amino acid residues (441 - 858)

[0223] Botulinum neurotoxin type C - amino acid residues (442 - 866)

[0224] Botulinum neurotoxin type D - amino acid residues (446 - 862)

[0225] Botulinum neurotoxin type E - amino acid residues (423 - 845)

[0226] Botulinum neurotoxin type F - amino acid residues (440 - 864)

[0227] Botulinum neurotoxin type G - amino acid residues (442 - 863)

[0228] Botulinum neurotoxin type X - amino acid residues (461 - 890)

[0229] Tetanus neurotoxin - amino acid residues (458 - 879)

[0230] For the recently identified BoNT / X, the translocation domain is reported to correspond to amino acids 460 - 890 thereof, where the L chain and H C border may vary by approximately 10 amino acids (e.g., 461 - 889 or 454 - 891). Preferably, the translocation domain of the engineered clostridial neurotoxin of the present invention is the BoNT / X translocation domain.

[0231] The reference sequences identified above should be considered as guidelines, as minor variations may occur depending on the subseryotype. For example, US2007 / 0166332 (incorporated herein by reference) cites slightly different clostridial sequences:

[0232] Botulinum neurotoxin type A - amino acid residues (A449 - K871)

[0233] Botulinum neurotoxin type B - amino acid residues (A442 - S858)

[0234] Botulinum neurotoxin type C - amino acid residues (T450 - N866)

[0235] Botulinum neurotoxin type D - amino acid residues (D446 - N862)

[0236] Botulinum neurotoxin type E - amino acid residues (K423 - K845)

[0237] Botulinum neurotoxin type F - amino acid residues (A440 - K864)

[0238] Botulinum neurotoxin type G - amino acid residues (S447 - S863)

[0239] Tetanus neurotoxin - amino acid residues (S458 - V879)

[0240] In the context of the present invention, various clostridial neurotoxin H N regions containing the translocation domain can be used in aspects of the present invention, provided that these active fragments can promote the release of non - cytotoxic proteases (e.g., clostridial L chain) from intracellular vesicles into the cytoplasm of target cells, thereby participating in the entire cellular mechanism by which clostridial neurotoxins proteolytically cleave substrates. The H N region of the clostridial neurotoxin heavy chain is approximately 410 - 430 amino acids in length and contains the translocation domain. Studies have shown that the entire length of the H N region of the clostridial neurotoxin heavy chain is not required for the translocation activity of the translocation domain. Thus, in the context of the present invention, the translocation domain can include clostridial neurotoxin H containing the translocation domain with 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 acidsN regions. Also covered are Clostridial neurotoxin Hs comprising a translocation domain and having a length 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 regions.

[0241] For more details on the genetic basis of toxin production by Clostridium botulinum and Clostridium tetani, see Henderson et al. (1997), The Clostridia: Molecular Biology and Pathogenesis, Academic press.

[0242] The term H N includes naturally occurring neurotoxin H N portions, and modified Hs having amino acid sequences and / or synthetic amino acid residues not found in nature, N provided that the modified H N portions still exhibit the above-described translocation function.

[0243] Alternatively, the translocation domain may be from a non-Clostridium. Examples of non-Clostridium (reference) translocation domain sources include, but are not limited to, 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 A type (Prior et al. Biochemistry (1992) 31, 3555-3559), the translocation domain of anthrax toxin (Blanke et al. Proc. Natl. Acad. Sci. USA (1996) 93, 8437-8442), various fusion peptides or hydrophobic peptides having a translocation 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). The translocation domain may reflect a translocation domain present in a naturally occurring protein or may include amino acid variations provided that these variations do not disrupt the translocation ability of the translocation domain.

[0244] Specific examples of viral (reference) translocation domains suitable for use in the present invention include certain translocation domains of viral-expressed membrane fusion proteins. For example, Wagner et al. (1992) and Murata et al. (1992) described the translocation (i.e., membrane fusion and vesicle formation) functions of a variety of fusion peptides and amphipathic peptides derived from the N-terminal region of influenza virus hemagglutinin. Other viral-expressed membrane fusion proteins known to have the desired translocation activity are the translocation domain of the fusion peptide of Semliki Forest Virus (SFV), the translocation domain of the glycoprotein G of vesicular stomatitis virus (VSV), the translocation domain of the SER virus F protein, and the translocation domain of the foamy virus envelope glycoprotein. Virally encoded spike proteins have particular applications in the context of the present invention, such as the G proteins of the E1 protein of SFV and the G protein of VSV.

[0245] The use of the (reference) translocation domains listed in the table (below) includes the 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 possesses the requisite translocation function. Variants may also contain one or more amino acid substitutions and / or amino acid deletions, insertions, or insertions-deletions (indels), so long as the variant has the necessary translocation function.

[0246]

[0247]

[0248] Clostridial neurotoxin H C Examples of domain reference sequences include:

[0249] BoNT / A-N872-L1296

[0250] BoNT / B-E859-E1291

[0251] BoNT / C1-N867-E1291

[0252] BoNT / D-S863-E1276

[0253] BoNT / E-R846-K1252

[0254] BoNT / F-K865-E1274

[0255] BoNT / G-N864-E1297

[0256] TeNT-I880-D1315

[0257] For the recently identified BoNT / X, the H C domain corresponds to amino acids 893 - 1306 thereof, where the domain boundaries may vary by about 25 amino acids (e.g., 868 - 1306 or 918 - 1306). Preferably, the H C domain of the engineered Clostridial neurotoxin of the present invention is the BoNT / XH C domain.

[0258] The Clostridial neurotoxins described herein may also comprise a translocation - promoting domain. This domain facilitates the delivery of a non - cytotoxic protease to 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.

[0259] By way of example, suitable translocation - promoting domains include envelope virus fusion peptide domains. For example, suitable fusion peptide domains include the influenza virus fusion peptide domain (e.g., the 23 - amino - acid influenza A virus fusion peptide domain), the alphavirus fusion peptide domain (e.g., the 26 - amino - acid Semliki Forest virus fusion peptide domain), the vesicular virus fusion peptide domain (e.g., the 21 - amino - acid vesicular stomatitis virus fusion peptide domain), the respiratory virus fusion peptide domain (e.g., the 25 - amino - acid Sendai virus fusion peptide domain), the measles virus fusion peptide domain (e.g., the 25 - amino - acid canine distemper virus fusion peptide domain), the avulavirus fusion peptide domain (e.g., the 25 - amino - acid Newcastle disease virus fusion peptide domain), the henipavirus fusion peptide domain (e.g., the 25 - amino - acid Hendra virus fusion peptide domain), the metapneumovirus fusion peptide domain (e.g., the 25 - amino - acid human metapneumovirus fusion peptide domain), or the spumavirus fusion peptide domain, such as the simian foamy virus fusion peptide domain; or fragments or variants thereof.

[0260] By further example, the translocation - promoting domain may comprise the H CN domain of a Clostridial neurotoxin or a fragment or variant thereof. More specifically, the H CN translocation - promoting domain of a Clostridial neurotoxin may have a length of at least 200 amino acids, at least 225 amino acids, at least 250 amino acids, at least 275 amino acids. In this regard, the H CN translocation - promoting domain of a Clostridial neurotoxin 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 (reference) examples include:

[0261] Botulinum neurotoxin type A - Amino acid residues (872 - 1110)

[0262] Botulinum neurotoxin type B - Amino acid residues (859 - 1097)

[0263] Botulinum neurotoxin type C - Amino acid residues (867 - 1111)

[0264] Botulinum neurotoxin type D - Amino acid residues (863 - 1098)

[0265] Botulinum neurotoxin type E - Amino acid residues (846 - 1085)

[0266] Botulinum neurotoxin type F - Amino acid residues (865 - 1105)

[0267] Botulinum neurotoxin type G - Amino acid residues (864 - 1105)

[0268] Botulinum neurotoxin type X - Amino acid residues (890 - 1121)

[0269] Tetanus neurotoxin - Amino acid residues (880 - 1127)

[0270] The above sequence positions may vary slightly according to the serotype / subtype, and for the appropriate (reference) Clostridial neurotoxin H CN More examples of domains include:

[0271] Botulinum neurotoxin type A - Amino acid residues (874 - 1110)

[0272] Botulinum neurotoxin type B - Amino acid residues (861 - 1097)

[0273] Botulinum neurotoxin type C - Amino acid residues (869 - 1111)

[0274] Botulinum neurotoxin type D - Amino acid residues (865 - 1098)

[0275] Botulinum neurotoxin type E - Amino acid residues (848 - 1085)

[0276] Botulinum neurotoxin type F - Amino acid residues (867 - 1105)

[0277] Botulinum neurotoxin type G - Amino acid residues (866 - 1105)

[0278] Tetanus neurotoxin - Amino acid residues (882 - 1127)

[0279] Any of the above promoting domains can be combined with any of the previously described translocation domain peptides suitable for use in the present invention. Thus, for example, a non-Clostridium promoting domain can be combined with a non-Clostridium translocation domain peptide or with a Clostridium translocation domain peptide. Alternatively, Clostridium neurotoxin H CN The translocation promoting domain can be combined with a non-Clostridium translocation domain peptide. Alternatively, Clostridium neurotoxin H CN The promoting domain can be combined with a Clostridium translocation domain peptide, examples of which include:

[0280] Botulinum neurotoxin type A - amino acid residues (449 - 1110)

[0281] Botulinum neurotoxin type B - amino acid residues (442 - 1097)

[0282] Botulinum neurotoxin type C - amino acid residues (450 - 1111)

[0283] Botulinum neurotoxin type D - amino acid residues (446 - 1098)

[0284] Botulinum neurotoxin type E - amino acid residues (423 - 1085)

[0285] Botulinum neurotoxin type F - amino acid residues (440 - 1105)

[0286] Botulinum neurotoxin type G - amino acid residues (447 - 1105)

[0287] Tetanus neurotoxin - amino acid residues (458 - 1127)

[0288] In some embodiments, the Clostridium neurotoxin of the present invention may lack the functional H C domain of the Clostridium neurotoxin. Thus, the Clostridium neurotoxin cannot bind to rat synaptosomal membranes in a binding assay (by the Clostridium H C component), as described by Shone et al. (1985) Eur. J. Biochem. 151, 75 - 82. The Clostridium neurotoxin may preferably lack the last 50 C-terminal amino acids of the Clostridium neurotoxin holotoxin. The Clostridium 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 the Clostridium neurotoxin holotoxin. Alternatively, H C the binding activity can be eliminated / reduced by mutagenesis - for example, for convenience of reference to BoNT / A, modification of one or two amino acid residues in the ganglioside binding pocket (mutation of W1266 to L and Y1267 to F) results in H CThe region loses its receptor binding function. Similar mutations can be made to the non-serotype A clostridial neurotoxin components, such as mutations in constructs based on botulinum B (W1262 changed to L and Y1263 changed to F) or constructs of botulinum E (W1224 changed to L and Y1225 changed to F). Other mutations in the active site can also achieve the same H C Receptor binding activity is eliminated, such as Y1267S in botulinum A toxin and corresponding highly conserved residues in other clostridial neurotoxins. Details of this mutation and other mutations are described in Rummel et al. (2004) (Molecular Microbiol. 51:631 - 634), which is incorporated herein by reference.

[0289] H of native clostridial neurotoxins C The peptide contains approximately 400 - 440 amino acid residues and consists of two functionally distinct domains, each domain being approximately 25 kDa, namely the N-terminal region (commonly referred to as H CN peptide or domain) and the C-terminal region (commonly referred to as H CC peptide or domain). This fact has been 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. Chem. 268:15, pp. 11188 - 11192; Rummel A (2007) PNAS 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. In addition, there is ample evidence that the C-terminal region (H CC ) consisting of 160 - 200 amino acid residues at the C-terminus is responsible for binding the clostridial neurotoxin to its native cell receptor, i.e., binding to the nerve endings at the neuromuscular junction - this fact has also been confirmed by the above-mentioned publications. Thus, references throughout this specification to a clostridial heavy chain lacking the functional heavy chain H C peptide (or domain), such that the heavy chain cannot bind to the cell surface receptor to which the native clostridial neurotoxin binds, means that the clostridial heavy chain only lacks the functional H CCPeptide. In other words, H CC The peptide region may be partially or completely absent, or otherwise modified (e.g., by conventional chemical or proteolytic treatment) to inactivate its natural binding ability to nerve endings at the neuromuscular junction.

[0290] Thus, the Clostridial neurotoxin H N peptide of the present invention may be C-terminally extended, i.e., it may be associated with all or part of the Clostridial neurotoxin H C domain (e.g., H CN , H CC or H C domain). As used herein, the Clostridial neurotoxin H N peptide of the present invention encompasses such C-terminally extended H N peptides that contain one or more amino acid residues from the Clostridial neurotoxin H C domain. Alternatively, the Clostridial neurotoxin H N peptide of the present invention may not be associated with (or lack) all or part of the Clostridial neurotoxin H C domain (e.g., H CN , H CC or H C domain).

[0291] Generally, if the Clostridial neurotoxin of the present invention or the Clostridial neurotoxin H N peptide of the present invention lacks all or part of the C-terminal peptide portion (H CC ) of the Clostridial neurotoxin, then it lacks the H C binding function of the native Clostridial neurotoxin. For example, a C-terminally extended Clostridial H N peptide may lack 40 amino acid residues, or 60 amino acid residues, or 80 amino acid residues, or 100 amino acid residues, or 120 amino acid residues, or 140 amino acid residues, or 150 amino acid residues, or 160 amino acid residues at the C-terminus of the heavy chain of the Clostridial neurotoxin. Alternatively, the Clostridial H N peptide of the present invention may lack the entire C-terminal peptide portion (H CC ) of the Clostridial neurotoxin, and thus lacks the H C binding function of the native Clostridial neurotoxin. For example, the Clostridial H N peptide may lack 165 amino acid residues, or 170 amino acid residues, or 175 amino acid residues, or 180 amino acid residues, or 185 amino acid residues, or 190 amino acid residues, or 195 amino acid residues at the C-terminus of the heavy chain of the Clostridial neurotoxin. Further for example, the Clostridial H N peptide of the present invention lacks the Clostridial H selected from the group consisting ofCC Reference sequences:

[0292] Clostridium botulinum type A neurotoxin - amino acid residues (Y1111 - L1296)

[0293] Clostridium botulinum type B neurotoxin - amino acid residues (Y1098 - E1291)

[0294] Clostridium botulinum type C neurotoxin - amino acid residues (Y1112 - E1291)

[0295] Clostridium botulinum type D neurotoxin - amino acid residues (Y1099 - E1276)

[0296] Clostridium botulinum type E neurotoxin - amino acid residues (Y1086 - K1252)

[0297] Clostridium botulinum type F neurotoxin - amino acid residues (Y1106 - E1274)

[0298] Clostridium botulinum type G neurotoxin - amino acid residues (Y1106 - E1297)

[0299] Clostridium botulinum type X neurotoxin - amino acid residues (Y1122 - D1306)

[0300] Tetanus neurotoxin - amino acid residues (Y1128 - D1315).

[0301] The reference sequences identified above should be considered as guidelines, as minor variations may occur depending on the sub - serotype.

[0302] The present invention is applicable to a variety of different types of clostridial neurotoxins. Thus, in the context of the present invention, the term "clostridial neurotoxin" encompasses 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 encompasses 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.

[0303] Botulinum neurotoxin (BoNT) is produced by Clostridium botulinum and exists in the form of a large protein complex, which is composed of the BoNT itself complexed with multiple accessory proteins. Currently, there are nine different types of botulinum neurotoxins, namely: botulinum neurotoxin serotypes A, B, C1, D, E, F, G, H, and X, and their structures and modes of action are similar. Different BoNT serotypes can be distinguished based on the inactivation caused by specific neutralizing antisera, and this serotype classification is related to the percentage of sequence identity at the amino acid level. According to the percentage of amino acid sequence identity, the BoNT proteins of a given serotype are further divided into different subtypes.

[0304] BoNT is absorbed in the gastrointestinal tract and, after entering the systemic circulation, binds to the presynaptic membrane of cholinergic nerve endings and blocks the release of its neurotransmitter acetylcholine. BoNT / B, BoNT / D, BoNT / F, and BoNT / G can cleave synaptobrevin / vesicle-associated membrane protein (VAMP); BoNT / C1, BoNT / A, and BoNT / E can cleave 25 kDa synaptosome-associated protein (SNAP-25); while BoNT / C1 can cleave syntaxin. BoNT / X has been found to be able to cleave SNAP-25, VAMP1, VAMP2, VAMP3, VAMP4, VAMP5, Ykt6, and syntaxin 1.

[0305] Tetanus toxin is produced by Clostridium tetani in a single serotype. Clostridium butyricum produces BoNT / E, while Clostridium baratii produces BoNT / F.

[0306] The term "clostridial neurotoxin" is also intended to cover 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 have been 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. For example, relative to the native (unmodified) clostridial neurotoxin sequence, a modified clostridial neurotoxin may have a modified amino acid sequence in one or more domains. Such modifications may alter functional aspects of the toxin, such as biological activity or persistence. Thus, the clostridial neurotoxin of the present invention can be a modified clostridial neurotoxin, or a derivative of a modified clostridial neurotoxin, or a derivative of a clostridial neurotoxin. Specifically, the engineered clostridial neurotoxin of the present invention can be an engineered modified clostridial neurotoxin, or a derivative of an engineered modified clostridial neurotoxin, or a derivative of an engineered clostridial neurotoxin.

[0307] A modified clostridial neurotoxin may have one or more modifications in the amino acid sequence of the heavy chain (e.g., modified H Cdomain), wherein the modified heavy chain binds to the target nerve cell with a higher or lower affinity than the native (unmodified) Clostridium neurotoxin. H C Such modifications in the domain can include modifying the H C residues in the ganglioside binding site or protein (SV2 or synaptotagmin) binding site of the domain, thereby altering the binding to ganglioside receptors and / or protein receptors of the target nerve cell. Examples of such modified Clostridium neurotoxins are described in WO 2006 / 027207 and WO 2006 / 114308, both of which are incorporated herein by reference in their entirety.

