Biosensor for real-time monitoring of clostridial neurotoxin production

Abstract

The present invention is a biosensor for real-time monitoring of clostridial neurotoxin production during bacterial fermentation processes. The biosensor comprises a surface for performing an analysis of binding events and kinetics, and immobilized on the surface is a binding substrate comprising: a donor fluorophore; an acceptor having an absorption spectrum that overlaps with an emission spectrum of the donor fluorophore; and a binding region that specifically binds to the clostridial neurotoxin; wherein the binding region is positioned between the donor fluorophore and the acceptor such that, upon activation of the donor fluorophore, resonance energy transfer is exhibited between the donor fluorophore and the acceptor.

Description

Technical Field

[0001] This invention relates to a biosensor and method for monitoring the production of Clostridium neurotoxins. Background Technology

[0002] Clostridium ( Clostridia Bacteria in clostridial bacteria produce highly potent and specific protein toxins that can poison neurons and other cells they are delivered to. Examples of such clostridial toxins include those produced by Clostridium tetani (Clostridium tetani). C. tetani Neurotoxin (TeNT) produced by Clostridium botulinum and neurotoxin produced by Clostridium botulinum C. botulinum Neurotoxins (BoNT) produced by *Clostridium pasteurellum* (see WO 2018 / 009903 A2) serotypes AG and X, and neurotoxins produced by *Clostridium pasteurellum* (see WO 2018 / 009903 A2). C. baratii ) and Clostridium butyricum ( C. butyricum The neurotoxins produced by tetanus toxin and botulinum toxin both work by inhibiting the function of affected neurons, particularly the release of neurotransmitters. While botulinum toxin acts at the neuromuscular junction and inhibits cholinergic transmission in the peripheral nervous system, tetanus toxin acts in the central nervous system.

[0003] In nature, clostridial neurotoxins (e.g., botulinum neurotoxin [BoNT]) are synthesized as single-chain polypeptides, which undergo post-translational modification via proteolytic cleavage events to form two polypeptide chains linked together by disulfide bonds. Cleavage occurs at specific cleavage sites (often called activation sites) located between cysteine ​​residues that provide the interchain disulfide bonds. It is this double-chain form that is the active form of the toxin. These two chains are referred to as the heavy chain (H chain) with a molecular weight of approximately 100 kDa and the light chain (L chain) with a molecular weight of approximately 50 kDa. The H chain contains the N-terminal translocation component (H... N (structural domain) and C-terminal targeting component (H) C (Structural domain). The cleavage site is located between the L chain and the translocation structural domain component. In H C After the domain binds to its target neuron and the bound toxin is internalized into the cell, H N The domain allows the L chain to translocate across the endosome membrane and into the cytosol, and the L chain provides protease function (also known as non-cytotoxic protease).

[0004] Non-cytotoxic proteases function by cleaving intracellular transport proteins called SNARE proteins (e.g., SNAP-25, VAMP, or synaptic fusion proteins). The acronym SNARE originates from the term soluble NSF attachment receptor (NSF). S oluble N SF A ttachment Re ceptor), where NSF refers to N-ethylmaleimide sensitizer (NSF). N -ethylmaleimide- S ensitive F SNARE proteins are essential for intracellular vesicle fusion and therefore for the transport of secreted molecules from cells via vesicles. The protease function is a zinc-dependent endopeptidase activity and exhibits high substrate specificity for SNARE proteins.

[0005] The use of clostridial neurotoxins in therapeutic and cosmetic treatments in humans and other mammals is expected to expand to a wider range of diseases and minor ailments where the properties of these toxins can benefit. In light of this, there is a growing demand for the large-scale manufacture of clostridial neurotoxins and their appropriate formulations.

[0006] Botulinum toxin (including type A toxin) is routinely obtained through a culturing and fermentation process. In addition to the botulinum toxin molecules, the resulting fermentation solution typically contains intact bacteria, lysed bacteria, culture medium nutrients, and fermentation byproducts. The fermentation solution is filtered to remove intact and / or lysed cell components, as well as optionally other fermentation medium residues, to produce a clear culture. The clear culture solution contains botulinum toxin molecules and various impurities, which can be removed to obtain concentrated, purified botulinum toxin (e.g., BoNT / A1) suitable for formulation into botulinum toxin pharmaceutical compositions.

[0007] Monitoring the generation of botulinum toxin during the culturing and fermentation process typically involves collecting samples from the fermentation solution and performing offline analysis. Offline analysis can be performed hours or even days after sample collection, and therefore does not allow for real-time monitoring of botulinum toxin generation. Furthermore, the testing methods required for offline monitoring are often slow, laborious, and expensive. For example, determining the concentration of botulinum toxin in a sample typically involves multiple steps (e.g., separating the toxin complex from residual impurities from the fermentation process) and involves manual handling of samples with inherent biosafety risks. In addition, batch processing of clostridium neurotoxins has several drawbacks, including potential variability in sensitivity due to the use of antibody batches for immunoprecipitation, and consequently reduced reproducibility and robustness.

[0008] Therefore, there is a need in the art for alternative and / or improved methods for monitoring the production of botulinum toxin during cultivation and fermentation processes. In particular, the present invention relates to biosensors and methods for real-time monitoring of botulinum toxin production. Summary of the Invention

[0009] The inventors have developed novel biosensors, binding substrates, and methods for rapid, real-time monitoring of Clostridium neurotoxin production during bacterial fermentation.

[0010] Advantageously, the biosensors, binding substrates, and methods of the present invention can be highly sensitive, thereby allowing the detection of picomolar concentrations of clostridial neurotoxins in the composition. The biosensors and methods of the present invention also advantageously allow the detection of clostridial neurotoxins in specific modified (e.g., oxidized), proteolytically activated, or aggregated forms (e.g., dimers and polymers, optionally with reduced potency). Therefore, biosensors can be used to determine whether a homogeneous population of clostridial neurotoxin peptides is produced during fermentation and / or to determine the proportion of clostridial neurotoxin variants produced.

[0011] The biosensors, binding substrates, and methods of the present invention have been specifically designed to require minimal steps and equipment, meaning they are suitable for use with said toxins that may cause serious and potentially fatal side effects in subjects exposed to the agent (e.g., in a biosafety level 3 environment).

[0012] Furthermore, the biosensors, binding substrates, and methods of the present invention overcome the challenges associated with the variability in the sensitivity of batches of reagents (e.g., monoclonal antibodies) used to monitor Clostridium neurotoxin production using conventional offline methods.

[0013] This invention provides a biosensor for real-time monitoring of clostridium neurotoxin production during bacterial fermentation. The biosensor includes a surface for analyzing binding events and kinetics, and a binding substrate is immobilized on the surface, the binding substrate comprising: a. Donor fluorophore; b. An acceptor having an absorption spectrum that overlaps with the emission spectrum of the donor fluorophore; and c. A binding region that specifically binds to the Clostridium neurotoxin; wherein the binding region is located between the donor fluorophore and the receptor, such that, upon activation of the donor fluorophore, a resonant energy transfer occurs between the donor fluorophore and the receptor.

[0014] It is also a binding substrate for real-time monitoring of clostridium neurotoxin production during bacterial fermentation, the binding substrate comprising: a. Donor fluorophore; b. An acceptor having an absorption spectrum that overlaps with the emission spectrum of the donor fluorophore; and c. A binding region that specifically binds to the Clostridium neurotoxin; wherein the binding region is located between the donor fluorophore and the receptor, such that, upon activation of the donor fluorophore, a resonant energy transfer occurs between the donor fluorophore and the receptor.

[0015] In some embodiments, the binding region includes one or more complementary sites for binding clostridium neurotoxin. The term "complementary site" is a term well-defined in the art that refers to an antigen-binding site of an antibody or antibody fragment.

[0016] In some embodiments, the binding region comprises one or more amino acid sequences that specifically bind to the Clostridium neurotoxin, and wherein the one or more amino acid sequences comprise the CDR sequence of one or more antibodies; preferably, wherein these antibodies are single-chain antibodies, such as nanobodies, single-chain variable fragments (scFv), single-chain Fab (scFab), microantibodies, or biantibodies; more preferably, wherein these single-chain antibodies are camel or shark nanobodies.

[0017] In some embodiments, the one or more amino acid sequences comprise a CDR sequence from a nanobody selected from the list comprising: ciA-H7, ciA-D1, ciA-H4, ciA-H11, ciA-C2, ciA-B5, ciA-F12, ciA-D12, ciA-A5, ciA-G5, ciB-H11, ciB-A11, ciB-B5, ciB-B9, ciA-H7 / B5, ciA-F12 / D12, and ciB-A11 / B5, or a combination thereof. For example, the one or more amino acid sequences may include sequences having at least 80% sequence identity with the full-length sequence of one or more nanobodies selected from the following list: ciA-H7, ciA-D1, ciA-H4, ciA-H11, ciA-C2, ciA-B5, ciA-F12, ciA-D12, ciA-A5, ciA-G5, ciB-H11, ciB-A11, ciB-B5, ciB-B9, ciA-H7 / B5, ciA-F12 / D12, and ciB-A11 / B5, or combinations thereof.

[0018] In some embodiments, the biosensor or binding substrate comprises one or more amino acid sequences comprising a CDR sequence from a nanobody capable of neutralizing clostridium neurotoxin toxicity, such as ciA-C2, ciA-H7, ciA-B5, and ciA-D1, or combinations thereof.

[0019] In some embodiments, the biosensor or binding substrate comprises one or more amino acid sequences that have at least 80% sequence identity with the full-length sequence of one or more nanobodies selected from the list below: ciA-C2, ciA-H7, ciA-B5, and ciA-D1, or combinations thereof.

[0020] The nanobodies of the present invention can be polymers, such as dimers, trimers or tetramers.

[0021] In some embodiments, the binding region comprises one or more amino acid sequences selected from (and optionally further comprising) sequences that specifically bind to one or more clostridial neurotoxins selected from: oxidized forms of clostridial neurotoxins (with reduced potency), improperly proteolytically activated forms of clostridial neurotoxins (with reduced potency), and / or aggregated forms of clostridial neurotoxins, including dimers and polymers (with reduced potency).

[0022] The biosensor and / or binding substrate of the present invention are advantageously highly sensitive. In some embodiments, the biosensor and / or binding substrate can be used to detect clostridium neurotoxins at concentrations less than 1 ng / ml, preferably less than 0.1 ng / ml.

[0023] A method for real-time monitoring of clostridial neurotoxin production during bacterial fermentation is also provided, the method comprising: a. culturing bacterial host cells capable of producing clostridial neurotoxin in a liquid culture medium; b. contacting the liquid culture medium with a biosensor of the present invention; c. exciting the donor fluorophore; and d. determining the resonance energy transfer at the biosensor relative to a control, wherein the difference in resonance energy transfer at the biosensor compared to the control indicates the presence of clostridial neurotoxin. Optionally, the bacterial host cells may be lysed before contacting the liquid culture medium with the biosensor.

[0024] A method for real-time monitoring of clostridial neurotoxin production during bacterial fermentation is also provided, the method comprising: a. transforming bacterial host cells capable of producing clostridial neurotoxins with an expression construct encoding the binding substrate of the present invention; b. culturing the bacterial host cells in a liquid culture medium; c. exciting the donor fluorophore; and d. determining the resonance energy transfer at the biosensor relative to a control, wherein the difference in resonance energy transfer at the biosensor compared to the control indicates the presence of clostridial neurotoxins.

[0025] In some embodiments, step d includes detecting donor fluorescence intensity at the biosensor, wherein an increase in donor fluorescence intensity at the biosensor compared to the control indicates the presence of clostridium neurotoxin.

[0026] In some embodiments, step d includes detecting the receptor fluorescence intensity at the biosensor, wherein a decrease in receptor fluorescence intensity at the biosensor compared to the control indicates the presence of clostridium neurotoxin.

[0027] In some embodiments, step d includes detecting the maximum emission value of the receptor and the maximum emission value of the donor fluorophore at the biosensor, wherein the offset of the emission value from the vicinity of the maximum emission value of the receptor to the vicinity of the maximum emission value of the donor fluorophore indicates the presence of clostridium neurotoxin.

[0028] In some embodiments, step d includes detecting the ratio of fluorescence amplitude near the maximum emission value of the receptor to the fluorescence amplitude near the maximum emission value of the donor fluorophore, wherein a decrease in the ratio at the biosensor compared to the control indicates the presence of clostridial neurotoxin. Detailed Implementation

[0029] In one aspect, the present invention provides a biosensor for real-time monitoring of Clostridium neurotoxin production during bacterial fermentation, the biosensor comprising a surface for analyzing binding events and kinetics, and a binding substrate immobilized on the surface, the binding substrate comprising: a. Donor fluorophore; b. An acceptor having an absorption spectrum that overlaps with the emission spectrum of the donor fluorophore; and c. A binding region that specifically binds to clostridium neurotoxin; wherein the binding region is located between the donor fluorophore and the receptor, such that, upon activation of the donor fluorophore, a resonant energy transfer occurs between the donor fluorophore and the receptor.

[0030] In one aspect, the present invention provides a biosensor for real-time monitoring of clostridium neurotoxin production during a bacterial fermentation process, the biosensor comprising a surface for analyzing binding events and kinetics, wherein the surface is configured to immobilize clostridium neurotoxin, and the biosensor further comprising a binding substrate, wherein the binding substrate comprises: a. Donor fluorophore; b. An acceptor having an absorption spectrum that overlaps with the emission spectrum of the donor fluorophore; and c. A binding region that specifically binds to clostridium neurotoxin; wherein the binding region is located between the donor fluorophore and the receptor, such that, upon activation of the donor fluorophore, a resonant energy transfer occurs between the donor fluorophore and the receptor.

[0031] In one aspect, the present invention provides a binding substrate for real-time monitoring of Clostridium neurotoxin production during bacterial fermentation, the binding substrate comprising: a. Donor fluorophore; b. An acceptor having an absorption spectrum that overlaps with the emission spectrum of the donor fluorophore; and c. A binding region that specifically binds to clostridium neurotoxin; wherein the binding region is located between the donor fluorophore and the receptor, such that, upon activation of the donor fluorophore, a resonant energy transfer occurs between the donor fluorophore and the receptor.

[0032] In one aspect, the present invention provides a method for real-time monitoring of Clostridium neurotoxin production during a bacterial fermentation process, the method comprising: a. Culture bacterial host cells capable of producing clostridium neurotoxin in liquid culture medium; b. Contact the liquid culture medium with the biosensor of the present invention; c. Excite the donor fluorophore; and d. Determine the resonant energy transfer at the biosensor relative to a control, wherein the difference in the resonant energy transfer at the biosensor compared to the control indicates the presence of clostridial neurotoxin.

[0033] In one aspect, the present invention provides a method for real-time monitoring of Clostridium neurotoxin production during a bacterial fermentation process, the method comprising: a. Transform bacterial host cells capable of producing clostridium neurotoxins using an expression construct encoding the binding substrate of this invention; b. Culture bacterial host cells in liquid culture medium; c. Excite the donor fluorophore; and d. Determine the resonant energy transfer at the biosensor relative to a control, wherein the difference in the resonant energy transfer at the biosensor compared to the control indicates the presence of clostridial neurotoxin.

[0034] The biosensor of the present invention enables rapid, real-time monitoring of clostridium neurotoxin production during bacterial fermentation. The biosensor comprises a surface (e.g., a support) and a binding substrate for analyzing binding events and / or kinetics.

[0035] In other aspects, the present invention provides a binding substrate for real-time monitoring of clostridium neurotoxin production during bacterial fermentation. In some embodiments, the binding substrate is not immobilized on a surface. For example, in some embodiments, the binding substrate can be expressed intracellularly in host cells capable of producing clostridium neurotoxin peptides. In some embodiments, a support is configured to capture or immobilize the binding substrate. In other embodiments, the support is configured to capture or immobilize clostridium neurotoxin peptides.

[0036] In some embodiments, the binding substrate includes a device for indicating the presence or absence of a clostridial neurotoxin polypeptide in the composition. Specifically, the binding substrate may comprise two distinct states, one indicating the presence of the clostridial neurotoxin polypeptide (e.g., via binding of the clostridial neurotoxin polypeptide to the binding substrate), and the other indicating the absence of the clostridial neurotoxin polypeptide (e.g., via the absence of binding of the clostridial neurotoxin polypeptide to the binding substrate).

[0037] Devices used to indicate the presence or absence of clostridial neurotoxins may comprise one or more modules (e.g., 1, 2, 3, 4, 5, or more modules). Preferably, the one or more modules are genetically encoded. For example, in a preferred embodiment, the one or more modules are expressed as a single-chain polypeptide. The term "single-chain" can refer to a single polypeptide molecule having a series of amino acid residues linked together by peptide bonds between the α-amino and carboxyl groups of adjacent residues. In other words, each enumerated element of the single-chain polypeptide can be linked to one or more other elements by peptide bonds. Exemplary modules may be selected from the detectable marker and clostridial neurotoxin binding region.

[0038] In a preferred embodiment, the one or more modules comprise one or more detectable markers. For example, the substrate may comprise 1, 2, 3, 4, 5 or more detectable markers, preferably 2. The detectable markers may be markers that can be visually detected by means of the optical properties of the markers. The detectable markers may be fluorescent markers (e.g., fluorophores). Many fluorescence techniques, including fluorescence microscopy, can be used to detect such markers.

[0039] In preferred embodiments, the binding substrate comprises one or more detectable markers, wherein the detectable markers are fluorophores. As used herein, the term fluorophore encompasses fluorescent proteins, bioluminescent proteins, fluorescent dyes (e.g., non-protein organic dyes), and quantum dots. Preferably, the fluorophore is a fluorescent protein. Preferably, the device for indicating the presence or absence of clostridium neurotoxin comprises at least two detectable markers (e.g., two fluorophores). For example, in a preferred embodiment, the binding substrate comprises a first detectable marker and a second detectable marker (e.g., a first fluorophore and a second fluorophore). In other embodiments, the binding substrate may comprise a first detectable marker (e.g., a fluorophore) and a portion that reduces the fluorescence intensity of the fluorophore under certain conditions, such as a fluorescence quencher or Au nanoparticles.

[0040] In some embodiments, the binding substrate comprises a first (donor) fluorophore and a second (acceptor) fluorophore. In a preferred embodiment, the donor and acceptor fluorophores are positioned such that, upon activation of the donor fluorophore, a resonance energy transfer (e.g., fluorescence (or Förster) resonance energy transfer, FRET) is exhibited between the donor fluorophore and the acceptor. As used herein, the term “FRET” refers to a nonradiative energy transfer between two fluorophores with different emission wavelengths, wherein the excitation energy of the excited-state donor fluorophore is transferred to the acceptor, and thus emission from the fluorescent acceptor or quenching of the fluorescent donor is observed (Lakowicz, JR Principles of Fluorescence Spectroscopy, 2nd ed., New York: Plenum Press, 1999). Therefore, as used herein, the term “donor” or “donor fluorophore” may refer to the fluorophore acting as the donor in the FRET phenomenon, and the term “acceptor” or “acceptor fluorophore” may refer to the fluorophore acting as the acceptor in the FRET phenomenon.

[0041] Preferably, the donor and acceptor fluorophores are functionally linked via one or more amino acid sequences that specifically bind to clostridium neurotoxin. The term "functional link" refers to a link between the donor and acceptor fluorophores in such a manner that any intervening sequence (e.g., a binding region) does not prevent the primary amino acid sequence of each fluorophore from forming a functional tertiary structure (e.g., a functional fluorophore). Methods for determining whether a given sequence functionally links the donor and acceptor fluorophores are known in the art. For example, those skilled in the art will be able to determine whether a given binding region functionally links the donor and acceptor fluorophores using visual detection, such as measuring FRET using fluorescence microscopy (e.g., by sensitized emission, acceptor photobleaching, and / or FLIM-FRET methods) or by measuring FRET using solution-based methods (e.g., using filters or a monochromatic spectrophotometer).

[0042] Preferably, the donor and acceptor fluorophores are separated by a binding region comprising one or more sequences that bind to the clostridium neurotoxin polypeptide. In other words, the binding region may contain one or more sequences (e.g., 1, 2, 3, 4, 5, or more sequences) that bind to the clostridium neurotoxin polypeptide. Preferably, the binding substrate is configured such that the binding of the clostridium neurotoxin to the binding region increases the distance between the donor and acceptor fluorophores. Preferably, the binding substrate is configured such that the binding of the clostridium neurotoxin to the binding region results in a change in fluorescence intensity from the donor and / or acceptor fluorophores. For example, in some embodiments, the donor and acceptor fluorophores are separated by an amino acid sequence that binds to the clostridium neurotoxin polypeptide in the presence of the clostridium neurotoxin.

[0043] In some embodiments, in the absence of the clostridial neurotoxin peptide, the binding region is not bound by the clostridial neurotoxin peptide, and the donor and acceptor fluorophores are in close proximity, causing a resonance energy transfer between the donor and acceptor fluorophores. However, in the presence of the clostridial neurotoxin peptide, the peptide binds to the binding region, thereby increasing the distance between the donor and acceptor fluorophores, thus reducing the resonance energy transfer between them. The resonance energy transfer between the donor and acceptor fluorophores can be detected as increased emission from the acceptor fluorophore and / or quenching from the donor fluorophore. Alternatively, the absence of resonance energy transfer between the donor and acceptor fluorophores can be detected as decreased emission from the acceptor or increased emission from the donor fluorophore.

[0044] In a preferred embodiment, the biosensor of the present invention comprises a plurality of binding substrates. In some embodiments, the binding substrates are identical. In other embodiments, the biosensor comprises two or more different binding substrates. The different binding substrates may contain different binding regions, such that a first plurality of binding substrates binds to a first group of clostridium neurotoxin peptides, and a second plurality of binding substrates binds to a second group of clostridium neurotoxin peptides.

