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WO2025141289A1 - Biocapteur pour le suivi en temps réel de la production de neurotoxine clostridiale - Google Patents

Biocapteur pour le suivi en temps réel de la production de neurotoxine clostridiale Download PDF

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Publication number
WO2025141289A1
WO2025141289A1 PCT/GB2024/053207 GB2024053207W WO2025141289A1 WO 2025141289 A1 WO2025141289 A1 WO 2025141289A1 GB 2024053207 W GB2024053207 W GB 2024053207W WO 2025141289 A1 WO2025141289 A1 WO 2025141289A1
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Prior art keywords
cia
clostridial neurotoxin
bont
binding
seq
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PCT/GB2024/053207
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English (en)
Inventor
Williams OLUGHU
Alison MASON
Karen Marie Polizzi
Ivet ANGELOVA
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Ipsen Biopharm Ltd
Ip2ipo Innovations Ltd
Original Assignee
Ipsen Biopharm Ltd
Imperial College Innovations Ltd
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Publication of WO2025141289A1 publication Critical patent/WO2025141289A1/fr
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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/33—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Clostridium (G)

Definitions

  • the present invention relates to biosensors for monitoring clostridial neurotoxin production and methods thereof.
  • Clostridia produce highly potent and specific protein toxins, which can poison neurons and other cells to which they are delivered.
  • Examples of such clostridial toxins include the neurotoxins produced by C. tetani (TeNT) and by C. botulinum (BoNT) serotypes A-G, and X (see WO 2018/009903 A2), as well as those produced by C. baratii and C. butyricum.
  • tetanus and botulinum toxins act by inhibiting the function of affected neurons, specifically the release of neurotransmitters. While botulinum neurotoxins act at the neuromuscular junction and inhibit cholinergic transmission in the peripheral nervous system, tetanus toxin acts in the central nervous system.
  • clostridial neurotoxins e.g. botulinum neurotoxins [BoNTs]
  • BoNTs botulinum neurotoxins
  • H-chain heavy chain
  • L-chain light chain
  • the H-chain comprises an N-terminal translocation component (H N domain) and a C-terminal targeting component (H C domain).
  • H N domain N-terminal translocation component
  • H C domain C-terminal targeting component
  • H N domain translocates the L-chain across the endosomal membrane and into the cytosol, and the L-chain provides a protease function (also known as a non- cytotoxic protease).
  • Non-cytotoxic proteases act by proteolytically cleaving intracellular transport proteins known as SNARE proteins (e.g. SNAP-25, VAMP, or Syntaxin).
  • SNARE derives from the term Soluble NSF Attachment Receptor, where NSF means N-ethylmaleimide-Sensitive Factor.
  • SNARE proteins are integral to intracellular vesicle fusion, and thus to secretion of molecules via vesicle transport from a
  • the protease function is a zinc-dependent endopeptidase activity and exhibits a high substrate specificity for SNARE proteins.
  • Botulinum toxins, including the type-A toxins, are conventionally obtained through a culturing and fermentation process.
  • Off-line analysis may be performed hours or even days after a sample is collected and therefore does not allow for the real-time monitoring of botulinum toxin production. Further, the testing methods required for off-line monitoring are generally slow, laborious and expensive. For example, determining the concentration of botulinum toxin in a sample typically uses multiple steps (e.g., to separate the toxin complex from residual impurities from the fermentation process) and involves the manual handling of samples having an inherent biosafety risk. Further, processing of clostridial neurotoxins in batches comes with several drawbacks, including potential differences in sensitivity due to the use of batches of antibodies for immunoprecipitation and thus reduced reproducibility and robustness.
  • the present invention relates to biosensors and methods for monitoring the production of botulinum toxins in real-time.
  • biosensors and methods for monitoring the production of botulinum toxins in real-time SUMMARY OF THE INVENTION
  • the present inventors have developed novel biosensors, binding substrates and methods for the rapid, real-time monitoring of clostridial neurotoxin production during a bacterial fermentation process.
  • the biosensors, binding substrates and methods of the invention may be highly sensitive, thereby allowing for the detection of picomolar concentrations of clostridial neurotoxins in a composition.
  • biosensors and methods of the invention advantageously also allow for the detection of specific modified (e.g., oxidised), proteolytically activated or aggregated forms (for example, dimers and multimers, optionally having reduced potency) of clostridial neurotoxins.
  • the biosensors may be used to determine whether the fermentation process produces a homogenous population of clostridial neurotoxin polypeptides and/or determine the proportion of clostridial neurotoxin variants produced.
  • the biosensors, binding substrates and methods of the invention have been specifically designed to require minimal steps and equipment, meaning that they are suitable for use with agents that may cause serious and potentially lethal side effects in subjects exposed to said agents (e.g., in biosafety level 3 environments).
  • biosensors, binding substrates and methods of the invention overcome the challenges associated with variability in the sensitivity of batches of reagents (e.g., monoclonal antibodies) used to monitor clostridial neurotoxin production using conventional off-line approaches.
  • the present invention provides a biosensor for real-time monitoring of clostridial neurotoxin production during a bacterial fermentation process, said biosensor comprising a surface for performing the analysis of binding events and kinetics, and onto which is immobilised a binding substrate comprising: a. a donor fluorophore; b. an acceptor having an absorbance spectrum overlapping the emission spectrum of the donor fluorophore; and c.