[0308] Most preferably, the BoNT / B H C domain further contains at least one amino acid residue substitution, insertion, insertion-deletion or deletion in the H CC subdomain, which, compared to the native BoNT / B sequence, has the effect of increasing the binding affinity of the BoNT / B neurotoxin for human Syt II. Suitable amino acid residue substitutions, insertions, insertion-deletions or deletions in the BoNT / B H CC subdomain have been disclosed in WO 2013 / 180799 and WO 2016 / 154534 (both incorporated herein by reference).

[0309] BoNT / B H CC Suitable amino acid residue substitutions, insertions, insertion-deletions or deletions in the subdomain can include substitution mutations selected from the group consisting of: V1118M; Y1183M; E1191M; E1191I; E1191Q; E1191T; S1199Y; S1199F; S1199L; S1201V; E1191C, E1191V, E1191L, E1191Y, S1199W, S1199E, S1199H, W1178Y, W1178Q, W1178A, W1178S, Y1183C, Y1183P and combinations thereof.

[0310] BoNT / B H CCSuitable amino acid residue substitutions, insertions, indels or deletions in the subdomain may also include combinations of two substitution mutations selected from the group consisting of: E1191M and S1199L, E1191M and S1199Y, E1191M and S1199F, E1191Q and S1199L, E1191Q and S1199Y, E1191Q and S1199F, E1191M and S1199W, E1191M and W1178Q, E1191C and S1199W, E1191C and S1199Y, E1191C and W1178Q, E1191Q and S1199W, E1191V and S1199W, E1191V and S1199Y, or E1191V and W1178Q.

[0311] BoNT / B H CC Suitable amino acid residue substitutions, insertions, indels or deletions in the subdomain may also include a combination of three substitution mutations: E1191M, S1199W and W1178Q.

[0312] Preferably, BoNT / B H CC Amino acid residue substitutions, insertions, indels or deletions in the subdomain include a combination of two substitution mutations: E1191M and S1199Y. Such modifications are present in the chimeric Clostridium neurotoxin of SEQ ID NO:118. E1191M may correspond to position 1204 of SEQ ID NO:118, while S1199Y may correspond to position 1212. Thus, SEQ ID NO:118 may contain 1204M and 1212Y.

[0313] The modification can be a modification when compared to the unmodified BoNT / B shown as SEQ ID NO:53, where the amino acid residue numbering is determined by alignment with SEQ ID NO:53. Since the presence of the methionine residue at position 1 of SEQ ID NO:53 (and the SEQ ID NOs corresponding to other Clostridial neurotoxin polypeptides described herein, including chimeric Clostridial neurotoxin polypeptides) is optional, those skilled in the art will consider the presence / absence of the methionine residue when determining the amino acid residue numbering. For example, when SEQ ID NO:53 includes methionine, the position numbering will be as defined above (e.g., E1191 will be E1191 of SEQ ID NO:53). Alternatively, when methionine is absent in SEQ ID NO:53, the amino acid residue numbering should be modified by -1 (e.g., E1191 in SEQ ID NO:53 will be E1190). Thus, the initial methionine amino acid residue of the polypeptide sequence of the chimeric Clostridial neurotoxin can be optional or absent. Similar considerations apply when the methionine is present / absent at position 1 of other polypeptide sequences described herein, and those skilled in the art will readily determine the correct amino acid residue numbering using conventional techniques in the art. Alignment can be performed using any method for determining sequence homology and / or % sequence identity described herein.

[0314] The modified Clostridial neurotoxin may have one or more modifications in the amino acid sequence of the light chain, such as modifications in the substrate binding or catalytic domain, which 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 US2011 / 0318385, both of which are incorporated herein by reference in their entirety.

[0315] The modified Clostridial neurotoxin can comprise 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 can comprise a leucine- or tyrosine-based motif, wherein the motif increases or decreases the biological activity and / or biological persistence of the modified Clostridial neurotoxin. Suitable leucine-based motifs include xDxxxLL, xExxxLL, xExxxIL, and xExxxLM (where x is any amino acid). Suitable tyrosine-based motifs include Y-x-x-Hy (where Hy is a hydrophobic amino acid). Examples of modified Clostridial neurotoxins comprising leucine- and tyrosine-based motifs are described in WO 2002 / 008268, which is incorporated herein by reference in its entirety.

[0316] The term "clostridial neurotoxin" is intended to encompass chimeric and hybrid clostridial neurotoxins. A hybrid clostridial neurotoxin contains at least a portion of the light chain from one clostridial neurotoxin or its subtype, and at least a portion of the 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 the heavy chain from another clostridial neurotoxin subtype. A chimeric clostridial neurotoxin may contain a portion (e.g., the binding domain) of the heavy chain of one clostridial neurotoxin subtype, while another portion of the heavy chain is from another clostridial neurotoxin subtype. Chimeric clostridial neurotoxins, particularly chimeric BoNTs, can be defined according to the serotype or sub-serotype of the four main domains of the neurotoxin: the L chain, H N 、H CN and H CC (as defined herein). For example, the (pre-engineered) LH N / A1-H C B1 chimera of SEQ ID NO:118 can be described as an AABB chimera. Similarly or alternatively, a therapeutic moiety may contain a light chain portion from a different clostridial neurotoxin. Such hybrid or chimeric clostridial neurotoxins can be used, for example, as a means of delivering the therapeutic benefits of such clostridial neurotoxins to patients who are immunoresistant to a given clostridial neurotoxin subtype, patients who may have a lower than average concentration of receptors for a given clostridial neurotoxin heavy chain binding domain, or patients who have protease-resistant variants of the membrane or vesicle toxin substrates (e.g., SNAP-25, VAMP, and synaptotagmin). Hybrid and chimeric clostridial neurotoxins are described in US 8,071,110, which is incorporated herein by reference in its entirety. Thus, the clostridial neurotoxin of the present invention can be a hybrid clostridial neurotoxin or a chimeric clostridial neurotoxin. Specifically, the engineered clostridial neurotoxin of the present invention can be an engineered hybrid clostridial neurotoxin or an engineered chimeric clostridial neurotoxin.

[0317] In some preferred embodiments, the clostridial neurotoxin is a BoNT / A (e.g., a BoNT / A hybrid or chimera) that contains at least one domain from a non-BoNT / A clostridial neurotoxin. By way of example, the clostridial neurotoxin of the present invention (which contains one or more endosomal protease cleavage sites) can include:

[0318] i. The BoNT / A L chain and non-BoNT / A H N and H C domains;

[0319] ii. The BoNT / A H N domain and non-BoNT / A L chain and H C domains

[0320] iii. BoNT / A H C domain and non-BoNT / A L chain and H N domain;

[0321] iv. BoNT / A L chain and H N domain and non-BoNT / A H C domain

[0322] v. BoNT / A L chain and H C domain and non-BoNT / A H N domain; or

[0323] vi. BoNT / A H N domain and H C domain and non-BoNT / A L chain.

[0324] By way of non-limiting example, the Clostridial neurotoxins of the invention (e.g., engineered Clostridial neurotoxins) include BoNT / A L chain and H N domain and BoNT / B H C domain (e.g., LH N / A1-H C / B1). Exemplary non-engineered LH N / A1-H C B1 chimeras that can be modified to include one or more endosomal protease cleavage sites according to the invention are given in SEQ ID NO:118. Exemplary engineered forms of the LH N / A1-H C B1 chimera of SEQ ID NO:118 are given in SEQ ID NO:159. The Clostridial neurotoxins of the invention (e.g., engineered Clostridial neurotoxins) can include BoNT / A L chain and H N domain and BoNT / C1 H C domain. The Clostridial neurotoxins of the invention (e.g., engineered Clostridial neurotoxins) can include BoNT / A L chain and H N domain and BoNT / D H C domain. The Clostridial neurotoxins of the invention (e.g., engineered Clostridial neurotoxins) can include BoNT / A L chain and H N domain and BoNT / E H C domain. The Clostridial neurotoxins of the invention (e.g., engineered Clostridial neurotoxins) can include BoNT / A L chain and H N domain and BoNT / F H C domain. The Clostridial neurotoxins of the invention (e.g., engineered Clostridial neurotoxins) can include BoNT / A L chain and H NDomain and BoNT / G H C domain. The Clostridial neurotoxin of the present invention (e.g., engineered Clostridial neurotoxin) may comprise BoNT / A L chain and H N domain and BoNT / X H C domain. The Clostridial neurotoxin of the present invention (e.g., engineered Clostridial neurotoxin) may comprise BoNT / A L chain and H N domain and TeNTH C domain.

[0325] For example, the Clostridial neurotoxin of the present invention (e.g., engineered Clostridial neurotoxin comprising one or more endosomal protease cleavage sites) may comprise:

[0326] i. BoNT / B L chain and non-BoNT / B H N and H C domain;

[0327] ii. BoNT / B H N domain and non-BoNT / B L chain and H C domain

[0328] iii. BoNT / B H C domain and non-BoNT / B L chain and H N domain;

[0329] iv. BoNT / B L chain and H N domain and non-BoNT / B H C domain

[0330] v. BoNT / B L chain and H C domain and non-BoNT / B H N domain; or

[0331] vi. BoNT / B H N domain and H C domain and non-BoNT / B L chain.

[0332] For example, the Clostridial neurotoxin of the present invention (e.g., engineered Clostridial neurotoxin comprising one or more endosomal protease cleavage sites) may comprise:

[0333] i. BoNT / C1 L chain and non-BoNT / C1 H N and H C domain;

[0334] ii. BoNT / C1 H N domain and non-BoNT / C1 L chain and H C domain

[0335] iii. BoNT / C1 H C domain and non-BoNT / C1 L-chain and H N domains;

[0336] iv. BoNT / C1 L-chain and H N domain and non-BoNT / C1 H C domains

[0337] v. BoNT / C1 L-chain and H C domain and non-BoNT / C1 H N domains; or

[0338] vi. BoNT / C1 H N domain and H C domains and non-BoNT / C1 L-chain.

[0339] Non-limiting examples include BoNT / C1 chimeras in which the non-BoNT / C1 element is from BoNT / D (i.e., BoNT / CD chimeras).

[0340] For example, the Clostridial neurotoxins of the present invention (e.g., engineered Clostridial neurotoxins comprising one or more endosomal protease cleavage sites) can comprise:

[0341] i. BoNT / D L-chain and non-BoNT / D H N and H C domains;

[0342] ii. BoNT / D H N domain and non-BoNT / D L-chain and H C domains

[0343] iii. BoNT / D H C domain and non-BoNT / D L-chain and H N domains;

[0344] iv. BoNT / D L-chain and H N domain and non-BoNT / D H C domains

[0345] v. BoNT / D L-chain and H C domain and non-BoNT / D H N domains; or

[0346] vi. BoNT / D H N domain and H C domains and non-BoNT / D L-chain.

[0347] Non-limiting examples include BoNT / D chimeras in which the non-BoNT / D element is from BoNT / C1 (i.e., BoNT / DC1 chimeras).

[0348] For example, the Clostridial neurotoxins of the invention (e.g., engineered Clostridial neurotoxins comprising one or more endosomal protease cleavage sites) can comprise:

[0349] i. BoNT / E L chain and non-BoNT / E H N and H C domain;

[0350] ii. BoNT / E H N domain and non-BoNT / E L chain and H C domain

[0351] iii. BoNT / E H C domain and non-BoNT / E L chain and H N domain;

[0352] iv. BoNT / E L chain and H N domain and non-BoNT / E H C domain

[0353] v. BoNT / E L chain and H C domain and non-BoNT / E H N domain; or

[0354] vi. BoNT / E H N domain and H C domain and non-BoNT / E L chain.

[0355] For example, the Clostridial neurotoxins of the invention (e.g., engineered Clostridial neurotoxins comprising one or more endosomal protease cleavage sites) can comprise:

[0356] i. BoNT / F L chain and non-BoNT / F H N and H C domain;

[0357] ii. BoNT / F H N domain and non-BoNT / F L chain and H C domain

[0358] iii. BoNT / F H C domain and non-BoNT / F L chain and H N domain;

[0359] iv. BoNT / F L chain and HN Domain and non-BoNT / F H C Domain

[0360] v. BoNT / F L chain and H C Domain and non-BoNT / F H N Domain; or

[0361] vi. BoNT / F H N Domain and H C Domain and non-BoNT / F L chain.

[0362] For example, the Clostridial neurotoxin of the present invention (e.g., an engineered Clostridial neurotoxin comprising one or more endosomal protease cleavage sites) may comprise:

[0363] i. BoNT / G L chain and non-BoNT / G H N and H C Domain;

[0364] ii. BoNT / G H N Domain and non-BoNT / G L chain and H C Domain

[0365] iii. BoNT / G H C Domain and non-BoNT / G L chain and H N Domain;

[0366] iv. BoNT / G L chain and H N Domain and non-BoNT / G H C Domain

[0367] v. BoNT / G L chain and H C Domain and non-BoNT / G H N Domain; or

[0368] vi. BoNT / G H N Domain and H C Domain and non-BoNT / G L chain.

[0369] For example, the Clostridial neurotoxin of the present invention (e.g., an engineered Clostridial neurotoxin comprising one or more endosomal protease cleavage sites) may comprise:

[0370] i. BoNT / X L chain and non-BoNT / X H N and H C Domain;

[0371] ii. BoNT / X H N Domain and non-BoNT / X L chain and HC Domain

[0372] iii. BoNT / X H C Domain and non-BoNT / X L chain and H N Domain;

[0373] iv. BoNT / X L chain and H N Domain and non-BoNT / X H C Domain

[0374] v. BoNT / X L chain and H C Domain and non-BoNT / X H N Domain; or

[0375] vi. BoNT / X H N Domain and H C Domain and non-BoNT / X L chain.

[0376] For example, the Clostridial neurotoxin of the present invention (e.g., an engineered Clostridial neurotoxin comprising one or more endosomal protease cleavage sites) may comprise:

[0377] i. TeNT L chain and non-TeNT H N and H C Domain;

[0378] ii. TeNT H N Domain and non-TeNT L chain and H C Domain

[0379] iii. TeNT H C Domain and non-TeNT L chain and H N Domain;

[0380] iv. TeNT L chain and H N Domain and non-TeNT H C Domain

[0381] v. TeNT L chain and H C Domain and non-TeNT H N Domain; or

[0382] vi. TeNT H N Domain and H C Domain and non-TeNT L chain.

[0383] The term "clostridial neurotoxin" may also encompass newly discovered botulinum neurotoxins and members of the botulinum neurotoxin-like protein family expressed by non-clostridial microorganisms, such as the enterococcus-encoded toxin with the closest sequence identity to BoNT / X; the Weissella oryzae-encoded toxin called BoNT / Wo (NCBI Ref Seq: WP_027699549.1), which cleaves VAMP2 at W89-W90; the Enterococcus faecium-encoded toxin (GenBank: OTO22244.1), which cleaves VAMP2 and SNAP25; the Chryseobacterium pipero-encoded toxin (NCBI Ref.Seq: WP_034687872.1) and the mosquito BoNT-like protein PMP1 (NCBI Ref.Seq: QEZ70852.1).

[0384] The term "clostridial neurotoxin" is intended to encompass retargeted clostridial neurotoxins. In retargeted clostridial neurotoxins, the clostridial neurotoxin is modified to include an exogenous ligand called the targeting moiety (TM) (i.e., not derived from the clostridial neurotoxin). The TM is selected to provide binding specificity to the desired target cell, and as part of the retargeting process, the native binding moiety of the clostridial neurotoxin (e.g., the H C domain or the H CCDomain). Retargeting techniques are described, for example, in the following: 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; WO2000 / 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 can be a retargeted Clostridial neurotoxin. Specifically, the engineered Clostridial neurotoxin of the present invention can be an engineered retargeted Clostridial neurotoxin. The engineered retargeted Clostridial neurotoxin of the present invention can comprise a TM present at the N-terminus or C-terminus of a single-chain neurotoxin, or the TM can be present in the center of the single-chain neurotoxin. In some preferred embodiments, the engineered retargeted Clostridial neurotoxin of the present invention can comprise a TM present at the N-terminus or C-terminus of a single-chain neurotoxin.

[0385] Engineering the retargeted Clostridial neurotoxin allows the use of TMs that are susceptible to proteolytic cleavage by proteases that are commonly used to activate recombinantly produced retargeted Clostridial neurotoxins, such as trypsin, Lys-C, and / or BoNT hydrolase. Thus, engineering the retargeted Clostridial neurotoxin to include one or more endosomal protease activation sites according to the present invention can result in increased stability compared to the corresponding retargeted Clostridial neurotoxin activated by conventional activating proteases (such as Lys-C, trypsin, and / or BoNT hydrolase). In some preferred embodiments, the engineered retargeted Clostridial neurotoxin comprises BoNT / A light chain (LC / A) and / or BoNT / A translocation domain (H N / A), particularly preferably LC / A and H N / A. In some preferred embodiments, the engineered retargeted Clostridial neurotoxin comprises BoNT / X light chain (LC / X) and / or BoNT / X translocation domain (H N / X), particularly preferably LC / X and H N / X. Such engineered retargeted BoNT / X is particularly preferred. Non-limiting examples of engineered retargeted clostridial neurotoxins include the neurotoxins of SEQ ID NO: 121, 160, 161, and 162.

[0386] The clostridial neurotoxins of the present invention (e.g., engineered clostridial neurotoxins) may lack the functional H C domain of the clostridial neurotoxin and also lack any functionally equivalent TM. Thus, the polypeptide lacks the native binding function of the clostridial neurotoxin and cannot bind to rat synaptosomal membranes in a binding assay (via the clostridial H C component, or via any functionally equivalent TM), as described by Shone et al. (1985) Eur. J. Biochem. 151, 75-82. Preferably, the TM is not a Wheat Germ Agglutinin (WGA) peptide. Thus, in some preferred embodiments, the clostridial neurotoxin is a retargeted clostridial neurotoxin, wherein the endogenous H C or H CC of the clostridial neurotoxin is replaced by an exogenous TM. Particularly preferred are embodiments in which the engineered clostridial neurotoxin is a retargeted clostridial neurotoxin, wherein the endogenous H C or H CC of the clostridial neurotoxin is replaced by an exogenous TM.