[0045] In some embodiments, a biosensor comprising one or more different binding substrates may be used in a method for real-time monitoring of oxidized clostridium neurotoxin production during a bacterial fermentation process. For example, in some embodiments, the biosensor may comprise a plurality of binding substrates, wherein at least a first plurality of binding substrates comprises a binding region containing a sequence specifically binding to an oxidized form of clostridium neurotoxin. In some embodiments, the biosensor may comprise a plurality of binding substrates, wherein a first plurality of binding substrates comprises a binding region containing a sequence specifically binding to a non-oxidized form of clostridium neurotoxin, and a second plurality of binding substrates comprises a binding region containing a sequence specifically binding to an oxidized form of clostridium neurotoxin (with reduced potency). Each plurality of binding substrates may further comprise different donor and acceptor fluorophores, enabling the plurality of binding substrates to distinguish the binding of a first group of clostridium neurotoxins to a second group (e.g., oxidized and non-oxidized clostridium neurotoxins).

[0046] In some embodiments, a biosensor comprising one or more different binding substrates may be used in a method for real-time monitoring of the production of incorrectly proteolytically activated clostridium neurotoxins during a bacterial fermentation process. For example, in some embodiments, the biosensor may comprise a plurality of binding substrates, wherein at least a first plurality of binding substrates comprises a binding region containing a sequence that specifically binds to an incorrectly proteolytically activated form of clostridium neurotoxin (e.g., with reduced potency). In some embodiments, the biosensor may comprise a plurality of binding substrates, wherein a first plurality of binding substrates comprises a binding region containing a sequence that specifically binds to proteolytically activated clostridium neurotoxins, and a second plurality of binding substrates comprises a binding region containing a sequence that specifically binds to an incorrectly proteolytically activated form of clostridium neurotoxin (e.g., with reduced potency). Each plurality of binding substrates may further comprise different donor and acceptor fluorophores, enabling the plurality of binding substrates to distinguish the binding of a first group of clostridium neurotoxins to a second group (e.g., proteolytically activated clostridium neurotoxins and incorrectly proteolytically activated clostridium neurotoxins).

[0047] In some embodiments, a biosensor comprising one or more different binding substrates may be used in a method for real-time monitoring of the production of aggregated forms of clostridial neurotoxins during a bacterial fermentation process. For example, in some embodiments, the biosensor may comprise a plurality of binding substrates, wherein at least a first plurality of binding substrates comprises a binding region containing a sequence that specifically binds to aggregated forms of clostridial neurotoxins (including dimers and multimers). In some embodiments, the biosensor may comprise a plurality of binding substrates, wherein a first plurality of binding substrates comprises a binding region containing a sequence that specifically binds to clostridial neurotoxin monomers, and a second plurality of binding substrates comprises a binding region containing a sequence that specifically binds to aggregated forms of clostridial neurotoxins (including dimers and multimers) (with reduced potency). Each plurality of binding substrates may further comprise different donor and acceptor fluorophores, enabling the plurality of binding substrates to distinguish the binding of a first group of clostridial neurotoxins to a second group (e.g., monomers and multimers).

[0048] Preferably, the binding of the clostridium neurotoxin peptide to the binding substrate increases donor fluorophore emission and / or decreases acceptor fluorophore emission in a concentration-dependent manner. In other words, a biosensor can be used to quantify the concentration of the clostridium neurotoxin peptide in the composition.

[0049] In a preferred embodiment, the donor and acceptor fluorophores are fluorescent proteins. Those skilled in the art can select suitable fluorescent proteins. For example, one or more fluorescent proteins may be selected from a list including: green fluorescent proteins (e.g., GFP, EGFP, Emerald, Superfolder GFP, Azami Green, mWasabi, TagGFP, TurboGFP, AcGFP, ZsGreen, T-Sapphire and their derivatives and variants), blue fluorescent proteins (e.g., EBFP, EBFP2, Azurite, mTagBFP and their derivatives and variants), cyan fluorescent proteins (e.g., ECFP, mECFP, Cerulean, mTurquoise, CyPet, AmCyan1, Midori-Ishi Cyan, TagCFP, mTFP1 (Teal) and their derivatives and variants), yellow fluorescent proteins (e.g., EYFP, Topaz, Venus, mCitrine, YPet, TagYFP, PhiYFP, ZsYellow1, mBanana and their derivatives and variants), and orange fluorescent proteins (e.g., Kusabira Orange, Kusabira...). Orange2, mOrange, mOrange2, dTomato, dTomato-Tandem, TagRFP, TagRFP-T, DsRed, DsRed2, DsRed-Express(T1), DsRed-Monomer, mTangerine and its derivatives and variants) and red fluorescent proteins (e.g., mRuby, mApple, mStrawberry, AsRed2, mRFP1, JRed, mCherry, HcRed1, mRaspberry, dKeima-Tandem, HcRed-Tandem, mPlum, AQ143 and their derivatives and variants). Other fluorescent proteins can be found in... https: / / www.fpbase.org / table / .

[0050] There are no particular limitations on FRET donor and acceptor pairs, and those skilled in the art will be able to select suitable FRET donor and acceptor pairs. For example, in most embodiments, the donor fluorophore and acceptor fluorophore will be different (e.g., with different excitation and emission wavelengths). Typically, FRET occurs between two closely spaced fluorophores with significant overlap (>30%) between the emission spectrum of the donor and the absorption spectrum of the acceptor. Furthermore, those skilled in the art will understand that, in order to maximize the FRET signal, the donor can be selected based on the quantum yield of the donor (e.g., by selecting a donor with a suitably high quantum yield, which is calculated as the number of emitted photons per absorbed photon) and / or an acceptor with a suitably high extinction coefficient (e.g., relating the amount of absorbed light at a given wavelength to the concentration of the fluorophore in solution).

[0051] Commonly used donor and acceptor pairs can be selected from cyan donor fluorophores and yellow acceptor fluorophores or green donor fluorophores and red acceptor fluorophores. In some embodiments, the donor fluorophore can be a green fluorescent protein (e.g., selected from mClover3, GFP, EGFP, Emerald, Superfolder GFP, Azami Green, mWasabi, TagGFP, TurboGFP, AcGFP, ZsGreen, T-Sapphire and their derivatives and variants), and the acceptor fluorophore can be a red fluorescent protein (e.g., mRuby, mApple, mStrawberry, AsRed2, mRFP1, JRed, mCherry, HcRed1, mRaspberry, dKeima-Tandem, HcRed-Tandem, mPlum, AQ143 and their derivatives and variants). In some embodiments, the donor fluorophore may be a cyan fluorescent protein (e.g., selected from ECFP, mECFP, Cerulean, mTurquoise, CyPet, AmCyan1, Midori-Ishi Cyan, TagCFP, mTFP1 (Teal) and its derivatives and variants), and the acceptor fluorophore may be a yellow fluorescent protein (e.g., selected from EYFP, Topaz, Venus, mCitrine, YPet, TagYFP, PhiYFP, ZsYellow1, mBanana and its derivatives and variants).

[0052] Exemplary non-limiting examples of FRET pairs may be selected from the list including: ECFP-EYFP, mTurquoise2-sEYFP, mTurquoise2-mVenus, EGFP-mCherry, Clover-mRuby2, mClover3-mRuby3, mNeonGreen-mRuby3, eqFP650-iRFP, mAmetrine-tdTomato, LSSmOrange-mKate2, EGFP-sREACh, EGFP-ShadowG, EGFP-activated PA-GFP, EGFP-Phanta, mTagBFP-sfGFP, mVenus-mKOκ, or CyOFP1-mCardinal. Alternatively, one or more online tools (including FRbase FRETCalculator) may be used. https: / / www.fpbase.org / fret / The appropriate FRET pair is selected. Preferably, the donor fluorophore is mClover3. Preferably, the acceptor fluorophore is mRuby3. Preferably, the donor fluorophore is mClover3 and the acceptor fluorophore is mRuby3.

[0053] In some embodiments, an increase in donor fluorescence intensity at the biosensor (e.g., compared to a negative control without clostridial neurotoxin) indicates the presence of clostridial neurotoxin. In some embodiments, a decrease in receptor fluorescence intensity at the biosensor (e.g., compared to a negative control without clostridial neurotoxin) indicates the presence of clostridial neurotoxin.

[0054] The donor fluorophore is preferably located at the N-terminus of the clostridial neurotoxin binding region. In some embodiments, the donor fluorophore is the N-terminal element that binds to the substrate. The donor fluorophore may comprise a polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 45. In some embodiments, the donor fluorophore may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity with SEQ ID NO: 45. In some embodiments, the donor fluorophore may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity with SEQ ID NO: 45, wherein the donor fluorophore has a peak excitation wavelength in the range of 499 to 510 nm, preferably about 506 nm, and / or a peak emission wavelength in the range of 512 nm to 522 nm, preferably 518 nm.

[0055] The donor fluorophore may consist of a polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 45. In some embodiments, the donor fluorophore may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity with SEQ ID NO: 45. In some embodiments, the donor fluorophore may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity with SEQ ID NO: 45, wherein the donor fluorophore has a peak excitation wavelength in the range of 499 to 510 nm, preferably about 506 nm, and / or a peak emission wavelength in the range of 512 nm to 522 nm, preferably 518 nm.

[0056] The receptor fluorophore (e.g., having an absorption spectrum that overlaps with the emission spectrum of the donor fluorophore) is preferably located at the C-terminus of the clostridial neurotoxin binding region. In some embodiments, the receptor is the C-terminal element that binds to the substrate. The receptor fluorophore may comprise a polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 46. In one embodiment, the receptor fluorophore may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity with SEQ ID NO: 46. In some embodiments, the donor fluorophore may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity with SEQ ID NO: 46, wherein the receptor fluorophore has a peak excitation wavelength in the range of 522 to 564 nm, preferably about 558 nm, and / or a peak emission wavelength in the range of 579 nm to 609 nm, preferably 592 nm.

[0057] The acceptor fluorophore may consist of a polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 46. In one embodiment, the acceptor fluorophore may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity with SEQ ID NO: 46. In some embodiments, the donor fluorophore may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity with SEQ ID NO: 46, wherein the acceptor fluorophore has a peak excitation wavelength in the range of 522 to 564 nm, preferably about 558 nm, and / or a peak emission wavelength in the range of 579 nm to 609 nm, preferably 592 nm.

[0058] In some embodiments, the binding substrate may further comprise additional elements, such as one or more purification tags. For example, the purification tag may be selected from epitope tags chosen from a list containing the following: his, FLAG, HA, V5, Myc, and Strep. Alternatively (or additionally), the purification tag may be selected from protein / domain tags chosen from a list containing GST, MBP, SUMO, CBP, Halo, Mus A, and FATT.

[0059] Alternatively (or additionally), the binding substrate may further comprise additional elements, such as one or more spacer sequences. For example, in some embodiments, the spacer sequence may be a sequence located between the donor fluorophore and the binding region. In some embodiments, the spacer sequence may be located between the acceptor fluorophore and the binding region. In some embodiments, the binding substrate comprises a first spacer sequence located between the donor fluorophore and the binding region and a second spacer sequence located between the acceptor fluorophore and the binding region. In the presence of more than one spacer, the spacers may have the same or different polypeptide sequences. The selection of appropriate spacer sequences and sizes is entirely within the capabilities of those skilled in the art. The spacers may have any suitable length, such as 3-20, 2-15, 5-15, or 4-8 amino acids. The spacers may contain (or consist of) glycine and serine residues.

[0060] The binding region may contain (or consist of) one or more sequences that specifically bind to a clostridium neurotoxin polypeptide. For example, the binding region may contain 1, 2, 3, 4, 5 or more sequences (or consist of) that specifically bind to a clostridium neurotoxin polypeptide. The sequences may be the same or different. Preferably, the sequences are different. As described herein, the binding region may contain (or consist of) a polymer of a sequence that binds to a single clostridium neurotoxin polypeptide. For example, when the binding region contains a sequence containing 2, 3 or 4 CDR sequences of antibodies, the antibodies may bind to different epitopes on the same clostridium neurotoxin polypeptide. Using a binding region containing more than one sequence that specifically binds to a single clostridium neurotoxin polypeptide can advantageously increase the binding affinity of the interaction (e.g., as by K...). D (Measured).

[0061] Clostridium neurotoxin peptides may contain (or consist of) sequences selected from BoNT / AG, BoNT / X, or TeNT. Clostridium neurotoxin peptides are described herein and may be selected from the group comprising wild-type peptides, modified peptides, or chimeric peptides and fragments thereof.

[0062] For example, the binding region can specifically bind to a polypeptide comprising a sequence having at least 70% sequence identity with any one of SEQ ID NO: 1 to 18. In some embodiments, the binding region can specifically bind to a polypeptide comprising a sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity with any one of SEQ ID NO: 1 to 18. Preferably, the binding region can specifically bind to a polypeptide comprising any one of SEQ ID NO: 1 to 18.

[0063] For example, the binding region can specifically bind to a polypeptide consisting of a sequence having at least 70% sequence identity with any one of SEQ ID NO: 1 to 18. In some embodiments, the binding region can specifically bind to a polypeptide consisting of a sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity with any one of SEQ ID NO: 1 to 18. Preferably, the binding region can specifically bind to a polypeptide consisting of any one of SEQ ID NO: 1 to 18.

[0064] For example, the binding region can specifically bind to a polypeptide comprising a sequence having at least 70% sequence identity with SEQ ID NO: 1. In some embodiments, the binding region can specifically bind to a polypeptide comprising a sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity with SEQ ID NO: 1. Preferably, the binding region can specifically bind to a polypeptide comprising SEQ ID NO: 1. For example, the binding region can specifically bind to a polypeptide composed of a sequence having at least 70% sequence identity with SEQ ID NO: 1. In some embodiments, the binding region can specifically bind to a polypeptide composed of a sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity with SEQ ID NO: 1. Preferably, the binding region can specifically bind to a polypeptide composed of SEQ ID NO: 1.

[0065] For example, the binding region can specifically bind to a polypeptide comprising a sequence having at least 70% sequence identity with SEQ ID NO: 13. In some embodiments, the binding region can specifically bind to a polypeptide comprising a sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity with SEQ ID NO: 13. Preferably, the binding region can specifically bind to a polypeptide comprising SEQ ID NO: 13. For example, the binding region can specifically bind to a polypeptide composed of a sequence having at least 70% sequence identity with SEQ ID NO: 13. In some embodiments, the binding region can specifically bind to a polypeptide composed of a sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity with SEQ ID NO: 13. Preferably, the binding region can specifically bind to a polypeptide composed of SEQ ID NO: 13.

[0066] In some embodiments, the binding region may comprise (or consist of) one or more sequences that specifically bind to the clostridial neurotoxin light chain (L) (e.g., as defined herein). In some embodiments, the binding region may comprise (or consist of) any sequence that specifically binds to the clostridial neurotoxin heavy chain (H) (e.g., as defined herein). In some embodiments, the binding region may comprise a clostridial neurotoxin translocation domain (H). N (e.g., as defined herein) any sequence (or composition thereof) that specifically binds to (e.g., as defined herein). In some embodiments, the binding region may contain a sequence that specifically binds to clostridium neurotoxin H. CN Any sequence (or composed of) that specifically binds to a domain (e.g., as defined herein). In some embodiments, the binding region may contain a binding domain that binds to Clostridium neurotoxin H. CC Any sequence (or composed of) that specifically binds to a domain (e.g., as defined herein).

[0067] For example, in some embodiments, the binding region may comprise (or consist of) one or more sequences that specifically bind to the BoNT / A light chain (L) (e.g., as defined herein). In some embodiments, the binding region may comprise (or consist of) any sequence that specifically binds to the BoNT / A heavy chain (H) (e.g., as defined herein). In some embodiments, the binding region may comprise a BoNT / A translocation domain (H). N (e.g., as defined herein) any sequence (or composition thereof) that specifically binds to. In some embodiments, the binding region may contain a sequence that specifically binds to BoNT / AH. CN Any sequence (or composed of) that specifically binds to a domain (e.g., as defined herein). In some embodiments, the binding region may contain elements that bind to BoNT / AH. CCAny sequence (or composed of) that specifically binds to a domain (e.g., as defined herein).

[0068] For example, in some embodiments, the binding region may comprise (or consist of) one or more sequences that specifically bind to the BoNT / B light chain (L) (e.g., as defined herein). In some embodiments, the binding region may comprise (or consist of) any sequence that specifically binds to the BoNT / B heavy chain (H) (e.g., as defined herein). In some embodiments, the binding region may comprise a BoNT / B translocation domain (H N (e.g., as defined herein) any sequence (or composition thereof) that specifically binds to. In some embodiments, the binding region may contain a sequence that specifically binds to BoNT / BH. CN Any sequence (or composed of) that specifically binds to a domain (e.g., as defined herein). In some embodiments, the binding region may contain elements specific to BoNT / BH. CC Any sequence (or composed of) that specifically binds to a domain (e.g., as defined herein).

[0069] In some embodiments, the binding region comprises one or more sequences that specifically bind to a chimeric clostridium neurotoxin polypeptide (e.g., mrBoNT / AB). In some embodiments, the binding region comprises one or more sequences that specifically bind to a modified clostridium neurotoxin polypeptide. In some embodiments, the modified clostridium neurotoxin polypeptide is an oxidized form of clostridium neurotoxin. In some embodiments, the binding region comprises one or more sequences that specifically bind to a malproteasome activated form of clostridium neurotoxin. In some embodiments, the binding region comprises one or more sequences that specifically bind to aggregated forms of clostridium neurotoxin (including dimers and multimers).

[0070] In some embodiments, one or more amino acid sequences comprise the CDR sequence of one or more antibodies. As used herein, the term “antibody” broadly refers to any immunoglobulin (Ig) molecule, such as a full-length “conventional” antibody comprising four polypeptide chains (e.g., two heavy (H) chains and two light (L) chains), as well as any functional fragment (e.g., an antigen-binding fragment), mutant, variant, or derivative thereof that retains the essential epitope-binding characteristics of an Ig molecule. Such mutant, variant, or derivative antibody entities are known in the art, and non-limiting examples thereof are discussed below.

[0071] In full-length antibodies, each heavy chain contains a heavy chain variable region (abbreviated as V in this paper). H The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated as V in this paper). LThe light chain constant region contains a domain, CL. H District and V L The region can be further subdivided into highly variable regions called complementarity-determining regions (CDRs), within which more conservative regions are interspersed, called frame regions (FRs). Each V H and V L It contains three CDRs and four FRs, which are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The antibody can be polyclonal (pAb) or monoclonal (mAb).

[0072] Alternatively, the term functional fragment (e.g., antigen-binding fragment) includes antibody fragments. Non-limiting examples of antibody fragments include Fab, Fv, scFv, dAb, Fd, Fab' or F(ab')2, tandem scFv, and biantibodies.

[0073] The antibodies of the present invention can be any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass or their functional fragments (e.g., antigen-binding fragments), mutants, variants or derivatives.

[0074] The antibodies of the present invention can be derived from any species (e.g., mice, humans, chickens, rats, rabbits, sheep, sharks, and camelids). Preferably, the antibodies of the present invention are camelid antibodies (e.g., from camels, dromedary camels, alpacas, llamas, or vicuñas) or cartilaginous fish (e.g., from sharks or rays) or their functional fragments (e.g., antigen-binding fragments), mutants, variants, or derivatives.

[0075] For example, as used herein, a “human antibody” is defined as an antibody having variable and constant regions derived from human immunoglobulin sequences, but it may, for example, contain amino acid residues in the CDR that are not encoded by human immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo).

[0076] In some embodiments, the antibody of the present invention may be a "CDR transplantation antibody"; defined as containing heavy chain and light chain variable region sequences from a species, but wherein V H and / or V L An antibody in which one or more CDR regions are replaced by CDR sequences of another species, such as an antibody with mouse heavy and light chain variable regions in which one or more mouse CDRs (e.g., CDR3 or all three CDRs) have been replaced by human CDR sequences.

[0077] In some embodiments, the antibody of the present invention may be a "humanized antibody"; defined as comprising a heavy chain and light chain variable region sequence derived from a non-human species (e.g., mouse), but wherein V H Sequence and / or V L At least a portion of the sequence has been altered to be more "human-like" (i.e., more similar to human germline variable sequences) antibodies. One type of humanized antibody is a CDR transplantation antibody, in which a human CDR sequence is introduced into a non-human antibody. H Sequence and V L The corresponding non-human CDR sequence is replaced in the sequence.

[0078] In some embodiments, the antibodies of the present invention may be “chimeric antibodies”; defined as antibodies comprising heavy and light chain variable region sequences from one species and constant region sequences from another species. The present invention covers chimeric antibodies having, for example, mouse heavy and light chain variable regions linked to human constant regions.

[0079] The antibodies of the present invention may be bispecific, dual-specific, or multispecific (e.g., binding to two or more different antigens). Preferably, the antibodies of the present invention will bind to the same antigen (e.g., binding to the same clostridium neurotoxin polypeptide).

[0080] It also covers antibody constructs, defined as polypeptides comprising one or more antigen-binding fragments of the present invention linked to a linker polypeptide or an immunoglobulin constant domain. The linker polypeptide comprises two or more amino acid residues linked by peptide bonds and is used to link one or more antigen-binding moieties.

[0081] Preferably, the binding region comprises a single-chain antibody or a multimer thereof (e.g., a dimer, trimer, or tetramer). Suitable single-chain antibodies include V of conventional antibodies (e.g., full-length human antibodies). H Domain and V L Domains, single-chain variable fragments (scFv), single-chain Fab (scFab), microantibodies, biantibodies or single-chain antibodies from camelids or cartilaginous fishes, and any functional fragments of a single-chain antibody that retain the basic epitope binding characteristics (e.g., antigen-binding fragments), mutants, variants or derivatives.

[0082] Functional single-chain antibodies derived from camelids are called HcAbs (heavy chain antibodies). HcAbs contain only the heavy chain (H) and not the light chain (L). HcAbs also lack the first constant domain (CH1) of the heavy chain. The N-terminal region contains a variable domain (called VHH) that binds to the specific antigen and two constant domains. The VHH in an HcAb is a structural and functional equivalent of the Fab fragment of a conventional antibody. Therefore, the antigen-binding site of an HcAb consists of only a single domain directly connected to the Fc domain via a hinge region. In preferred embodiments, the binding region comprises a camelid VHH or a camelid HcAb (or a combination thereof).