  • binding region that specifically binds to the clostridial neurotoxin; wherein said binding region being positioned between the donor fluorophore and the acceptor such that, following activation of the donor fluorophore, resonance energy transfer is exhibited between said donor fluorophore and said acceptor.
  • a binding substrate for real-time monitoring of clostridial neurotoxin production during a bacterial fermentation process comprising: a. a donor fluorophore; b. an acceptor having an absorbance spectrum overlapping the emission spectrum of the donor fluorophore; and c.
  • the binding region comprises one or more paratopes that bind(s) clostridial neurotoxin.
  • paratope is a well-defined term in the art that refers to the antigen-binding site of an antibody or an antibody fragment.
  • the binding region comprises one or more amino acid sequence(s) that specifically bind(s) to the clostridial neurotoxin, and wherein said one or more amino acid sequence(s) comprise the CDR sequences of one or more antibodies; preferably, wherein the antibodies are single-chain antibodies such as nanobodies, single-chain variable fragments (scFvs), single-chain Fab (scFab), minibodies or diabodies; further preferably wherein the single-chain antibodies are camelid or shark nanobodies.
  • scFvs single-chain variable fragments
  • scFab single-chain Fab
  • said one or more amino acid sequence(s) comprises the CDR sequences from nanobodies 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.
  • said one or more amino acid sequence(s) may comprise a sequence having at least 80% sequence identity to the full-length sequence of one or more 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.
  • the biosensor or binding substrate comprise one or more amino acid sequence comprises the CDR sequences from nanobodies capable of neutralising clostridial neurotoxin toxicity such as ciA-C2, ciA-H7, ciA-B5 and ciA-D1, or combinations thereof.
  • the biosensor or substrate comprise one or more amino acid sequence comprising a sequence having at least 80% sequence identity to the full-length sequence of one or more nanobody selected from the list comprising: ciA-C2, ciA-H7, ciA-B5 and ciA-D1, or combinations thereof.
  • the nanobody of the invention may be multimer, such as a dimer, trimer or tetramer.
  • the binding region comprises one or more amino acid sequence(s) selected from (and optionally further comprises) a sequence that specifically binds to a clostridial neurotoxin selected from the one or more of: an oxidised form of clostridial neurotoxin (having reduced potency), an incorrect proteolytically activated form of a clostridial neurotoxin (having reduced potency), and/or aggregated forms of clostridial neurotoxin, including dimers and multimers (having reduced potency).
  • the biosensor and/or binding substrate of the invention are advantageously highly sensitive.
  • the biosensor and/or binding substrate may be used to detect clostridial neurotoxins at a concentration of less than 1ng/ ml, preferably less than 0.1ng/ ml.
  • a method for real-time monitoring of clostridial neurotoxin production during a bacterial fermentation process comprising: a. culturing a bacterial host cell capable of producing a clostridial neurotoxin in a liquid medium; b. contacting said liquid medium with a biosensor of the invention; c. exciting said donor fluorophore; and d.
  • the bacterial host cell may be lysed prior to contacting the liquid medium with a biosensor.
  • a method for real-time monitoring of clostridial neurotoxin production during a bacterial fermentation process comprising: a. transforming a bacterial host cell capable of producing a clostridial neurotoxin with an expression construct encoding a binding substrate of the invention; b. culturing the bacterial host cell in a liquid medium; c.exciting said donor fluorophore; and d.
  • step d comprises donor fluorescence intensity at said biosensor, wherein increased donor fluorescence intensity of at said biosensor as compared to said control is indicative of the presence of clostridial neurotoxin.
  • step d comprises detecting acceptor fluorescence intensity at said biosensor, wherein decreased acceptor fluorescence intensity at said biosensor as compared to said control is indicative of the presence of clostridial neurotoxin.
  • step d comprises detecting an acceptor emission maximum and a donor fluorophore emission maximum at said biosensor, wherein a shift in emission maxima from near said acceptor emission maximum to near said donor fluorophore emission maximum is indicative of the presence of clostridial neurotoxin.
  • step d comprises detecting the ratio of fluorescence amplitudes near an acceptor emission maximum to the fluorescence amplitudes near a donor fluorophore emission maximum, wherein a decreased ratio at said biosensor as compared to the control is indicative of the presence of clostridial neurotoxin.
  • the invention provides a biosensor for real-time monitoring of clostridial neurotoxin production during a bacterial fermentation process, said biosensor comprising a surface for performing the analysis of binding events and kinetics, and onto which is immobilised a binding substrate comprising: a. a donor fluorophore; b. an acceptor having an absorbance spectrum overlapping the emission spectrum of the donor fluorophore; and c.
  • the invention provides a biosensor for real-time monitoring of clostridial neurotoxin production during a bacterial fermentation process, said biosensor comprising a surface for performing the analysis of binding events and kinetics, wherein the surface is configured for immobilisation of a neurotoxin, and further comprising a binding substrate, wherein the binding substrate comprises: a. a donor fluorophore; b.
  • the invention provides a binding substrate for real-time monitoring of clostridial neurotoxin production during a bacterial fermentation process comprising: a. a donor fluorophore; b. an acceptor having an absorbance spectrum overlapping the emission spectrum of the donor fluorophore; and c.
  • the invention provides a method for real-time monitoring of clostridial neurotoxin production during a bacterial fermentation process, said method comprising: a. culturing a bacterial host cell capable of producing a clostridial neurotoxin in a liquid medium; b. contacting said liquid medium with a biosensor of the invention; c. exciting said donor fluorophore; and d.