[0387] The clostridial neurotoxins of the present invention (e.g., engineered clostridial neurotoxins) may comprise an LH N polypeptide (e.g., engineered LH N polypeptide), i.e., a polypeptide comprising or consisting of a clostridial L chain and a clostridial H N domain, as defined herein.

[0388] Clostridial neurotoxins (e.g., engineered clostridial neurotoxins) may comprise an LH N polypeptide (e.g., engineered LH N polypeptide) and a targeting moiety (TM).

[0389] The present invention also encompasses clostridial neurotoxins having additional non-native protease cleavage sites. Such sites require exogenous proteases for cleavage, thereby allowing better control of the timing and location of the cleavage event. Non-native protease cleavage sites that may be employed in clostridial neurotoxins include:

[0390] TEV (Tobacco Etch virus) (ENLYFQ↓G) (SEQ ID NO: 114)

[0392] Thrombin (LVPR↓GS) (SEQ ID NO: 115)

[0393] PreScission (LEVLFQ↓GP)(SEQ ID NO:116).

[0394] Additional protease cleavage sites include recognition sequences that are cleaved by non-cytotoxic proteases, such as the light chain of Clostridial neurotoxins. These recognition sequences include SNARE (e.g., SNAP-25, synaptobrevin, VAMP) protein recognition sequences that are cleaved by non-cytotoxic proteases, such as the light chain of Clostridial neurotoxins. Clostridial neurotoxins containing non-native protease cleavage sites are described in US 7,132,259, EP 1206554-B2, and US 2007 / 0166332, which are incorporated herein by reference in their entireties. The term protease cleavage site also encompasses introns, which are self-cleaving sequences. The self-splicing reaction is controllable, for example, by varying the concentration of an existing reducing agent.

[0395] The present invention also encompasses Clostridial neurotoxins that contain a "destructive cleavage site". In such Clostridial neurotoxins, a non-native protease cleavage site is incorporated at a position within the Clostridial neurotoxin that is selected such that cleavage at that position will reduce the activity of the Clostridial neurotoxin or inactivate it. If the Clostridial neurotoxin migrates to a non-target location after administration, the destructive protease cleavage site may be susceptible to local protease cleavage. Suitable non-native protease cleavage sites include those described above. Clostridial neurotoxins containing a destructive cleavage site are described in WO 2010 / 094905 and WO2002 / 044199, both of which are incorporated herein by reference in their entireties.

[0396] The Clostridial neurotoxins of the present invention (e.g., engineered Clostridial neurotoxins), particularly their light chain components, can be pegylated, which helps to increase stability, such as the duration of action of the light chain component. Pegylation is particularly preferred when the light chain contains a BoNT / A, B, or C1 protease. Pegylation preferably involves adding polyethylene glycol to the N-terminus of the light chain component. For example, the N-terminus of the light chain can be extended by one or more amino acid (e.g., cysteine) residues, which can be the same or different. One or more of said amino acid residues can be linked (e.g., covalently linked) to its own PEG molecule. An example of this technique is described in WO2007 / 104567, which is incorporated herein by reference in its entirety.

[0397] The chimeric Clostridial neurotoxin of the present invention may not contain a therapeutic or diagnostic agent (e.g., nucleic acid, protein, peptide, or small molecule therapeutic or diagnostic agent) other than the light chain and the heavy chain. For example, in one embodiment, the chimeric Clostridial neurotoxin may not contain a covalently or non-covalently associated therapeutic or diagnostic agent. Thus, the chimeric Clostridial neurotoxin of the present invention is preferably not used as a delivery vehicle for other therapeutic or diagnostic agents.

[0398] In embodiments where the chimeric Clostridial neurotoxin described herein has a tag (e.g., His-tag) and / or linker for purification, the tag and / or linker are optional.

[0399] The Clostridial neurotoxin of the present invention (e.g., engineered Clostridial neurotoxin) may not contain the complex proteins present in the native Clostridial neurotoxin complex.

[0400] The Clostridial neurotoxin of the present invention (e.g., engineered Clostridial neurotoxin) can be produced using recombinant nucleic acid technology. Thus, the engineered Clostridial neurotoxin (as described above) can be a recombinant engineered Clostridial neurotoxin. The single-chain Clostridial neurotoxin (as described herein) can be a recombinant single-chain neurotoxin.

[0401] Compared to the tolerance to the corresponding (pre-engineered) Clostridial neurotoxin, the tolerance to the engineered Clostridial neurotoxin of the present invention (i.e., reduction of off-target and / or adverse effects) can be increased. Specifically, compared to the tolerance to the corresponding (pre-engineered) Clostridial neurotoxin when administering the pre-engineered Clostridial neurotoxin (e.g., in a double-stranded form), the tolerance to the engineered Clostridial neurotoxin of the present invention can be increased. The tolerance can be quantified / determined as described below.

[0402] The engineered clostridial neurotoxin of the present invention may have equal or enhanced potency compared to the corresponding (pre-engineered) clostridial neurotoxin. Specifically, the potency of the engineered clostridial neurotoxin of the present invention may be equal to or enhanced compared to the potency of the corresponding (pre-engineered) clostridial neurotoxin when administered in the double-stranded form of the pre-engineered clostridial neurotoxin. As used herein, the term "equal potency" means that the potency of the engineered clostridial neurotoxin is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, up to about 100% of the potency of the corresponding (pre-engineered) clostridial neurotoxin. Preferably, as used herein, "equal potency" means that the potency of the engineered clostridial neurotoxin is at least about 95%, at least about 99%, at least about 100%, at least about 101%, up to about 105% of the potency of the corresponding (pre-engineered) clostridial neurotoxin. As used herein, the term "enhanced potency" means that the potency of the engineered clostridial neurotoxin is at least about 10%, at least about 15%, at least about 20%, at least about 25% higher compared to the potency of the corresponding (pre-engineered) clostridial neurotoxin. Any suitable assay can be used to measure potency, and conventional examples of assays are described herein.

[0403] Compared to the safety profile and / or therapeutic window of the corresponding (pre-engineered) clostridial neurotoxin, the engineered clostridial neurotoxin of the present invention generally has an improved safety profile and / or therapeutic window. Without being bound by theory, this may be due to its increased tolerance and / or equal or enhanced potency. Specifically, when the pre-engineered clostridial neurotoxin is administered (e.g., in the double-stranded form), the engineered clostridial neurotoxin of the present invention may have an improved safety profile and / or therapeutic window compared to the safety profile and / or therapeutic window of the corresponding (pre-engineered) clostridial neurotoxin.

[0404] One way to define these favorable properties (representing an increase in the therapeutic index) is based on the safety ratio (for clinical applications) or the tolerance index (TI, in animal models, which can be calculated as described below) of the engineered clostridial toxin. In this regard, the adverse effects of clostridial neurotoxins (such as those caused by the spread of the neurotoxin away from the site of administration) can be experimentally evaluated by measuring the percentage of body weight loss in relevant animal models (e.g., mice, where body weight loss is detected within seven days after administration). Depending on the target cell of interest, the expected targeting effect of the clostridial toxin can be experimentally evaluated by any suitable technique. Suitable assays are known in the art, and it is routine for those of ordinary skill in the art to select an appropriate assay for a given target cell type. For the clostridial neurotoxins of the present invention that target motor neurons, the Digital Abduction Score (DAS) assay, a method for measuring muscle paralysis, can be used. The DAS assay can be performed by injecting 20 μl of the engineered clostridial toxin formulated in gelatin phosphate buffer into the gastrocnemius / soleus muscle complex of a mouse, and then evaluating the digital abduction score using the Aoki method (Aoki KR, Toxicon 39:1815-1820; 2001). In the DAS assay, the mouse tail is briefly suspended to elicit a characteristic startle response, and the mouse will extend its hindlimbs and abduct its digits. After injecting the clostridial toxin, the degree of digital abduction is scored on a five-point scale (0 = normal to 4 = maximum reduction in digital abduction and leg extension). For the clostridial neurotoxins of the present invention that target other neuronal subtypes, any suitable assay known in the art can be used. SNARE cleavage assays can also be used to evaluate the activity of the engineered clostridial neurotoxins of the present invention, and examples thereof are described in detail in the art (e.g., Western blotting). Assays that detect and / or quantify the effect of the engineered clostridial neurotoxin on the release of marker signaling molecules can also be used. Specific marker signaling molecules can be selected based on the cell type targeted by the engineered clostridial neurotoxin. For example, the signaling molecule may be a hormone, substance P, CGRP, glutamate, glycine, depending on whether the targeted cell is related to hormone secretion or pain-sensing neurons. For the treatment of pain, animal studies can be used to evaluate whether there is a greater tolerance to noxious stimuli. Typical in vivo assays will measure different types of pain (e.g., mechanical, cold, heat), and the readings may be behavioral (e.g., licking / biting the treatment site or withdrawing from noxious stimuli), or may involve the use of the von Frey test. Any suitable nociception test can be used, and examples of such tests are well known in the art.

[0405] The safety ratio or TI of a Clostridial neurotoxin can be expressed as the ratio between the amount of toxin required to achieve a 10% body weight loss (measured at the peak effect within the first seven days after dosing in mice) and the amount of toxin required to achieve a DAS score of 2. Thus, a high safety ratio or TI score is desirable and indicates that the toxin can effectively paralyze the target muscle with few adverse off-target effects. The engineered toxins of the present invention can have a higher safety ratio and / or TI than the safety ratio and / or TI of an equivalent unmodified (before engineering) single-chain Clostridial neurotoxin. The calculation of TI may vary depending on the experimental model used.

[0406] For example, in the DAS mouse model, the engineered Clostridial toxin of the present invention has a TI of at least 8 (e.g., at least 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50), where TI is calculated as: - the toxin dose (pg / mouse) required for a 10% body weight change divided by the DAS ED 50 (pg / mouse)[ED 50 = the dose required to produce a DAS score of 2].

[0407] For clinical applications, the safety ratio can be calculated.

[0408] The present invention provides a nucleic acid (e.g., DNA or RNA) comprising a nucleic acid sequence encoding a Clostridial neurotoxin (e.g., an engineered Clostridial neurotoxin) as described herein. The nucleic acid sequence can be prepared as part of an expression vector, wherein the nucleic acid is operably linked to a promoter. Preferably, the nucleic acid can be prepared as part of a DNA expression vector comprising a promoter and a terminator.

[0409] Preferably, the vector has a promoter selected from:

[0410]

[0411] Alternatively, the promoter can preferably be selected from:

[0412]

[0413] The nucleic acid molecules of the present invention can be prepared using any suitable method known in the art. Thus, the nucleic acid molecules can be prepared using chemical synthesis techniques. Alternatively, the nucleic acid molecules of the present invention can be prepared using molecular biology techniques.

[0414] The nucleic acid molecules and expression vectors of the present invention can preferably be designed on a computer and then synthesized by conventional synthesis techniques, including conventional DNA synthesis techniques.

[0415] Optionally, the above nucleic acid sequence information is modified to achieve codon bias according to the final host cell (e.g., E. coli) expression system to be employed.

[0416] The present invention provides nucleotide sequences encoding the engineered Clostridial neurotoxins of the present invention. The nucleotide sequences of the present invention encode polypeptides comprising one or more endoprotease cleavage sites as described herein.

[0417] The nucleotide sequence may comprise a sequence having at least 70% sequence identity with SEQ ID NO: 163, and wherein the nucleotide encoding the BoNT / C activation loop (SEQ ID NO: 164) is replaced with a nucleic acid encoding one or more endoprotease sites or an exogenous activation loop comprising said one or more endosomal cleavage sites. The nucleotide sequence may comprise a sequence having at least 80% or 90% sequence identity with SEQ ID NO: 163, wherein the nucleotide encoding the BoNT / C activation loop (SEQ ID NO: 164) is replaced with a nucleic acid encoding one or more endoprotease sites or an exogenous activation loop comprising said one or more endosomal cleavage sites. Preferably, the nucleotide sequence comprises SEQ ID NO: 163 (more preferably, consists of), wherein the nucleotide encoding the BoNT / C activation loop (SEQ ID NO: 164) is replaced with a nucleic acid encoding one or more endoprotease sites or an exogenous activation loop comprising said one or more endosomal cleavage sites. Non-limiting examples of nucleic acids encoding exogenous activation loops that may replace SEQ ID NO: 164 within SEQ ID NO: 163 include SEQ ID NO: 165, 166, and 167. Accordingly, non-limiting examples of nucleic acids encoding exemplary engineered Clostridial neurotoxins (particularly engineered retargeted BoNT / X) include SEQ ID NO: 168, 169, and 170.

[0418] The nucleotide sequence may encode an engineered Clostridial neurotoxin having at least 70% sequence identity with one or more of SEQ ID NO: 121 or 159 - 162. The nucleotide sequence may encode an engineered Clostridial neurotoxin having at least 80% or 90% sequence identity with one or more of SEQ ID NO: 121 or 159 - 162. Preferably, the nucleotide sequence encodes an engineered Clostridial neurotoxin comprising any one of SEQ ID NO: 121 or 159 - 162 (more preferably consisting of).

[0419] The terms "nucleotide sequence" and "nucleic acid" and "polynucleotide" are used synonymously herein. Preferably, the nucleotide sequence is a DNA sequence.

[0420] The present invention provides a method for generating a single-chain (engineered) Clostridial neurotoxin protein having a light chain and a heavy chain, the method comprising expressing a polynucleotide or expression vector as described herein in a suitable host cell and recovering the expressed engineered Clostridial neurotoxin. Recovering the expressed engineered Clostridial neurotoxin may comprise lysing the host cell to provide a host cell homogenate containing the single-chain (engineered) Clostridial neurotoxin protein, and / or isolating the single-chain (engineered) Clostridial neurotoxin protein. The method may further comprise the step of introducing the polynucleotide or expression vector as described herein into a host cell. Suitable host cells include bacterial cell lines for the recombinant production of Clostridial neurotoxins, in particular Escherichia coli cells.

[0421] The present invention provides a method for proteolytically processing the (engineered) Clostridial neurotoxin of the present invention into the corresponding double-chain Clostridial neurotoxin, the method comprising contacting the (engineered) Clostridial neurotoxin with one or more endosomal proteases, thereby generating a double-chain Clostridial neurotoxin (e.g., wherein the light chain and the heavy chain are joined together by a disulfide bond).

[0422] Accordingly, the present invention provides a double-chain Clostridial neurotoxin obtainable by the method of the present invention.

[0423] As used herein, the term "obtainable" also encompasses the term "obtained". Preferably, the term "obtainable" means obtained.

[0424] Activation of Engineered Clostridial Neurotoxins

[0425] The present invention provides a method for proteolytically processing the engineered Clostridial neurotoxin of the present invention into the corresponding double-chain Clostridial neurotoxin, the method comprising contacting the engineered Clostridial neurotoxin with one or more endosomal proteases, thereby generating a double-chain Clostridial neurotoxin. The contacting can be in vitro, ex vivo or in vivo, preferably in vivo. Accordingly, the therapeutic methods and uses of the present invention may comprise in vivo activation of the engineered Clostridial neurotoxin of the present invention by cleavage at one or more endosomal protease activation sites by expression of one or more endosomal proteases within the target cell.

[0426] Accordingly, the method of the present invention may further comprise contacting the engineered Clostridial neurotoxin with one or more endosomal proteases, thereby generating the corresponding double-chain engineered Clostridial neurotoxin. Preferably, the contacting occurs in vivo.

[0427] The present invention also provides a method for proteolytically processing a single-chain Clostridial neurotoxin into a corresponding double-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 endosomal proteases; wherein the single-chain Clostridial neurotoxin has an activation loop comprising one or more endosomal protease cleavage sites as described herein or consisting of the endosomal protease cleavage sites (e.g., any one or more of SEQ ID NOs: 1 to 38, 130-152 or 171-187; 12 to 38, 130-152 or 171-187; 1 to 34, 130-152 or 171-187; or 12 to 34, 130-152 or 171-187); and wherein the one or more endosomal proteases hydrolyze the peptide bonds of the activation loop, thereby producing a double-chain Clostridial neurotoxin. Preferably, the contacting occurs in vivo.

[0428] The present invention encompasses contacting a single-chain Clostridial neurotoxin (e.g., an engineered Clostridial neurotoxin of the present invention) with one or more endosomal proteases, wherein the one or more endosomal proteases are capable of hydrolyzing the peptide bonds in the activation loop of the single-chain Clostridial neurotoxin, thereby producing a double-chain Clostridial neurotoxin. Preferably, the contacting occurs in vivo.

[0429] The contacting can occur under any suitable conditions that cause more than 30%, 40%, 50% or 60% (preferably more than 70%) of the single-chain Clostridial neurotoxin to be proteolytically processed into the corresponding double-chain Clostridial neurotoxin, with no or substantially no hydrolysis of peptide bonds outside the activation loop of the Clostridial neurotoxin. "Substantially no hydrolysis" can mean that less than 5%, 4%, 3%, 2% or 1% of the contacted Clostridial neurotoxin contains peptide bonds outside the activation loop that have been hydrolyzed by one or more endosomal proteases in the method of the present invention.

[0430] 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 objectives. Conventional techniques can be used, such as visual analysis by SDS-PAGE (e.g., staining with Coomassie Brilliant Blue or a dye of similar sensitivity) or spectroscopic techniques (e.g., mass spectrometry) of the reaction product after the contacting, to empirically determine the optimization of such conditions.

[0431] When evaluated by SDS-PAGE (e.g., staining with Coomassie Brilliant Blue or a dye of similar sensitivity), the method of the present invention preferably produces only the L-chain and H-chain of the Clostridial neurotoxin.