[0083] Functional single-chain antibodies produced by cartilaginous fish are known as novel immunoglobulin antigen receptors (IgNARs). IgNARs have a homodimeric structure comprising two heavy-chain polypeptides, each containing a single variable domain (VNAR) and five constant domains. In a preferred embodiment, the binding region comprises (or is composed of) a VNAR derived from cartilaginous fish (e.g., shark IgNAR).

[0084] VHH and VNAR, which are derived from camelids and cartilaginous fish respectively, are also known as nanobodies or nanoantibody (Nb).

[0085] Any single-chain antibody or its functional fragment (e.g., VHH or VNAR), mutant, variant, or derivative that retains the basic epitope binding characteristics of the single-chain antibody can be used in this invention, as long as the antibody binds to clostridial neurotoxin. Suitable single-chain antibodies are known in the art. For example, a single-chain antibody may comprise three CDR sequences (e.g., CDR1, CDR2, and CDR3) of one of the exemplary single-chain camelid antibodies shown in Table 1. In some embodiments, the single-chain antibody of this invention comprises three CDR sequences (e.g., CDR1, CDR2, and CDR3) and has at least 70% sequence identity with any of the single-chain camelid antibodies shown in Table 1; optionally, the antibody retains the binding specificity shown in Table 1. For example, in some embodiments, the single-chain antibody of the present invention comprises three CDR sequences (e.g., CDR1, CDR2, and CDR3) and has at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity with any of the single-chain camelid antibodies shown in Table 1; optionally, the antibody retains the binding specificity shown in Table 1. Preferably, the single-chain antibody of the present invention is selected from Table 1. Furthermore, those skilled in the art will be able to determine whether the alternative single-chain antibody binds to clostridium neurotoxin using conventional experimental methods.

[0086] Table 1: Exemplary Single-Chain Camelidae Antibodies In some embodiments, the binding region comprises a CDR sequence from a nanobody selected from the list comprising: ciA-H7, ciA-D1, ciA-H4, ciA-H11, ciA-C2, ciA-B5, ciA-F12, ciA-D12, ciA-A5, ciA-G5, ciB-H11, ciB-A11, ciB-B5, ciB-B9, ciA-H7 / B5, ciA-F12 / D12, and ciB-A11 / B5, or a combination thereof. Preferably, the binding region comprises three CDR sequences from at least one nanobody selected from the list comprising: ciA-H7, ciA-D1, ciA-H4, ciA-H11, ciA-C2, ciA-B5, ciA-F12, ciA-D12, ciA-A5, ciA-G5, ciB-H11, ciB-A11, ciB-B5, ciB-B9, ciA-H7 / B5, ciA-F12 / D12, and ciB-A11 / B5, or combinations thereof.

[0087] In some embodiments, the binding region comprises a sequence having at least 70% sequence identity with the full-length sequence of one or more nanobodies selected from the list below: ciA-H7, ciA-D1, ciA-H4, ciA-H11, ciA-C2, ciA-B5, ciA-F12, ciA-D12, ciA-A5, ciA-G5, ciB-H11, ciB-A11, ciB-B5, ciB-B9, ciA-H7 / B5, ciA-F12 / D12, and ciB-A11 / B5, or combinations thereof. For example, in some embodiments, the binding region comprises a sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity with the full-length sequence of one or more nanobodies selected from the list below: ciA-H7, ciA-D1, ciA-H4, ciA-H11, ciA-C2, ciA-B5, ciA-F12, ciA-D12, ciA-A5, ciA-G5, ciB-H11, ciB-A11, ciB-B5, ciB-B9, ciA-H7 / B5, ciA-F12 / D12, and ciB-A11 / B5. Preferably, the binding region comprises a full-length sequence of one or more nanobodies selected from the list below: ciA-H7, ciA-D1, ciA-H4, ciA-H11, ciA-C2, ciA-B5, ciA-F12, ciA-D12, ciA-A5, ciA-G5, ciB-H11, ciB-A11, ciB-B5, ciB-B9, ciA-H7 / B5, ciA-F12 / D12, and ciB-A11 / B5, or combinations thereof.

[0088] In some embodiments, the single-chain antibody of the present invention comprises three CDR sequences (e.g., CDR1, CDR2, and CDR3) and has at least 70% sequence identity with any one of SEQ ID NO: 22 to 44; optionally, the antibody retains the binding specificity shown in Table 1. For example, in some embodiments, the single-chain antibody of the present invention comprises three CDR sequences (e.g., CDR1, CDR2, and CDR3) and has at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity with any one of SEQ ID NO: 22 to 44; optionally, the antibody retains the binding specificity shown in Table 1. Preferably, the single-chain antibody of the present invention is selected from any one of SEQ ID NO: 22 to 44. Furthermore, those skilled in the art will be able to determine whether alternative single-chain antibodies bind to clostridium neurotoxin using conventional experimental methods.

[0089] In some embodiments, the single-chain antibody of the present invention comprises three CDR sequences (e.g., CDR1, CDR2, and CDR3) and has at least 70% sequence identity with SEQ ID NO: 22; optionally, the antibody retains the binding specificity shown in Table 1. For example, in some embodiments, the single-chain antibody of the present invention comprises three CDR sequences (e.g., CDR1, CDR2, and CDR3) and has at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity with SEQ ID NO: 22; optionally, the antibody retains the binding specificity shown in Table 1. Preferably, the single-chain antibody of the present invention comprises SEQ ID NO: 22.

[0090] In some embodiments, the single-chain antibody of the present invention comprises three CDR sequences (e.g., CDR1, CDR2, and CDR3) and has at least 70% sequence identity with SEQ ID NO: 23; optionally, the antibody retains the binding specificity shown in Table 1. For example, in some embodiments, the single-chain antibody of the present invention comprises three CDR sequences (e.g., CDR1, CDR2, and CDR3) and has at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity with SEQ ID NO: 23; optionally, the antibody retains the binding specificity shown in Table 1. Preferably, the single-chain antibody of the present invention comprises SEQ ID NO: 23.

[0091] In some embodiments, the single-chain antibody of the present invention comprises three CDR sequences (e.g., CDR1, CDR2, and CDR3) and has at least 70% sequence identity with SEQ ID NO: 24; optionally, the antibody retains the binding specificity shown in Table 1. For example, in some embodiments, the single-chain antibody of the present invention comprises three CDR sequences (e.g., CDR1, CDR2, and CDR3) and has at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity with SEQ ID NO: 24; optionally, the antibody retains the binding specificity shown in Table 1. Preferably, the single-chain antibody of the present invention comprises SEQ ID NO: 24.

[0092] In some embodiments, the single-chain antibody of the present invention comprises three CDR sequences (e.g., CDR1, CDR2, and CDR3) and has at least 70% sequence identity with SEQ ID NO: 25; optionally, the antibody retains the binding specificity shown in Table 1. For example, in some embodiments, the single-chain antibody of the present invention comprises three CDR sequences (e.g., CDR1, CDR2, and CDR3) and has at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity with SEQ ID NO: 25; optionally, the antibody retains the binding specificity shown in Table 1. Preferably, the single-chain antibody of the present invention comprises SEQ ID NO: 25.

[0093] In some embodiments, the single-chain antibody of the present invention comprises three CDR sequences (e.g., CDR1, CDR2, and CDR3) and has at least 70% sequence identity with SEQ ID NO: 26; optionally, the antibody retains the binding specificity shown in Table 1. For example, in some embodiments, the single-chain antibody of the present invention comprises three CDR sequences (e.g., CDR1, CDR2, and CDR3) and has at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity with SEQ ID NO: 26; optionally, the antibody retains the binding specificity shown in Table 1. Preferably, the single-chain antibody of the present invention comprises SEQ ID NO: 26.

[0094] In preferred embodiments, antibodies (e.g., VHH and VNAR derived from camelids or cartilaginous fish) are capable of neutralizing clostridium neurotoxin toxicity. Suitable neutralizing antibodies are known in the art. Furthermore, those skilled in the art will be able to determine whether an antibody is a neutralizing antibody using conventional experimental methods. In some embodiments, when the antibody is a multimer, only one of the antibodies may be neutralizing. For example, a dimer may comprise a first neutralizing antibody and a second non-neutralizing antibody. In some embodiments, the potency of an antibody can be enhanced by combining two or more antibodies (e.g., neutralizing and non-neutralizing antibodies such as ciA-B5 and ciA-D12).

[0095] In particularly preferred embodiments, the antibody is selected from ciA-H7 (SEQ ID NO: 24 or 25), ciA-C2 (SEQ ID NO: 22 or 23), ciA-D12 (SEQ ID NO: 29), ciA-B5 (SEQ ID NO: 27), and their multimers (e.g., dimers, trimers, or tetramers). In these antibodies, ciA-H7 binds to the BoNT / A light chain (L), and ciA-B5 binds to the BoNT / A translocation domain (H). N ) combines, and ciA-C2 and BoNT / A in H CC Domain and H CN Binding occurs at regions between the domains. Alternatively, the antibody may be a dimer comprising ciA-B5 and ciA-D12 (e.g., ciA-B5-D12, SEQ ID NO: 26), wherein each single-chain antibody binds simultaneously to BoNT / A.

[0096] The antibodies of this invention are not limited to specific methods of generation or production. Therefore, this invention provides antibodies already manufactured by antibody-secreting hybridomas, as well as antibodies generated by recombinant cells that have been transformed or transfected with one or more nucleic acids encoding the antibody. Preferably, the recombinant antibody is a single-chain antibody (e.g., a camelid VHH or camelid HcAb and / or a cartilaginous fish VNAR or cartilaginous fish IgNAR). Such hybridomas, recombinant cells, and nucleic acids form part of this invention.

[0097] The antibodies of the present invention, or their antigen-binding fragments, exhibit selectivity or specificity for specific epitopes of clostridium neurotoxin polypeptides or clostridium neurotoxin polypeptides as described herein. In terms of specificity, it will be understood that the antibody binds to the target molecule (in this case, a clostridium neurotoxin polypeptide) but shows no significant cross-reactivity with non-clostridium neurotoxin polypeptides. In some embodiments, antibodies specific to a specific clostridium neurotoxin epitope of the present invention will not show significant cross-reactivity with other clostridium neurotoxin epitopes. For example, antibodies specific to the BoNT / A heavy chain may not show significant cross-reactivity with the BoNT / B heavy chain. Cross-reactivity can be assessed by any suitable method. If an antibody binds to another clostridium neurotoxin epitope or a non-clostridium neurotoxin polypeptide with at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 100% of its binding strength to the clostridium neurotoxin epitope, then the cross-reactivity of the antibody with that clostridium neurotoxin epitope to another molecule can be considered significant. Binding affinity can be quantified in any suitable manner, such as by K... D Quantitative.

[0098] In some embodiments, the support is configured to immobilize the binding substrate onto the support. Immobilizing the binding substrate onto the support advantageously allows for convenient washing of any unbound complex and easy removal of the supernatant, while minimizing the loss of the bound complex. For example, immobilizing the binding substrate onto the support advantageously allows for convenient washing of any clostridium neurotoxin peptides (e.g., BoNT / A) that do not bind to the immobilized binding substrate, and easy removal of the supernatant, while minimizing the loss of the immobilized binding substrate-clostridium neurotoxin complex. Immobilization methods are known in the art.

[0099] In other embodiments, a support is configured to immobilize the clostridium neurotoxin polypeptide onto the support. Immobilizing the clostridium neurotoxin polypeptide onto the support advantageously allows for convenient washing of any unbound complexes and easy removal of the supernatant, while minimizing the loss of bound complexes. For example, immobilizing the clostridium neurotoxin polypeptide onto the support advantageously allows for convenient washing of any binding substrates that do not bind to the immobilized clostridium neurotoxin polypeptide and easy removal of the supernatant, while minimizing the loss of the immobilized clostridium neurotoxin-binding substrate complex. Immobilization methods are known in the art.

[0100] Supports can take many forms. For example, any suitable surface for immobilizing peptides (e.g., binding substrates or clostridial neurotoxins) can be used. Non-limiting examples of suitable supports include beads, plates, columns, chips, or reaction vessels.

[0101] In a preferred embodiment, the support is an amine-reactive second-generation (AR2G) support. Those skilled in the art will understand that the AR2G support contains a high density of carboxylic acids that can be activated by reacting with EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) and s-NHS (N-hydroxysulfosuccinimide) to generate highly reactive NHS esters. NHS esters react rapidly with the primary amines of biomolecules such as proteins and peptides to form highly stable amide bonds. The resulting immobilized biomolecules can be used for screening or kinetic experiments. Successful regeneration (e.g., using an acidic buffer, such as a buffer having a pH of about 1 to about 2, preferably about 1.0 to 1.7) will remove the analyte-binding coupler and leave the immobilized target protein intact on the biosensor surface.

[0102] Therefore, in some embodiments, the binding substrate is immobilized on the support by forming a stable amine bond between the NHS ester on the amine reactive second generation (AR2G) support and the primary amine in the binding substrate. Preferably, the binding substrate is immobilized on the amine reactive second generation (AR2G) support. In some embodiments, the binding substrate is configured to bind to the AR2G support. Alternatively, the binding substrate is configured to bind to the support via a tag. In some embodiments, a tag (such as a GST tag) is present on the binding substrate, and a suitable binding partner (e.g., glutathione or a derivative thereof) is present on the support.

[0103] Alternatively, the clostridial neurotoxin peptide can be immobilized on the support by forming a stable amine bond between the NHS ester on the amine-reactive second-generation (AR2G) support and the primary amine in the clostridial neurotoxin peptide. Preferably, the clostridial neurotoxin peptide is immobilized on the amine-reactive second-generation (AR2G) support. In some embodiments, the AR2G support is configured for binding to the clostridial neurotoxin peptide. Alternatively, the clostridial neurotoxin peptide is configured for binding to the support via a tag. In some embodiments, the tag (such as a GST tag) is present on the binding substrate, and a suitable binding partner (e.g., glutathione or a derivative thereof) is present on the support.

[0104] The biosensors of the present invention can be used to monitor binding events and kinetics in real time. Real-time monitoring facilitates the rapid optimization and control of biological processes. In some embodiments, real-time monitoring can be at-line, on-line, or in-line. The biosensors of the present invention can be used to monitor binding events and kinetics at-line. The biosensors of the present invention can be used to monitor binding events and kinetics on-line. The biosensors of the present invention can be used to monitor binding events and kinetics in-line.

[0105] Near-line measurements typically involve removing a sample and then analyzing it manually or using an automated sampling device near the production process. In-line measurements typically involve transferring the sample from the manufacturing process via a bypass flow and returning it to the bioreactor. In-line measurements enable continuous monitoring and thus control of the production process. Finally, in-line (or in-situ) measurements are typically performed directly in a bioreactor equipped with process sensors.

[0106] In contrast, offline measurements typically involve removing the sample from the bioreactor before analysis. Generally, sample pretreatment (e.g., filtration and / or dilution) is required between the removal from the bioreactor and the analysis. Besides the complexities involved in manual processing, a major drawback of offline measurements is the time delay, which leads to reduced measurement frequency and potentially lower product yield.

[0107] The binding substrate of the present invention can be produced by expressing a nucleic acid encoding the binding substrate in a suitable host cell. For example, the host cell can be a bacterial cell, such as *Escherichia coli* (E. coli). E. coli The host cells can be either DH5α cells or BL21 cells; or bacteria of the genus Clostridium, such as Clostridium botulinum. The binding substrate can then be isolated from the host cells using standard techniques.

[0108] The nucleic acid molecule encoding the binding substrate of the present invention is also described.

[0109] The nucleic acid encoding the binding substrate of this invention can encode: A polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 45; preferably, a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity with SEQ ID NO: 45; most preferably, a polypeptide sequence comprising SEQ ID NO: 45; and A polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 46; preferably, a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity with SEQ ID NO: 46; most preferably, a polypeptide sequence comprising SEQ ID NO: 46.

[0110] The nucleic acid encoding the binding substrate of this invention can encode: A polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 45; preferably, a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity with SEQ ID NO: 45; most preferably, a polypeptide sequence comprising SEQ ID NO: 45; A polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 22 or 23; preferably, a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity with SEQ ID NO: 22 or 23; most preferably, a polypeptide sequence comprising SEQ ID NO: 22 or 23; and A polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 46; preferably, a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity with SEQ ID NO: 46; most preferably, a polypeptide sequence comprising SEQ ID NO: 46.

[0111] The nucleic acid encoding the binding substrate of this invention can encode: A polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 45; preferably, a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity with SEQ ID NO: 45; most preferably, a polypeptide sequence comprising SEQ ID NO: 45; A polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 22 or 23, wherein the CDR sequence is as defined in SEQ ID NO: 23; preferably, a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity with SEQ ID NO: 22 or 23, wherein the CDR sequence is as defined in SEQ ID NO: 23; most preferably, a polypeptide sequence comprising SEQ ID NO: 22 or 23; and A polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 46; preferably, a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity with SEQ ID NO: 46; most preferably, a polypeptide sequence comprising SEQ ID NO: 46.

[0112] The binding substrate of the present invention may comprise: A polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 45; preferably, a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity with SEQ ID NO: 45; most preferably, a polypeptide sequence comprising SEQ ID NO: 45; and A polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 46; preferably, a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity with SEQ ID NO: 46; most preferably, a polypeptide sequence comprising SEQ ID NO: 46.

[0113] The binding substrate of the present invention may comprise: A polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 45; preferably, a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity with SEQ ID NO: 45; most preferably, a polypeptide sequence comprising SEQ ID NO: 45; A polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 22 or 23; preferably, a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity with SEQ ID NO: 22 or 23; most preferably, a polypeptide sequence comprising SEQ ID NO: 22 or 23; and A polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 46; preferably, a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity with SEQ ID NO: 46; most preferably, a polypeptide sequence comprising SEQ ID NO: 46.

[0114] The binding substrate of the present invention may comprise: A polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 45; preferably, a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity with SEQ ID NO: 45; most preferably, a polypeptide sequence comprising SEQ ID NO: 45; A polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 22 or 23, wherein the CDR sequence is as defined in SEQ ID NO: 23; preferably, a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity with SEQ ID NO: 22 or 23, wherein the CDR sequence is as defined in SEQ ID NO: 23; most preferably, a polypeptide sequence comprising SEQ ID NO: 22 or 23; and A polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 46; preferably, a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity with SEQ ID NO: 46; most preferably, a polypeptide sequence comprising SEQ ID NO: 46.

[0115] The concentration of clostridial neurotoxin polypeptide in a composition can be determined using the method of the present invention. To obtain the concentration, the method can be performed using at least a second, third, and / or fourth composition comprising different concentrations of clostridial neurotoxin polypeptide and determining the resonance energy transfer value of the at least second, third, and / or fourth composition.

[0116] In some embodiments, the biosensor enables the detection of clostridium neurotoxins at concentrations less than 5 ng / ml, less than 4 ng / ml, less than 3 ng / ml, less than 2 ng / ml, less than 1 ng / ml, less than 0.9 ng / ml, less than 0.8 ng / ml, less than 0.7 ng / ml, less than 0.6 ng / ml, less than 0.5 ng / ml, less than 0.4 ng / ml, less than 0.3 ng / ml, less than 0.2 ng / ml, or less than 0.1 ng / ml. Preferably, the biosensor enables the detection of clostridium neurotoxins at concentrations less than 0.1 ng / ml.

[0117] In some instances, the term "biosensor" is used herein. However, this is to indicate that more than one "biosensor" may be present when the method is performed. It is not intended to necessarily indicate the presence of two or more different types of biosensors, although this is covered. Thus, in some embodiments, the biosensor may be of one type, for example, all biosensors comprising a surface for analyzing binding events and kinetics, and immobilizing on this surface a plurality of substantially identical binding substrates comprising: (i) a first donor fluorophore; (ii) a clostridium neurotoxin binding region; and (iii) a receptor having an absorption spectrum overlapping the emission spectrum of the donor fluorophore. In other embodiments, the biosensor may be of one type, for example, all biosensors comprising a surface for analyzing binding events and kinetics, and immobilizing on this surface a clostridium neurotoxin polypeptide and individually a plurality of substantially identical binding substrates comprising (i) a first donor fluorophore; (ii) a clostridium neurotoxin binding region; and (iii) a receptor having an absorption spectrum overlapping the emission spectrum of the donor fluorophore. Preferably, the biosensor is of one type.

[0118] Similarly, in some cases, the term "binding substrate" is used herein. However, this is to indicate that more than one "binding substrate" may be present when the method is performed. It is not intended to necessarily indicate the presence of two or more different types of binding substrates, although this is covered. Thus, the binding substrate can be of one type, for example, all binding substrates are substantially identical and comprise: (i) a first donor fluorophore; (ii) a clostridium neurotoxin binding region; and (iii) a receptor having an absorption spectrum that overlaps with the emission spectrum of the donor fluorophore. Preferably, the binding substrate is of one type.

[0119] In one aspect, the present invention relates to a method for real-time (e.g., in-line, beside-line, or on-line) monitoring of clostridial neurotoxin production during a bacterial fermentation process. In some embodiments, the present invention relates to a method for in-line monitoring of clostridial neurotoxin production during a bacterial fermentation process. In some embodiments, the present invention relates to a method for beside-line monitoring of clostridial neurotoxin production during a bacterial fermentation process. In some embodiments, the present invention relates to a method for on-line monitoring of clostridial neurotoxin production during a bacterial fermentation process.

[0120] The method of the present invention may further include the step of culturing host cells capable of producing clostridial neurotoxin. Preferably, the host cells are bacterial host cells (e.g., *Escherichia coli* or bacteria of the genus *Clostridium* such as *Clostridium botulinum*). In some embodiments, the nucleic acid sequence encoding clostridial neurotoxin is modified against codon bias according to the expression system of the final host cell to be used (e.g., *Escherichia coli* or bacteria of the genus *Clostridium* such as *Clostridium botulinum*). Preferably, clostridial neurotoxin is produced by culturing bacterial host cells capable of producing clostridial neurotoxin (e.g., *Escherichia coli* or bacteria of the genus *Clostridium* such as *Clostridium botulinum*) in a liquid culture medium.