  • the invention provides a method for real-time monitoring of clostridial neurotoxin production during a bacterial fermentation process, said method comprising: a. transforming a bacterial host cell capable of producing a clostridial neurotoxin with an expression construct encoding a binding substrate of the invention; b. culturing the bacterial host cell in a liquid medium; c. exciting said donor fluorophore; and d.
  • the biosensor of the invention enables rapid, real-time monitoring of clostridial neurotoxin production during a bacterial fermentation process.
  • the biosensor comprises a surface for performing the analysis of binding events and/or kinetics (e.g., a support) and a binding substrate.
  • the invention provides a binding substrate for real-time monitoring of clostridial neurotoxin production during a bacterial fermentation process.
  • the binding substrate is not immobilised to a surface.
  • the binding substrate may be expressed intracellularly in a host cell that is capable of producing a clostridial neurotoxin polypeptide.
  • the support is configured to capture or immobilise the binding substrate.
  • the support is configure to capture or immobilise clostridial neurotoxin polypeptides.
  • the binding substrate comprises a means for indicating the presence or absence of clostridial neurotoxin polypeptides in a composition.
  • the binding substrate may comprise two different states, wherein one state indicates the presence of clostridial neurotoxin polypeptides (e.g., via the binding of clostridial neurotoxin polypeptides to the binding substrate) and another state indicating the absence of clostridial neurotoxin polypeptides (e.g., via the absence of binding of clostridial neurotoxin polypeptides to the binding substrate).
  • the means for indicating the presence or absence of a clostridial neurotoxin may comprise one or more modules (e.g., 1, 2, 3, 4, 5 or more modules).
  • the one or more modules are genetically encoded.
  • the one or more modules are expressed as a single-chain polypeptide.
  • the term “single-chain” may refer to a single polypeptide molecule having a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues.
  • each recited element of the single-chain polypeptide may be connected to the other element(s) by means of a peptide bond.
  • Exemplary modules may be selected from detectable labels and clostridial neurotoxin binding regions.
  • the one or more comprise one or more detectable label.
  • the binding substrate may comprise 1, 2, 3, 4, 5 or more detectable labels, preferably 2 detectable labels.
  • the detectable label may be a label that can be detected visually by way of the label’s optical properties.
  • a detectable label may be a fluorescent label (e.g., a fluorophore). Such a label may be detected using any number of fluorescent techniques including fluorescent microscopy.
  • the binding substrate comprises one or more detectable label, wherein the detectable label is a fluorophore.
  • the term fluorophore encompasses fluorescent proteins, bioluminescent proteins, fluorescent dyes (e.g., non- protein organic dyes) and quantum dots.
  • the fluorophore is a fluorescent protein.
  • the means for indicating the presence or absence of a clostridial neurotoxin comprises at least two detectable labels (e.g., two fluorophores).
  • the binding substrate comprises a first and second detectable label (e.g., a first and second fluorophore).
  • the binding substrate may comprise a first detectable label (e.g., a fluorophore) and a moiety that reduces the fluorescence intensity of the fluorophore under certain conditions, such as a fluorescence quencher or Au-nano particle.
  • the binding substrate comprises a first (donor) fluorophore and a second (acceptor) fluorophore.
  • the donor fluorophore and acceptor fluorophore are positioned such that following activation of the donor fluorophore, resonance energy transfer (e.g., fluorescence (or Förster) resonance energy transfer, FRET) is exhibited between said donor fluorophore and said acceptor.
  • resonance energy transfer e.g., fluorescence (or Förster) resonance energy transfer, FRET
  • FRET refers to non-radiative energy transfer between two fluorophores having different emission wavelengths, in which the excitation energy of a donor fluorophore in an excited state is transferred to an acceptor, and thus emission from the fluorescence acceptor, or the quenching of the fluorescence donor is observed (Lakowicz, J.R.
  • the terms “donor” or “donor fluorophore” may refer to a fluorophore acting as a donor in the FRET phenomenon
  • the terms “acceptor” or “acceptor fluorophore” may refer to a fluorophore acting as an acceptor in the FRET phenomenon.
  • the donor fluorophore and the acceptor fluorophore are functionally connected by one or more amino acid sequence that specifically binds to a clostridial neurotoxin.
  • the term “functionally connect” refers to joining the donor fluorophore and the acceptor fluorophores in such a way that any intervening sequence (e.g., a binding region) does not prevent the primary amino acid sequence of each fluorophore forming a functional tertiary structure (e.g., a functional fluorophore). Methods for determining whether a given sequence functionally connects a donor and acceptor fluorophore are known in the art.
  • the skilled person will be able to determine whether a given binding region functionally connects a donor and acceptor fluorophore using for example, visual detection using fluorescent microscopy to measure FRET (e.g., by sensitized emission, acceptor photobleaching and/or FLIM-FRET methods) or solution-based methods to measure FRET (e.g using filter or monochromatic spectrophotometers).
  • the donor fluorophore and acceptor fluorophore are separated by a binding region comprising one or more sequences that bind to clostridial neurotoxin polypeptides.
  • the binding region may comprise one or more sequences (e.g., 1, 2, 3, 4, 5 or more sequences) that bind to clostridial neurotoxin polypeptides.
  • the binding substrate is configured such that binding of a clostridial neurotoxin to the binding region increases the distance between the donor fluorophore and the acceptor fluorophore.