[0432] In the method of the present invention, proteolytic processing by one or more endosomal proteases generally produces fewer than 5 degradation products of the Clostridial neurotoxin L chain or H chain, more preferably fewer than 4, 3, 2 or 1 degradation product. Preferably, the L chain and H chain produced by the method of the present invention are full-length L chain and H chain.

[0433] Generally, processing of each of the one or more endosomal proteases in the method of the present invention hydrolyzes 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer or a single peptide bond within the engineered Clostridial neurotoxin, preferably one or two peptide bonds. Preferably, processing of each of the one or more endosomal proteases in the method of the present invention hydrolyzes 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer or a single peptide bond within the activation loop of the engineered Clostridial neurotoxin, preferably one or two peptide bonds. When two or more endosomal protease cleavage sites are present within the engineered Clostridial neurotoxin of the present invention, the total number of peptide bonds that can be hydrolyzed by two or more endosomal proteases is generally 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer. Preferably, a single peptide bond is hydrolyzed by each of the two or more endosomal proteases. Exemplary endosomal protease cleavage sites are described herein, indicating the position of the hydrolyzed peptide bond.

[0434] For in vitro activation of an engineered Clostridial neurotoxin by one or more endosomal proteases of the present invention, any suitable activation conditions can be used. Determining suitable conditions is routine practice for those of ordinary skill in the art. By way of non-limiting example, about 2 μg to about 5 μg of cathepsin L can be used per 0.1 - 1 mg of engineered Clostridial neurotoxin and activation can be carried out at room temperature (about 21°C) for 1 - 3 hours. By way of other non-limiting examples, about 10 μg to about 25 μg of AEP can be used per 0.1 - 1 mg of engineered Clostridial neurotoxin and activation can be carried out at room temperature (about 21°C) for 1 - 3 hours. Other temperatures (e.g., about 4°C or about 37°C) can be used, with the amount of endosomal protease increased / decreased accordingly.

[0435] Many cells endogenously express one or more endosomal proteases within endosomes and / or lysosomes. As used herein, the term endosome encompasses lysosomes. Thus, one or more endosomal proteases expressed by the cell are generally present intracellularly. Accordingly, the step of contacting the Clostridial neurotoxin according to the invention with one or more endosomal proteases can occur intracellularly in the cell treated with the Clostridial neurotoxin. In other words, the contact of the Clostridial neurotoxin according to the invention with one or more endosomal proteases may involve one or more endosomal proteases endogenously present in the target cell. Thus, after administration of the Clostridial neurotoxin to an individual, the contact of the Clostridial neurotoxin according to the invention with one or more endosomal proteases can occur in vivo. When the contacting step occurs in vivo, it generally involves one or more endosomal proteases endogenously present in one or more cells in the tissue or organ to be treated according to the invention.

[0436] The invention also provides a double-stranded Clostridial neurotoxin obtainable by the method of the invention. Since activation of the double-stranded form occurs by cleavage at one or more endosomal protease cleavage sites as described herein, the C-terminal and N-terminal cleavage ends of the resulting double-stranded Clostridial neurotoxin differ in sequence from the corresponding (pre-engineered) Clostridial neurotoxin. In contrast, conventional trypsin cleavage of (pre-engineered) BoNT / A will produce a double-stranded form with an LC having a C-terminus ending in the sequence TSK and an HC having an N-terminus starting with ALNDLC. These double-stranded Clostridial neurotoxins can be used in the therapies described herein. Unless otherwise stated, all disclosures herein related to therapeutic indications and formulations in the context of the engineered or single-chain Clostridial neurotoxins of the invention apply equally and without reservation to the double-stranded Clostridial neurotoxins obtainable by the method of the invention.

[0437] Therapies and formulations

[0438] The Clostridial neurotoxin of the invention is applicable to medicine and / or cosmetics. In use, since the engineered Clostridial neurotoxin of the invention can be cleaved in vivo by one or more endosomal proteases as described herein, the Clostridial neurotoxin is preferably administered in single-chain form. Alternatively, the engineered Clostridial neurotoxin of the invention can be administered in double-stranded form (e.g., obtainable by the method of the invention).

[0439] The (engineered) Clostridial neurotoxin of the present invention can be used for preventing or treating certain medical or cosmetic diseases and disorders. Thus, in another aspect, the present invention provides the (engineered) Clostridial neurotoxin as described above for medical use. In addition, as described herein, the present invention relates to a single-chain Clostridial neurotoxin for preventing or treating certain medical or cosmetic diseases and disorders, wherein the single-chain Clostridial neurotoxin is administered to a subject. In addition, as described herein, the present invention relates to a double-chain Clostridial neurotoxin obtainable by the method of the present invention for preventing or treating certain medical or cosmetic diseases and disorders, wherein the double-chain Clostridial neurotoxin obtainable by the method of the present invention is administered to a subject. Thus, in another aspect, the present invention provides the (engineered) Clostridial neurotoxin as described above for medical use.

[0440] Accordingly, the present invention provides a Clostridial neurotoxin (e.g., an engineered Clostridial neurotoxin) as described above, for preventing or treating a disease or disorder selected from the following: disorders 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., spastic torticollis), cosmetic applications (cosmesis) of cell / muscle disabling (via SNARE downregulation or inactivation), neuromuscular disorders or diseases of eye movement (e.g., concomitant strabismus, vertical strabismus, lateral rectus paralysis, nystagmus, dysthyroid myopathy), writer's cramp, bruxism, Wilson's disease, tremor, convulsion, segmental myoclonus, spasm, spasm due to chronic multiple sclerosis, spasm leading to abnormal bladder control, hostility, back spasm, cramp, levator ani syndrome, spina bifida, tardive dyskinesia, Parkinson's disease, stuttering, hemifacial spasm, eyelid disorders, cerebral palsy, focal spasm, spastic colitis, neurogenic bladder, pelvic floor dyssynergia, limb spasm, convulsion, tremor, bruxism, anal fissure, achalasia, dysphagia, lacrimation, hyperhidrosis, hypersalivation, hypersecretion of the gastrointestinal tract, muscle pain (e.g., pain caused by muscle spasm), headache (e.g., tension headache or migraine), phantom pain (e.g., phantom limb pain), glabellar lines, skin wrinkles, cancer, uterine disorders, urogenital disorders, urogenital nerve disorders, bladder pain syndrome, interstitial cystitis, chronic neurogenic inflammation, and smooth muscle disorders. In some cases, the disorder 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 method of the present invention, for treating or preventing the above-mentioned diseases or disorders. Preferably, the composition of the present invention can be used for preventing or treating a disease or disorder selected from the following: limb spasm (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).

[0441] When the Clostridium neurotoxin of the present invention (e.g., an engineered Clostridium neurotoxin) contains a BoNT / X sequence (or a portion thereof), the Clostridium neurotoxin is capable of targeting other types of secretory cells other than neurons because it can 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 can be innate immune cells, including but not limited to natural killer cells, mast cells, eosinophils, basophils, macrophages, neutrophils, and dendritic cells. Secretory immune cells that secrete antibodies (e.g., white blood cells) may also be targets of the Clostridium neurotoxins of the present disclosure. Non-limiting examples of antibody-secreting cells include but are not limited to plasma B cells, plasma cells, plasmocytes, and effector B cells. In some embodiments, the Clostridium neurotoxin can modulate the immune response. Accordingly, the therapeutic use of the Clostridium neurotoxins of the present invention for treating disorders associated with unwanted secretion, preferably unwanted immune secretion, is further contemplated herein. Disorders associated with unwanted immune secretion include but are not limited to: inflammation, psoriasis, allergy, hemophagocytic lymphohistiocytosis, and alcoholic pancreatic disease.

[0442] The present invention also provides the use of a Clostridium neurotoxin as described above (e.g., an engineered Clostridium neurotoxin or a double-stranded Clostridium neurotoxin obtainable by the method of the present invention) in the manufacture of a medicament for a method of preventing or treating a disease or disorder as described herein.

[0443] The present invention also provides a method of treating a disease or disorder as described herein, the method comprising administering to a subject in need thereof a therapeutically effective amount of a Clostridium neurotoxin as described above (e.g., an engineered Clostridium neurotoxin or a double-stranded Clostridium neurotoxin obtainable by the method of the present invention).

[0444] The present invention also provides a non-therapeutic use of a composition as described herein for treating aesthetic or cosmetic disorders. For cosmetic or aesthetic use, the individual being treated is preferably not suffering from a disease or disorder, such as a disease or disorder associated with unwanted neural activity and as described above. More preferably, the individual is a healthy individual, i.e., an individual not suffering from any disease. Preferably, the composition of the present invention can be used to prevent or treat upper facial wrinkles - glabellar wrinkles, lateral canthal wrinkles, and / or intradermal wrinkles.

[0445] The present invention provides a pharmaceutical composition, which comprises the (engineered) Clostridium neurotoxin or double-stranded Clostridium neurotoxin of the present invention and a pharmaceutically acceptable carrier, excipient, adjuvant, propellant and / or salt. Preferably, the (engineered) Clostridium neurotoxin is in single-chain form (for example, engineered to comprise one or more endosomal protease cleavage sites). The pharmaceutical composition of the present invention can be a liquid composition (or formulation) or a solid composition (or formulation).

[0446] The present invention also provides a cosmetic composition, which comprises the (engineered) Clostridium neurotoxin of the present invention or the double-stranded Clostridium 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 Clostridium neurotoxin (such as an engineered Clostridium neurotoxin or a double-stranded Clostridium neurotoxin obtainable by the method of the present invention) for preventing or alleviating a cosmetic indication indicative of the application of botulinum neurotoxin. The present invention also provides the use of a cosmetic composition comprising a Clostridium neurotoxin (such as an engineered Clostridium neurotoxin or a double-stranded Clostridium neurotoxin obtainable by the method of the present invention) for preventing or alleviating a cosmetic indication indicative of the application of botulinum neurotoxin. Preferably, the (engineered) Clostridium neurotoxin is in single-chain form (for example, engineered to comprise one or more endosomal protease cleavage sites). The cosmetic composition of the present invention can be a liquid composition (or formulation) or a solid composition (or formulation).

[0447] The Clostridium neurotoxin of the present invention (such as an engineered Clostridium neurotoxin) can be formulated for oral, parenteral, continuous infusion, inhalation or topical application. Compositions suitable for injection can be in the form of a solution, suspension or emulsion, or can be in the form of a dry powder to be dissolved or suspended in a suitable vehicle before use.

[0448] The liquid composition of the present invention can be (i) a solution before lyophilization, (ii) a reconstituted solution, or (iii) a solution not intended for lyophilization and / or not reconstituted after lyophilization. The liquid composition of type (iii) can also be referred to as a "ready-to-use" composition or "ready-to-use" solution, because they are manufactured and formulated as liquids and sold in liquid form. Unless explicitly stated to the contrary, all disclosures related to liquid formulations herein apply to any liquid formulation, including solutions before lyophilization, reconstituted solutions and ready-to-use compositions. The liquid composition can be packaged according to the amount (especially the absolute weight) of the chimeric Clostridium neurotoxin of the present invention as described herein. The liquid composition can be packaged so as to allow for up to 15 injections from a single container.

[0449] The liquid composition can be packaged according to the amount (especially the absolute weight) of the engineered Clostridium neurotoxin of the present invention as described herein.

[0450] In cases where local delivery of a Clostridium neurotoxin (e.g., an engineered Clostridium neurotoxin) is desired, the Clostridium neurotoxin (e.g., an engineered Clostridium neurotoxin) can be formulated as a cream (e.g., for topical application) or for subcutaneous injection.

[0451] Local delivery methods can include aerosols or other sprays (e.g., nebulizers). In this regard, aerosol formulations of Clostridium neurotoxins (e.g., engineered Clostridium neurotoxins) are capable of being delivered to the lungs and / or other nasal and / or bronchial or respiratory passages.

[0452] The Clostridium neurotoxins of the present invention (e.g., engineered Clostridium neurotoxins) can be administered to a patient by intrathecal or epidural injection at the level of the spinal segment involved in the innervation of the affected organ in the spine.

[0453] The preferred route of administration is by laparoscopy and / or local injection, particularly intramuscular injection.

[0454] The dosage range for administration of the compositions of the present invention is the dosage range that produces the desired therapeutic effect. A therapeutically effective dose refers to the amount of the chimeric neurotoxin used in the compositions of the present invention that prevents, ameliorates, or treats the accompanying symptoms of the diseases or disorders mentioned herein. The therapeutic efficacy and toxicity of compounds are generally determined in the art by standard pharmaceutical procedures in cell culture and / or experimental animals, including, for example, ED 50 (50% population effective treatment dose) and LD 50 (50% population lethal dose). The dose ratio between the therapeutic effect and the toxic effect is the therapeutic index, and it can be expressed as LD 50 / ED 50 ratio. Generally, it should be understood that the required dosage range for a given drug depends 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 disorder, contraindications (if any), and the judgment of the attending physician.

[0455] Regarding the engineered neurotoxin compositions of the present invention, suitable single unit doses (also referred to as unit doses), i.e., the dose administered at each 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 engineered neurotoxin, 25,000 pg of engineered neurotoxin, or 36,000 pg of engineered neurotoxin.

[0456] Treatment may include administering injections at multiple injection sites (usually not more than 20, preferably not more than 15 injection sites), and administering a single unit dose at each injection site.

[0457] The liquid dosage form is generally prepared using a Clostridium neurotoxin (e.g., an engineered Clostridium neurotoxin) and a pyrogen-free sterile vehicle. Depending on the vehicle and concentration used, the Clostridium neurotoxin (e.g., an engineered Clostridium neurotoxin) may be dissolved or suspended in the vehicle. When preparing a solution, the Clostridium neurotoxin (e.g., an engineered Clostridium neurotoxin) may be dissolved in the vehicle, and if desired, the solution may be made isotonic by adding sodium chloride and sterilized using aseptic techniques by filtration through a sterile filter, and then filled into a suitable sterile vial or ampoule and sealed. Alternatively, if the solution stability is sufficient, the solution in a sealed container may also be sterilized by an autoclave. Advantageously, additives such as buffers, solubilizers, stabilizers, preservatives or bactericides, suspending or emulsifying agents, and / or local anesthetics may be dissolved in the vehicle.

[0458] The dry powder may be dissolved or suspended in a suitable vehicle before use, which may be prepared by loading pre-sterilized components into a sterile container using aseptic techniques within a sterile area. Alternatively, the components may be dissolved into a suitable container using aseptic techniques in a sterile area. The product is then lyophilized and the container is sealed aseptically.

[0459] The preparation of parenteral suspensions suitable for intramuscular, subcutaneous, or intradermal injection is substantially the same, except that the sterile components are suspended in a sterile vehicle rather than dissolved, and sterilization cannot be achieved by filtration. The components may be separated in a sterile state, or may be sterilized after separation, for example by gamma-ray sterilization.

[0460] Advantageously, the composition includes a suspending agent, such as polyvinylpyrrolidone, to promote the uniform distribution of the components.

[0461] Administration according to the present invention may utilize a variety of delivery techniques, including particulate encapsulation, viral delivery systems, or high-pressure aerosol impingement.

[0462] The disclosures related to the various methods of the present invention are intended to be equally applicable to other methods, Clostridium neurotoxins, such as engineered Clostridium neurotoxins (whether in single-chain or double-chain form), uses, or pharmaceutical compositions, and their medical uses, and vice versa.

[0463] Sequence homology

[0464] Any of a variety of sequence alignment methods can be used to determine the percentage identity, including but not limited to global methods, local methods, and hybrid methods such as the segment method. The scheme for determining the percentage identity is a routine procedure within the capabilities of those skilled in the art. Global methods align the sequences of molecules from beginning to end and determine the best alignment by summing the scores of individual residue pairs and applying 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 WaIIe et al., Align-M - A New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bioinformatics: 1428-1435 (2004).

[0465] Thus, the 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 using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum 62" scoring matrix of Henikoff and Henikoff (supra), and the alignment score is optimized as follows (amino acids are represented by the standard single letter code).

[0466] The "percent sequence identity" between two or more nucleic acid or amino acid sequences is a function of the number of positions shared by the sequences. Thus, the identity % can be calculated as the number of identical nucleotides / amino acids divided by the total number of nucleotides / amino acids, multiplied by 100. The calculation of the sequence identity % may also take into account the number of gaps, and the length of each gap that needs to be introduced to optimize the alignment of two or more sequences. The sequence comparison between two or more sequences and the determination of the percent identity can be performed using specific mathematical algorithms familiar to those skilled in the art (such as BLAST).

[0467] Alignment score for determining sequence identity

[0468]

[0469] Then calculate the percent identity as follows:

[0470] Total number of identical matches

[0471] __________________________________________× 100

[0472] [The length of the longer sequence plus the number of gaps introduced into the longer sequence in order to align the two sequences]

[0473] Substantially homologous polypeptides are characterized by having one or more amino acid substitutions, deletions or additions. These changes are preferably minor, 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, generally 1 to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide up to about 20-25 residues or an affinity tag.

[0474] Conservative amino acid substitutions

[0475] Basic: Arginine

[0476] Lysine

[0477] Histidine

[0478] Acidic: Glutamic acid

[0479] Aspartic acid

[0480] Polar: Glutamine

[0481] Asparagine

[0482] Hydrophobic: Leucine

[0483] Isoleucine

[0484] Valine

[0485] Aromatic: Phenylalanine

[0486] Tryptophan

[0487] Tyrosine

[0488] Small: Glycine

[0489] Alanine

[0490] Serine

[0491] Threonine

[0492] Methionine

[0493] 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) can replace the amino acid residues of the polypeptides of the present invention. A limited number of non-conservative amino acids, amino acids not encoded by the genetic code, and non-natural amino acids can replace the polypeptide amino acid residues. The polypeptides of the present invention can also contain non-naturally occurring amino acid residues.