[0121] In some embodiments, a method for real-time monitoring of Clostridium neurotoxin production includes the step of contacting a culture medium (e.g., a liquid culture medium) with the biosensor of the present invention. In some embodiments, In some embodiments, clostridial neurotoxin is secreted into the fermentation solution. In other embodiments, where clostridial neurotoxin is not secreted into the fermentation solution, the method may further include the step of releasing clostridial neurotoxin from host cells. Means for releasing clostridial neurotoxin from host cells are known in the art and include both mechanical and non-mechanical means of cell disruption or lysis.

[0122] A method for real-time (e.g., in-line, sideline, or in-line) monitoring of clostridial neurotoxin production during bacterial fermentation is also provided, the method comprising transforming bacterial host cells capable of producing clostridial neurotoxins with an expression construct encoding the binding substrate of the present invention. Thus, the binding substrate is expressed in the host cells.

[0123] The method of the present invention may further include the step of culturing transformed host cells. Preferably, the transformed host cells are bacterial host cells (e.g., *Escherichia coli* or bacteria of the genus *Clostridium* such as *Clostridium botulinum*). In some embodiments, the nucleic acid sequence encoding clostridium neurotoxin is modified against codon bias according to the expression system of the transformed host cell (e.g., *Escherichia coli* or bacteria of the genus *Clostridium* such as *Clostridium botulinum*) to be used.

[0124] The method of the present invention further includes the step of exciting a donor fluorophore in the biosensor. In some embodiments, the excitation of the donor fluorophore requires exposure of the donor fluorophore to radiation having a wavelength selected from the donor excitation radiation range. In some embodiments, the excitation wavelength is specifically selected to avoid directly exciting the acceptor fluorophore, and thereby reducing crosstalk and background signal.

[0125] The method of the present invention may further include the step of measuring the characteristics of the biosensor. In a preferred embodiment, the method includes the step of determining the resonant energy transfer at the biosensor. As indicated above, this can be determined by measuring the fluorescence intensity of the donor and / or acceptor. Preferably, this can be determined by measuring the fluorescence intensity of the acceptor.

[0126] Clostridium neurotoxins in a sample are detected by measuring the emission from the donor fluorophore and the acceptor using a fluorescence analysis system. The emission from the fluorescent donor and acceptor is measured using a fluorescence analysis system, and the emission from the fluorescent donor and acceptor changes if the amount or concentration of the clostridium neurotoxin changes. Therefore, this invention can be used to measure changes in the amount or concentration of clostridium neurotoxins.

[0127] FRET can be measured by a variety of different methods, each of which will be known to those skilled in the art. Exemplary, non-limiting methods for measuring FRET include sensitized emission, acceptor photobleaching, fluorescence lifetime imaging microscopy FRET (FLIMFRET), fluorophore donor spectral imaging, and isomorphic FRET and polarization anisotropy imaging. For example, sensitized emission typically involves exciting a donor fluorophore and collecting signals from both the donor and acceptor fluorophores using specially selected emission filters. Using this method, acceptor fluorescence increases in the presence of a donor, while donor fluorescence decreases in the presence of an acceptor. FRET can then be measured using the ratio of fluorescence intensity changes. Alternatively, acceptor photobleaching is based on a technique that quenches donor fluorescence during FRET when some donor fluorescence energy is transferred to the acceptor. In FLIM FRET, donor fluorescence is quenched through FRET interactions, and the amount of quenching can be calculated by measuring the reduction in the fluorescence decay time of the donor molecule. Spectroscopic imaging involves recording the entire emission spectrum of both donor and acceptor fluorescence at donor excitation. Spectroscopic imaging is based on the principle that overlapping spectra can be separated not only by their emission peaks but also by their different overall shapes. Finally, similar to conventional FRET, isomorphic FRET involves the transfer of excited-state energy between fluorophores. However, the fluorophores selected in isomorphic FRET are identical.

[0128] Preferably, determining the relative resonant energy transfer at the biosensor includes the step of comparing the resonant energy transfer with a control, wherein the difference in resonant energy transfer at the biosensor compared with the control indicates the presence of clostridial neurotoxin.

[0129] In some embodiments, a suitable control may be a negative control (e.g., a composition not containing clostridium neurotoxin peptides). A negative control may be a negative reference standard. A negative reference standard may correspond to a value that has been theoretically or experimentally determined and represents a negative result in the method of the present invention. This value may have been determined before carrying out the method of the present invention, or it may be determined concurrently with or after carrying out the method of the present invention.

[0130] In some embodiments (e.g., when the control is a negative control), an increase in donor fluorescence intensity at the biosensor compared to the control can indicate the presence of clostridial neurotoxin. In contrast, a decrease in donor fluorescence intensity at the biosensor compared to the control can allow determination that the composition does not contain (or contains undetectable amounts of) clostridial neurotoxin peptides.

[0131] In some embodiments (e.g., when the control is a negative control), a decrease in receptor fluorescence intensity at the biosensor compared to the control can indicate the presence of clostridial neurotoxin. In some embodiments, when the control is a negative control, the same level of receptor fluorescence intensity compared to the negative control indicates that the tested composition does not contain a clostridial neurotoxin peptide. In such cases, this can allow determination that the composition does not contain a clostridial neurotoxin peptide.

[0132] In some embodiments, a suitable control may be a positive control (e.g., a composition containing a known or predetermined concentration of a clostridium neurotoxin polypeptide (e.g., a clostridium neurotoxin polypeptide capable of binding to the binding region).

[0133] In some embodiments (e.g., when the control is a positive control), an increase in donor fluorescence intensity at the biosensor compared to the control may indicate a higher amount or concentration of clostridial neurotoxin. Conversely, a decrease in donor fluorescence intensity at the biosensor compared to the control may allow determination that the composition contains a lower amount or concentration of clostridial neurotoxin peptides.

[0134] In some embodiments, when the control is a positive control, a decrease in receptor fluorescence intensity at the biosensor compared to the control may indicate a higher amount or concentration of clostridial neurotoxin. In some embodiments, when the control is a positive control, having the same level of receptor fluorescence intensity compared to the positive control may allow determination that the composition being tested contains a known or predetermined concentration of clostridial neurotoxin peptide.

[0135] In some embodiments, the method of the present invention includes the step of comparing the donor and / or acceptor fluorescence intensity of a test composition with the donor and / or acceptor fluorescence intensity of a control composition.

[0136] The degree of difference can be quantified to indicate the quantity of clostridial neurotoxin peptides. The degree of difference can also be quantified to indicate the concentration of clostridial neurotoxin peptides. For example, preferably, the higher the donor fluorescence compared to a negative control, the higher the quantity of clostridial neurotoxin peptides. Preferably, the higher the donor fluorescence compared to a negative control, the higher the concentration of clostridial neurotoxin peptides. Alternatively, preferably, the lower the receptor fluorescence compared to a negative control, the higher the quantity of clostridial neurotoxin peptides. Preferably, the lower the receptor fluorescence compared to a negative control, the higher the concentration of clostridial neurotoxin peptides.

[0137] In some embodiments, the step of determining the resonant energy transfer at the biosensor (e.g., relative to a control) includes detecting the maximum emission of the receptor and the maximum emission of the donor fluorophore at the biosensor, optionally wherein an offset of the emission maximum from near the maximum emission of the receptor to near the maximum emission of the donor fluorophore indicates the presence of clostridium neurotoxin.

[0138] In some embodiments, the step of determining the resonant energy transfer at the biosensor (e.g., relative to a control) includes detecting the ratio of fluorescence amplitude near the maximum emission value of the receptor to the fluorescence amplitude near the maximum emission value of the donor fluorophore, optionally wherein a decrease in the ratio at the biosensor compared to a control indicates the presence of clostridium neurotoxin.

[0139] As used herein, the term “different” (and related terms such as “change,” “variable,” and “differential” and their synonyms) may mean a difference that is significantly different from a comparison object (e.g., a control as described herein). A difference (and related terms such as “change,” “variable,” and “differential” and their synonyms) may mean a statistically significant difference when compared to a comparison object (e.g., a control as described herein). A “significant difference” may be a difference of at least 5%, 10%, 15%, 20%, 25%, or 30% when compared to a comparison object (e.g., a control as described herein). The term “no difference” (and related terms such as “unchanged” and “same” and their synonyms) may mean that there is no significant difference when compared to a comparison object (e.g., a control as described herein). No difference (and related terms such as “unchanged” and “same” and their synonyms) may mean that there is no statistically significant difference when compared to a comparison object (e.g., a control as described herein). As used herein, the term "lower" (and related terms such as "less than") may mean at least 10%, 25%, 20%, 50%, 75%, 100%, 150%, or 200% lower than a comparison object (e.g., a control as described herein). The term "lower" (and related terms such as "less than") may mean statistically significantly lower than a comparison object (e.g., a control as described herein). As used herein, the term "higher" (and related terms such as "above") may mean at least 10%, 25%, 20%, 50%, 75%, 100%, 150%, or 200% higher than a comparison object (e.g., a control as described herein). The term "higher" (and related terms such as "above") may mean statistically significantly higher than a comparison object (e.g., a control as described herein).

[0140] In some cases, the terms "clostridium neurotoxin polypeptide" and "clostridium neurotoxin H" are mentioned in this article. CC "Structural domain" (respectively). However, this is to indicate that when the method is performed, (e.g., in the composition) more than one clostridium neurotoxin polypeptide or clostridium neurotoxin H may be present. CC Domains (separately). It is not intended to necessarily indicate the presence of two or more different types of clostridium neurotoxin polypeptides or clostridium neurotoxin H. CCStructural domains (separately), although this is covered. Therefore, clostridium neurotoxin polypeptides or clostridium neurotoxin H CC The domains can be of one type (e.g., all clostridium neurotoxin peptides are BoNT / A peptides, or all clostridium neurotoxin H...). CC All structural domains are BoNT / AH CC Domains [respectively], or multiple types (e.g., a portion of a clostridium neurotoxin polypeptide is a BoNT / B polypeptide and a portion is a BoNT / A polypeptide, or clostridium neurotoxin H...). CC Part of the structural domain is BoNT / AH CC The structural domain and part of it is BoNT / BH CC (Structural domains [respectively]). Preferably, the clostridium neurotoxin polypeptide is of one type. Preferably, the clostridium neurotoxin H CC A domain is one type. The same applies to instances where "botulinum neurotoxin peptide" is mentioned in this article.

[0141] The clostridium neurotoxin according to the present invention may comprise botulinum neurotoxin or tetanus neurotoxin (TeNT) H CC Structural domain. The clostridium neurotoxin of the present invention may contain BoNT / AH. CC Structural domain, BoNT / BH CC Structural domain, BoNT / C1H CC Structural domain, BoNT / DH CC Structural domain, BoNT / EH CC Structural domain, BoNT / FH CC Structural domain, BoNT / GH CC Structural domain, BoNT / XH CC Domain or TeNT H CC Structural domain. Preferably, the clostridial neurotoxin of the present invention comprises BoNT / BH. CC Structural domain or BoNT / AH CC Structural domain, preferably BoNT / BH CC Structural domain.

[0142] The clostridium neurotoxin according to the present invention may comprise botulinum neurotoxin or tetanus neurotoxin (TeNT) H C Structural domain. The clostridium neurotoxin of the present invention may contain BoNT / AH. C Structural domain, BoNT / BH C Structural domain, BoNT / C1 H C Structural domain, BoNT / DH C Structural domain, BoNT / EH CStructural domain, BoNT / FH C Structural domain, BoNT / GH C Structural domain, BoNT / XH C Domain or TeNT H C Structural domain. Preferably, the clostridial neurotoxin of the present invention comprises BoNT / BH. C Structural domain or BoNT / AH C Structural domain, preferably BoNT / BH C Structural domain.

[0143] The term "clostridium neurotoxin" encompasses toxins produced by *Clostridium botulinum* (botulinum neurotoxin serotypes A, B, C1, D, E, F, G, and X), toxins produced by *Clostridium tetani* (tetanus neurotoxin), toxins produced by *Clostridium butyricum* (botulinum neurotoxin serotype E), and toxins produced by *Clostridium pasteurellii* (botulinum neurotoxin serotype F). Reference BoNT / A sequence is shown as SEQ ID NO: 1. Reference BoNT / B sequence is shown as SEQ ID NO: 2. Reference BoNT / C1 (also referred to herein as BoNT / C) sequence is shown as SEQ ID NO: 3. Reference BoNT / D sequence is shown as SEQ ID NO: 4. Reference BoNT / E sequence is shown as SEQ ID NO: 5. Reference BoNT / F sequence is shown as SEQ ID NO: 6. Reference BoNT / G sequence is shown as SEQ ID NO: 7. Reference BoNT / X sequence is shown as SEQ ID NO: 8. Reference TeNT sequence is shown as SEQ ID NO: 9. The term "clostridium neurotoxin" can also encompass newly discovered members of the botulinum neurotoxin protein family expressed by non-clostridium microorganisms, such as the toxin encoded by Enterococcus (which has the closest sequence identity to BoNT / X), and Milweissella var. voles called BoNT / Wo. Weissella oryzae The toxin encoded by ) (NCBI reference sequence: WP_027699549.1) (which cleaves VAMP2 at W89-W90), Enterococcus faecalis ( Enterococcus faecium The toxin encoded by ) (GenBank: OTO22244.1) (which cleaves VAMP2 and SNAP25) and Piperichus chrysogenum ( Chryseobacterium pipero The toxin is encoded as WP_034687872.1 (NCBI reference sequence: WP_034687872.1).

[0144] Therefore, the clostridial neurotoxin can be selected from BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, BoNT / X, and TeNT (tetanus neurotoxin). Therefore, the compositions of the present invention can contain BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, BoNT / X, or TeNT. Therefore, the clostridial neurotoxin polypeptide can be a BoNT / A polypeptide, a BoNT / B polypeptide, a BoNT / C polypeptide, a BoNT / D polypeptide, a BoNT / E polypeptide, a BoNT / F polypeptide, a BoNT / G polypeptide, a BoNT / X polypeptide, or a TeNT polypeptide. Preferably, the clostridial neurotoxin is a botulinum neurotoxin, such as a botulinum neurotoxin selected from BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, and BoNT / X. Therefore, the compositions of the present invention may contain BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, or BoNT / X. Therefore, the clostridium neurotoxin polypeptide may be a BoNT / A polypeptide, a BoNT / B polypeptide, a BoNT / C polypeptide, a BoNT / D polypeptide, a BoNT / E polypeptide, a BoNT / F polypeptide, a BoNT / G polypeptide, or a BoNT / X polypeptide.

[0145] Clostridium neurotoxin is composed of two polypeptide chains: a heavy chain (H chain) with a molecular weight of approximately 100 kDa and a light chain (L chain) with a molecular weight of approximately 50 kDa. The H chain contains a C-terminal targeting component (receptor-binding domain or H+). C (structural domain) and N-terminal translocation component (H) N (Structural domains). Botulinum neurotoxin (BoNT) is produced by Clostridium botulinum as a large protein complex, which consists of BoNTs themselves complexed with various accessory proteins. Currently, there are eight different classes of botulinum neurotoxin: serotypes A, B, C1, D, E, F, G, and X, all with similar structures and modes of action. Different BoNT serotypes can be distinguished based on inactivation by specific neutralizing antisera, where this serotype classification is related to the percentage of sequence identity at the amino acid level. Based on the percentage of amino acid sequence identity, the BoNT protein of a given serotype is further subdivided into different subtypes.

[0146] BoNT is absorbed in the gastrointestinal tract and, upon entering systemic circulation, binds to the presynaptic membrane of cholinergic nerve endings, preventing the release of the neurotransmitter acetylcholine. BoNT / B, BoNT / D, BoNT / F, and BoNT / G cleave small synaptic vesicle protein / vesicle-associated membrane protein (VAMP); BoNT / C1, BoNT / A, and BoNT / E cleave 25 kDa synaptic-associated protein (SNAP-25); and BoNT / C1 cleaves synaptic fusion protein. BoNT / X has been found to cleave SNAP-25, VAMP1, VAMP2, VAMP3, VAMP4, VAMP5, Ykt6, and synaptic fusion protein 1. Tetanus toxin is produced by Clostridium tetani in a single serotype. Clostridium butyricum produces BoNT / E, while Clostridium pasteurellium produces BoNT / F.

[0147] Examples of L-chain reference sequences include: Type A botulinum neurotoxin: amino acid residues 1-448 Botulinum neurotoxin type B: Amino acid residues 1-440 C1 type botulinum neurotoxin: amino acid residues 1-441 Botulinum neurotoxin type D: Amino acid residues 1-445 Botulinum neurotoxin type E: Amino acid residues 1-422 Type F botulinum neurotoxin: amino acid residues 1-439 G-type botulinum neurotoxin: amino acid residues 1-441 Tetanus neurotoxin: amino acid residues 1-457 For the recently identified BoNT / X, the L chain has been reported to correspond to its amino acid 1-439, where the L chain boundary may vary by about 25 amino acids (e.g., 1-414 or 1-464).

[0148] The BoNT / AL chain may contain a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% of amino acid residues 1-448 of SEQ ID NO: 1. The BoNT / BL chain may contain a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% of amino acid residues 1-440 of SEQ ID NO: 2. The BoNT / C1 L chain may contain a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% of amino acid residues 1-441 of SEQ ID NO: 3. The BoNT / DL chain may contain a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% of amino acid residues 1-445 of SEQ ID NO: 4. The BoNT / EL chain may contain a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% of the amino acid residues 1-422 of SEQ ID NO: 5. The BoNT / FL chain may contain a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% of the amino acid residues 1-439 of SEQ ID NO: 6. The BoNT / GL chain may contain a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% of the amino acid residues 1-441 of SEQ ID NO: 7. The BoNT / XL chain may contain a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% of the amino acid residues 1-439 of SEQ ID NO: 8. The TeNT L chain may contain a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% of amino acid residues 1-457 of SEQ ID NO: 9.

[0149] The reference sequences used for identification above should be considered as guidelines, as slight variations may occur depending on the subserotype. By way of example, US 2007 / 0166332 (incorporated hereinforcingly in its entirety) references a slightly different Clostridium sequence: Botulinum neurotoxin type A: Amino acid residue M1-K448 Botulinum neurotoxin type B: Amino acid residue M1-K441 C1 type botulinum neurotoxin: amino acid residue M1-K449 Type D botulinum neurotoxin: amino acid residue M1-R445 Type E botulinum neurotoxin: amino acid residue M1-R422 Type F botulinum neurotoxin: amino acid residue M1-K439 G-type botulinum neurotoxin: amino acid residue M1-K446 Tetanus neurotoxin: Amino acid residue M1-A457 The translocation domain is a fragment of the H chain of the clostridial neurotoxin, which roughly corresponds to the amino-terminal half of the H chain, or corresponds to the domain of that fragment in the complete H chain. In one embodiment, the H of the H chain... C The function can be achieved by missing H C The amino acid sequence is removed (at the DNA synthesis level, or post-synthetic level, by treatment with nucleases or proteases). Alternatively, H can be removed by chemical or biological treatment. C Functional inactivation. Therefore, in some embodiments, the H chain may not be able to bind to the binding sites on target cells to which the natural clostridium neurotoxin (i.e., the whole toxin) binds.

[0150] Suitable examples of (reference) translocation domains include: Type A botulinum neurotoxin - amino acid residues (449-871) Botulinum neurotoxin type B - amino acid residues (441-858) Type C botulinum neurotoxin - amino acid residues (442-866) Type D botulinum neurotoxin - amino acid residues (446-862) Type E botulinum neurotoxin - amino acid residues (423-845) Type F botulinum neurotoxin - amino acid residues (440-864) G-type botulinum neurotoxin - amino acid residues (442-863) Tetanus neurotoxin - amino acid residues (458-879) BoNT / AH N The domain may contain a polypeptide sequence comprising at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% of amino acid residues 449-871 of SEQ ID NO: 1. BoNT / BH N The domain may contain a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% of amino acid residues 441-858 of SEQ ID NO:2. BoNT / C1 H NThe domain may contain a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% of amino acid residues 442-866 of SEQ ID NO: 3. BoNT / DH N The domain may contain a polypeptide sequence comprising at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% of amino acid residues 446-862 of SEQ ID NO: 4. BoNT / EH N The domain may contain a polypeptide sequence comprising at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% of amino acid residues 423-845 of SEQ ID NO: 5. BoNT / FH N The domain may contain a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% of amino acid residues 440-864 of SEQ ID NO: 6. BoNT / GH N The domain may contain a polypeptide sequence comprising at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% of amino acid residues 442-863 of SEQ ID NO: 7. BoNT / XH N The domain may contain a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% of amino acid residues 440-892 of SEQ ID NO: 8. TeNT H N The domain may contain a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% of amino acid residues 458-879 of SEQ ID NO: 9.

[0151] The reference sequences used for identification above should be considered as guidelines, as slight variations may occur depending on the subserotype. By way of example, US 2007 / 0166332 (incorporated hereby by reference) uses a slightly different Clostridium sequence: Type A botulinum neurotoxin - amino acid residues (A449-K871) Botulinum neurotoxin type B - amino acid residues (A442-S858) Type C botulinum neurotoxin - amino acid residues (T450-N866) Type D botulinum neurotoxin - amino acid residues (D446-N862) Type E botulinum neurotoxin - amino acid residues (K423-K845) Type F botulinum neurotoxin - amino acid residues (A440-K864) G-type botulinum neurotoxin - amino acid residues (S447-S863) Tetanus neurotoxin - amino acid residues (S458-V879) In the context of this invention, various clostridium neurotoxins H containing translocation domains N The region can be used in aspects of the present invention. H from the heavy chain of clostridium neurotoxin. N The region is approximately 410-430 amino acids long and contains a translocation domain. Studies have shown that the H from the clostridial neurotoxin heavy chain... N The entire length of the region is not essential for the translocation activity of the translocation domain. Therefore, aspects of this embodiment may include clostridial neurotoxin H comprising a translocation domain having a length of, for example, at least 350 amino acids, at least 375 amino acids, at least 400 amino acids, and at least 425 amino acids. N Region. Other aspects of this embodiment may include clostridium neurotoxin H comprising a translocation domain 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 district.