  • the binding substrate is configured such that binding of a clostridial neurotoxin to the binding region results in a change in the fluorescence intensity from the donor fluorophore and/or the acceptor fluorophore.
  • the donor and acceptor fluorophores are separated by an amino acid sequence that binds to clostridial neurotoxin polypeptides in the presence of said clostridial neurotoxin.
  • the binding region in the absence of clostridial neurotoxin polypeptides, the binding region is unbound by clostridial neurotoxin polypeptides and the donor and acceptor fluorophores are in close proximity such that resonance energy transfer between said donor fluorophore and said acceptor occurs.
  • the binding substrates are identical.
  • the biosensor comprises two or more different binding substrates.
  • the different binding substrates may comprise different regions such that a first plurality of binding substrates binds to a first population of clostridial neurotoxin polypeptides and a second plurality of binding substrates binds to a second population of clostridial neurotoxin polypeptides.
  • a biosensor comprising one or more different binding substrates may be used in a method for real-time monitoring of oxidised clostridial neurotoxin production during a bacterial fermentation process.
  • FRET pairs may be selected from the list comprising: 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-mKO ⁇ or CyOFP1-mCardinal.
  • suitable FRET pairs may be selected using one or several online tools, including FRbase FRET Calculator (https://www.fpbase.org/fret/).
  • the donor fluorophore is mClover3.
  • the acceptor fluorophore is Preferably, the donor fluorophore is mClover3 and the acceptor fluorophore is mRuby3.
  • increased donor fluorescence intensity e.g., compared to a negative control comprising no clostridial neurotoxin
  • a donor fluorophore may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 45, wherein the donor fluorophore has a peak excitation wavelength within the range of from 499 to 510 nm, preferably about 506 nm and/or a peak emission wavelength within the range of from 512 nm to 522 nm, preferably 518 nm.
  • a donor fluorophore may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 45.
  • a donor fluorophore may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 45. In some embodiments, a donor fluorophore may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 45, wherein the donor fluorophore has a peak excitation wavelength within the range of from 499 to 510 nm, preferably about 506 nm and/or a peak emission wavelength within the range of from 512 nm to 522 nm, preferably 518 nm.
  • a donor fluorophore may comprise a polypeptide sequence having least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 46, wherein the acceptor fluorophore has a peak excitation wavelength within the range of from 522 to 564 nm, preferably about 558 nm and/or a peak emission wavelength within the range of from 579 nm to 609 nm, preferably 592 nm.
  • An acceptor fluorophore may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 46.
  • an acceptor fluorophore may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 46.
  • a donor fluorophore may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 46, wherein the acceptor fluorophore has a peak excitation wavelength within the range of from 522 to 564 nm, preferably about 558 nm and/or a peak emission wavelength within the range of from 579 nm to 609 nm, preferably 592 nm.
  • the binding substrate may further comprise additional elements such as one or more purification tags.
  • the purification tag may be selected from an epitope tag selected from the list comprising: his, FLAG, HA, V5, Myc, and Strep.
  • the purification tag may be selected from a protein/domain tag selected from the list comprising GST, MBP, SUMO, CBP, Halo, Mus A and FATT.
  • the binding substrate may further comprise additional elements such as one or more spacer sequence.
  • a spacer sequence may be a sequence positioned between the donor fluorophore and the binding region.
  • a spacer sequence may be positioned between the acceptor fluorophore and the binding region.
  • the binding substrate comprises a first spacer sequence positioned between the donor fluorophore and the binding region and a second spacer sequence positioned between the acceptor fluorophore and the binding region.
  • the spacers may have the same or different polypeptide sequences. It is well-within the skilled person’s capability to select an appropriate spacer sequence and size.
  • a spacer may be of any suitable length, such as 3-20, 2-15, 5-15 or 4-8 amino acids in length.
  • a spacer may comprise (or consist of) glycine and serine residues.
  • the binding region may comprise (or consist of) one or more sequence that binds specifically to a clostridial neurotoxin polypeptide.
  • the binding region may comprise (or consist of) 1, 2, 3, 4, 5 or more sequences that bind specifically to a clostridial neurotoxin polypeptide.
  • the sequences may be or different.
  • the sequences are different.
  • the binding region may comprise (or consist of) a multimer of sequences that bind to a single clostridial neurotoxin polypeptide.
  • the binding region comprises a sequence comprising the CDR sequences of 2, 3 or 4 antibodies, the antibodies may bind to different epitopes on the same clostridial neurotoxin polypeptide.
  • binding region comprising more than one sequence that binds specifically to a single clostridial neurotoxin polypeptide advantageously may increase the binding affinity of the interaction (e.g., as measured by K D ).
  • the clostridial neurotoxin polypeptide may comprise (or consist of) a sequence selected from any one of BoNT/A-G, BoNT/X or TeNT.
  • the clostridial neurotoxin polypeptides are described herein and may be selected from the group comprising wild-type polypeptides, modified or chimeric polypeptide and fragments thereof.
  • the binding region may bind specifically to a polypeptide comprising a sequence having at least 70% sequence identity to any one of SEQ ID NO: 1 to 18.
  • the binding region may bind specifically to a polypeptide comprising a sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to any one of SEQ ID NO: 1 to 18.
  • the binding region may bind specifically a polypeptide comprising any one of SEQ ID NO: 1 to 18.