[0494] Non-naturally occurring amino acids include, but are not limited to, trans-3-methylproline, 2,4-methylene-proline, cis-4-hydroxyproline, trans-4-hydroxy-proline, N-methylglycine, allothreonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethyl-homocysteine, nitro-glutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine. Several methods are known in the art for incorporating non-naturally occurring amino acid residues into proteins. For example, an in vitro system can be employed, in which chemically aminoacylated suppressor tRNAs are used to suppress nonsense mutations. Amino acid synthesis and tRNA aminoacylation methods are known in the art. Transcription and translation of a plasmid containing a nonsense mutation are carried out in a cell-free system comprising an Escherichia coli S30 extract and commercially available enzymes and other reagents. The protein is purified by chromatography. See, for example, 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. 271:19991-8, 1996). In a third method, Escherichia coli cells are cultured in the absence of the natural amino acid to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturally occurring amino acid is incorporated into the polypeptide in place of its natural counterpart. 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 scope of substitution (Wynn and Richards, Protein Sci. 2:395-403, 1993).

[0495] A limited number of non-conservative amino acids, amino acids not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids can replace the amino acid residues of the polypeptides of the present invention.

[0496] The essential amino acids in the polypeptides of the present invention can be identified according to 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, such as by techniques including nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutagenesis of putative contact-site amino acids. See, for example, de Vos et al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identity of essential amino acids can also be inferred by analysis of homology to related components of the polypeptides of the present invention (such as translocation or protease components).

[0497] Known mutagenesis and screening methods can be used, 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), to make and test multiple amino acid substitutions. Briefly, these authors disclosed methods of simultaneously randomizing two or more positions in a polypeptide, selecting functional polypeptides, and then sequencing the mutagenized polypeptides to determine the range of allowable 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).

[0498] Sequence information

[0499] When the starting Met amino acid residue or the corresponding start codon is indicated in any of the following SEQ ID NOs, the residue / codon is optional.

[0500] SEQ ID NO:1 - Cathepsin L cleavage site consensus sequence

[0501] Leu Xaa1 Xaa2 Xaa3 Xaa4 Xaa5 Xaa6

[0502] wherein: Xaa1 is L, V, F, I or Y

[0503] Xaa2 is G, K or A (G or K is preferred)

[0504] Xaa3 is A, G or S

[0505] Xaa4 is P or E

[0506] Xaa5 is P or G

[0507] Xaa6 is P, D or E

[0508] SEQ ID NO:2 - Cathepsin L cleavage site consensus sequence

[0509] Xaa1 Xaa2 Xaa3 Xaa4 Xaa5 Xaa6

[0510] Wherein: Xaa1 is G or M

[0511] Xaa2 is F, V, Y, I or L

[0512] Xaa3 is G, Q or T (G or Q is preferred)

[0513] Xaa4 is G or H (G is preferred)

[0514] Xaa5 is P or H

[0515] Xaa6 is H, P or G

[0516] SEQ ID NO:3 - Cathepsin L cleavage site consensus sequence

[0517] Leu Xaa1 Xaa2 Xaa3 Xaa4 Xaa5 Xaa6

[0518] Wherein: Xaa1 is L, V, F, I or Y

[0519] Xaa2 is G or K

[0520] Xaa3 is A, G or S

[0521] Xaa4 is P or E

[0522] Xaa5 is P or G

[0523] Xaa6 is P, D or E

[0524] SEQ ID NO:4 - Cathepsin L cleavage site consensus sequence

[0525] Xaa1 Xaa2 Xaa3 Gly Xaa4 Xaa5

[0526] Wherein: Xaa1 is G or M

[0527] Xaa2 is F, V, Y, I or L

[0528] Xaa3 is G or Q

[0529] Xaa4 is P or H

[0530] Xaa5 is H, P or G

[0531] SEQ ID NO:5 - Cathepsin B cleavage site consensus sequence

[0532] Leu Xaa1 Xaa2 Xaa3 Xaa4 Xaa5 Gly

[0533] Wherein: Xaa1 is A, V or F

[0534] Xaa2 is G or A (G is preferred)

[0535] Xaa3 is G, L or F

[0536] Xaa4 is A or V

[0537] Xaa5 is G or A (G is preferred)

[0538] SEQ ID NO:6 - Cathepsin B cleavage site consensus sequence

[0539] Xaa1 Xaa2 Gly Xaa3 Xaa4 Gly

[0540] Wherein: Xaa1 is G, L or P

[0541] Xaa2 is A, V, F or Y

[0542] Xaa3 is F or G (F is preferred)

[0543] Xaa4 is V or A

[0544] SEQ ID NO:7 - Cathepsin B cleavage site consensus sequence

[0545] Leu Xaa1 Gly Xaa2 Xaa3 Gly Gly

[0546] Wherein: Xaa1 is A, V or F

[0547] Xaa2 is G, L or F

[0548] Xaa3 is A or V

[0549] SEQ ID NO:8 - Cathepsin B cleavage site consensus sequence

[0550] Xaa1 Xaa2 Gly Phe Xaa3 Gly

[0551] wherein: Xaa1 is G, L or P

[0552] Xaa2 is A, V, F or Y

[0553] Xaa3 is V or A

[0554] SEQ ID NO:9 - Cathepsin D cleavage site consensus sequence

[0555] Leu Xaa1 Xaa2 Xaa3 Xaa4 Xaa5 Xaa6

[0556] wherein: Xaa1 is E or L

[0557] Xaa2 is V or E (V is preferred)

[0558] Xaa3 is L or F

[0559] Xaa4 is I, L or F

[0560] Xaa5 is V or A

[0561] Xaa6 is L or E

[0562] SEQ ID NO:10 - Cathepsin D cleavage site consensus sequence

[0563] Leu Xaa1 Val Xaa2 Xaa3 Xaa4 Xaa5

[0564] wherein: Xaa1 is E or L

[0565] Xaa2 is L or F

[0566] Xaa3 is I, L or F

[0567] Xaa4 is V or A

[0568] Xaa5 is L or E

[0569] SEQ ID NO:11 - AEP cleavage site consensus sequence

[0570] Xaa1 Xaa2 Glu Xaa3 Xaa4 Glu Xaa5

[0571] wherein: Xaa1 is E or A

[0572] Xaa2 is A or G

[0573] Xaa3 is N or D

[0574] Xaa4 is G or S

[0575] Xaa5 is L or A

[0576] SEQ ID NO:12 - Cathepsin L cleavage site

[0577] STSQKSIVAYTMSLGADSS

[0578] SEQ ID NO:13 - Cathepsin L cleavage site

[0579] LFRGGHHPD

[0580] SEQ ID NO:14 - Cathepsin L cleavage site

[0581] ELVTPARDFGHFGLS

[0582] SEQ ID NO:15 - Cathepsin L cleavage site

[0583] QAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQA

[0584] SEQ ID NO:16 - Cathepsin L cleavage site

[0585] STSQKSIVAYTMSLGADSSTGFGTNE

[0586] SEQ ID NO:17 - Cathepsin L cleavage site

[0587] LFRGGHHPDTGFGTNE

[0588] SEQ ID NO:18 - Cathepsin L cleavage site

[0589] ELVTPARDFGHFGLSTGFGTNE

[0590] SEQ ID NO:19 - Cathepsin L cleavage site

[0591] QAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNE

[0592] SEQ ID NO:20 - Cathepsin B cleavage site

[0593] LFGFVG

[0594] SEQ ID NO:21 - Cathepsin D cleavage site

[0595] ALVEKLLELKKK

[0596] SEQ ID NO: 22 - AEP Cleavage Site

[0597] QEAANERQQ

[0598] SEQ ID NO: 23 - AEP Cleavage Site

[0599] SGLTNIKTE

[0600] SEQ ID NO: 24 - AEP Cleavage Site

[0601] PDLKNVKSK

[0602] SEQ ID NO: 25 - AEP Cleavage Site

[0603] PGGGNKKIE

[0604] SEQ ID NO: 26 - AEP Cleavage Site

[0605] QLGKNEEGA

[0606] SEQ ID NO: 27 - Cathepsin L Cleavage Site

[0607] QKVGKAMYAP

[0608] SEQ ID NO: 28 - Cathepsin B Cleavage Site

[0609] GFLG

[0610] SEQ ID NO: 29 - Cathepsin D Cleavage Site

[0611] TVIVITLVMLKKKQ

[0612] SEQ ID NO: 30 - Cathepsin D Cleavage Site

[0613] PVETDSEEQPYLEMDL

[0614] SEQ ID NO: 31 - Cathepsin D Cleavage Site

[0615] LEGMELIVSQVHPETKENEIYPVWSGLP

[0616] SEQ ID NO: 32 - Cathepsin D Cleavage Site

[0617] QKEYALLYKLDIEP

[0618] SEQ ID NO: 33 - Cathepsin D Cleavage Site

[0619] SLAEEEVVIRSED

[0620] SEQ ID NO:34 - AEP Cleavage Site

[0621] ERNSNLVGAA

[0622] SEQ ID NO:35 - Multiple Endosomal Protease Cleavage Sites

[0623]

[0624] The bold and underlined residues identify the P1 residues within the endosomal protease cleavage sites

[0625] SEQ ID NO:36 - Multiple Endosomal Protease Cleavage Sites

[0626]

[0627] The bold and underlined residues identify the P1 residues within the endosomal protease cleavage sites

[0628] SEQ ID NO:37 - Multiple Endosomal Protease Cleavage Sites

[0629]

[0630] The bold and underlined residues identify the P1 residues within the endosomal protease cleavage sites

[0631] SEQ ID NO:38 - Multiple Endosomal Protease Cleavage Sites

[0632]

[0633] SEQ ID NO:39 - Amino Acid Sequence of Human Cathepsin L1 (UniProt Accession No. P07711)

[0634] MNPTLILAAFCLGIASATLTFDHSLEAQWTKWKAMHNRLYGMNEEGWRRAVWEKNMKMIELHNQEYREGKHSFTMAMNAFGDMTSEEFRQVMNGFQNRKPRKGKVFQEPLFYEAPRSVDWREKGYVTPVKNQGQCGSCWAFSATGALEGQMFRKTGRLISLSEQNLVDCSGPQGNEGCNGGLMDYAFQYVQDNGGLDSEESYPYEATEESCKYNPKYSVANDTGFVDIPKQEKALMKAVATVGPISVAIDAGHESFLFYKEGIYFEPDCSSEDMDHGVLVVGYGFESTESDNNKYWLVKNSWGEEWGMGGYVKMAKDRRNHCGIASAASYPTV

[0635] SEQ ID NO:40 - Amino acid sequence of human cathepsin B (UniProt accession number P07858)

[0636] MWQLWASLCCLLVLANARSRPSFHPLSDELVNYVNKRNTTWQAGHNFYNVDMSYLKRLCGTFLGGPKPPQRVMFTEDLKLPASFDAREQWPQCPTIKEIRDQGSCGSCWAFGAVEAISDRICIHTNAHVSVEVSAEDLLTCCGSMCGDGCNGGYPAEAWNFWTRKGLVSGGLYESHVGCRPYSIPPCEHHVNGSRPPCTGEGDTPKCSKICEPGYSPTYKQDKHYGYNSYSVSNSEKDIMAEIYKNGPVEGAFSVYSDFLLYKSGVYQHVTGEMMGGHAIRILGWGVENGTPYWLVANSWNTDWGDNGFFKILRGQDHCGIESEVVAGIPRTDQYWEKI

[0637] SEQ ID NO:41 - Amino acid sequence of human cathepsin D (UniProt accession number P07339)

[0638] MQPSSLLPLALCLLAAPASALVRIPLHKFTSIRRTMSEVGGSVEDLIAKGPVSKYSQAVPAVTEGPIPEVLKNYMDAQYYGEIGIGTPPQCFTVVFDTGSSNLWVPSIHCKLLDIACWIHHKYNSDKSSTYVKNGTSFDIHYGSGSLSGYLSQDTVSVPCQSASSASALGGVKVERQVFGEATKQPGITFIAAKFDGILGMAYPRISVNNVLPVFDNLMQQKLVDQNIFSFYLSRDPDAQPGGELMLGGTDSKYYKGSLSYLNVTRKAYWQVHLDQVEVASGLTLCKEGCEAIVDTGTSLMVGPVDEVRELQKAIGAVPLIQGEYMIPCEKVSTLPAITLKLGGKGYKLSPEDYTLKVSQAGKTLCLSGFMGMDIPPPSGPLWILGDVFIGRYYTVFDRDNNRVGFAEAARL

[0639] SEQ ID NO:42 - Amino acid sequence of human cathepsin K (UniProt accession number P43235)

[0640] MWGLKVLLLPVVSFALYPEEILDTHWELWKKTHRKQYNNKVDEISRRLIWEKNLKYISIHNLEASLGVHTYELAMNHLGDMTSEEVVQKMTGLKVPLSHSRSNDTLYIPEWEGRAPDSVDYRKKGYVTPVKNQGQCGSCWAFSSVGALEGQLKKKTGKLLNLSPQNLVDCVSENDGCGGGYMTNAFQYVQKNRGIDSEDAYPYVGQEESCMYNPTGKAAKCRGYREIPEGNEKALKRAVARVGPVSVAIDASLTSFQFYSKGVYYDESCNSDNLNHAVLAVGYGIQKGNKHWIIKNSWGENWGNKGYILMARNKNNACGIANLASFPKM

[0641] SEQ ID NO:43 - Amino acid sequence of human cathepsin S (UniProt accession number P25774)

[0642] MKRLVCVLLVCSSAVAQLHKDPTLDHHWHLWKKTYGKQYKEKNEEAVRRLIWEKNLKFVMLHNLEHSMGMHSYDLGMNHLGDMTSEEVMSLMSSLRVPSQWQRNITYKSNPNRILPDSVDWREKGCVTEVKYQGSCGACWAFSAVGALEAQLKLKTGKLVSLSAQNLVDCSTEKYGNKGCNGGFMTTAFQYIIDNKGIDSDASYPYKAMDQKCQYDSKYRAATCSKYTELPYGREDVLKEAVANKGPVSVGVDARHPSFFLYRSGVYYEPSCTQNVNHGVLVVGYGDLNGKEYWLVKNSWGHNFGEEGYIRMARNKGNHCGIASFPSYPEI

[0643] SEQ ID NO:44 - Amino acid sequence of human AEP (UniProt accession number Q99538)

[0644] MVWKVAVFLSVALGIGAVPIDDPEDGGKHWVVIVAGSNGWYNYRHQADACHAYQIIHRNGIPDEQIVVMMYDDIAYSEDNPTPGIVINRPNGTDVYQGVPKDYTGEDVTPQNFLAVLRGDAEAVKGIGSGKVLKSGPQDHVFIYFTDHGSTGILVFPNEDLHVKDLNETIHYMYKHKMYRKMVFYIEACESGSMMNHLPDNINVYATTAANPRESSYACYYDEKRSTYLGDWYSVNWMEDSDVEDLTKETLHKQYHLVKSHTNTSHVMQYGNKTISTMKVMQFQGMKRKASSPVPLPPVTHLDLTPSPDVPLTIMKRKLMNTNDLEESRQLTEEIQRHLDARHLIEKSVRKIVSLLAASEAEVEQLLSERAPLTGHSCYPEALLHFRTHCFNWHSPTYEYALRHLYVLVNLCEKPYPLHRIKLSMDHVCLGHY

[0645] SEQ ID NO:45 (BoNT / A1 - UniProt P10845)

[0646]

[0647]

[0648] In some embodiments, valine 27 may be replaced by alanine, as shown in SEQ ID NO:117.

[0649] The endogenous activation loop is underlined with a dashed line.

[0650] SEQ ID NO:46 (BoNT / A2 - GenBank accession number X73423.1)

[0651]

[0652]

[0653] The endogenous activation loop is underlined with a dashed line.

[0654] SEQ ID NO:47 (BoNT / A3 - GenBank accession number DQ185900.1)

[0655]

[0656]

[0657] The endogenous activation loop is underlined with a dashed line.

[0658] SEQ ID NO:48 (BoNT / A4 - GenBank accession number EU341307.1)

[0659]

[0660]

[0661] The endogenous activation loop is underlined with a dashed line.

[0662] SEQ ID NO:49 (BoNT / A5 - GenBank accession number EU679004.1)

[0663]

[0664]

[0665] The endogenous activation loop is underlined with a dashed line.

[0666] SEQ ID NO:50 (BoNT / A6 - GenBank accession number FJ981696.1)

[0667]

[0668]

[0669] The endogenous activation loop is underlined with a dotted line.

[0670] SEQ ID NO:51 (BoNT / A7 - GenBank accession number JQ954969.1)

[0671]

[0672]

[0673] The endogenous activation loop is underlined with a dotted line.

[0674] SEQ ID NO:52 (BoNT / A8 - GenBank accession number KM233166.1)

[0675]

[0676]

[0677] The endogenous activation loop is underlined with a dotted line.

[0678] SEQ ID NO:53 (BoNT / B1 - UniProt P10844)

[0679]

[0680] The endogenous activation loop is underlined with a dotted line.

[0681] SEQ ID NO:54 (BoNT / B2 - GenBank accession number AB084152.1)

[0682]

[0683] The endogenous activation loop is underlined with a dotted line.

[0684] SEQ ID NO:55 (BoNT / B3 - GenBank accession number EF028400.1)

[0685]

[0686]

[0687] The endogenous activation loop is underlined with a dotted line.

[0688] SEQ ID NO:56 (BoNT / B4 - GenBank accession number EF051570.1)

[0689]

[0690]

[0691] The endogenous activation loop is underlined with a dotted line.

[0692] SEQ ID NO:57 (BoNT / B5 - GenBank accession number EF033130.1)

[0693]

[0694]

[0695] The endogenous activation loop is underlined with a dotted line.

[0696] SEQ ID NO:58 (BoNT / B6 - GenBank accession number AB302852.1)

[0697]

[0698]

[0699] The endogenous activation loop is underlined with a dotted line.

[0700] SEQ ID NO:59 (BoNT / B7 - GenBank accession number JQ354985.1)

[0701]

[0702]

[0703] The endogenous activation loop is underlined with a dotted line.

[0704] SEQ ID NO:60 (BoNT / B8 - GenBank accession number JQ964806.1)

[0705]

[0706]

[0707] The endogenous activation loop is underlined with a dotted line.