[0152] Regarding Clostridium botulinum ( Clostridium botulinum For further details on the genetic basis of tetanus toxin production, see Henderson et al. (1997). The Clostridia: Molecular Biology and Pathogenesis [Clostridium: Molecular Biology and Pathogenic Mechanisms], Academic press [Academic Publishing House]

[0153] Term H N Covering naturally occurring neurotoxin H N Partially, and modified H with amino acid sequences and / or synthetic amino acid residues not found in nature. N Partial. In one embodiment, the modified H N Some still exhibit the aforementioned translocation function.

[0154] Clostridium neurotoxin receptor binding domain (H C Examples of reference sequences include: BoNT / A - N872-L1296 BoNT / B - E859-E1291 BoNT / C1 - N867-E1291 BoNT / D - S863-E1276 BoNT / E - R846-K1252 BoNT / F - K865-E1274 BoNT / G - N864-E1297 TeNT - I880-D1315 Regarding the recently identified BoNT / X, H C The domains have been reported to correspond to amino acids 893-1306, with the domain boundaries possibly varying by about 25 amino acids (e.g., 868-1306 or 918-1306).

[0155] BoNT / AH C The domain may contain a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% of amino acid residues 872-1296 of SEQ ID NO: 1. BoNT / BH C The domain may contain a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% of amino acid residues 859-1291 of SEQ ID NO:2. BoNT / C1 H C The domain may contain a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% of amino acid residues 867-1291 of SEQ ID NO: 3. BoNT / DH C The domain may contain a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% of amino acid residues 863-1276 of SEQ ID NO: 4. BoNT / EH C The domain may contain a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% of amino acid residues 846-1252 of SEQ ID NO: 5. BoNT / FH C The domain may contain a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% of amino acid residues 865-1274 of SEQ ID NO: 6. BoNT / GH C The domain may contain a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% of amino acid residues 864-1297 of SEQ ID NO: 7. BoNT / XH CThe domain may contain a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% of amino acid residues 893-1306 of SEQ ID NO: 8. TeNT H C The domain may contain a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9% or 100% of amino acid residues 880-1315 of SEQ ID NO: 9.

[0156] Clostridium neurotoxin H chain (e.g., H) C The domain portion may further include a translocation-promoting domain (or a fragment thereof may be a translocation-promoting domain fragment). This domain promotes L-chain delivery to the cytosol of target cells and is described, for example, in WO 08 / 008803 and WO 08 / 008805, each of which is incorporated herein by reference.

[0157] By way of example, the translocation-promoting domain can contain clostridium neurotoxin H. CN A domain or a fragment or variant thereof. More specifically, Clostridium neurotoxin H... CN The translocation-promoting domain can have a length of at least 200 amino acids, at least 225 amino acids, at least 250 amino acids, or at least 275 amino acids. In this regard, Clostridium neurotoxin H... CN The translocation-promoting domain preferably has a length of up to 200 amino acids, up to 225 amino acids, up to 250 amino acids, or up to 275 amino acids. Specific (reference) examples include: Type A botulinum neurotoxin - amino acid residues (872-1110) Botulinum neurotoxin type B - amino acid residues (859-1097) Type C botulinum neurotoxin - amino acid residues (867-1111) Type D botulinum neurotoxin - amino acid residues (863-1098) Type E botulinum neurotoxin - amino acid residues (846-1085) Type F botulinum neurotoxin - amino acid residues (865-1105) G-type botulinum neurotoxin - amino acid residues (864-1105) Tetanus neurotoxin - amino acid residues (880-1127) The above sequence positions may vary slightly depending on serotype / subtype, and appropriate (reference) Clostridium neurotoxin H CN Other examples of structural domains include: Type A botulinum neurotoxin - amino acid residues (874-1110) Botulinum neurotoxin type B - amino acid residues (861-1097) Type C botulinum neurotoxin - amino acid residues (869-1111) Type D botulinum neurotoxin - amino acid residues (865-1098) Type E botulinum neurotoxin - amino acid residues (848-1085) Type F botulinum neurotoxin - amino acid residues (867-1105) G-type botulinum neurotoxin - amino acid residues (866-1105) Tetanus neurotoxin - amino acid residues (882-1127) Any of the above-described promoting domains can be combined with any previously described translocation domain peptide suitable for use in this invention. Thus, by way of 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 non-clostridium translocation domain peptides. Alternatively, clostridium neurotoxin H... CN The promoting domain can be combined with Clostridium translocation domain peptides, examples of which include: Type A botulinum neurotoxin - amino acid residues (449-1110) Botulinum toxin type B - amino acid residues (442-1097) Type C botulinum neurotoxin - amino acid residues (450-1111) Type D botulinum neurotoxin - amino acid residues (446-1098) Type E botulinum neurotoxin - amino acid residues (423-1085) Type F botulinum neurotoxin - amino acid residues (440-1105) G-type botulinum neurotoxin - amino acid residues (447-1105) Tetanus neurotoxin - amino acid residues (458-1127) H of natural clostridium neurotoxin C Peptides contain approximately 400-440 amino acid residues and are composed of two functionally distinct domains, each approximately 25 kDa (i.e., the N-terminal region (often called the H-terminal region)). CN Peptide or domain) and C-terminal region (often called H) CCIt consists of peptides or domains. This fact is corroborated by the following publications, each of which is incorporated herein by reference in its entirety: Umland TC (1997) Nat. Struct. Biol. [Nature Structural Biology] 4: 788-792; Herreros J (2000) Biochem. J. [Journal of Biochemistry] 347: 199-204; Halpern J (1993) J. Biol. Chem. [Journal of Biochemistry] 268: 15, pp. 11188-11192; Rummel A (2007) PNAS [Proceedings of the National Academy of Sciences] 104: 359-364; Lacey DB (1998) Nat. Struct. Biol. [Nature Structural Biology] 5: 898-902; Knapp (1998) Am. Cryst. Assoc. Abstract Papers [Abstract Papers of the American Crystallographic Society] 25: 90; Swaminathan and Eswaramoorthy (2000) Nat. Struct. Biol. [Nature Structural Biology] 7: 1751-1759; and Rummel A (2004) Mol. Microbiol. [Molecular Microbiology] 51(3), 631-643. Furthermore, there is ample evidence that the C-terminal region (H... CC (Its C-terminus consists of 160-200 amino acid residues) responsible for the binding of clostridial neurotoxins to their natural cellular receptors, namely, to nerve endings at the neuromuscular junction – a fact also confirmed by the aforementioned publications. Therefore, throughout this specification, the lack of a functional heavy chain H is mentioned. C The fact that the peptide (or domain) prevents the heavy chain from binding to cell surface receptors that bind to natural clostridium neurotoxins means that the clostridium heavy chain merely lacks functional H. CC Peptide. 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 reduce its natural binding ability to nerve endings at the neuromuscular junction.

[0158] The following provides H CC Reference sequence: Type A botulinum neurotoxin - amino acid residues (Y1111-L1296) Type B botulinum neurotoxin - amino acid residues (Y1098-E1291) Type C botulinum neurotoxin - amino acid residues (Y1112-E1291) Type D botulinum neurotoxin - amino acid residues (Y1099-E1276) Type E botulinum neurotoxin - amino acid residues (Y1086-K1252) Type F botulinum neurotoxin - amino acid residues (Y1106-E1274) G-type botulinum neurotoxin - amino acid residues (Y1106-E1297) Tetanus neurotoxin - amino acid residues (Y1128-D1315).

[0159] The reference sequences used for the above identification should be considered as guides, as slight variations may occur depending on the subserotype.

[0160] BoNT / AH CC The domain may contain a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% of the amino acid residues 1111-1296 of SEQ ID NO: 1. BoNT / BH CC The domain may contain a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% of amino acid residues 1098-1291 of SEQ ID NO: 2. BoNT / C1 H CC The domain may contain a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% of amino acid residues 1112-1291 of SEQ ID NO: 3. BoNT / DH CC The domain may contain a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% of amino acid residues 1099-1276 of SEQ ID NO:4. BoNT / EH CC The domain may contain a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% of amino acid residues 1086-1252 of SEQ ID NO: 5. BoNT / FH CC The domain may contain a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% of amino acid residues 1106-1274 of SEQ ID NO: 6. BoNT / GH CCThe domain may contain a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% of amino acid residues 1106-1297 of SEQ ID NO: 7. TeNT H CC The domain may contain a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% of amino acid residues 1128-1315 of SEQ ID NO: 9.

[0161] The term "clostridium neurotoxin" is also intended to cover modified clostridium neurotoxins and their derivatives, including but not limited to those described below. Modified clostridium neurotoxins or their derivatives may contain one or more modified amino acids, or may contain one or more inserted amino acids not present in the natural (unmodified) form of clostridium neurotoxin, compared to the sequence of the natural (unmodified) clostridium neurotoxin. By way of example, modified clostridium neurotoxins may have a modified amino acid sequence in one or more domains relative to the sequence of the natural (unmodified) clostridium neurotoxin. Such modifications can modify functional aspects of the toxin, such as biological activity or persistence. Therefore, in one embodiment, the clostridium neurotoxin of the present invention is a modified clostridium neurotoxin, or a modified clostridium neurotoxin derivative, or a clostridium neurotoxin derivative.

[0162] Modified clostridium neurotoxins can be present in the amino acid sequence of the heavy chain (such as modified H). C The domain contains one or more modifications, wherein the modified heavy chain binds to target nerve cells with a higher or lower affinity than the natural (unmodified) clostridium neurotoxin. C Such modifications in a structural domain can include modifications to H C Residues in the ganglioside-binding site or protein (SV2 or synaptic binding protein) binding site of the domain alter the binding to ganglioside receptors and / or protein receptors on target nerve cells. Examples of such modified clostridium neurotoxins are described in WO 2006 / 027207 and WO 2006 / 114308, both of which are hereby incorporated by reference in their entirety.

[0163] Therefore, BoNT / AH CC The structural domain is preferably a modified BoNT / AH. CC Structural domains, more preferably modified BoNT / AH CStructural domain. Therefore, preferably, the clostridial neurotoxin according to the invention is a modified BoNT / A. Preferably, the modified clostridial neurotoxin comprises the one or more modifications when compared to an equivalent unmodified clostridial neurotoxin lacking one or more modifications that increase the isoelectric point of the clostridial neurotoxin. Suitable modified clostridial neurotoxins are described below and in WO 2015 / 004461 A1 and WO 2016 / 110662 A1, which are incorporated herein by reference. Exemplary sequences include SEQ ID NO: 10-13 (preferably SEQ ID NO: 10 - mrBoNT / A) as described herein.

[0164] The modified BoNT / A can be a modified BoNT / A containing one or more amino acid residues selected from the following: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN991, GLU 992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274, and THR 1277. Compared to known BoNT / A, this modified BoNT / A can exhibit reduced or absent side effects. The modified BoNT / A can exhibit increased tissue retention properties, thereby providing increased potency and / or duration of action, and can allow for the use of reduced doses (or increased doses without any additional adverse reactions) compared to known clostoxin therapeutics, thus providing additional advantages.

[0165] Modifications can be made when compared to BoNT / A as shown in SEQ ID NO: 1, where the amino acid residue numbering is determined by comparison with SEQ ID NO: 1. Since the presence of a methionine residue at position 1 of SEQ ID NO: 1 (and SEQ ID NO corresponding to the modified BoNT / A peptide described herein) 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, if SEQ ID NO: 1 contains methionine, the position numbering would be as defined above (e.g., ASN 886 would be ASN 886 of SEQ ID NO: 1). Alternatively, if methionine is not present in SEQ ID NO: 1, the amino acid residue numbering should be modified by -1 (e.g., ASN 886 would be ASN 885 of SEQ ID NO: 1). Similar considerations apply when methionine is present / absent at position 1 of other peptide sequences described herein, and those skilled in the art will readily determine the correct amino acid residue numbering using conventional techniques in the art.

[0166] The alignment described herein for determining amino acid residue numbers can be performed using any of the methods described herein for determining sequence homology and / or sequence identity %

[0167] The one or more amino acid residues indicated by the above modifications are one or more surface-exposed amino acid residues.

[0168] The modified BoNT / A may include modifications at one or more amino acid residues selected from the following: ASN886, ASN 930, ASN 954, SER 955, GLN 991, ASN 1025, ASN 1026, ASN 1052, ASN 1188, ASP1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274, and THR 1277.

[0169] When used in the context of modified BoNT / A, the term "one or more amino acid residues" preferably means at least 2, 3, 4, 5, 6, or 7 of the indicated one or more amino acid residues. Therefore, a modified BoNT / A may contain at least 2, 3, 4, 5, 6, or 7 (preferably 7) modifications at the indicated one or more amino acid residues. A modified BoNT / A may contain 1-30, 3-20, or 5-10 amino acid modifications. More preferably, when used in the context of modified BoNT / A, the term "one or more amino acid residues" means all of the indicated one or more amino acid residues.

[0170] Preferably, when compared with SEQ ID NO: 1, the modified BoNT / A does not contain any other amino acid modifications except for one or more amino acid modifications at one or more indicated amino acid residues.

[0171] Modifications can be selected from: i. Replace the acidic amino acid residues exposed on the surface with basic amino acid residues; ii. Replace the surface-exposed acidic amino acid residues with uncharged amino acid residues; iii. Replace uncharged surface-exposed amino acid residues with basic amino acid residues; iv. Inserting basic amino acid residues; and v. lacking acidic amino acid residues exposed on the surface.

[0172] The modifications indicated above produce a modified BoNT / A that, compared to the corresponding unmodified BoNT / A (e.g., SEQ ID NO: 1), has an increased positive surface charge and an increased isoelectric point. Without being bound by theory, it is believed that the increased net positive charge promotes electrostatic interactions between the peptide and anionic extracellular components, thereby promoting peptide binding to the cell surface and thus increasing retention and / or duration of action at the application site.

[0173] The isoelectric point (pI) is a specific property of a given protein. As is well known in the art, proteins are made up of a specific sequence of amino acids (also called amino acid residues when referring to proteins). Each amino acid in the standard set of twenty amino acids has a different side chain (or R group), meaning that each amino acid residue in a protein exhibits different chemical properties, such as charge and hydrophobicity. These properties can be influenced by the surrounding chemical environment, such as temperature and pH. The overall chemical character of a protein will depend on the sum of these different factors.

[0174] Certain amino acid residues (described in detail below) have ionizable side chains that can exhibit a charge depending on the surrounding pH. Whether such a side chain is charged at a given pH depends on the pKa of the relevant ionizable portion, where pKa is the negative logarithm of the acid dissociation constant (Ka) of the specified proton from the conjugate base.

[0175] For example, acidic residues such as aspartic acid and glutamic acid have side-chain carboxylic acid groups with a pKa value of approximately 4.1 (the exact pKa value can depend on temperature, ionic strength, and the microenvironment of the ionizable group). Therefore, these side chains exhibit a negative charge at a pH of 7.4 (often referred to as "physiological pH"). At lower pH values, these side chains will protonate and lose their charge.

[0176] In contrast, basic residues (such as lysine and arginine) have nitrogen-containing side chains with pKa values ​​of approximately 10⁻¹². Therefore, these side chains exhibit a positive charge at pH 7.4. At higher pH values, these side chains will undergo deprotonation and lose their charge.

[0177] Therefore, the total (net) charge of a protein molecule depends on the number of acidic and basic residues present in the protein (and their surface exposure) as well as the surrounding pH. Changing the surrounding pH changes the total charge on the protein. Thus, for each protein, there exists a given pH at which the amount of positive and negative charges are equal, and the protein does not exhibit a total net charge. This point is called the isoelectric point (pI). The isoelectric point is a standard concept in protein biochemistry that will be familiar to those skilled in the art.

[0178] Therefore, the isoelectric point (pI) is defined as the pH at which a protein exhibits zero net charge. An increase in pI means that a higher pH is required for a protein to exhibit zero net charge. Thus, an increase in pI indicates an increase in the net positive charge of the protein at a given pH. Conversely, a decrease in pI means that a lower pH is required for a protein to exhibit zero net charge. Thus, a decrease in pI indicates a decrease in the net positive charge of the protein at a given pH.

[0179] Methods for determining the pI of a protein are known in the art and will be familiar to those skilled in the art. By way of example, the pI of a protein can be calculated from the average pKa value of each amino acid present in the protein (“calculated pI”). Such calculations can be performed using computer programs known in the art, such as the Compute pI / MW Tool from ExPASy (https: / / web.expasy.org / compute_pi / ), which is a preferred method for calculating pI according to the present invention. The same calculation techniques / programs should be used to compare the pI values ​​of different molecules.

[0180] Where appropriate, the calculated pI (“observed pI”) of a protein can be experimentally confirmed using isoelectric focusing. This technique uses electrophoresis to separate proteins based on their pI. Isoelectric focusing is typically performed using a gel with a fixed pH gradient. When an electric field is applied, the protein migrates through the pH gradient until it reaches a pH at which it has zero net charge; this point is the protein's pI. The results provided by isoelectric focusing are typically of relatively low resolution, and therefore the inventors have found that results provided by calculated pI (as described above) are more suitable for use.

[0181] Throughout this specification, unless otherwise stated, “pI” means “calculated pI”.

[0182] The pI of a protein can be increased or decreased by altering the number of basic and / or acidic groups displayed on the protein surface. This can be achieved by modifying one or more amino acids in the protein. For example, an increase in pI can be provided by reducing the number of acidic residues or by increasing the number of basic residues.

[0183] The modified BoNT / A of the present invention may have a pI value that is at least 0.2, 0.4, 0.5, or 1 pI unit higher than that of BoNT / A (e.g., SEQ ID NO: 1). Preferably, the modified BoNT / A may have a pI of at least 6.6 (e.g., at least 6.8).

[0184] The properties of 20 standard amino acids are shown in the table below: The following amino acids are considered to be charged amino acids: aspartic acid (negative), glutamic acid (negative), arginine (positive), and lysine (positive).

[0185] At pH 7.4, the side chains of aspartic acid (pKa 3.1) and glutamic acid (pKa 4.1) have negative charges, while the side chains of arginine (pKa 12.5) and lysine (pKa 10.8) have positive charges. Aspartic acid and glutamic acid are referred to as acidic amino acid residues. Arginine and lysine are referred to as basic amino acid residues.

[0186] The following amino acids are considered to be uncharged polar amino acids (meaning they can participate in hydrogen bond formation): asparagine, glutamine, histidine, serine, threonine, tyrosine, cysteine, methionine, and tryptophan.

[0187] The following amino acids are considered to be uncharged hydrophobic amino acids: alanine, valine, leucine, isoleucine, phenylalanine, proline, and glycine.

[0188] In amino acid insertion, additional amino acid residues (amino acid residues that are not normally present) are incorporated into the BoNT / A polypeptide sequence, thus increasing the total number of amino acid residues in the sequence. In amino acid deletion, amino acid residues are removed from the clostoxin amino acid sequence, thus reducing the total number of amino acid residues in the sequence.

[0189] Preferably, the modification is a substitution that advantageously maintains the same number of amino acid residues in the modified BoNT / A. In amino acid substitution, an amino acid residue forming part of the BoNT / A polypeptide sequence is replaced with a different amino acid residue. The substituted amino acid residue can be one of 20 standard amino acids, as described above. Alternatively, the substituted amino acid in amino acid substitution can be a non-standard amino acid (an amino acid that is not part of the standard set of the aforementioned 20 amino acids). By way of example, the substituted amino acid can be a basic non-standard amino acid, such as L-ornithine, L-2-amino-3-guanidinopropionic acid, or the D-isomers of lysine, arginine, and ornithine. Methods for introducing non-standard amino acids into proteins are known in the art and include recombinant protein synthesis using an *E. coli* auxotrophic expression host.

[0190] In one embodiment, the substitution is selected from: replacing an acidic amino acid residue with a basic amino acid residue, replacing an acidic amino acid residue with an uncharged amino acid residue, and replacing an uncharged amino acid residue with a basic amino acid residue. In one embodiment, the substitution is replacing an acidic amino acid residue with an uncharged amino acid residue, and replacing an acidic amino acid residue with its corresponding uncharged amide amino acid residue (i.e., replacing aspartic acid with asparagine and glutamic acid with glutamine).

[0191] Preferably, the basic amino acid residue is a lysine residue or an arginine residue. In other words, the substitution is with lysine or arginine. Most preferably, the modification is with lysine substitution.

[0192] After modification according to the present invention, the modified BoNT / A is preferably able to bind to the target cell receptor bound by the unmodified BoNT / A (e.g., SEQ ID NO: 1).

[0193] Preferably, the modified BoNT / A used in this invention comprises the compound located at clostoxin H. CNThe modified BoNT / A contains amino acid modifications between 4 and 40 amino acids in the domain. The modified BoNT / A preferably also has a pI of at least 6.6. The modified BoNT / A preferably comprises modifications of at least four amino acids selected from the following: ASN 886, ASN 930, ASN 954, SER 955, GLN 991, ASN 1025, ASN 1026, and ASN 1052, wherein the modification includes substituting an amino acid with a lysine or arginine residue. For example, the modified BoNT / A may comprise modifications of at least five amino acids selected from the following: ASN 886, ASN 930, ASN 954, SER 955, GLN 991, ASN 1025, ASN 1026, ASN 1052, and GLN 1229, wherein the modification includes substituting an amino acid with a lysine or arginine residue.

[0194] Methods for modifying proteins by substitution, insertion, or deletion of amino acid residues are known in the art. By way of example, amino acid modifications can be introduced by modifying the DNA sequence encoding a polypeptide (e.g., encoding unmodified BoNT / A or a fragment thereof). This can be achieved using standard molecular cloning techniques (e.g., by site-directed mutagenesis, where a short strand of DNA (oligonucleotide) encoding one or more desired amino acids is used to replace the original coding sequence with a polymerase, or by inserting / deleting portions of the gene with various enzymes (e.g., ligases and restriction endonucleases). Alternatively, the modified gene sequence can be chemically synthesized.