  • the binding region may bind specifically to a polypeptide consisting of a sequence having at least 70% sequence identity to any one of SEQ ID NO: 1 to 18.
  • the binding region may bind specifically a polypeptide consisting of a sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to any one of SEQ ID NO: 1 to 18.
  • the binding region may bind specifically to a polypeptide consisting of any one of SEQ ID NO: 1 to 18.
  • the binding region may bind specifically to a polypeptide comprising a sequence having at least 70% sequence identity to SEQ ID NO: 1.
  • the binding region may bind specifically to a polypeptide comprising a sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 1.
  • the binding region may bind specifically a polypeptide comprising SEQ ID NO: 1.
  • the binding region may bind specifically to a polypeptide consisting of a sequence having at least 70% sequence identity to SEQ ID NO: 1.
  • the binding region may bind specifically to a polypeptide consisting of a sequence having at least 70% sequence identity to SEQ ID NO: 13.
  • the binding region may bind specifically a polypeptide consisting of a sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 13.
  • the binding region may bind specifically to a polypeptide consisting of SEQ ID NO: 13.
  • the binding region may comprise (or consist of) one or more sequence that binds specifically to a clostridial neurotoxin light chain (L)(e.g., as defined herein).
  • the binding region may comprise (or consist of) any sequence that binds specifically to a clostridial neurotoxin heavy chain (H) (e.g., as defined herein). In some embodiments, the binding region may comprise (or consist of) any sequence that binds specifically to a clostridial neurotoxin translocation domain (H N ) (e.g., as defined herein). In some embodiments, the binding region may comprise (or consist of) any sequence that binds specifically to a clostridial neurotoxin H CN domain (e.g., as defined herein).
  • the binding region may comprise (or consist of) any sequence that binds specifically to a BoNT/B translocation domain (H N ) (e.g., as defined herein). In some embodiments, the binding region may comprise (or consist of) any sequence that binds specifically to a BoNT/B H CN domain (e.g., as defined herein). In some embodiments, the binding region may comprise (or consist of) any sequence that binds specifically to a BoNT/B H CC domain (e.g., as defined herein). In some embodiments, the binding region comprises one or more sequence(s) that bind specifically to a chimeric clostridial neurotoxin polypeptide (e.g., mrBoNT/AB).
  • H N BoNT/B translocation domain
  • the binding region may comprise (or consist of) any sequence that binds specifically to a BoNT/B H CN domain (e.g., as defined herein). In some embodiments, the binding region may comprise (or consist of) any sequence that
  • IgNARs have a homodimeric structure comprising two heavy chain polypeptides, each comprising a single variable domain (VNAR) and five constant domains.
  • the binding region comprises (or consists of) an VNAR from cartilaginous fish (e.g., a shark IgNAR).
  • VHHs and VNARs derived from camelid and cartilaginous fish, respectively, are also known as nanobodies or nanoantibodies (Nbs).
  • the binding region comprises a sequence having at least 70% sequence identity to the full-length sequence of one or more 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.
  • the binding region comprises a sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to the full- length sequence of one or more 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.
  • a single-chain antibody of the invention comprises the three CDR sequences (e.g., CDR1, CDR2 and CDR3) and has at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 22; optionally wherein the antibody retains the binding specificity as shown in Table 1.
  • a single-chain antibody of the invention comprises SEQ ID NO: 22.
  • a single-chain antibody of the invention comprises the three CDR sequences (e.g., CDR1, CDR2 and CDR3) and has at least 70% sequence identity to SEQ ID NO: 23; optionally wherein the antibody retains the binding specificity as shown in Table 1.
  • a single-chain antibody of the invention comprises the three CDR sequences (e.g., CDR1, CDR2 and CDR3) and has at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 24; optionally wherein the antibody retains the binding specificity as shown in Table 1.
  • a single-chain antibody of the invention comprises SEQ ID NO: 24.
  • a single-chain antibody of the invention comprises the three CDR sequences (e.g., CDR1, CDR2 and CDR3) and has at least 70% sequence identity to SEQ ID NO: 25; optionally wherein the antibody the binding specificity as shown in Table 1.
  • a single-chain antibody of the invention comprises the three CDR sequences (e.g., CDR1, CDR2 and CDR3) and has at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 26; optionally wherein the antibody retains the binding specificity as shown in Table 1.
  • a single-chain antibody of the invention comprises SEQ ID NO: 26.
  • the antibodies e.g., a VHH and VNAR derived from camelid or cartilaginous fish
  • Suitable neutralising antibodies are known in the art.
  • a BoNT/B H N domain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 441-858 of SEQ ID NO: 2.
  • a BoNT/C1 H N domain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 442-866 of SEQ ID NO: 3.
  • a BoNT/D H N domain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 446-862 of SEQ ID NO: 4.
  • a BoNT/E H N domain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 423-845 of SEQ ID NO: 5.
  • a BoNT/F H N domain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 440-864 of SEQ ID NO: 6.
  • a BoNT/G H N domain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 442-863 of SEQ ID NO: 7.
  • H N can include clostridial neurotoxin H N regions comprising a translocation domain having a length of, for example, at most 350 amino acids, at most 375 amino acids, at most 400 amino acids and at most 425 amino acids.
  • H N embraces naturally-occurring neurotoxin H N portions, and modified H N portions having amino acid sequences that do not occur in nature and/ or synthetic amino acid residues. In one embodiment said modified H N portions still demonstrate the above-mentioned translocation function.