[0708] SEQ ID NO:61 (BoNT / C1 - UniProt P18640)

[0709]

[0710]

[0711] The endogenous activation loop is underlined with a dotted line.

[0712] SEQ ID NO:62 (BoNT / D - UniProt P19321)

[0713]

[0714]

[0715] The endogenous activation loop is underlined with a dotted line.

[0716] SEQ ID NO:63 (BoNT / C - GenBank accession number AB200360.1)

[0717]

[0718]

[0719] The endogenous activation loop is underlined with a dotted line.

[0720] SEQ ID NO:64 (BoNT / D - GenBank accession number AB745660.1)

[0721]

[0722]

[0723] The endogenous activation loop is underlined with a dotted line.

[0724] SEQ ID NO:65 (BoNT / E - UniProt Q00496)

[0725]

[0726]

[0727] The endogenous activation loop is underlined with a dotted line.

[0728] SEQ ID NO:66 (BoNT / E1 - GenBank accession number GQ244314.1)

[0729]

[0730]

[0731] The endogenous activation loop is underlined with a dotted line.

[0732] SEQ ID NO:67 (BoNT / E2 - GenBank accession number EF028404.1)

[0733]

[0734] The endogenous activation loop is underlined with a dashed line.

[0735] SEQ ID NO:68 (BoNT / E3 - GenBank accession number EF028403.1)

[0736]

[0737] The endogenous activation loop is underlined with a dashed line.

[0738] SEQ ID NO:69 (BoNT / E4 - GenBank accession number AB088207.1)

[0739]

[0740] The endogenous activation loop is underlined with a dashed line.

[0741] SEQ ID NO:70 (BoNT / E5 - GenBank accession number AB037711.1)

[0742]

[0743]

[0744] The endogenous activation loop is underlined with a dashed line.

[0745] SEQ ID NO:71 (BoNT / E6 - GenBank accession number AM695759.1)

[0746]

[0747]

[0748] The endogenous activation loop is underlined with a dashed line.

[0749] SEQ ID NO:72 (BoNT / E7 - GenBank accession number JN695729.1)

[0750]

[0751]

[0752] The endogenous activation loop is underlined with a dashed line.

[0753] SEQ ID NO:73 (BoNT / E8 - GenBank accession number JN695730.1)

[0754]

[0755]

[0756] The endogenous activation loop is underlined with a dashed line.

[0757] SEQ ID NO:74 (BoNT / E9 - GenBank accession number JX424534.1)

[0758]

[0759]

[0760] The endogenous activation loop is underlined with a dashed line.

[0761] SEQ ID NO:75 (BoNT / E10 - GenBank accession number KF861917.1)

[0762]

[0763]

[0764] The endogenous activation loop is underlined with a dashed line.

[0765] SEQ ID NO:76 (BoNT / E11 - GenBank accession number KF861875.1)

[0766]

[0767]

[0768] The endogenous activation loop is underlined with a dashed line.

[0769] SEQ ID NO:77 (BoNT / E12 - GenBank accession number KM370319.1)

[0770]

[0771]

[0772] The endogenous activation loop is underlined with a dashed line.

[0773] SEQ ID NO:78 (BoNT / F1 - UniProt A7GBG3)

[0774]

[0775]

[0776] The endogenous activation loop is underlined with a dotted line.

[0777] SEQ ID NO:79 (BoNT / F2 - GenBank accession number GU213209.1)

[0778]

[0779]

[0780] The endogenous activation loop is underlined with a dotted line.

[0781] SEQ ID NO:80 (BoNT / F3 - GenBank accession number GU213227.1)

[0782]

[0783]

[0784] The endogenous activation loop is underlined with a dotted line.

[0785] SEQ ID NO:81 (BoNT / F4 - GenBank accession number GU213214.1)

[0786]

[0787]

[0788] The endogenous activation loop is underlined with a dotted line.

[0789] SEQ ID NO:82 (BoNT / F5 - GenBank accession number GU213211.1)

[0790]

[0791]

[0792] The endogenous activation loop is underlined with a dotted line.

[0793] SEQ ID NO:83 (BoNT / F6 - GenBank accession number M92906.1)

[0794]

[0795] The endogenous activation loop is underlined with a dotted line.

[0796] SEQ ID NO:84 (BoNT / F7 - GenBank accession number GU213233.1)

[0797]

[0798] The endogenous activation loop is underlined with a dotted line.

[0799] SEQ ID NO:85 (BoNT / G - UniProt Q60393)

[0800]

[0801]

[0802] The endogenous activation loop is underlined with a dotted line.

[0803] SEQ ID NO:86 (BoNT / F A - GenBank accession number KGO15617.1)

[0804]

[0805]

[0806] The endogenous activation loop is underlined with a dotted line.

[0807] SEQ ID NO:87 (polypeptide sequence of BoNT / X)

[0808]

[0809]

[0810] The endogenous activation loop is underlined with a dotted line.

[0811] SEQ ID NO:88 (TeNT - UniProt P04958)

[0812]

[0813]

[0814] The endogenous activation loop is underlined with a dotted line.

[0815] SEQ ID NO:89 (activation loop of BoNT / D)

[0816] CLRLTKNSRDDSTC

[0817] SEQ ID NO:90 (activation loop of BoNT / DC)

[0818] CLRLTRNSRDDSTC

[0819] SEQ ID NO:91 (activation loops of BoNT / C1 and CD)

[0820] CHKAIDGRSLYNKTLDC

[0821] SEQ ID NO:92 (BoNT / A4 activation loop)

[0822] CVRGIITSKTKSLDEGYNKALNELC

[0823] SEQ ID NO:93 (BoNT / A5 and A7 activation loops)

[0824] CVRGIITSKTKSLDEGYNKALNDLC

[0825] SEQ ID NO:94 (BoNT / A1 and A6 activation loops)

[0826] CVRGIITSKTKSLDKGYNKALNDLC

[0827] SEQ ID NO:95 (BoNT / A3 activation loop)

[0828] CVRGIIPFKTKSLDEGYNKALNYLC

[0829] SEQ ID NO:96 (BoNT / A2 and A8 activation loops)

[0830] CVRGIIPFKTKSLDEGYNKALNDLC

[0831] SEQ ID NO:97 (BoNT / H activation loop)

[0832] CSNSNTKNSLC

[0833] SEQ ID NO:98 (BoNT / E1 to E5, E9 and E12 activation loops)

[0834] CKNIVSVKGIRKSIC

[0835] SEQ ID NO:99 (BoNT / E11 activation loop)

[0836] CTNIFSPKGIRKSIC

[0837] SEQ ID NO:100 (BoNT / E7, E8 and E10 activation loops)

[0838] CKNIVFSKGITKSIC

[0839] SEQ ID NO:101 (BoNT / E6 activation loop)

[0840] CKNIVFSKGIRKSIC

[0841] SEQ ID NO:102 (BoNT / F7 activation loop)

[0842] CKSIVSKKGTKNSLC

[0843] SEQ ID NO:103 (BoNT / F5 activation loop)

[0844] CLNSSFKKNTKKPLC

[0845] SEQ ID NO:104 (BoNT / F1 and F6 activation loops)

[0846] CKSVIPRKGTKAPPRLC

[0847] SEQ ID NO:105 (BoNT / F4 activation loop)

[0848] CKSIIPRKGTKAPPRLC

[0849] SEQ ID NO:106 (BoNT / F2 and F3 activation loops)

[0850] CKSIIPRKGTKQSPSLC

[0851] SEQ ID NO:107 (TeNT activation loop)

[0852] CKKIIPPTNIRENLYNRTASLTDLGGELC

[0853] SEQ ID NO:108 (BoNT / G activation loop)

[0854] CKPVMYKNTGKSEQC

[0855] SEQ ID NO:109 (BoNT / B4 activation loop)

[0856] CKSVKVPGIC

[0857] SEQ ID NO:110 (BoNT / B2, B3, B6 and B8 activation loops)

[0858] CKSVRAPGIC

[0859] SEQ ID NO:111 (BoNT / B1, B5 and B7 activation loops)

[0860] CKSVKAPGIC

[0861] SEQ ID NO:112 (BoNT / X Activation Loop)

[0862] CPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTNVSYPCSLLNGC

[0863] SEQ ID NO:113 Metal-Coordinating SNARE Cleavage Motif

[0864] HEXXH

[0865] SEQ ID NO:114 (TEV Cleavage Site)

[0866] ENLYFQG

[0867] SEQ ID NO:115 (Thrombin Cleavage Site)

[0868] LVPRGS

[0869] SEQ ID NO:116 (PreScission Cleavage Site)

[0870] LEVLFQGP

[0871] SEQ ID NO:117 (BoNT / A GenBank Accession No. AF488749.1)

[0872]

[0873]

[0874] The endogenous activation loop is underlined with a dotted line.

[0875] SEQ ID NO:118: Non-Engineered BoNT / AB Chimera

[0876]

[0877]

[0878] The SytII-binding mutations E1191M and S1199Y are bold and underlined.

[0879] The endogenous activation loop is underlined with a dotted line.

[0880] SEQ ID NO:119: LC / A1-Cloop-H N / A1

[0881]

[0882]

[0883] LC / A1 is italicized

[0884] The BoNT / C activation loop is bolded and underlined

[0885] H N / A1 is at the C-terminus of the C activation loop and is neither underlined nor italicized

[0886] SEQ ID NO:120: LC / X-Cloop-H N / X

[0887]

[0888]

[0889] LC / X is italicized

[0890] The BoNT / C activation loop is bolded and underlined

[0891] H N / X is at the C-terminus of the C activation loop and is neither underlined nor italicized

[0892] The 2x AP linker is underlined with a dotted line

[0893] SEQ ID NO:121: LC / X-EndoSite-H N / X

[0894]

[0895]

[0896] LC / X is italicized

[0897] The EndoSite activation loop is bolded and underlined

[0898] H N / X is at the C-terminus of the C activation loop and is neither underlined nor italicized

[0899] The 2x AP linker is underlined with a dotted line

[0900] SEQ ID NO:122 (Additional protease cleavage site)

[0901] xDxxxLL

[0902] x is any amino acid

[0903] SEQ ID NO:123 (Additional protease cleavage site)

[0904] xExxxLL

[0905] x is any amino acid

[0906] SEQ ID NO:124 (Additional protease cleavage site)

[0907] xExxxIL

[0908] x is any amino acid

[0909] SEQ ID NO:125 (Additional protease cleavage site)

[0910] xExxxLM

[0911] x is any amino acid

[0912] SEQ ID NO:126 (Influenza virus hemagglutinin translocation domain)

[0913] GLFGAIAGFIENGWEGMIDGWYG

[0914] SEQ ID NO:127 (Exemplary Endosite exogenous activation loop)

[0915] AEP cleavage sites (QEAANERQQ, PDLKNVKS, and SGLTNIKTE) are shown in bold

[0916] CathB cleavage sites (DLFGFVGL and GFVGLFRG) are double underlined

[0917] CathL cleavage sites (QAKKDFFSSHPLREPVNATED, ELVTPARD, RDFGHFGL, and GLFRGHHP) are dot underlined

[0918] SEQ ID NO:128 (Exemplary Endosite exogenous activation loop)

[0919] AEP cleavage sites (QLGKNEEG and SGLTNIKTE) are shown in bold

[0920] CathB cleavage sites (GLFGFVGL and GFVGLFRG) are double underlined

[0921] CathL cleavage sites (GLFRGHHP, ELVTPARD, and RDFGHFGL) are dot underlined

[0922] SEQ ID NO:129 (Exemplary Endosite exogenous activation loop)

[0923]

[0924] The AEP cleavage sites (PGGGNKKIE and PDLKNVKSK) are shown in bold

[0925] The CathB cleavage sites (DLFGFVGL and GFVGLFRG) are double underlined

[0926] The CathL cleavage sites (GLFRGHHP and ELVTPARD) are underlined

[0927] SEQ ID NO:130 (Exemplary endosomal protease (AEP) cleavage site)

[0928] PDLKNVKS

[0929] SEQ ID NO:131 (Exemplary endosomal protease (CathB) cleavage site)

[0930] DLFGFVGL

[0931] SEQ ID NO:132 (Exemplary endosomal protease (CathB) cleavage site)

[0932] GFVGLFRG

[0933] SEQ ID NO:133 (Exemplary endosomal protease (CathB) cleavage site)

[0934] GSGLFGFVGGSG

[0935] SEQ ID NO:134 (Exemplary endosomal protease (CathB) cleavage site)

[0936] LFGFVGLFGFVG

[0937] SEQ ID NO:135 (Exemplary endosomal protease (CathB) cleavage site)

[0938] LFGFVGLFGFVGLFGFVG

[0939] SEQ ID NO:136 (Exemplary endosomal protease (CathB) cleavage site)

[0940] GLFGFVGL

[0941] SEQ ID NO:137 (Exemplary endosomal protease (CathL) cleavage site)

[0942] QAKKDFFSSHPLREPVNATED

[0943] SEQ ID NO:138 (Exemplary Endosomal Protease (CathL) Cleavage Site)

[0944] ELVTPARD

[0945] SEQ ID NO:139 (Exemplary Endosomal Protease (CathL) Cleavage Site)

[0946] RDFGHFGL

[0947] SEQ ID NO:140 (Exemplary Endosomal Protease (CathL) Cleavage Site)

[0948] GLFRGHHP

[0949] SEQ ID NO:141 (Exemplary Endosomal Protease (CathL) Cleavage Site)

[0950] GSGLFRGHHPDGSG

[0951] SEQ ID NO:142 (Exemplary Endosomal Protease (CathL) Cleavage Site)

[0952] LFRGHHPDLFRGHHPD

[0953] SEQ ID NO:143 (Exemplary Endosomal Protease (CathL) Cleavage Site)

[0954] ELVTPARDFGHFGLS

[0955] SEQ ID NO:144 (Exemplary Endosomal Protease (CathL) Cleavage Site)

[0956] QAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFLGTNE

[0957] SEQ ID NO:145 (Exemplary Endosomal Protease (CathL) Cleavage Site)

[0958] LFRGHHPDSTSQKSIVAYTMSLGADSS

[0959] SEQ ID NO:146 (Exemplary Endosomal Protease (CathL) Cleavage Site)

[0960] STSQKSIVAYTMSLGADSSLFRGHHPD

[0961] SEQ ID NO:147 (Exemplary Endosomal Protease (CathL) Cleavage Site)

[0962] STSQKSIVAYTMSLGADSSSTSQKSIVAYTMSLGADSS

[0963] SEQ ID NO:148 (Exemplary endosomal protease (CathL) cleavage site)

[0964] LFRGHHPDLFRGHHPDLFRGHHPD

[0965] SEQ ID NO:149 (Exemplary endosomal protease (CathL) cleavage site)

[0966] ELVTPARDFGHFGLSELVTPARDFGHFGLS

[0967] SEQ ID NO:150 (Exemplary endosomal protease (CathL) cleavage site)

[0968] STSQKSIVAYTMSLGADSSELVTPARDFGHFGLSLFRGHHPD

[0969] SEQ ID NO:151 (Exemplary endosomal protease (AEP) cleavage site)

[0970] QLGKNEEG

[0971] SEQ ID NO:152 (Exemplary endosomal protease (CathD) cleavage site)

[0972] GERGFFYTPKT

[0973] SEQ ID NO:153 GS spacer consensus sequence

[0974] (Gly-Gly-Gly-Gly-Ser) n

[0975] SEQ ID NO:154 GS5 spacer

[0976] GGGGS

[0977] SEQ ID NO:155 GS10 spacer

[0978] GGGGSGGGGS

[0979] SEQ ID NO:156 GS15 spacer

[0980] GGGGSGGGGSGGGGS

[0981] SEQ ID NO:157 GS20 spacer

[0982] GGGGSGGGGSGGGGSGGGGS

[0983] SEQ ID NO:158 GS25 Spacer

[0984] GGGGSGGGGSGGGGSGGGGSGGGGS

[0985] SEQ ID NO:159 LH N / A1-H C Exemplary Engineered Forms of B1 Chimeras

[0986]

[0987]

[0988] LC / A is italicized

[0989] The EndoSite Activation Loop is bolded and underlined

[0990] H N / A is at the C-terminus of the EndoSite Activation Loop and is neither underlined nor italicized

[0991] SEQ ID NO:160: Exemplary LC / X-EndoSite-H N / X

[0992]

[0993]

[0994] LC / X is italicized

[0995] The EndoSite Activation Loop is bolded and underlined

[0996] H N / X is at the C-terminus of the C Activation Loop and is neither underlined nor italicized

[0997] 2x AP Linker is dotted underlined

[0998] SEQ ID NO:161: Exemplary LC / X-EndoSite-H N / X

[0999]

[1000] LC / X is italicized

[1001] The EndoSite Activation Loop is bolded and underlined

[1002] H N / X is at the C-terminus of the C activation loop and is neither underlined nor italicized

[1003] The 2x AP linker is underlined with a dotted line

[1004] SEQ ID NO:162: Exemplary LC / X-EndoSite-H N / X

[1005]

[1006] LC / X is italicized

[1007] The EndoSite activation loop is bold and underlined

[1008] H N / X is at the C-terminus of the C activation loop and is neither underlined nor italicized

[1009] The 2x AP linker is underlined with a dotted line

[1010] SEQ ID NO:163: Nucleic acid encoding LC / X-Cloop-H N Exemplary nucleic acid encoding / X

[1011]

[1012]

[1013] The nucleic acid encoding Cloop is bold and underlined

[1014] The nucleic acid encoding the 2xAP linker is underlined with a dotted line

[1015] SEQ ID NO:164: Exemplary nucleic acid Cloop

[1016] tgtcataaagccattgatggtcgcagcctgtataacaaaaccctggat

[1017] SEQ ID NO:165: Exemplary nucleic acid encoding the exogenous activation loop of SEQ ID NO:127