[0195] The modified BoNT / A may comprise a polypeptide sequence having at least 70% sequence identity with any one of SEQ ID NO: 10-13. In one embodiment, the modified BoNT / A may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity with any one of SEQ ID NO: 10-13. Preferably, the modified BoNT / A may comprise any one of SEQ ID NO: 10-13. The modified BoNT / A may consist of a polypeptide sequence having at least 70% sequence identity with any one of SEQ ID NO: 10-13. In one embodiment, the modified BoNT / A may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity with any one of SEQ ID NO: 10-13. Preferably, the modified BoNT / A may consist of any one of SEQ ID NO: 10-13. Of the listed SEQ ID NOs, SEQ ID NO: 10 is the most preferred. Those skilled in the art will understand that, in the event that the polypeptide sequence of the modified BoNT / A changes in terms of sequence identity % compared to a given SEQ ID NO, at least one of the modifications (e.g., modifications that increase pI) still exists in the variant modified BoNT / A (e.g., it is unmodified).

[0196] Therefore, the compositions of the present invention preferably comprise modified BoNT / A, such as the modified BoNT / A described above. In some embodiments, the clostridium neurotoxin polypeptide is a modified BoNT / A polypeptide, the L chain is a BoNT / AL chain, and the H chain is a modified BoNT / A polypeptide. N The structural domain is BoNT / AH N domain, and H CC Structural domains (e.g., H) C The structural domain is a modified BoNT / AH CC Structural domains (e.g., modified BoNT / AH) CC (structural domain).

[0197] The modified BoNT / A may contain substitutions at one or more (preferably two or more, three or more, four or more, five or more, or six or more, more preferably all) positions of 930, 955, 991, 1026, 1052, 1229, and 886. Preferably, the modified BoNT / A contains lysine or arginine (more preferably lysine) at one or more of positions of 930, 955, 991, 1026, 1052, 1229, and 886. In one embodiment, the modified BoNT / A contains lysine or arginine (more preferably lysine) at at least two, three, four, five, six, or all of positions of 930, 955, 991, 1026, 1052, 1229, and 886. Most preferably, the modified BoNT / A contains lysine or arginine (more preferably lysine) at all positions 930, 955, 991, 1026, 1052, 1229 and 886.

[0198] Clostridium neurotoxins may comprise (or consist of) heterozygous or chimeric clostridium neurotoxins. A heterozygous clostridium neurotoxin comprises at least a portion of a light chain from one clostridium neurotoxin or its subtype and at least a portion of a heavy chain from another clostridium neurotoxin or its subtype. In one embodiment, a heterozygous clostridium neurotoxin may comprise an entire light chain from one clostridium neurotoxin subtype and a heavy chain from another clostridium neurotoxin subtype. In another embodiment, a chimeric clostridium neurotoxin may contain a portion of a heavy chain from one clostridium neurotoxin subtype (e.g., a binding domain), wherein another portion of the heavy chain originates from another clostridium neurotoxin subtype. Similarly or alternatively, a therapeutic element may comprise light chain portions from different clostridium neurotoxins. Such heterozygous or chimeric clostridial neurotoxins can be used as a means of delivering the therapeutic benefits of such clostridial neurotoxins to subjects who are immune to a given clostridial neurotoxin subtype, to subjects whose receptor concentrations against the heavy chain binding domain of a given clostridial neurotoxin may be below average, or to subjects who may have protease-resistant variants of membrane or vesicular toxin substrates (e.g., SNAP-25, VAMP, and synaptic fusion proteins). Heterozygous and chimeric clostridial neurotoxins are described in US 8,071,110, which is hereby incorporated by reference in its entirety.

[0199] The clostridium neurotoxin of the present invention may contain BoNT / BH CC Clostridium neurotoxins containing the BoNT / BH domain, preferably. C Chimeric Clostridium neurotoxins containing a translocation domain. Therefore, in a particularly preferred embodiment, the clostridium neurotoxin of the present invention can be a chimeric clostridium neurotoxin comprising a BoNT / A light chain and a translocation domain (LH).N (H) and BoNT / B receptor binding domain (H) C A structural domain (preferably composed of it). Most preferably, the BoNT / BH C The domain contains the following substitutions E1191M and S1199Y. Suitable chimeric clostridium neurotoxins may be those taught in WO 2017 / 191315 A1, which is incorporated herein by reference. Such preferred sequences include SEQ ID NO:14-18, wherein SEQ ID NO:14 is the most preferred.

[0200] BoNT / A LH N The structural domain can be used with BoNT / BH C The structural domains are covalently connected. The chimera BoNT / A is also referred to herein as "BoNT / AB" or "BoNT / AB chimera".

[0201] LH N The C-terminal amino acid residue of the domain can correspond to the LH that separates BoNT / A. N and H C 3 of the structural domain 10 The first amino acid residue of the helix, and H C The N-terminal amino acid residue of the domain can correspond to the LH that separates BoNT / B. N and H C 3 of the structural domain 10 The second amino acid residue of the helix.

[0202] This article mentions "LH separated by BoNT / A" N and H C 3 of the structural domain 10 The first amino acid residue of the helix refers to the part that separates LH. N and H C 3 of the structural domain 10 The N-terminal residue of the helix.

[0203] This article mentions "LH separated by BoNT / B" N and H C 3 of the structural domain 10 The second amino acid residue of the helix refers to the residue separating LH. N and H C 3 of the structural domain 10 The amino acid residue following the N-terminal residue of the helix.

[0204] 3 10 Helices are a type of secondary structure found in proteins and polypeptides; other types include α-helices, β-sheets, and anti-rotations. 10The amino acids in the helix are arranged in a right-handed helical structure, where each turn is completed by three residues and ten atoms, which are separated by intramolecular hydrogen bonds. Each amino acid corresponds to a 120° rotation in the helix (i.e., the helix has three residues per turn) and a translation of 2.0 Å (= 0.2 nm) along the helical axis, and has 10 atoms in the ring formed by forming hydrogen bonds. Most importantly, the NH group of the amino acid forms a hydrogen bond with the C=O group of the amino acid three residues ahead; this repeating i + 3 → i hydrogen bond formation defines 3 10 Spiral. 3 10 Helices are a standard concept in structural biology that is familiar to those skilled in the art.

[0205] This 3 10 The helix corresponds to the four residues that form the actual helix and two cap (or transition) residues (one at each end of the four residues). As used herein, the term "separated LH" refers to... N and H C 3 of the structural domain 10 The "helix" is composed of these 6 residues.

[0206] Structural analysis and sequence alignment were performed to identify the LH-separated segments. N and H C 3 of the structural domain 10 Spiral. This 3 10 The spiral ends at its N-terminus (i.e., at LH). N The C-terminal portion of the structural domain is surrounded by an α-helix, and at its C-terminus (i.e., at H...) C The N-terminal portion of the structural domain is surrounded by a β chain. 3 10 The first (N-terminal) residue (cap or transition residue) of the helix also corresponds to the C-terminal residue of this α-helix.

[0207] Separate LH N and H C 3 of the structural domain 10 The helix can be determined, for example, by publicly available crystal structures of botulinum neurotoxins, such as botulinum neurotoxins A1 and B1, which are 3BTA (http: / / www.rcsb.org / pdb / explore / explore.do?structureId=3BTA) and 1EPW (http: / / www.rcsb.org / pdb / explore / explore.do?structureId=1EPW), respectively.

[0208] Alternatively, publicly available computer simulation modeling and comparison tools can be used to determine LH levels that are isolated from other neurotoxins. N and H C 3 of the structural domain10 The location of the helix is ​​listed in various tools and services, such as the homology modeling server LOOPP (Learning, Observing and Outputting Protein Patterns, http: / / loopp.org), PHYRE (Protein Homology / analogY Recognition Engine, http: / / www.sbg.bio.ic.ac.uk / phyre2 / ), Rosetta (https: / / www.rosettacommons.org / ), the protein stacking server SuperPose (http: / / wishart.biology.ualberta.ca / superpose / ), the alignment program Clustal Omega (http: / / www.clustal.org / omega / ), and many other tools / services listed in the Internet Resources for Molecular and Cell Biologists (http: / / molbiol-tools.ca / ). Specifically, "H" N / H CN "The area around the connector is structurally highly conserved, making it an ideal region for superimposing different serotypes."

[0209] For example, the following methods can be used to determine this 3 10 Helical sequences in other neurotoxins: 1. Based on the BoNT / A1 crystal structure (3BTA.pdb), the structure homology modeling tool LOOP (http: / / loopp.org) was used to obtain the predicted structures of other BoNT serotypes; 2. Edit the resulting structure (pdb) file to include only H. CN The N-terminus of the domain and approximately 80 residues preceding it (which is H) N (part of the structural domain), thus preserving the structurally highly conserved "H" N / H CN "district; 3. Use the protein stacking server SuperPose (http: / / wishart.biology.ualberta.ca / superpose / ) to stack each serotype onto the 3BTA.pdb structure; 4. Examine the overlaid PDB file to locate H in BoNT / A1. C3 at the beginning of the structural domain 10 The helix was then used to identify the corresponding residues in other serotypes; 5. Use Clustal Omega to compare with other BoNT serotype sequences to check if the corresponding residues are correct.

[0210] The LH determined by this method is provided below. N H C and 3 10 Examples of spiral structural domains: Using structural analysis and sequence alignment, it was found that the LH cells were separated. N and H C 3 of the structural domain 10 The β-chain following the helix is ​​a conserved structure in all botulinum toxins and tetanus neurotoxins, and when separated from LH... N and H C 3 of the structural domain 10 The first residue of the helix begins at the 8th residue (e.g., at residue 879 in BoNT / A1).

[0211] BoNT / AB chimeras can contain H from BoNT / B C LH from BoNT / A are covalently connected to the structural domain. N structural domain • Among them, LH N The C-terminal amino acid residue of the domain corresponds to the H located at BoNT / A. C The β chain at the start (N-terminus) of the domain extends to the eighth amino acid residue at the N-terminus, and •where H C The N-terminal amino acid residue of the domain corresponds to the H located in BoNT / B. C The β chain from the start (N-terminus) of the domain extends to the seventh amino acid residue at the N-terminus.

[0212] BoNT / AB chimeras can contain H from BoNT / B C LH from BoNT / A are covalently connected to the structural domain. N structural domain • Among them, LH N The C-terminal amino acid residue of the domain corresponds to the LH located at BoNT / A. N The C-terminal amino acid residue of the α-helix at the end of the domain (C-terminus), and •where H C The N-terminal amino acid residue of the domain corresponds to the LH located immediately adjacent to BoNT / B. NThe C-terminal amino acid residue of the α-helix at the end (C-terminus) of the domain.

[0213] The fundamental principle of the BoNT / AB chimera design process is to attempt to ensure that the secondary structure is not damaged, thereby minimizing any changes to the tertiary structure. Not wanting to be bound by theory, it is assumed that by not destroying the tertiary structure... 10 The four core amino acid residues of the helix ensure the optimal conformation of the chimeric neurotoxin.

[0214] LH from BoNT / A N The domain may correspond to amino acid residues 1 to 872 of SEQ ID NO: 1 or a polypeptide sequence having at least 70% sequence identity with it. LH from BoNT / A N The domain may correspond to amino acid residues 1 to 872 of SEQ ID NO: 1 or a polypeptide sequence having at least 80%, 90%, or 95% sequence identity with it. Preferably, LH from BoNT / A N The domain corresponds to amino acid residues 1 to 872 of SEQ ID NO: 1.

[0215] H from BoNT / B C The domain may correspond to amino acid residues 860 to 1291 of SEQ ID NO: 2 or a polypeptide sequence having at least 70% sequence identity with it. H from BoNT / B C The domain may correspond to amino acid residues 860 to 1291 of SEQ ID NO: 2 or a polypeptide sequence having at least 80%, 90%, or 95% sequence identity with it. Preferably, the H domain is derived from BoNT / B. C The domain corresponds to amino acid residues 860 to 1291 of SEQ ID NO: 2.

[0216] Preferably, LH N The structural domain corresponds to amino acid residues 1 to 872 of BoNT / A (SEQ ID NO: 1), and H C The domain corresponds to amino acid residues 860 to 1291 of BoNT / B (SEQ ID NO: 1).

[0217] Preferably, BoNT / BH C The structure domain is further contained in H CC At least one amino acid residue in a domain (e.g., a subdomain) is substituted, added, or deleted, which has the effect of increasing the binding affinity of BoNT / B neurotoxin to human Syt II compared to the native BoNT / B sequence. BoNT / BH CCSuitable substitutions, additions or deletions of amino acid residues in the domain have been disclosed in WO 2013 / 180799 and WO 2016 / 154534 (both of which are incorporated herein by reference).

[0218] BoNT / BH CC Suitable amino acid residue substitutions, additions, or deletions in the domain 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.

[0219] BoNT / BH CC Suitable amino acid residue substitutions, additions, or deletions in the domain further include combinations of two substitution mutations selected from the group consisting of: E1191M and S1199L, E1191M and S1199Y, E1191M and S1199F, E1191Q and S1199L, E1191Q and S1199Y, E1191Q and S1199F, E1191M and S1199W, E1191M and W1178Q, E1191C and S1199W, E1191C and S1199Y, E1191C and W1178Q, E1191Q and S1199W, E1191V and S1199W, E1191V and S1199Y, or E1191V and W1178Q.

[0220] BoNT / BH CC Suitable amino acid residue substitutions, additions, or deletions in the domain also include combinations of three substitution mutations: E1191M, S1199W, and W1178Q.

[0221] Preferably, BoNT / BH CC Appropriate amino acid residue substitutions, additions, or deletions in the domain include combinations of two substitution mutations, namely E1191M and S1199Y.

[0222] Modifications can be made when compared to the unmodified BoNT / B shown as SEQ ID NO: 2, where the amino acid residue numbering is determined by comparison with SEQ ID NO: 2. Since the presence of the methionine residue at position 1 of SEQ ID NO: 2 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, if SEQ ID NO: 2 contains methionine, the position numbering would be as defined above (e.g., E1191 would be E1191 of SEQ ID NO: 2). Alternatively, if methionine is not present in SEQ ID NO: 2, the amino acid residue numbering should be modified by -1 (e.g., E1191 would be E1190 of SEQ ID NO: 2). Similar considerations apply when 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.

[0223] The chimeric clostridium neurotoxin may comprise a polypeptide sequence having at least 70% sequence identity with any one of SEQ ID NO: 14-18. In one embodiment, the chimeric clostridium neurotoxin may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity with any one of SEQ ID NO: 14-18. Preferably, the chimeric clostridium neurotoxin may comprise any one of SEQ ID NO: 14-18. The chimeric clostridium neurotoxin may consist of a polypeptide sequence having at least 70% sequence identity with any one of SEQ ID NO: 14-18. In one embodiment, the chimeric clostridium neurotoxin may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity with any one of SEQ ID NO: 14-18. Preferably, the chimeric clostridium neurotoxin may consist of any one of SEQ ID NO: 14-18. Of the listed SEQ ID NOs, SEQ ID NO: 14 is the most preferred. Those skilled in the art will understand that, compared to a given SEQ ID NO, it contains at least one BoNT / BH CC In cases where the polypeptide sequence of a chimeric Clostridium neurotoxin with a mutated domain varies in terms of sequence identity percentage, at least one BoNT / BH... CC Domain mutations (preferably E1191M and S1199Y) are present in variant chimeric Clostridium neurotoxins (e.g., they are unmodified).

[0224] Therefore, the compositions of the present invention most preferably comprise chimeric clostridium neurotoxins, such as the chimeric clostridium neurotoxins described above. In some embodiments, the clostridium neurotoxin polypeptide is a chimeric clostridium neurotoxin polypeptide, the L chain is a BoNT / AL chain, and the H chain is a... N The structural domain is BoNT / AH N domain, and H CC Structural domains (e.g., H) C The structural domain is BoNT / BH CC Structural domains (e.g., BoNT / BH) CC (structural domain).

[0225] In another embodiment, the clostridium neurotoxin of the present invention may be a chimeric clostridium neurotoxin comprising a BoNT / X light chain and a translocation domain (LH). N The receptor-binding domain (H) from different (i.e., non-BoNT / X) Clostridium neurotoxins C (domain) or part thereof. Suitable chimeric and / or heterozygous clostridium neurotoxins may be the clostridium neurotoxins taught in WO 2020 / 065336 A1, which is incorporated herein by reference.

[0226] In embodiments where the clostridium neurotoxin described herein has a label (e.g., a His label) and / or adapter for purification, the label and / or adapter are optional.

[0227] The clostridium neurotoxin of the present invention may not contain the complex protein present in naturally occurring clostridium neurotoxin complexes.

[0228] The clostridial neurotoxin of the present invention can be generated using recombinant nucleic acid technology. Therefore, in one embodiment, the clostridial neurotoxin (as described above) is a recombinant clostridial neurotoxin.

[0229] In one embodiment, a nucleic acid (e.g., DNA) comprising a nucleic acid sequence encoding a clostridium neurotoxin is provided. In one embodiment, the nucleic acid sequence is prepared as part of a DNA vector comprising a promoter and a terminator. The nucleic acid sequence may be selected from any nucleic acid sequence described herein.

[0230] In a preferred embodiment, the carrier has a promoter selected from the following: In another preferred embodiment, the vector has a promoter selected from the following: Nucleic acid molecules can be prepared using any suitable method known in the art. Therefore, chemical synthesis techniques can be used to prepare nucleic acid molecules. Alternatively, molecular biology techniques can be used to prepare the nucleic acid molecules of this invention.

[0231] The DNA constructs of the present invention are preferably designed by computer simulation and then synthesized by conventional DNA synthesis techniques.

[0232] Depending on the final host cell (e.g., E. coli) expression system to be used, the above nucleic acid sequence information may be modified for codon bias.

[0233] The terms "nucleotide sequence" and "nucleic acid" are used synonymously herein. Preferably, the nucleotide sequence is a DNA sequence.

[0234] The clostridium neurotoxin of the present invention is preferably present as a double-stranded clostridium neurotoxin, wherein the L chain is bonded to the H chain (or a component thereof, such as H) via a disulfide bond. N (structural domain) connection. Therefore, the clostridial neurotoxin of the present invention can be any clostridial neurotoxin or variant thereof that has been cleaved by a protease in its activation loop (at one or more sites) (indicated by the sequence identity % with a given SEQ ID NO).

[0235] Clostridium neurotoxin preferably comprises an L-chain and an H-chain linked by disulfide bonds, and can be obtained by cleaving a polypeptide containing at least 70% sequence identity with SEQ ID NO: 10 at one or more sites in its activation loop using a protease. In one embodiment, clostridium neurotoxin comprises an L-chain and an H-chain linked by disulfide bonds, and can be obtained by cleaving a polypeptide containing at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity with SEQ ID NO: 10 at one or more sites in its activation loop using a protease. Preferably, clostridium neurotoxin comprises an L-chain and an H-chain linked by disulfide bonds, and can be obtained by cleaving a polypeptide containing SEQ ID NO: 10 at one or more sites in its activation loop using a protease.

[0236] The clostridial neurotoxin most preferably comprises an L-chain and an H-chain linked by disulfide bonds, and can be obtained by cleaving a polypeptide containing at least 70% sequence identity with SEQ ID NO: 14 at one or more sites in its activation loop using a protease. In one embodiment, the clostridial neurotoxin comprises an L-chain and an H-chain linked by disulfide bonds, and can be obtained by cleaving a polypeptide containing at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity with SEQ ID NO: 14 at one or more sites in its activation loop using a protease. Preferably, the clostridial neurotoxin comprises an L-chain and an H-chain linked by disulfide bonds, and can be obtained by cleaving a polypeptide containing SEQ ID NO: 14 at one or more sites in its activation loop using a protease.

[0237] The protease used to cleave the activation loop is preferably Lys-C. Suitable proteases and methods for cleaving the activation loop to produce double-stranded Clostridium neurotoxin are taught in WO 2014 / 080206, WO 2014 / 079495 and EP 2677029 A2, which are incorporated herein by reference.

[0238] Suitable activation loop sequences are shown in the table below: Lys-C can cleave the activation loop at the C-terminus of one or more lysine residues present in the activation loop. In cases where Lys-C cleaves the activation loop more than once, those skilled in the art will understand that the small peptide of the activation loop of the double-stranded Clostridium neurotoxin may not be present when compared to SEQ ID NO shown herein.

[0239] This invention provides a method for producing a single-stranded Clostridium neurotoxin having a light chain and a heavy chain, the method comprising expressing the nucleic acid described herein in an expression host, lysing host cells to provide a host cell homogenate containing the single-stranded Clostridium neurotoxin, and isolating the single-stranded Clostridium neurotoxin. In one aspect, this invention provides a method for proteolytically treating the Clostridium neurotoxin described herein, the method comprising contacting the Clostridium neurotoxin with a protease that hydrolyzes peptide bonds in the activation loop of the Clostridium neurotoxin, thereby converting the (single-stranded) Clostridium neurotoxin into a corresponding double-stranded Clostridium neurotoxin (e.g., wherein the light chain and heavy chain are linked together by disulfide bonds).

[0240] Therefore, the present invention provides a double-stranded Clostridium neurotoxin that can be obtained by the method of the present invention.

[0241] In one aspect, the present invention provides a method for producing a therapeutic or cosmetic Clostridium neurotoxin composition, the method comprising: (a) Obtaining the results according to the method of the invention, and formulating and / or packaging the composition for therapeutic or cosmetic purposes when the concentration of clostridium neurotoxin is the same as or higher than that of the positive control; or (b) When the concentration of clostridium neurotoxin is lower than that of the positive control, the composition is further fermented and formulated and / or packaged for further purified composition for therapeutic or cosmetic use.

[0242] The compositions of the present invention may comprise a first clostridium neurotoxin formulation comprising one or more pharmaceutically acceptable carriers, excipients, adjuvants, propellants, and / or salts.