  • H C clostridial neurotoxin receptor binding domain reference sequences
  • BoNT/X the H C has been reported as corresponding to amino acids 893-1306 thereof, with the domain boundary potentially varying by approximately 25 amino acids (e.g.868-1306 or 918-1306).
  • a BoNT/A H C domain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 872-1296 of SEQ ID NO: 1.
  • a BoNT/B H C domain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 859-1291 of SEQ ID NO: 2.
  • a BoNT/C1 H C domain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 867-1291 of SEQ ID NO: 3.
  • a BoNT/D H C domain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 863-1276 of SEQ ID NO: 4.
  • a BoNT/E H C domain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 846-1252 of SEQ ID NO: 5.
  • a BoNT/F H C domain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 865-1274 of SEQ ID NO: 6.
  • a BoNT/G H C domain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 864-1297 of SEQ ID NO: 7.
  • a BoNT/X H C domain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 893-1306 of SEQ ID NO: 8.
  • a TeNT H C domain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 880-1315 of SEQ ID NO: 9.
  • a clostridial neurotoxin H-chain e.g.
  • the H C domain portion may further comprise a translocation facilitating domain (or a fragment thereof may be translocation facilitating domain fragment).
  • Said domain facilitates delivery of the L-chain into the cytosol of the target cell and are described, for example, in WO 08/008803 and WO 08/008805, each of which is herein incorporated by reference thereto.
  • a translocation facilitating domain may comprise a clostridial neurotoxin H CN domain or a fragment or variant thereof.
  • a clostridial neurotoxin H CN translocation facilitating domain may have a length of at least 200 amino acids, at least 225 amino acids, at least 250 amino acids, at least 275 amino acids.
  • a non-clostridial facilitating domain may be combined with a non- clostridial translocation domain peptide or with clostridial translocation domain peptide.
  • a clostridial neurotoxin H CN translocation facilitating domain may be combined with a non-clostridial translocation domain peptide.
  • a clostridial heavy-chain lacking a functional heavy chain H C peptide (or domain) such that the heavy-chain is incapable of binding to cell surface receptors to which a native clostridial neurotoxin binds means that the clostridial heavy-chain simply lacks a functional H CC peptide.
  • the H CC peptide region may be either partially or wholly deleted, or otherwise modified (e.g. through conventional chemical or proteolytic treatment) to reduce its native binding ability for nerve terminals at the neuromuscular junction.
  • a BoNT/C1 H CC domain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 1112-1291 of SEQ ID NO: 3.
  • a BoNT/D H CC domain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 1099-1276 of SEQ ID NO: 4.
  • a BoNT/E H CC domain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 1086-1252 of SEQ ID NO: 5.
  • a BoNT/F H CC domain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 1106-1274 of SEQ ID NO: 6.
  • a BoNT/G H CC domain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 1106-1297 of SEQ ID NO: 7.
  • a TeNT H CC domain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 1128-1315 of SEQ ID NO: 9.
  • clostridial neurotoxin is also intended to embrace modified clostridial neurotoxins and derivatives thereof, including but not limited to those described below.
  • a modified clostridial neurotoxin or derivative may contain one or more amino acids that has been modified as compared to the native (unmodified) form of the clostridial neurotoxin, or may contain one or more inserted amino acids that are not present in the native (unmodified) form of the clostridial neurotoxin.
  • a modified clostridial neurotoxin may have modified amino acid sequences in one or more domains relative to the native (unmodified) clostridial neurotoxin sequence. Such modifications may modify functional aspects of the toxin, for example biological activity or persistence.
  • the clostridial neurotoxin of the invention is a modified clostridial neurotoxin, or a modified clostridial neurotoxin derivative, or a clostridial neurotoxin derivative.
  • a modified clostridial neurotoxin may have one or more modifications in the amino acid sequence of the heavy chain (such as a modified H C domain), wherein said modified heavy chain binds to target nerve cells with a higher or lower affinity than the native (unmodified) clostridial neurotoxin.
  • said modified clostridial neurotoxin comprises one or more modifications that increases the isoelectric point of the clostridial neurotoxin when compared to an equivalent unmodified clostridial neurotoxin lacking said one or more modifications.
  • 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 NOs: 10-13 (preferably SEQ ID NO: 10 – mrBoNT/A) described herein.
  • a modified BoNT/A may be one that comprises a modification at one or more amino acid residue(s) selected from: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, 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.
  • Such a modified BoNT/A may demonstrate a reduction in, or absence of, side effects compared to the use of known BoNT/A.
  • Said modified BoNT/A may exhibit increased tissue retention properties, thereby providing increased potency and/or duration of action and can allow for reduced dosages to be used compared to known clostridial toxin therapeutics (or increased dosages without any additional adverse effects), thus providing further advantages.
  • the modification may be a modification when compared to a BoNT/A shown as SEQ ID NO: 1, wherein the amino acid residue numbering is determined by alignment with SEQ ID NO: 1.
  • SEQ ID NO: 1 includes a methionine
  • the position numbering will be as defined above (e.g. ASN 886 will be ASN 886 of SEQ ID NO: 1).
  • the amino acid residue numbering should be modified by -1 (e.g. ASN 886 will be ASN 885 of SEQ ID NO: 1).
  • a modified BoNT/A may comprise a modification at one or more amino acid residue(s) selected from: ASN 886, ASN 930, ASN 954, SER 955, GLN 991, ASN 1025, ASN 1026, ASN 1052, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274 and THR 1277.