[1018] TGTCAGGAAGCCGCTAACGAGAGACAGCAGGCCAAGAAAGACTTTTTTAGTAGTCATCCTCTGAGAGAA CCCGTGAATGCGACTGAAGATCCCGATCTGAAGAACGTTAAAAGCGGGCTCACAAATATCAAAACCGAGCTCGTTAC CCCGGCTCGTGACCTGTTTGGCTTCGTAGGATTATTCCGTGGACACCATCCAGACTGC

[1019] SEQ ID NO:166: Exemplary nucleic acid encoding the exogenous activation loop of SEQ ID NO:128

[1020] TGCCAGTTAGGTAAAAACGAAGAAGGCTTGTTTGGCTTCGTCGGTCTTTTTAGAGGCCACCATCCGGAC GAACTTGTTACTCCTGCGCGCGATTTTGGGCATTTCGGTCTGTCTGGACTCACCAATATTAAAACCGAATGT

[1021] SEQ ID NO:167: Exemplary nucleic acid encoding the exogenous activation loop of SEQ ID NO:129

[1022] TGTCCTGGTGGGGGTAACAAGAAAATTGAGCTCGTCACCCCCGCGCGTGATCTGTTTGGATTTGTTGGG CTGTTCCGTGGCCACCATCCTGACTTAAAAAACGTTAAAAGCAAATGT

[1023] SEQ ID NO:168: Exemplary nucleic acid encoding an engineered retargeted BoNT / X comprising the exogenous activation loop of SEQ ID NO:127

[1024]

[1025]

[1026] The nucleic acid encoding Cloop is in bold and underlined

[1027] The nucleic acid encoding the 2xAP linker is underlined with a dotted line

[1028] SEQ ID NO:169: Exemplary nucleic acid encoding an engineered retargeted BoNT / X comprising the exogenous activation loop of SEQ ID NO:128

[1029]

[1030] The nucleic acid encoding Cloop is in bold and underlined

[1031] The nucleic acid encoding the 2xAP linker is underlined with a dotted line

[1032] SEQ ID NO:170: Exemplary nucleic acid encoding an engineered retargeted BoNT / X comprising the exogenous activation loop of SEQ ID NO:129

[1033]

[1034]

[1035] The nucleic acid encoding Cloop is in bold and underlined

[1036] The nucleic acid encoding the 2xAP linker is underlined with a dotted line

[1037] SEQ ID NO:171: Exemplary cathepsin L cleavage site

[1038] GYYSTTIRYQATGFGTNE

[1039] SEQ ID NO:172: Exemplary cathepsin L cleavage site

[1040] LFRGHHPD

[1041] SEQ ID NO:173: Exemplary cathepsin L cleavage site

[1042] GLFRGHHPD

[1043] SEQ ID NO:174: Exemplary cathepsin L cleavage site

[1044] QAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNEP

[1045] SEQ ID NO:175: Exemplary cathepsin L cleavage site

[1046] QAKKDFFSSHPL

[1047] SEQ ID NO:176: Exemplary cathepsin L cleavage site

[1048] REPVNATEDPSSGYYS

[1049] SEQ ID NO:177: Exemplary cathepsin L cleavage site

[1050] TTIRYQATGFGTNE

[1051] SEQ ID NO:178: Exemplary cathepsin L cleavage site

[1052] TTIRYQATGFGTNEP

[1053] SEQ ID NO:179: Exemplary cathepsin L cleavage site

[1054] EVDLLIGSS

[1055] SEQ ID NO:180: Exemplary cathepsin L cleavage site

[1056] EVDLLIGSSGE

[1057] SEQ ID NO:181: Exemplary cathepsin B cleavage site

[1058] GLAGFLGG

[1059] SEQ ID NO:182: Exemplary cathepsin B cleavage site

[1060] GLFGFVGG

[1061] SEQ ID NO:183: Exemplary cathepsin B cleavage site

[1062] TVGSFGFE

[1063] SEQ ID NO:184: Exemplary cathepsin B cleavage site

[1064] TVGSFGFEGG

[1065] SEQ ID NO:185: Exemplary cathepsin D cleavage site

[1066] LASLLELPEFLLFLQ

[1067] SEQ ID NO:186: Exemplary cathepsin D cleavage site

[1068] GLTTELFSPVD

[1069] SEQ ID NO:187: Exemplary AEP cleavage site

[1070] LERNSNLVGAA

[1071] SEQ ID NO:188: Core cathepsin L cleavage motif

[1072] MSLGADSS

[1073] SEQ ID NO:189: Core cathepsin L cleavage motif

[1074] LFRGHHP

[1075] SEQ ID NO:190: Core cathepsin L cleavage motif

[1076] GLFRGHHP

[1077] SEQ ID NO:191: Core cathepsin L cleavage motif

[1078] KDFFSSHP

[1079] SEQ ID NO:192: Core cathepsin L cleavage motif

[1080] EPVNATED

[1081] SEQ ID NO:193: Core cathepsin L cleavage motif

[1082] TGFGTNE

[1083] SEQ ID NO:194: Core cathepsin L cleavage motif

[1084] TGFGTNEP

[1085] SEQ ID NO:195: Core cathepsin L cleavage motif

[1086] QKVGKAMY

[1087] SEQ ID NO:196: Core cathepsin L cleavage motif

[1088] LLIGSS

[1089] SEQ ID NO:197: Core cathepsin L cleavage motif

[1090] LLIGSSGE

[1091] SEQ ID NO:198: Core cathepsin B cleavage motif

[1092] GSFGFE

[1093] SEQ ID NO:199: Core cathepsin B cleavage motif

[1094] GSFGFEGG

[1095] SEQ ID NO:200: Core cathepsin D cleavage motif

[1096] VEKLLELK

[1097] SEQ ID NO:201: Core cathepsin D cleavage motif

[1098] VITLVMLK

[1099] SEQ ID NO:202: Core cathepsin D cleavage motif

[1100] GMELIVSQ

[1101] SEQ ID NO:203: Core cathepsin D cleavage motif

[1102] QPYLEMDL

[1103] SEQ ID NO:204: Core cathepsin D cleavage motif

[1104] EYALLYKL

[1105] SEQ ID NO:205: Core cathepsin D cleavage motif

[1106] LAEEEVVI

[1107] SEQ ID NO:206: Core cathepsin D cleavage motif

[1108] LASLLELP

[1109] SEQ ID NO:207: Core cathepsin D cleavage motif

[1110] TTELFSPV

[1111] SEQ ID NO:208: Core AEP cleavage motif

[1112] EAANERQQ

[1113] SEQ ID NO:209: Core AEP cleavage motif

[1114] GLTNIKTE

[1115] SEQ ID NO:210: Core AEP cleavage motif

[1116] DLKNVKSK

[1117] SEQ ID NO:211: Core AEP cleavage motif

[1118] GGGNKKIE

[1119] SEQ ID NO:212: Core AEP cleavage motif

[1120] LGKNEEGA

[1121] SEQ ID NO:213: Core AEP cleavage motif

[1122] ERNSNLV

[1123] SEQ ID NO:214: Core AEP cleavage motif

[1124] LERNSNLV

[1125] Example

[1126] The present invention will be further illustrated by the following examples, which are intended to exemplify the invention only and in no way limit the invention. In addition to the data presented below, the contents of PCT / GB2022 / 050756, particularly the examples section, are incorporated herein by reference. Although this case involves an engineered Clostridium neurotoxin containing a furin cleavage site rather than an endosomal protease cleavage site, it contains data obtained from an engineered Clostridium neurotoxin having common features with the engineered Clostridium neurotoxin of the present invention, as well as related synthetic methods.

[1127] Example 1 - Design and generation of BoNT engineered to contain an endoprotease cleavage site

[1128] Engineered BoNTs are produced by determining the required amino acid sequence, reverse translating it into the corresponding nucleic acid sequence, and then performing codon optimization for recombinant expression in bacteria.

[1129] The resulting gene sequence is examined to ensure that common restriction sites (NdeI, XhoI, BamHI, HindIII, NcoI, and EcoRI) are not present in the sequence. An initiation codon is added at the 5' end, a His tag (optionally cleavable) and a termination codon are added at 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) are added to enable subcloning into an expression vector.

[1130] The gene sequence of the engineered BoNT is then subcloned into the pK8 vector (which contains a kanamycin resistance gene, a T7 promoter, a T7 terminator, an origin of replication from pBR322, and a multiple cloning site). The plasmid of each engineered BoNT is then amplified in the Escherichia coli strain DH5α under kanamycin selection and extracted by miniprep using standard molecular biology techniques.

[1131] An Escherichia coli expression strain BL21 carrying λDE3 is then transformed with the plasmid DNA, spread onto agar supplemented with kanamycin, and incubated overnight at 37°C. The colonies are then harvested and used to prepare a glycerol stock. A 100 mL modified TB medium supplemented with kanamycin is then inoculated using a stab, and then incubated overnight at 37°C, shaken at 225 RPM to provide aeration. Several baffled conical flasks, each containing up to 1 L of the same nutrient medium and antibiotic, are inoculated with 10 mL of this starter culture. The cultures are grown under the same conditions for several hours until the optical density (A600) ≥ 0.6, and then the incubator temperature is set to 16°C. One hour later, the cultures are induced to express the engineered BoNT by adding IPTG. After 20 hours, the cells are harvested by centrifugation and stored at -80°C until use.

[1132] Thaw the cells in 50 mM Tris pH 7.4 containing 0.25 M NaCl (5 mL / g cells) and solubilize at 4 °C by passing twice through a cell homogenizer at 20 kPSI or by sonication (10 x 30 s on / off). Remove cell debris by centrifugation and load the clarified supernatant onto a nickel affinity column pre-equilibrated with 50 mM Tris pH 7.4 containing 0.5 M NaCl (“Buffer NA”) using an FPLC system (GE). Wash the column with Buffer NA until A280 reaches a stable baseline. Collect the wash and eluted proteins from the column over 25 column volumes (CV) using a linear gradient of 0 - 0.5 M imidazole in Buffer NA, collecting 3 mL fractions

[1133] All collected materials were stored at 4 °C while analyzing the samples by stained SDS PAGE (Invitrogen). Pool the fractions showing a strong protein band at the MW of the target molecule calculated according to the protein marker and measure the total protein concentration using a Nanodrop (Thermo Fisher).

[1134] Desalt the pooled fractions 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 to a stable baseline, elute the protein using a linear gradient of 0 - 0.5 M NaCl in Buffer QA over 25 CV. Analyze the samples by SDS-PAGE and desalt the fractions containing the pure target into 50 mM HEPES pH 7.2 containing 150 mM NaCl, then aliquot and store at -80 °C. Analysis of the final product sample in the presence and absence of DTT by SDS PAGE showed a single band

[1135] The above method was used to generate three engineered BoNTs with the structure LC / A1-Cloop-H N / A1-EndoSite-TM-His6: EndoSite = CatDReo (BIO4934), Ebo (BIO4935), and CatBL (BIO4945). All three of these engineered BoNTs contain the same targeting moiety (TM), BoNT / A1 light chain (LC / A1), and BoNT / A1 translocation domain (H N / A1), but have different endosomal protease cleavage sites and their combinations. The pre-engineered LC / A1-Cloop-H of each of these engineered BoNTs NThe / A1 sequence is SEQ ID NO: 119. BIO4934 contains an endoprotease cleavage site of SEQ ID NO: 35, which itself contains one cleavage site of cathepsin D and two cleavage sites of cathepsin L. BIO4935 contains an endoprotease cleavage site of SEQ ID NO: 36, which itself contains two cleavage sites of cathepsin L. BIO4945 contains an endoprotease cleavage site of SEQ ID NO: 37, which itself contains one cleavage site of cathepsin B and two cleavage sites of cathepsin L.

[1136] By pH control, samples of each of CatDReo (BIO4934), Ebo (BIO4935), and CatBL (BIO4945) were incubated in 1 mM DTT with PBS at pH 7.2 or 50 mM MES at pH 5 without the corresponding endoprotease at room temperature for 3 hours, and then reduced for analysis.

[1137] As Figure 1 shown in N A, when electrophoresed on Coomassie blue gels, for CatDReo (BIO4934), Ebo (BIO4935), and CatBL (BIO4945), the bands corresponding to the full-length engineered BoNT were darker at pH 7.2 compared to pH 5. At both pH values, the intensities of the LC / A1 bands and the H

[1138] / A1-EndoSite-TM-His6 bands were similar. Weak bands were observed at >20 kDa in CatDReo (BIO4934), Ebo (BIO4935), and CatBL (BIO4945), indicating the presence of impurities. Notably, no visible bands were present at approximately 15 kDa (at pH 7.2 or pH 5), indicating that cleavage at the EndoSite was zero / below the limit of detection / negligible for protein detection in the absence of the corresponding endoprotease. Figure 1 B) and anti-His tag (data not shown) antibodies. As Figure 1 shown in N B, using the anti-LC / A1 antibody, there was no difference in the signal intensities of the full-length band or the LC / A1 band and the H N / A1-EndoSite-TM-His6 had cross-reactivity, most likely due to overexposure of the Western blot. Nevertheless, no visible band was observed at approximately 15 kDa (at pH 7.2 or pH 5), indicating that cleavage at EndoSite was zero / below the limit of detection / negligible in the absence of the corresponding endosomal protease.

[1139] Using anti-His tag antibody, it was again confirmed that buffer pH had no significant effect on CatDReo (BIO4934), Ebo (BIO4935), and CatBL (BIO4945). Some faint bands were observed at <20 kDa. It was hypothesized that these bands might be artifactual cross-reactivity or impurities, or trace truncated products that could only be detected by overexposed Western blots.

[1140] Example 2 - BoNT engineered to contain a cathepsin L1 cleavage site can be effectively cleaved by cathepsin L1 Cleaved

[1141] Three engineered BoNTs (LC / A1-Cloop-H N / A1-EndoSite-TM-His6, where EndoSite = CatDReo (BIO4934), Ebo (BIO4935), or CatBL (BIO4945)) generated in Example 1 were studied.

[1142] The ability of cathepsin L to cleave BIO4934, BIO4935, and BIO4945 was tested by incubating 90 μg / mL mg / mL of each engineered BoNT with serial dilutions of cathepsin L1 at room temperature in 50 mM MES pH 5 for 2 hours, reducing with DTT, and resolving by SDS PAGE for Coomassie blue staining and Western blot analysis.

[1143] BIO4934, BIO4935, and BIO4945 were each approximately 118 kDa (composed of approximately 100 kDa LH N / A + approximately 18 kDa TM-HT).

[1144] BIO4934, BIO4935, and BIO4945 were all sensitive to cathepsin L, and after cleavage, pairs of approximately 50 kDa (LC / A and H N / A upon reduction) bands and >15 kDa TM-HT bands were generated. Cleavage of each of the BIO4934, BIO4935, and BIO4945 engineered BoNTs was concentration-dependent, and a certain degree of cleavage could be achieved even with low concentrations of cathepsin L1 (14 ng / mL). Exemplary data for BIO4934 and BIO4935 are shown respectively inFigure 2A and 2B in

[1145] Thus, these data indicate that cathepsin L can be successfully used to cleave and thus activate engineered BoNTs BIO4934, BIO4935, and BIO4945.

[1146] Example 3 - BoNT engineered to contain a cathepsin B cleavage site can be effectively cleaved by cathepsin B Cleaved

[1147] The sensitivity of CatBL (BIO4945) engineered BoNT generated in Example 1 to cathepsin B was investigated.

[1148] The ability of cathepsin B to cleave BIO4945 was tested by incubating 0.3 mg / mL CatBL (BIO4945) with serial dilutions of cathepsin B at room temperature in 50 mM MES pH 5 for 2 h, reducing with DTT, and resolving by SDS PAGE for Coomassie blue staining and Western blot analysis.

[1149] As Figure 3 shown, BIO4945 is sensitive to cathepsin B, and cleavage generates paired bands of approximately 50 kDa (LC / A and H N / A upon reduction) and >15 kDa TM-HT bands. Cleavage of BIO4945 engineered BoNT is concentration-dependent, and even low concentrations of cathepsin B (5 ng / mL) can achieve a certain degree of cleavage. Thus, these data indicate that cathepsin B can be successfully used to cleave and thus activate engineered BoNT BIO4935.

[1150] Example 4 - BoNT engineered to contain an asparaginyl endopeptidase cleavage site can be effectively cleaved by asparaginyl endopeptidase Cleaved

[1151] Another engineered BoNT (LC / A1-Cloop-H N / A1) with the same TM, LC / A1, and H N / A1 as described in Examples 1 to 3, but with an AEP cleavage site (LC / A1-Cloop-H

[1152] / A1-EndoSite-TM-His6, where EndoSite = AEP (BIO4938)) was generated and studied. BIO4938 contains the endoprotease cleavage site of SEQ ID NO:38, which itself contains five AEP cleavage sites.

[1153] 150 μg / mL BIO4938 was incubated with serial dilutions of AEP at room temperature in 50 mM MES pH 5 for approximately 2 h and reduced for analysis.BIO4938 is approximately 119 kDa (composed of approximately 100 kDa LH N / A+ approximately 19 kDa AEP-TM-HT).

[1154] As Figure 4 shown, BIO4938 is sensitive to AEP and upon cleavage yields paired bands of approximately 50 kDa (LC / A and H N / A upon reduction) and >15 kDa TM-HT bands. Cleavage of engineered BoNT by BIO4938 is concentration-dependent and even low concentrations of AEP B (7 ng / mL) can achieve a certain degree of cleavage.