[0243] In one aspect, therapeutic or cosmetic clostridium neurotoxin compositions are provided that can be obtained by the methods of the present invention, optionally wherein the therapeutic or cosmetic clostridium neurotoxin compositions are packaged.

[0244] As used in this article, the term “available” also encompasses the term “obtained”.

[0245] In one aspect, the present invention provides an isolated binding substrate. In some embodiments, the isolated binding substrate binds to a clostridium neurotoxin polypeptide.

[0246] In one aspect, the present invention provides an isolated complex comprising a clostridial neurotoxin that binds to a binding substrate. In some embodiments, the binding substrate is immobilized onto a support (e.g., a surface for analyzing binding events and kinetics). In other embodiments, a clostridial neurotoxin peptide is immobilized onto a support (e.g., a surface for analyzing binding events and kinetics).

[0247] The isolated binding substrate can complex with clostridial neurotoxin, in which the clostridial neurotoxin binds to the binding site.

[0248] Isolated binding substrates can refer to binding substrates that have been isolated from cells. Such binding substrates may have been recombined and isolated using standard techniques. Therefore, in some embodiments, the term "isolated binding substrate" is intended to encompass binding substrates in the in vitro environment. Preferably, as described herein in general with respect to binding substrates, the isolated binding substrate is immobilized on a support.

[0249] In one aspect, the present invention provides the use of a binding substrate for the isolation of clostridium neurotoxins for determining the presence or absence of clostridium neurotoxin polypeptides contained in a composition, wherein the isolated binding substrate comprises a binding region.

[0250] In one aspect, the present invention provides a method for producing a therapeutic or cosmetic Clostridium neurotoxin composition, the method comprising: (a) Obtaining the result according to the method of the invention; and (b) When the composition contains a clostridium neurotoxin polypeptide, the composition is formulated and / or packaged for therapeutic or cosmetic use, optionally wherein the composition contains at least a predetermined concentration of the clostridium neurotoxin polypeptide; or (c) When it is determined that the composition does not contain clostridium neurotoxin polypeptides or when the concentration of clostridium neurotoxin is below a predetermined value, the composition is further fermented; and (d) Formulating and / or packaging further purified compositions for therapeutic or cosmetic purposes.

[0251] In some embodiments, formulation may include selecting one or more preferred pharmaceutically acceptable carriers, excipients, adjuvants, propellants and / or salts for the formulation of clostridial neurotoxins.

[0252] In one embodiment, where the terms "obtaining the result of a method or measurement" or "obtaining multiple results of a method or measurement" are used herein, the method for obtaining said one or more results may be performed as part of the method of the present invention.

[0253] In one aspect, the present invention provides a kit comprising: (a) Separated bound substrate; (b) A surface used for combined event and dynamic analysis; and (b) optionally, a device for detecting the binding of botulinum neurotoxin to a binding substrate; and / or (c)Optionally, its instruction manual.

[0254] In one aspect, the present invention provides a kit comprising: (a) Separated binding substrate; and (b) optionally, a device for detecting the binding of botulinum neurotoxin to a binding substrate; and / or (c)Optionally, its instruction manual.

[0255] The embodiments related to the various methods of the present invention are intended to be equally applicable to alternative methods, products and / or uses, and vice versa.

[0256] sequence homology The percentage of identity can be determined using any of a variety of sequence alignment methods, including, but not limited to, global, local, and hybrid methods, such as the segmentation method. The scheme for determining the percentage of identity is a conventional procedure within the capabilities of those skilled in the art. Global methods align sequences from the beginning to the end of the molecule and determine the optimal alignment by summing the scores of individual residue pairs and applying a vacancy penalty. Non-restrictive methods include, for example, CLUSTAL W, see 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) NucleicAcids Research 4673-4680 (1994); and iterative refinement, see Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein. Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments, 264(4) J. MoI. Biol. 823-838. (1996). Local methods align sequences by identifying one or more conserved motifs shared by all input sequences.Non-restrictive methods include, for example, Match-box, see, for example, Eric Depiereux and Ernest Feytmans, Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8(5) CABIOS [Computer Applications in the Biological Sciences] 501-509 (1992); Gibbs sampling, see, for example, CE Lawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131) Science [Science] 208-214 (1993); Align-M, see, for example, Ivo Van Waille et al., Align-M - A New Algorithm for Multiple Alignment of Highly Divergent Sequences [Align-M - [A new algorithm for multiple alignment of highly variable sequences], 20(9) Bioinformatics: 1428-1435 (2004).

[0257] Therefore, the percentage of sequence identity is determined using conventional methods. See, for example, Altschul et al., Bull. Math. Bio. [Bulletin of Mathematical Biology] 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 89:10915-19, 1992. In short, the alignment score of two amino acid sequences is optimized by comparing them using a vacancy opening penalty of 10, a vacancy extension penalty of 1, and the “blosum 62” scoring matrix of Henikoff and Henikoff (ibid.), as described below (amino acids are indicated by standard single-letter codes); preferably, this method is used to align sequences with the SEQ ID NO described herein to define amino acid position numbers, as described herein.

[0258] The "sequence identity percentage" between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by the sequences. Therefore, 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 sequence identity % can also take into account the number of vacancies and the length of each vacancy that needs to be introduced to optimize the alignment of two or more sequences. Sequence comparisons and the determination of the identity percentage between two or more sequences can be performed using specific mathematical algorithms (such as BLAST) familiar to those skilled in the art.

[0259] Alignment score used to determine sequence identity Then the percentage of identity is calculated as follows: Essentially homologous polypeptides are characterized by having one or more amino acid substitutions, deletions, or additions. These changes are preferably small in nature, i.e., conserved amino acid substitutions (see below) and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions typically of 1 to 30 amino acids; and small amino-terminal or carboxyl-terminal extensions, such as amino-terminal methionine residues, small linker peptides of up to about 20-25 residues, or affinity tags.

[0260] Conservative amino acid substitution Alkaline: Arginine Lysine Histidine Acidic: Glutamic acid Aspartic acid Polar: glutamine Asparagine Hydrophobic: Leucine Isoleucine Valine Aromatic group: Phenylalanine Tryptophan Tyrosine Small: Glycine alanine Serine threonine Methionine In addition to the 20 standard amino acids, non-standard amino acids (such as 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaleine, and α-methylserine) may also replace amino acid residues in the polypeptides of the present invention. A limited number of non-conserved amino acids, amino acids not encoded by the genetic code, and non-natural amino acids may replace amino acid residues in the polypeptides. The polypeptides of the present invention may also contain non-naturally occurring amino acid residues.

[0261] Non-naturally occurring amino acids include, but are not limited to, trans-3-methylproline, 2,4-methyl-bridged-proline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allothreonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, piperidinic acid, tert-leucine, valine, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine. Several methods for incorporating non-naturally occurring amino acid residues into proteins are known in the art. For example, an in vitro system in which chemically ammoniated repressive tRNA can be used to suppress nonsense mutations can be employed. Methods for synthesizing amino acids and ammoniated tRNA are known in the art. Transcription and translation of plasmids containing nonsense mutations are performed in a cell-free system containing *E. 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 the second method, translation is performed in Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylated repressive tRNA (Turcatti et al., J. Biol. Chem. 271:19991-8, 1996). In the third method, *E. coli* cells are cultured in the absence of the natural amino acid to be substituted (e.g., phenylalanine) and in the presence of one or more desired non-natural amino acids (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The non-natural amino acids are incorporated into the polypeptide to replace their natural counterparts. See Koide et al., *Biochem.* 33:7470-6, 1994. Naturally occurring amino acid residues can be converted to non-natural species through in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, *Protein Sci.* 2:395-403, 1993).

[0262] A limited number of non-conserved amino acids, amino acids not encoded by the genetic code, non-naturally occurring amino acids, and non-natural amino acids can replace the amino acid residues of the polypeptides of the present invention.

[0263] Essential amino acids in the peptides of the present invention can be identified using 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, along with mutagenesis of the amino acids at the presumed contact sites. 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 be inferred from homology analysis with related components of the peptides of the present invention (e.g., translocation or protease components).

[0264] Multiple amino acid substitutions can be performed and tested using known mutagenesis and screening methods, 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). In short, these authors disclosed methods for simultaneously randomizing two or more positions in a peptide, selecting functional peptides, and then sequencing the mutagenized peptide to determine the profile of permissible substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., U.S. Patent No. 5,223,409; Huse, WIPO Publication WO 92 / 06204) and regional directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

[0265] 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 pertains. Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20th edition, John Wiley and Sons, New York (1994), and Hale and Marham, THE HARPER COLLINSDICTIONARY OF BIOLOGY, Harper Perennial, New York (1991) provide general dictionaries for those skilled in the art of the matter regarding the many terms used in this disclosure.

[0266] This disclosure is not limited to the exemplary methods and materials disclosed herein, and any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of embodiments of this disclosure. Numerical ranges include the numbers that define the range. Unless otherwise indicated, any nucleic acid sequence is written from left to right in a 5' to 3' direction; amino acid sequences are written from left to right in an amino to carboxyl direction.

[0267] The headings provided herein are not intended to limit any aspect or embodiment of this disclosure.

[0268] Amino acids are referred to herein by name, three-letter abbreviation, or single-letter abbreviation. As used herein, the term "protein" includes proteins, polypeptides, and peptides. As used herein, the term "amino acid sequence" is synonymous with the terms "polypeptide" and / or "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" are used interchangeably herein. Conventional single-letter and three-letter codes for amino acid residues may be used in this disclosure and claims. Three-letter codes for amino acids are defined according to the Joint Commission on Biochemical Nomenclature (JCBN) of IUPACIUB. It should also be understood that, due to the degeneracy of the genetic code, a polypeptide can be encoded by more than one nucleotide sequence.

[0269] Other definitions of terms may appear throughout the specification. Before describing exemplary embodiments in more detail, it should be understood that this disclosure is not limited to the specific embodiments described, and therefore these may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of this disclosure will be defined only by the appended claims.

[0270] Where a range of values ​​is provided, it should be understood that, unless the context explicitly states otherwise, each intermediate value (to one-tenth of a unit of the lower limit) between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intermediate value within the stated range and any other stated value or intermediate value within that range is covered in this disclosure. The upper and lower limits of these smaller ranges may be independently included in or excluded from the range, and each range (where any, no, or both limits are included in the smaller range) is also covered in this disclosure, subject to any limits specifically excluded from the stated range. Where a stated range includes one or two limits, the range excluding any one or both of those included limits is also included in this disclosure.

[0271] It must be noted that, unless the context clearly specifies otherwise, the singular forms “a,” “an,” and “the” as used herein and in the appended claims include plural indicators. Thus, for example, reference to “a clostridium neurotoxin” includes a plurality of such candidates, and reference to “the clostridium neurotoxin” includes reference to one or more clostridium neurotoxins and their equivalents known to those skilled in the art, and so on.

[0272] Only disclosures of the publications discussed herein prior to the filing date of this application are provided. Nothing herein should be construed as an admission that such publications constitute prior art as claimed in the appended claims.

[0273] sequence list The residue / codon is optional, provided that the initial Met amino acid residue or the corresponding initial codon is indicated in any of the following SEQ ID NOs.

[0274] SEQ ID NO: 1 - Peptide sequence of natural BoNT / A (rBoNT / A) Peptide sequence of SEQ ID NO: 2 - BoNT / B SEQ ID NO: 3 - Polypeptide sequence of BoNT / C Peptide sequence of SEQ ID NO: 4 - BoNT / D Peptide sequence of SEQ ID NO: 5 - BoNT / E Peptide sequence of SEQ ID NO: 6 - BoNT / F SEQ ID NO: 7 - Polypeptide sequence of BoNT / G Peptide sequence of SEQ ID NO: 8 - BoNT / X SEQ ID NO: 9 - Peptide sequence of TeNT The polypeptide sequence of SEQ ID NO: 10 - mrBoNT / A SEQ ID NO: 11 - Peptide sequence of cationic BoNT / A variant 2 SEQ ID NO: 12 - Peptide sequence of cationic BoNT / A variant 3 SEQ ID NO: 13 - Peptide sequence of cationic BoNT / A variant 4 The polypeptide sequence of SEQ ID NO: 14 - mrBoNT / AB SEQ ID NO: 15 - Peptide sequence of BoNT / AB variant 2 SEQ ID NO: 16 - Peptide sequence of BoNT / AB variant 3 SEQ ID NO: 17 - Peptide sequence of BoNT / AB variant 4 SEQ ID NO: 18 - Peptide sequence of BoNT / AB variant 5 SEQ ID NO: 19 - Full-length human SV2a polypeptide sequence SEQ ID NO: 20 - Full-length human SV2b polypeptide sequence SEQ ID NO: 21 - Full-length human SV2c polypeptide sequence The polypeptide sequence of SEQ ID NO: 22 - Cia-C2 SEQ ID NO: 23 - Peptide sequence of alternative Cia-C2 (Genbank: HQ700705, AEJ91546.1) The polypeptide sequence of SEQ ID NO: 24 - CiaH7 The polypeptide sequence of SEQ ID NO: 25 - CiaH7 (Genbank: HQ700708, AEJ91549.1). The polypeptide sequence of SEQ ID NO: 26 - CiaD12 / B5 The polypeptide sequence of SEQ ID NO: 27 - ciA-B5 (Genbank: HQ700704, AEJ91545.1) The polypeptide sequence of SEQ ID NO: 28 - ciA-F12 (Genbank: HQ700706, AEJ91547.1) The polypeptide sequence of SEQ ID NO: 29 - ciA-D12 (Genbank: HQ700702, AEJ91543.1) The polypeptide sequence of SEQ ID NO: 30 - ciA-A5 (Genbank: HQ700703, AEJ91544.1) The polypeptide sequence of SEQ ID NO: 31 - ciA-G5 (Genbank: HQ700707, AEJ91548.1) The polypeptide sequence of SEQ ID NO: 32 - ciB-H11 (Genbank: HQ700712, AEJ91553.1) The polypeptide sequence of SEQ ID NO: 33 - ciB-A11 (Genbank: HQ700709, AEJ91550.1) The polypeptide sequence of SEQ ID NO: 34 - ciB-B5 (Genbank: HQ700711, AEJ91552.1) The polypeptide sequence of SEQ ID NO: 35 - ciB-B9 (Genbank: HQ700710, AEJ91551.1) The polypeptide sequence of SEQ ID NO: 36 - ciA-B5 (Genbank: HQ700704, AEJ91545.1) The polypeptide sequence of SEQ ID NO: 37 - ciA-F12 (Genbank: HQ700706, AEJ91547.1) The polypeptide sequence of SEQ ID NO: 38 - ciA-D12 (Genbank: HQ700702, AEJ91543.1) The polypeptide sequence of SEQ ID NO: 39 - ciA-A5 (Genbank: HQ700703, AEJ91544.1) The polypeptide sequence of SEQ ID NO: 40 - ciA-G5 (Genbank: HQ700707, AEJ91548.1) The polypeptide sequence of SEQ ID NO: 41 - ciB-H11 (Genbank: HQ700712, AEJ91553.1) The polypeptide sequence of SEQ ID NO: 42 - ciB-A11 (Genbank: HQ700709, AEJ91550.1). The polypeptide sequence of SEQ ID NO: 43 - ciB-B5 (Genbank: HQ700711, AEJ91552.1) The polypeptide sequence of SEQ ID NO: 44 - ciB-B9 (Genbank: HQ700710, AEJ91551.1) The polypeptide sequence of SEQ ID NO: 45 - mClover3 SEQ ID NO: 46 - Peptide sequence of mRuby3 SEQ ID NO: 1 - Peptide sequence of natural BoNT / A (rBoNT / A) Peptide sequence of SEQ ID NO: 2 - BoNT / B SEQ ID NO: 3 - Polypeptide sequence of BoNT / C Peptide sequence of SEQ ID NO: 4 - BoNT / D Peptide sequence of SEQ ID NO: 5 - BoNT / E Peptide sequence of SEQ ID NO: 6 - BoNT / F SEQ ID NO: 7 - Polypeptide sequence of BoNT / G Peptide sequence of SEQ ID NO: 8 - BoNT / X SEQ ID NO: 9 - Peptide sequence of TeNT The polypeptide sequence of SEQ ID NO: 10 - mrBoNT / A SEQ ID NO: 11 - Peptide sequence of cationic BoNT / A variant 2 SEQ ID NO: 12 - Peptide sequence of cationic BoNT / A variant 3 SEQ ID NO: 13 - Peptide sequence of cationic BoNT / A variant 4 The polypeptide sequence of SEQ ID NO: 14 - mrBoNT / AB SEQ ID NO: 15 - Peptide sequence of BoNT / AB variant 2 SEQ ID NO: 16 - Peptide sequence of BoNT / AB variant 3 SEQ ID NO: 17 - Peptide sequence of BoNT / AB variant 4 SEQ ID NO: 18 - Peptide sequence of BoNT / AB variant 5 SEQ ID NO: 19 - Full-length human SV2a polypeptide sequence SEQ ID NO: 20 - Full-length human SV2b polypeptide sequence SEQ ID NO: 21 - Full-length human SV2c polypeptide sequence The polypeptide sequence of SEQ ID NO: 22 - Cia-C2 SEQ ID NO: 23 - Peptide sequence of alternative Cia-C2 (Genbank: HQ700705, AEJ91546.1) Bold = CDR1 Underlined = CDR2 Italics = CDR3 The polypeptide sequence of SEQ ID NO: 24 - CiaH7 SEQ ID NO: 25 - Peptide sequence of the alternative CiaH7 (Genbank: HQ700708, AEJ91549.1) The polypeptide sequence of SEQ ID NO: 26 - CiaD12 / B5 The polypeptide sequence of SEQ ID NO: 27 - ciA-B5 (Genbank: HQ700704, AEJ91545.1) The polypeptide sequence of SEQ ID NO: 28 - ciA-F12 (Genbank: HQ700706, AEJ91547.1) The polypeptide sequence of SEQ ID NO: 29 - ciA-D12 (Genbank: HQ700702, AEJ91543.1) The polypeptide sequence of SEQ ID NO: 30 - ciA-A5 (Genbank: HQ700703, AEJ91544.1) The polypeptide sequence of SEQ ID NO: 31 - ciA-G5 (Genbank: HQ700707, AEJ91548.1) The polypeptide sequence of SEQ ID NO: 32 - ciB-H11 (Genbank: HQ700712, AEJ91553.1) The polypeptide sequence of SEQ ID NO: 33 - ciB-A11 (Genbank: HQ700709, AEJ91550.1) The polypeptide sequence of SEQ ID NO: 34 - ciB-B5 (Genbank: HQ700711, AEJ91552.1) The polypeptide sequence of SEQ ID NO: 35 - ciB-B9 (Genbank: HQ700710, AEJ91551.1) The polypeptide sequence of SEQ ID NO: 36 - ciA-B5 (Genbank: HQ700704, AEJ91545.1) The polypeptide sequence of SEQ ID NO: 37 - ciA-F12 (Genbank: HQ700706, AEJ91547.1) The polypeptide sequence of SEQ ID NO: 38 - ciA-D12 (Genbank: HQ700702, AEJ91543.1) The polypeptide sequence of SEQ ID NO: 39 - ciA-A5 (Genbank: HQ700703, AEJ91544.1) The polypeptide sequence of SEQ ID NO: 40 - ciA-G5 (Genbank: HQ700707, AEJ91548.1) The polypeptide sequence of SEQ ID NO: 41 - ciB-H11 (Genbank: HQ700712, AEJ91553.1) The polypeptide sequence of SEQ ID NO: 42 - ciB-A11 (Genbank: HQ700709, AEJ91550.1). The polypeptide sequence of SEQ ID NO: 43 - ciB-B5 (Genbank: HQ700711, AEJ91552.1) The polypeptide sequence of SEQ ID NO: 44 - ciB-B9 (Genbank: HQ700710, AEJ91551.1) The polypeptide sequence of SEQ ID NO: 45 - mClover3 SEQ ID NO: 46 - Peptide sequence of mRuby3 Attached Figure Description

[0275] Embodiments of the present invention will now be described by way of example only, with reference to the following figures and examples.

[0276] Figure 1 An exemplary expression builder is shown.

[0277] Figure 2 This demonstrates that FRET occurs in the absence of binding between BoNT / A and the exemplary binding substrate.

[0278] Figure 3 The expression of the binding substrate containing camel antibody is shown.

[0279] Figure 4 This is a schematic diagram showing protein fixation onto the AR2G support.

[0280] Figure 5 The binding kinetics of glycosylated SV2c are shown.

[0281] Figure 6 The binding kinetics of anti-BoNT / A (rabbit polyclonal antibody) are shown.

[0282] Figure 7 The binding kinetics of mClover3-ciA-C2-mRuby3 are shown.

[0283] Figure 8 The binding kinetics of mClover3-ciA-D12-ciA-B5-mRuby3 are shown.

[0284] Figure 9 The binding kinetics of mClover3-ciA-H7-mRuby3 are shown.

[0285] Figure 10 The binding kinetics of mClover3-SV2C 529-566-mRuby3 are shown.

[0286] Figure 11 The binding kinetics of mClover3-SV2C 454-580-mRuby3 are shown.

[0287] Figure 12 The binding kinetics of mClover3-SV2C 566-580-mRuby3 are shown.

[0288] Example Example 1: SV2 connector variant library for BoNT / A testing Materials and Methods Design an expression construct that encodes a binding substrate containing a donor fluorophore (mClover3), a binding region, and an acceptor fluorophore (mRuby3), and clone it into the pRSETa plasmid using Gibson assembly (see [link to pRSETa plasmid]). Figure 1 The expression constructs containing binding regions are selected from the table below.

[0289] Table 2: SV2 junction zone library The resulting plasmid was used to transform DH5α cells. Colonies were picked, and the presence of the plasmid was confirmed by PCR and sequencing. The expression construct was expressed in BL21 cells, and the resulting binding substrate was isolated.

[0290] Design 0 (i.e., amino acids 529-566 containing the full-length SV2c (SEQ ID NO: 21)) was selected for further analysis.