  • the term “one or more amino acid residue(s)” when used in the context of a modified BoNT/A preferably means at least 2, 3, 4, 5, 6 or 7 of the indicated amino acid residue(s).
  • a modified BoNT/A may comprise at least 2, 3, 4, 5, 6 or 7 (preferably 7) modifications at the indicated amino acid residue(s).
  • an amino acid residue that forms part of the BoNT/A polypeptide sequence is replaced with a different amino acid residue.
  • the replacement amino acid residue may be one of the 20 standard amino acids, as described above.
  • the replacement amino acid in an amino acid substitution may be a non-standard amino acid (an amino acid that is not part of the standard set of 20 described above).
  • the replacement amino acid may be a basic non-standard amino acid, e.g. L-Ornithine, L-2-amino-3-guanidinopropionic acid, or D-isomers of Lysine, Arginine and Ornithine).
  • the C-terminal amino acid residue of the LH N domain may correspond to the first amino acid residue of the 3 10 helix separating the LH N and H C domains of BoNT/A
  • the N-terminal amino acid residue of the H C domain may correspond to the second amino acid residue of the 3 10 helix separating the LH N and H C domains in BoNT/B.
  • Reference herein to the “first amino acid residue of the 3 10 helix separating the LH N and H C domains of BoNT/A” means the N-terminal residue of the 3 10 helix separating the LH N and H C domains.
  • a BoNT/AB chimera may comprise an LH N domain from BoNT/A covalently linked to a H C domain from BoNT/B, • wherein the C-terminal amino acid of the LH N domain corresponds to the C- terminal amino acid residue of the ⁇ -helix located at the end (C-term) of LH N domain of BoNT/A, and • wherein the N-terminal amino acid residue of the H C domain corresponds to the amino acid residue immediately C-terminal to the C-terminal amino acid residue of the ⁇ -helix located at the end (C-term) of LH N domain of BoNT/B.
  • the LH N domain from BoNT/A 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 thereto.
  • the LH N domain from BoNT/A corresponds to amino acid residues 1 to 872 of SEQ ID NO: 1.
  • the H C domain from BoNT/B may correspond to amino acid residues 860 to 1291 of SEQ ID NO: 2, or a polypeptide sequence having at least 70% sequence identity thereto.
  • the H C domain from BoNT/B 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 thereto.
  • the H C domain from BoNT/B corresponds to amino acid residues 860 to 1291 of SEQ ID NO: 2.
  • the LH N domain corresponds to amino acid residues 1 to 872 of BoNT/A (SEQ ID NO: 1) and the H C domain corresponds to amino acid residues 860 to 1291 of BoNT/B (SEQ ID NO: 1).
  • a BoNT/B H C domain further comprises at least one amino acid residue substitution, addition or deletion in the H CC domain (e.g. subdomain) which has the effect of increasing the binding affinity of BoNT/B neurotoxin for human Syt II as compared to the natural BoNT/B sequence.
  • Suitable amino acid residue substitution, addition or deletion in the BoNT/B H CC domain have been disclosed in WO 2013/180799 and in WO 2016/154534 (both herein incorporated by reference).
  • Suitable amino acid residue substitution, addition or deletion in the BoNT/B H CC 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.
  • Suitable amino acid residue substitution, addition or deletion in the BoNT/B H CC 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.
  • Suitable amino acid residue substitution, addition or deletion in the BoNT/B H CC domain also include a combination of three substitution mutations which are E1191M, S1199W and W1178Q.
  • the suitable amino acid residue substitution, addition or deletion in the BoNT/B H CC domain includes a combination of two substitution mutations which are E1191M and S1199Y.
  • the modification may be a modification when compared to unmodified BoNT/B shown as SEQ ID NO: 2, wherein the amino acid residue numbering is determined by alignment with SEQ ID NO: 2.
  • SEQ ID NO: 2 As the presence of a methionine residue at position 1 of SEQ ID NO: 2 is optional, the skilled person will take the presence/absence of the methionine residue into account when determining amino acid residue numbering.
  • SEQ ID NO: 2 includes a methionine
  • the position numbering will be as defined above (e.g. E1191 will be E1191 of SEQ ID NO: 2).
  • the methionine is absent from SEQ ID NO: 2 the amino acid residue numbering should be modified by -1 (e.g. E1191 will be E1190 of SEQ ID NO: 2).
  • E1191 will be E1190 of SEQ ID NO: 2
  • a chimeric clostridial neurotoxin may comprise a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 14-18.
  • a chimeric clostridial neurotoxin may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to any one of NOs: 14-18.
  • a chimeric clostridial neurotoxin may comprise any one of SEQ ID NOs: 14-18.
  • a chimeric clostridial neurotoxin may consist of a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 14-18.
  • a chimeric clostridial neurotoxin may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to any one of SEQ ID NOs: 14-18.
  • a chimeric clostridial neurotoxin may consist of any one of SEQ ID NOs: 14-18. Of the recited SEQ ID NOs, SEQ ID NO: 14 is most preferred.
  • a composition of the invention most preferably comprises a chimeric clostridial neurotoxin, such as a chimeric clostridial neurotoxin as described above.
  • non-BoNT/X clostridial neurotoxin.
  • a suitable chimeric and/or hybrid clostridial neurotoxin may be one taught in WO 2020/065336 A1, which is incorporated herein by reference.