[1155] Example 5 - Design and generation of a retargeted BoNT / X with an endogenously activated loop engineered to contain an endoprotease cleavage site ​

[1156] Repeat the method of Example 1 to generate engineered BoNTs derived from re-targeted BoNT / X molecules. Engineered BoNT / X is derived from re-targeted BoNT / X having the structure LC / X-Cloop-H N / X-2xAP linker-TM-tag, where LC / X-Cloop-H N / X-2xAP linker has the sequence shown in SEQ ID NO: 120 and the tag is a cleavable affinity purification tag, such as a His-tag. Alternatively, engineered BoNT / X is derived from re-targeted BoNT / X having an N-terminal tag, which has the structure tag-LC / X-Cloop-H N / X-2xAP linker-TM. The tag is optionally cleavable. The BoNT / C activation loop is replaced with a selected EndoSite activation loop (e.g., SEQ ID NOs: 127 - 129). Thus, the engineered re-targeted BoNT / X has the structure LC / X-EndoSite-H N / X-2xAP linker-TM-tag or tag-LC / X-EndoSite-H N / X-2xAP linker-TM, where LC / X-EndoSite-H N / X-2xAP linker has the sequences shown in SEQ ID NOs: 160 - 162. Thus, LC / X-EndoSite-H N / X-2xAP is sensitive to one or more of AEP, cathepsin B, and / or cathepsin L.

[1157] A set concentration of LC / X-Cloop-H N / X-2xAP linker-TM-tag or tag-LC / X-EndoSite-H N / X-2xAP linker-TM was treated with serial dilutions of AEP, cathepsin B, or cathepsin L and incubated for 2 hours at room temperature. The samples were reduced with DTT and analyzed by SDS PAGE and western blotting (anti-tag) to evaluate cleavage of EndoSite by AEP, cathepsin B, or cathepsin L compared to untreated samples. Protein gels showed a decrease in intensity of the single-chain single band and the appearance of two smaller bands representing the active double-strand with an increase in intensity. Anti-tag blots showed similar results except that the smaller of the two bands was missing as this was the unlabeled LC.

[1158] Target cells (e.g., primary cortical neurons) were treated with serial dilutions of LC / X-Cloop-H N / X-2xAP linker-TM-tag or tag-LC / X-EndoSite-H N / X-2xAP linker-TM and incubated for 24 hours. Thereafter, the cells were harvested and lysed with 1xNuPAGE buffer, DTT, and Benzonase. The lysates were then analyzed by western blotting to determine if the substrate was cleaved by LC / X (e.g., VAMP2, VAMP4, or Ykt6), and the disappearance of the substrate band and the appearance of the cleavage fragment band were measured by densitometry. The amount of cleaved substrate was expressed as a percentage of the total amount of uncleaved and cleaved substrate, and the target molecule concentration (EC50) required to cause half-maximal cleavage of the substrate was calculated by non-linear regression. This was compared to the cleavage activity of the corresponding molecule without EndoSite (e.g., Cloop, LC / X-Cloop-H N / X-2xAP linker-TM-tag or native Xloop, LC / X-Xloop-H N / X-2xAP linker-TM-tag or tag-LC / X-Xloop-H N / X-2xAP linker-TM).

[1159] Single-chain LC / X-EndoSite-H N / X-2xAP linker-TM-tag or tag-LC / X-EndoSite-H N / X-2xAP linker-TM showed cleavage of the substrate, while the corresponding single-chain non-EndoSite re-targeted BoNT / X (LC / X-Cloop-H N / X-2xAP linker-TM-tag, tag-LC / X-Cloop-H N / X-2xAP linker-TM, LC / X-Xloop-H N / X-2xAP linker-TM-tag or tag-LC / X-Xloop-HN / X-2xAP joint - TM) showed no lysis or minimal lysis.

[1160] All publications mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the methods and systems described herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications to the manner in which the invention is practiced will be apparent to those skilled in biochemistry and biotechnology or related fields and are intended to be within the scope of the following claims.

Claims

1. An engineered Clostridial neurotoxin, the engineered Clostridial neurotoxin comprising an endosomal protease cleavage site, wherein cleavage at the cleavage site gives rise to the production of a double-stranded form of the engineered Clostridial neurotoxin.

2. The engineered Clostridial neurotoxin according to claim 1, wherein the endosomal protease cleavage site is a cleavage site specific for the following enzyme: (a) asparaginyl endopeptidase (AEP); or (b) cathepsin, optionally cathepsin L1, B, D, K or S.

3. The engineered Clostridial neurotoxin according to claim 1 or 2, wherein the endosomal protease cleavage site comprises or consists of the following: (a) an AEP core motif selected from SEQ ID NO: 208, 209, 210, 211, 212, 213 and / or 214; (b) a cathepsin L core motif selected from SEQ ID NO: 138, 188, 189, 190, 191, 192, 193, 194, 195, 196 and / or 197; (c) a cathepsin B core motif selected from SEQ ID NO: 20, 181, 198 and / or 199; and / or (d) a cathepsin D core motif selected from SEQ ID NO: 200, 201, 202, 203, 204, 205, 206 and / or 207.

4. The engineered Clostridial neurotoxin according to any one of the preceding claims, wherein: (a) the endosomal protease cleavage site comprises or consists of one or more of the following: SEQ ID NO: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186 and / or 187; (b) the endosomal protease cleavage site is a cathepsin L cleavage site comprising or consisting of one or more of the following: SEQ ID NO: 12, 13, 14, 15, 16, 17, 18, 19, 27, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 171, 172, 173, 174, 175, 176, 177, 178, 179 and / or 180; (c) The endoprotease cleavage site is a cathepsin B cleavage site comprising or consisting of one or more of the following: SEQ ID NO: 20, 28, 131, 132, 133, 134, 135, 136, 181, 182, 183, and / or 184; (d) The endoprotease cleavage site is a cathepsin D cleavage site comprising or consisting of one or more of the following: SEQ ID NO: 21, 29, 30, 31, 32, 33, 152, 185, and / or 186; and / or (e) The endoprotease cleavage site is an AEP cleavage site comprising or consisting of one or more of the following: SEQ ID NO: 22, 23, 24, 25, 26, 34, 130, 151, and / or 187.

5. The engineered Clostridial neurotoxin according to any one of the preceding claims, wherein the engineered Clostridial neurotoxin comprises an exogenous activation loop, the exogenous activation loop comprising or consisting of any one of the following: SEQ ID NO: 35, 36, 37, 38, 127, 128, and / or 129.

6. The engineered Clostridial neurotoxin according to any one of the preceding claims, wherein the endogenous activation loop of the Clostridial neurotoxin or a part thereof has been replaced by an endoprotease cleavage site.

7. The engineered Clostridial neurotoxin according to claim 6, wherein the endogenous neurotoxin activation loop is one or more selected from SEQ ID NO: 89 to 112.

8. The engineered Clostridial neurotoxin according to any one of the preceding claims, wherein the 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) A chimeric BoNT or a hybrid BoNT.

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

10. The engineered Clostridial neurotoxin according to any one of the preceding claims, wherein the engineered Clostridial neurotoxin is a single-chain Clostridial neurotoxin, the single-chain Clostridial neurotoxin: (a) is encoded by a nucleotide sequence having at least 70% sequence identity with SEQ ID NO: 163, wherein SEQ ID NO: 164 within SEQ ID NO: 163 is optionally replaced by a nucleotide sequence encoding at least one endoprotease cleavage site selected from SEQ ID NO: 165, 166, and 167; and / or (b) comprising a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 120, wherein SEQ ID NO: 91 within SEQ ID NO: 120 is optionally replaced by at least one endoprotease cleavage site or an exogenous activation loop selected from SEQ ID NOs: 127 - 129.

11. The engineered clostridial neurotoxin according to any one of the preceding claims, wherein the engineered clostridial neurotoxin is a retargeted clostridial neurotoxin, and the endogenous H C or H CC of the clostridial neurotoxin is replaced by an exogenous targeting moiety (TM).

12. An engineered Clostridial neurotoxin comprising an endoprotease cleavage site, said engineered Clostridial neurotoxin comprising a polypeptide sequence having at least 70% sequence identity, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% sequence identity to SEQ ID NOs: 160 - 162.

13. A method for proteolytically processing an engineered Clostridial neurotoxin according to any one of claims 1 to 12 into a corresponding double - chain Clostridial neurotoxin, said method comprising contacting said engineered Clostridial neurotoxin with an endoprotease specific for said endoprotease cleavage site to produce a double - chain Clostridial neurotoxin.

14. A double - chain Clostridial neurotoxin obtainable by the method according to claim 13.

15. A polynucleotide encoding an engineered Clostridial neurotoxin according to any one of claims 1 to 11 as claimed in claim 12.

16. An expression vector comprising the polynucleotide according to claim 15 operably linked to a promoter.

17. The polynucleotide according to claim 15 or the expression vector according to claim 16, wherein the polynucleotide or expression vector: (a) comprises a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 163 or consists of said nucleotide sequence, wherein SEQ ID NO: 164 within SEQ ID NO: 163 is optionally replaced by a nucleotide sequence encoding at least one endoprotease cleavage site selected from SEQ ID NOs: 165, 166 and 167; and / or (b) encodes a polypeptide sequence having at least 70% sequence identity to one or more of SEQ ID NOs: 120, wherein SEQ ID NO: 91 within SEQ ID NO: 120 is optionally replaced by at least one endoprotease cleavage site or an exogenous activation loop selected from SEQ ID NOs: 127 - 129.

18. A method for producing an engineered Clostridial neurotoxin as claimed in any one of claims 1 to 12, said method comprising the steps of: Expressing the polynucleotide according to claim 15 or 17 or the expression vector according to claim 16 or 17 in a cell and recovering the expressed engineered Clostridial neurotoxin.

19. The method according to claim 18, said method further comprising the step of introducing the polynucleotide according to claim 15 or 17 or the expression vector according to claim 16 or 17 into said cell.

20. A cell expressing an engineered Clostridial neurotoxin according to any one of claims 1 to 12.

21. The cell according to claim 20, wherein the cell comprises the polynucleotide according to claim 15 or 17 or the expression vector according to claim 16 or 17.

22. A pharmaceutical composition comprising the engineered Clostridial neurotoxin according to any one of claims 1 to 12 or the double-stranded Clostridial neurotoxin according to claim 14, and a pharmaceutically acceptable carrier, excipient, diluent, adjuvant, propellant and / or salt.

23. The engineered Clostridial neurotoxin according to any one of claims 1 to 12, the double-stranded Clostridial neurotoxin according to claim 14 or the pharmaceutical composition according to claim 22, for use in a method of preventing or treating a disease or disorder indicated for treatment with botulinum neurotoxin, wherein optionally, the disease or disorder is selected from disorders associated with unwanted immune secretion, strabismus, blepharospasm, squint, dystonia (such as spastic dystonia, oromandibular dystonia, focal dystonia, tardive dystonia, laryngeal dystonia, limb dystonia, cervical dystonia), torticollis (such as spastic torticollis), cosmetic applications (cosmesis) of beauty therapy that benefit from cell / muscle disablement (via SNARE downregulation or inactivation), neuromuscular disorders or diseases of eye movement (such as concomitant strabismus, vertical strabismus, lateral rectus paralysis, nystagmus, thyrotoxic myopathy), writer's cramp, bruxism, Wilson's disease, tremor, tic, segmental myoclonus, spasm, spasm caused by chronic multiple sclerosis, spasm leading to abnormal bladder control, hostility, back spasm, cramp, levator ani syndrome, spina bifida, tardive dyskinesia, Parkinson's disease, stuttering, hemifacial spasm, eyelid disorders, cerebral palsy, focal spasm, spastic colitis, neurogenic bladder, pelvic floor dyssynergia, limb spasm, tic, tremor, bruxism, anal fissure, achalasia, dysphagia, lacrimation, hyperhidrosis, hypersalivation, hypersecretion of the gastrointestinal tract, muscle pain (such as pain caused by muscle spasm), headache (such as tension headache or migraine), phantom pain (such as phantom limb pain), glabellar lines, skin wrinkles, cancer, uterine disorders, urogenital disorders, urogenital nerve disorders, bladder pain syndrome, interstitial cystitis, chronic neurogenic inflammation and smooth muscle disorders.

24. Use of an engineered Clostridium neurotoxin as described in any one of claims 1 to 12, a double-stranded Clostridium neurotoxin as described in claim 14, or a pharmaceutical composition as described in claim 22, for the manufacture of a medicament for preventing or treating a disease or disorder indicated for treatment with botulinum neurotoxin, wherein optionally, the disease or disorder is selected from disorders associated with unwanted immune secretion, strabismus, blepharospasm, squint, dystonia (such as spastic dystonia, oromandibular dystonia, focal dystonia, tardive dystonia, laryngeal dystonia, limb dystonia, cervical dystonia), torticollis (such as spastic torticollis), cosmetic applications (cosmesis) of beauty therapy that benefits from cell / muscle disability (via SNARE downregulation or inactivation), neuromuscular disorders or diseases of eye movement (such as concomitant strabismus, vertical strabismus, lateral rectus palsy, nystagmus, dysthyroid myopathy), writer's cramp, bruxism, Wilson's disease, tremor, tic, segmental myoclonus, spasm, spasm caused by chronic multiple sclerosis, spasm leading to abnormal bladder control, hostility, back spasm, cramp, levator ani syndrome, spina bifida, tardive dyskinesia, Parkinson's disease, stuttering, hemifacial spasm, eyelid disorders, cerebral palsy, focal spasm, spastic colitis, neurogenic bladder, pelvic floor dyssynergia, limb spasm, tic, tremor, bruxism, anal fissure, achalasia, dysphagia, lacrimation, hyperhidrosis, hypersalivation, hypersecretion of the gastrointestinal tract, muscle pain (such as pain caused by muscle spasm), headache (such as tension headache or migraine), phantom pain (such as phantom limb pain), glabellar lines, skin wrinkles, cancer, uterine disorders, urogenital disorders, urogenital nerve disorders, bladder pain syndrome, interstitial cystitis, chronic neurogenic inflammation, and smooth muscle disorders.

25. Use of an engineered Clostridium neurotoxin or a pharmaceutical composition according to the use described in claim 23, or an engineered Clostridium neurotoxin or a pharmaceutical composition according to claim 24, wherein the Clostridium neurotoxin is administered to a subject in single-chain form.

26. Use of an engineered Clostridium neurotoxin or a pharmaceutical composition according to the use described in claim 23 or 25, or an engineered Clostridium neurotoxin or a pharmaceutical composition according to claim 24 or 25, wherein the Clostridium neurotoxin or the pharmaceutical composition is substantially free of the double-stranded form of the Clostridium neurotoxin.

27. The use of an engineered Clostridium neurotoxin or pharmaceutical composition according to the use described in claim 23, 25 or 26, or the use of an engineered Clostridium neurotoxin or pharmaceutical composition according to any one of claims 24 to 26, wherein the Clostridium neurotoxin or the pharmaceutical composition comprises less than 400 pg of double-stranded Clostridium neurotoxin per 100 ng of single-stranded Clostridium neurotoxin, or less than 300 pg of double-stranded Clostridium neurotoxin per 100 ng of single-stranded Clostridium neurotoxin, or less than 200 pg of double-stranded Clostridium neurotoxin per 100 ng of single-stranded Clostridium neurotoxin, or less than 100 pg of double-stranded Clostridium neurotoxin per 100 ng of single-stranded Clostridium neurotoxin, or less than 50 pg of double-stranded Clostridium neurotoxin per 100 ng of single-stranded Clostridium neurotoxin.

28. A cosmetic composition, the cosmetic composition comprising an engineered Clostridium neurotoxin according to any one of claims 1 to 12 or a double-stranded Clostridium neurotoxin according to claim 14, and a cosmetically acceptable carrier, excipient, diluent, adjuvant, propellant and / or salt.

29. The use of the cosmetic composition according to claim 28 for preventing or alleviating a cosmetic indication indicative of the application of botulinum neurotoxin.

30. The cosmetic composition according to claim 28 or the use of the cosmetic composition according to claim 28, wherein the Clostridium neurotoxin is administered to a subject in single-chain form.

31. The cosmetic composition according to claim 28 or 30 or the use of the cosmetic composition according to claim 29 or 30, wherein the Clostridium neurotoxin or the cosmetic composition is substantially free of the double-stranded form of the Clostridium neurotoxin.

32. The cosmetic composition according to claim 28, 30 or 31 or the use of the cosmetic composition according to any one of claims 29 to 31, wherein the Clostridium neurotoxin or the cosmetic composition comprises less than 400 pg of double-stranded Clostridium neurotoxin per 100 ng of single-stranded Clostridium neurotoxin, or less than 300 pg of double-stranded Clostridium neurotoxin per 100 ng of single-stranded Clostridium neurotoxin, or less than 200 pg of double-stranded Clostridium neurotoxin per 100 ng of single-stranded Clostridium neurotoxin, or less than 100 pg of double-stranded Clostridium neurotoxin per 100 ng of single-stranded Clostridium neurotoxin, or less than 50 pg of double-stranded Clostridium neurotoxin per 100 ng of single-stranded Clostridium neurotoxin.

33. A method for proteolytically processing a single-chain Clostridium neurotoxin into the corresponding double-stranded Clostridium neurotoxin, the method comprising: (a) providing a single-chain Clostridium neurotoxin; and (b) contacting the single-chain Clostridium neurotoxin with an endosomal protease; wherein the single-chain Clostridium neurotoxin has an activation loop comprising or consisting of a polypeptide sequence as defined in any one of claims 3 to 5. wherein the endosomal protease hydrolyzes the peptide bond of the activation loop, thereby producing a double-stranded Clostridium neurotoxin.

34. The method according to claim 33, wherein the activation loop comprises or consists of a polypeptide sequence as defined in claim 4 or 5.

35. The method according to claim 33 or 34, wherein the single-chain Clostridium neurotoxin: (a) is an engineered Clostridium neurotoxin according to any one of claims 1 to 10; (b) is encoded by a nucleotide sequence having at least 70% sequence identity with SEQ ID NO: 163, wherein SEQ ID NO: 164 within SEQ ID NO: 163 is optionally replaced by a nucleotide sequence encoding at least one endosomal protease cleavage site selected from SEQ ID NO: 165, 166, and 167; and / or (c) comprises a polypeptide sequence having at least 70% sequence identity with one or more of SEQ ID NO: 120, wherein SEQ ID NO: 91 within SEQ ID NO: 120 is optionally replaced by at least one endosomal protease cleavage site or an exogenous activation loop selected from SEQ ID NO: 127 - 129.

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