[0291] As a proof-of-concept, the binding of mClover3-SV2c-mRuby3 to the substrate was evaluated to determine whether fluorescence energy transfer occurred between the mClover3 donor and the mRuby3 acceptor in the presence and absence of BoNT / A. Specifically, in the presence of BoNT / A, no emission from the mRuby3 acceptor was detected. However, in the absence of BoNT / A, emission from mRuby3 peaked at a wavelength of 590 nm (see [link to study]). Figure 2 ).

[0292] Example 2: VHH nanobody for BoNT / A detection Select appropriate neutralizing Camelidae VHH nanobodies based on their ability to bind to BoNT / A.

[0293] Three different expression constructs were designed, each containing a donor fluorophore (mClover3), a binding region, and an acceptor fluorophore (mRuby3), and cloned into the pRSETa plasmid using Gibson assembly. Specifically, three different constructs were selected, each containing a binding region comprising CiA-H7, ciA-C2, and a dimer (containing ciA-D12 and ciA-B5) (ciA-B5-D12).

[0294] The resulting plasmid was used to transform DH5α cells. Colonies were picked, and the presence of the plasmid was confirmed by PCR and sequencing. Expression of the expression construct was performed in BL21 cells. The resulting binding substrate was isolated and separated using SDS-PAGE. Figure 3 Coomassie blue staining for each construct is shown. Specifically, the expected bands were observed at 67.6 kD for ciA-C2, 66 kD for ciA-H7, and 81.2 kD for the ciA-B5-D12 dimer.

[0295] Example 3: Analysis combined with dynamics Materials and Methods BoNT / A is immobilized onto an amine-reactive second-generation (AR2G) biolayer interferometry (BLI) platform (Octet® BLI platform) via EDC-catalyzed amide bond formation. Specifically, a covalent bond is formed between the reactive amine on BoNT / A and the carboxyl-terminated biosensor surface. Covalent immobilization immobilizes BoNT / A onto the AR2G biosensor surface for use in conjunction with event analysis and kinetic characterization. A schematic diagram showing BoNT / A immobilized onto an AR2G support and subsequently coupled with the biosensor of the present invention is shown in [illustration missing]. Figure 4 middle.

[0296] The binding of immobilized BoNT / A with each of the following analytes was measured using biolayer interferometry (BLI) (Octet® BLI platform): 1. Glycosylated SV2c; 2. Anti-BoNT / A (rabbit polyclonal antibody; 150 kDa; stock solution concentration: 2 mg / mL); 3. mClover3-ciA-C2-mRuby3 (67.6 kDa; 1.5 mL; stock solution concentration: 1.7 mg / mL). 4. mClover3-ciA-D12-ciA-B5-mRuby3 (81.2 kDa; 300 µL; stock solution concentration: 10 mg / mL). 5. mClover3-ciA-H7-mRuby3 (66 kDa; 1 mL; stock solution concentration: 6.8 mg / mL); 6. mClover3-SV2C 529-566-mRuby3 (59 kDa; 100 µL; stock solution concentration: 8.4 mg / mL); 7. mClover3-SV2C 454-580-mRuby3 (69 kDa; 200 µL; stock solution concentration: 0.2 mg / mL); and 8. mClover3-SV2C 566-580-mRuby3 (55.9 kDa; 220 µL; stock solution concentration: 1.1 mg / mL).

[0297] result Glycosylated SV2c The binding kinetics between captured BoNT / A and SV2c are summarized in Table 3 and Figure 5 Specifically, the binding of BoNT / A to glycosylated SV2C during the association phase is slow and gradual. As assessed using a sensor map, an unusually slow dissociation from BoNT / A was found at pH 7.4, with a clear indication of increased recombination during the dissociation phase.

[0298] Table 3: Binding kinetics of glycosylated SV2c The above results confirm the effectiveness of this method, as the Kd during association is comparable to that in the literature.

[0299] Anti-BoNT / A (rabbit polyclonal antibody) The binding kinetics between captured BoNT / A and anti-BoNT / A (rabbit polyclonal antibody) are summarized in Table 4 and Figure 6 Specifically, the binding of BoNT / A to anti-BoNT / A during the association phase is slow and gradual. As assessed using a sensor map, dissociation from BoNT / A was found to be exceptionally slow at pH 7.4.

[0300] Table 4: Binding kinetics of anti-BoNT / A (rabbit polyclonal antibody) The results above demonstrate that BoNT / A folds into its native conformation, thereby allowing anti-BoNTA antibodies to bind to their BoNT / A epitopes.

[0301] mClover3-ciA-C2-mRuby3 The binding kinetics between captured BoNT / A and VHH ciA-C2 are summarized in Table 5 and Figure 7 Specifically, during the association phase, the binding of BoNT / A to VHH ciA-C2 was initially rapid, followed by a slow, gradual association rate. As assessed using a sensor plot, dissociation from BoNT / A was found to be exceptionally slow at pH 7.4, where approximately 54% of the initial analyte / ligand interaction was detected.

[0302] Table 5: Binding kinetics of mClover3-ciA-C2-mRuby3 Compared to binding substrates containing the SV2 sequence, binding substrates containing mClover3-CIaC2-mRuby3 showed approximately a 3-fold increase in affinity for BoNT / A.

[0303] mClover3-ciA-D12-ciA-B5-mRuby3 The binding kinetics between captured BoNT / A and VHH ciA-D12 / B5 are summarized in Table 6 and Figure 8 Specifically, during the association phase, the binding of BoNT / A to VHH ciA-D12 / B5 was initially rapid, followed by a slow, gradual association rate. As assessed using a sensor plot, dissociation from BoNT / A was found to be exceptionally slow at pH 7.4, where approximately 100% of the initial analyte / ligand interaction was detected.

[0304] Table 6: Binding kinetics of mClover3-ciA-D12-ciA-B5-mRuby3 mClover3-ciA-H7-mRuby3 The binding kinetics between captured BoNT / A and VHH ciA-H7 are summarized in Table 7 and Figure 9 Specifically, during the association phase, the binding of BoNT / A to VHH ciA-H7 was initially rapid, followed by a slow, gradual association rate. As assessed using a sensor plot, dissociation from BoNT / A was found to be exceptionally slow at pH 7.4, where approximately 50% of the initial analyte / ligand interaction was detected.

[0305] Table 7: Binding kinetics of mClover3-ciA-H7-mRuby3 mClover3-SV2C 529-566-mRuby3 The binding kinetics between the captured BoNT / A and mClover3-SV2C 529-566-mRuby3 are summarized in Table 8 and Figure 10 Specifically, during the association phase, the binding of BoNT / A to mClover3-SV2C 529-566-mRuby3 was initially rapid, followed by a slow, gradual association rate. As assessed using a sensor plot, dissociation from BoNT / A was found to be exceptionally slow at pH 7.4, with approximately 6.22% of the initial analyte / ligand interaction detected.

[0306] Table 8: Binding kinetics of mClover3-SV2C 529-566-mRuby3 mClover3-SV2C 454-580-mRuby3 The binding kinetics between the captured BoNT / A and mClover3-SV2C 454-580-mRuby3 are summarized in Table 9 and Figure 11 Specifically, during the association phase, the binding of BoNT / A to mClover3-SV2C 454-580-mRuby3 was initially rapid, followed by a slow, gradual association rate. As assessed using a sensor plot, dissociation from BoNT / A was found to be exceptionally slow at pH 7.4, with approximately 11% of the initial analyte / ligand interaction detected.

[0307] Table 9: Binding kinetics of mClover3-SV2C 454-580-mRuby3 mClover3-SV2C 566-580-mRuby3 The binding kinetics between the captured BoNT / A and mClover3-SV2C 566-580-mRuby3 are summarized in Table 10 and Figure 12 Specifically, during the association phase, the binding of BoNT / A to mClover3-SV2C 566-580-mRuby3 was initially rapid, followed by a slow, gradual association rate. As assessed using a sensor plot, dissociation from BoNT / A was found to be exceptionally slow at pH 7.4, with approximately 12.3% of the initial analyte / ligand interaction detected.

[0308] Table 10: Binding kinetics of mClover3-SV2C 566-580-mRuby3 in conclusion Based on the above results, and especially the binding affinity (K) D mClover3-ciA-C2-mRuby3 was selected as the best-performing binding substrate. Importantly, the inventors have demonstrated that binding substrates containing donor and acceptor fluorophores, as well as binding regions containing clostridium neurotoxin nanobodies, can bind to BoNT / A with high affinity. Specifically, the affinity is significantly greater than that achieved using comparative binding substrates containing SV2c binding regions. Therefore, the resulting highly sensitive biosensor and binding substrate can be used to monitor clostridium neurotoxin production during bacterial fermentation in real time.

[0309] All publications mentioned in the foregoing specification are incorporated herein by reference. Various modifications and variations to 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 conjunction 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 carried out, which will be apparent to those skilled in the art of biochemistry and biotechnology or related fields, are intended to be within the scope of the following claims.

[0310] Aspects of the present invention 1. A biosensor for real-time monitoring of clostridium neurotoxin production during bacterial fermentation, the biosensor comprising a surface for analyzing binding events and kinetics, wherein the surface is configured to immobilize clostridium neurotoxin, and the biosensor further comprising a binding substrate, wherein the binding substrate comprises: a. Donor fluorophore; b. An acceptor having an absorption spectrum that overlaps with the emission spectrum of the donor fluorophore; and c. A binding region that specifically binds to the Clostridium neurotoxin; wherein the binding region is located between the donor fluorophore and the receptor, such that, upon activation of the donor fluorophore, a resonant energy transfer occurs between the donor fluorophore and the receptor.

[0311] 2. A binding substrate for real-time monitoring of clostridium neurotoxin production during bacterial fermentation, the binding substrate comprising: a. Donor fluorophore; b. An acceptor having an absorption spectrum that overlaps with the emission spectrum of the donor fluorophore; and c. A binding region that specifically binds to the Clostridium neurotoxin; wherein the binding region is located between the donor fluorophore and the receptor, such that, upon activation of the donor fluorophore, a resonant energy transfer occurs between the donor fluorophore and the receptor.

[0312] 3. The biosensor according to aspect 1 or the binding substrate according to aspect 2, wherein the binding region comprises one or more complementary sites for binding clostridium neurotoxin.

[0313] 4. The biosensor or binding substrate according to aspect 3, wherein the one or more complementary sites comprise one or more amino acid sequences that specifically bind to the Clostridium neurotoxin, and wherein the one or more amino acid sequences comprise the CDR sequence of one or more antibodies; preferably, wherein these antibodies are single-chain antibodies, such as nanobodies, single-chain variable fragments (scFv), single-chain Fab (scFab), microantibodies, or biantibodies; more preferably, wherein these single-chain antibodies are camel or shark nanobodies.

[0314] 5. The biosensor or binding substrate according to aspect 4, wherein the one or more amino acid sequences comprise a CDR sequence from a nanobody selected from the list comprising: ciA-H7, ciA-D1, ciA-H4, ciA-H11, ciA-C2, ciA-B5, ciA-F12, ciA-D12, ciA-A5, ciA-G5, ciB-H11, ciB-A11, ciB-B5, ciB-B9, ciA-H7 / B5, ciA-F12 / D12 and ciB-A11 / B5 or combinations thereof.

[0315] 6. The biosensor or binding substrate according to aspect 5, wherein the one or more amino acid sequences comprise a sequence having at least 80% sequence identity with the full-length sequence of one or more nanobodies selected from the list below: ciA-H7, ciA-D1, ciA-H4, ciA-H11, ciA-C2, ciA-B5, ciA-F12, ciA-D12, ciA-A5, ciA-G5, ciB-H11, ciB-A11, ciB-B5, ciB-B9, ciA-H7 / B5, ciA-F12 / D12, and ciB-A11 / B5, or combinations thereof.

[0316] 7. The biosensor or binding substrate according to aspect 4, wherein the one or more amino acid sequences comprise a CDR sequence from a nanobody capable of neutralizing clostridium neurotoxin toxicity, such nanobodies as ciA-C2, ciA-H7, ciA-B5, and ciA-D1, or combinations thereof.

[0317] 8. The biosensor or binding substrate according to aspect 7, wherein the one or more amino acid sequences comprise a sequence having at least 80% sequence identity with the full-length sequence of one or more nanobodies selected from the list below: ciA-C2, ciA-H7, ciA-B5, and ciA-D1, or combinations thereof.

[0318] 9. The biosensor or binding substrate according to any one of aspects 5 to 8, wherein the nanobody is a dimer, trimer or tetramer; optionally, wherein the nanobody comprises ciA-B5-D12 or a combination thereof.

[0319] 10. A biosensor according to any one of aspects 1 or 3-9, or a binding substrate according to any one of aspects 2-9, wherein the one or more amino acid sequences are selected from (and optionally further comprise) sequences that specifically bind to one or more clostridium neurotoxins selected from: Oxidized form of clostridium neurotoxin (with reduced potency). Incorrectly activated proteolytic forms of clostridium neurotoxin (with reduced potency). Clostridium neurotoxins in aggregate form, including dimers and polymers (with reduced potency).

[0320] 11. The biosensor according to any one of aspects 1 or 3-10, or the binding substrate according to any one of aspects 2-10, wherein the clostridial neurotoxin is detectable at a concentration of less than 1 ng / ml, preferably less than 0.1 ng / ml.

[0321] 12. A method for real-time monitoring of clostridium neurotoxin production during bacterial fermentation, the method comprising: a. Culture bacterial host cells capable of producing clostridium neurotoxin in liquid culture medium; b. Contact the liquid culture medium with the biosensor according to any one or more of the foregoing aspects; c. Excite the donor fluorophore; and d. Determine the resonant energy transfer at the biosensor relative to a control, wherein the difference in the resonant energy transfer at the biosensor compared to the control indicates the presence of clostridial neurotoxin.

[0322] 13. The method according to aspect 12, wherein the clostridial neurotoxin is immobilized on the surface of the biosensor.

[0323] 14. The method according to aspect 13, wherein after being fixed to the support, the clostridial neurotoxin is brought into contact with the binding substrate.

[0324] 15. The method according to any one of aspects 12 to 14, wherein the bacterial host cell is lysed before the liquid culture medium is brought into contact with the biosensor.

[0325] 16. A method for real-time monitoring of clostridium neurotoxin production during bacterial fermentation, the method comprising: a. Transform bacterial host cells capable of producing clostridial neurotoxins using an expression construct encoding the binding substrate according to any one of aspects 2-11; b. Culture the bacterial host cells in a liquid culture medium; c. Excite the donor fluorophore; and d. Determine the resonant energy transfer at the biosensor relative to a control, wherein the difference in the resonant energy transfer at the biosensor compared to the control indicates the presence of clostridial neurotoxin.

[0326] 17. The method according to any one of aspects 12 to 16, wherein step d includes detecting donor fluorescence intensity at the biosensor, wherein an increase in donor fluorescence intensity at the biosensor compared to the control indicates the presence of clostridium neurotoxin.

[0327] 18. The method according to any one of aspects 12 to 16, wherein step d includes detecting the receptor fluorescence intensity at the biosensor, wherein a decrease in the receptor fluorescence intensity at the biosensor compared to the control indicates the presence of clostridium neurotoxin.

[0328] 19. The method according to any one of aspects 12 to 16, wherein step d includes detecting a maximum emission value of the receptor and a maximum emission value of the donor fluorophore at the biosensor, wherein an offset of the emission maximum value from the vicinity of the maximum emission value of the receptor to the vicinity of the maximum emission value of the donor fluorophore indicates the presence of clostridium neurotoxin.

[0329] 20. The method according to any one of aspects 12 to 16, wherein step d comprises detecting the ratio of fluorescence amplitude near the maximum emission value of the receptor to the fluorescence amplitude near the maximum emission value of the donor fluorophore, wherein a decrease in the ratio at the biosensor compared to the control indicates the presence of clostridium neurotoxin.

Claims

1. A biosensor for real-time monitoring of clostridium neurotoxin production during bacterial fermentation, the biosensor comprising a surface for analyzing binding events and kinetics, and a binding substrate immobilized on the surface, the binding substrate comprising: a. Donor fluorophore; b. An acceptor having an absorption spectrum that overlaps with the emission spectrum of the donor fluorophore; and c. A binding region that specifically binds to the Clostridium neurotoxin; wherein the binding region is located between the donor fluorophore and the receptor, such that, upon activation of the donor fluorophore, a resonant energy transfer occurs between the donor fluorophore and the receptor.

2. A binding substrate for real-time monitoring of clostridium neurotoxin production during bacterial fermentation, the binding substrate comprising: a. Donor fluorophore; b. An acceptor having an absorption spectrum that overlaps with the emission spectrum of the donor fluorophore; and c. A binding region that specifically binds to the Clostridium neurotoxin; wherein the binding region is located between the donor fluorophore and the receptor, such that, upon activation of the donor fluorophore, a resonant energy transfer occurs between the donor fluorophore and the receptor.

3. The biosensor of claim 1 or the binding substrate of claim 2, wherein the binding region comprises one or more complementary sites for binding clostridium neurotoxin.

4. The biosensor or binding substrate of claim 3, wherein the one or more complementary sites comprise one or more amino acid sequences that specifically bind to the Clostridium neurotoxin, and wherein the one or more amino acid sequences comprise a CDR sequence of one or more antibodies; preferably, wherein these antibodies are single-chain antibodies, such as nanobodies, single-chain variable fragments (scFv), single-chain Fab (scFab), microantibodies, or biantibodies; more preferably, wherein these single-chain antibodies are camel or shark nanobodies.

5. The biosensor or binding substrate of claim 4, wherein the one or more amino acid sequences comprise a CDR sequence from a nanobody selected from the list comprising: ciA-H7, ciA-D1, ciA-H4, ciA-H11, ciA-C2, ciA-B5, ciA-F12, ciA-D12, ciA-A5, ciA-G5, ciB-H11, ciB-A11, ciB-B5, ciB-B9, ciA-H7 / B5, ciA-F12 / D12, and ciB-A11 / B5, or combinations thereof.

6. The biosensor or binding substrate of claim 5, wherein the one or more amino acid sequences comprise a sequence having at least 80% sequence identity with the full-length sequence of one or more nanobodies selected from the list below: ciA-H7, ciA-D1, ciA-H4, ciA-H11, ciA-C2, ciA-B5, ciA-F12, ciA-D12, ciA-A5, ciA-G5, ciB-H11, ciB-A11, ciB-B5, ciB-B9, ciA-H7 / B5, ciA-F12 / D12, and ciB-A11 / B5, or combinations thereof.

7. The biosensor or binding substrate of claim 4, wherein the one or more amino acid sequences comprise a CDR sequence from a nanobody capable of neutralizing clostridium neurotoxin toxicity, such nanobodies as ciA-C2, ciA-H7, ciA-B5, and ciA-D1, or combinations thereof.

8. The biosensor or binding substrate of claim 7, wherein the one or more amino acid sequences comprise a sequence having at least 80% sequence identity with the full-length sequence of one or more nanobodies selected from the list below: ciA-C2, ciA-H7, ciA-B5, and ciA-D1, or combinations thereof.

9. The biosensor or binding substrate according to any one of claims 5 to 8, wherein the nanobody is a dimer, trimer, or tetramer; optionally, wherein the nanobody comprises ciA-B5-D12 or a combination thereof.

10. The biosensor according to any one of claims 1 or 3-9, or the binding substrate according to any one of claims 2-9, wherein the one or more amino acid sequences are selected from (and optionally further comprise) sequences that specifically bind to one or more clostridium neurotoxins selected from: Oxidized form of clostridium neurotoxin (with reduced potency). Incorrectly activated proteolytic forms of clostridium neurotoxin (with reduced potency). Clostridium neurotoxins in aggregate form, including dimers and polymers (with reduced potency).

11. The biosensor according to any one of claims 1 or 3-10, or the binding substrate according to any one of claims 2-10, wherein the clostridial neurotoxin is detectable at a concentration of less than 1 ng / ml, preferably less than 0.1 ng / ml.

12. A method for real-time monitoring of clostridium neurotoxin production during bacterial fermentation, the method comprising: a. Culture bacterial host cells capable of producing clostridium neurotoxin in liquid culture medium; b. Contact the liquid culture medium with the biosensor according to any one or more of the preceding claims; c. Excite the donor fluorophore; as well as d. Determine the resonant energy transfer at the biosensor relative to a control, wherein the difference in the resonant energy transfer at the biosensor compared to the control indicates the presence of clostridial neurotoxin.

13. The method of claim 12, wherein the bacterial host cell is lysed before the liquid culture medium is brought into contact with the biosensor.

14. A method for real-time monitoring of clostridium neurotoxin production during bacterial fermentation, the method comprising: a. Transforming bacterial host cells capable of producing clostridium neurotoxins using an expression construct encoding the binding substrate according to any one of claims 2-11; b. Culture the bacterial host cells in a liquid culture medium; c. Excite the donor fluorophore; as well as d. Determine the resonant energy transfer at the biosensor relative to a control, wherein the difference in the resonant energy transfer at the biosensor compared to the control indicates the presence of clostridial neurotoxin.

15. The method according to any one of claims 12 to 14, wherein step d comprises detecting donor fluorescence intensity at the biosensor, wherein an increase in donor fluorescence intensity at the biosensor compared to the control indicates the presence of clostridium neurotoxin.

16. The method according to any one of claims 12 to 14, wherein step d comprises detecting the receptor fluorescence intensity at the biosensor, wherein a decrease in the receptor fluorescence intensity at the biosensor compared to the control indicates the presence of clostridium neurotoxin.

17. The method according to any one of claims 12 to 14, wherein step d comprises detecting the maximum emission value of the receptor and the maximum emission value of the donor fluorophore at the biosensor, wherein the offset of the emission maximum value from the vicinity of the maximum emission value of the receptor to the vicinity of the maximum emission value of the donor fluorophore indicates the presence of clostridium neurotoxin.

18. The method according to any one of claims 12 to 14, wherein step d comprises detecting the ratio of fluorescence amplitude near the maximum emission value of the receptor to the fluorescence amplitude near the maximum emission value of the donor fluorophore, wherein a decrease in the ratio at the biosensor compared to the control indicates the presence of clostridium neurotoxin.

Images