  • a clostridial neurotoxin described herein has a tag for purification (e.g. a His-tag) and/or a linker, said tag and/or linker are optional.
  • the clostridial neurotoxins of the present invention may be free from the complexing proteins that are present in a naturally occurring clostridial neurotoxin complex.
  • the clostridial neurotoxins of the present invention can be produced using recombinant nucleic acid technologies.
  • a clostridial neurotoxin (as described above) is a recombinant clostridial neurotoxin.
  • a nucleic acid (for a DNA) comprising a nucleic acid sequence encoding a clostridial neurotoxin is provided.
  • 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 of the nucleic acid sequences described herein.
  • the vector has a promoter selected from: Promoter Induction Agent Typical Induction Condition Tac (hybrid) IPTG 0.2 mM (0.05-2.0mM) AraBAD L-arabinose 0.2% (0.002-0.4%) T7-lac operator IPTG 0.2 mM (0.05-2.0mM)
  • the vector has a promoter selected from: Promoter Induction Agent Typical Induction Condition Tac (hybrid) IPTG 0.2 mM (0.05-2.0mM) AraBAD L-arabinose 0.2% (0.002-0.4%) T7-lac operator IPTG 0.2 mM (0.05-2.0mM) T5-lac operator IPTG 0.2 mM (0.05-2.0mM)
  • the nucleic acid molecules may be made using any suitable process known in the art.
  • the nucleic acid molecules may be made using chemical synthesis techniques.
  • the nucleic acid molecules of the invention may be made using molecular biology techniques.
  • the DNA construct of the present invention is preferably designed in silico, and then synthesised by conventional DNA synthesis techniques.
  • the above-mentioned nucleic acid sequence information is optionally modified for codon- biasing according to the ultimate host cell (e.g. E. coli) expression system that is to be employed.
  • the terms “nucleotide sequence” and “nucleic acid” are used synonymously herein.
  • the nucleotide sequence is a DNA sequence.
  • a clostridial neurotoxin of the invention is preferably present as a di-chain clostridial neurotoxin in which the L-chain is linked to the H-chain (or component thereof, e.g. the H N domain) via a di-sulphide bond.
  • a clostridial of the invention may be any clostridial neurotoxin or variant (expressed by way of % sequence identity to a given SEQ ID NO) herein that has been cleaved by a protease in its activation loop (at one or more sites).
  • a clostridial neurotoxin preferably comprises an L-chain and H-chain, wherein the L-chain and H-chain are joined by a di-sulphide bond and are obtainable by (e.g. obtained by) cleaving a polypeptide comprising at least 70% sequence identity to SEQ ID NO: 10 with a protease in its activation loop at one more sites.
  • a clostridial neurotoxin comprises an L-chain and H-chain, wherein the L-chain and H-chain are joined by a di-sulphide bond and are obtainable by (e.g.
  • a clostridial neurotoxin comprises an L-chain and H-chain, wherein the L-chain and H-chain are joined by a di-sulphide bond and are obtainable by (e.g. obtained by) cleaving a polypeptide comprising SEQ ID NO: 10 with a protease in its activation loop at one more sites.
  • a clostridial neurotoxin most preferably comprises an L-chain and H-chain, wherein the L-chain and H-chain are joined by a di-sulphide bond and are obtainable by (e.g. obtained by) cleaving a polypeptide comprising at least 70% sequence identity to SEQ ID NO: 14 with a protease in its activation loop at one more sites.
  • a clostridial neurotoxin comprises an L-chain and H-chain, wherein the L-chain and H-chain are joined by a di-sulphide bond and are obtainable by (e.g.
  • a clostridial neurotoxin comprises an L-chain and H-chain, wherein the L-chain and H-chain are joined by a di-sulphide bond and are obtainable by (e.g. obtained by) cleaving a polypeptide comprising SEQ ID NO: 14 with a protease in its activation loop at one more sites.
  • the protease used to cleave the activation loop is preferably Lys-C.
  • Suitable proteases and method for cleaving activation loops to produce di-chain clostridial neurotoxins are taught in WO 2014/080206, WO2014/079495, and EP2677029A2, which are incorporated herein by reference.
  • Suitable activation loop sequences are shown in the table below: Protein id aa aa s tart end BoNT/D_AB012112 437 450 BoNT/DC_AB745660 437 450 BoNT/C1_X62389 437 453 BoNT/CD_AB200360 437 453 BoNT/A4_EU341307 430 454 BoNT/A7_JQ954969 454 BoNT/A6_FJ981696 454 BoNT/A1_AF488749 454 BoNT/A5_EU679004 430 454 BoNT/A3_DQ185900 426 450 BoNT/A2_X73423 454 BoNT/A8_KM233166 430 454 BoNT/H
  • the invention provides a method of producing a single-chain clostridial neurotoxin having a light chain and a heavy chain, the method comprising expressing a nucleic acid described herein in an expression host, lysing the host cell to provide a host cell homogenate containing the single-chain clostridial neurotoxin, and isolating the single-chain clostridial neurotoxin.
  • the present invention provides a method of proteolytically processing a clostridial neurotoxin described herein, the method comprising contacting the clostridial neurotoxin with a protease that hydrolyses a peptide bond in the activation loop of the clostridial neurotoxin, thereby converting the (single-chain) clostridial neurotoxin into a corresponding di-chain clostridial neurotoxin (e.g. wherein the light chain and heavy chain are joined together by a disulphide bond).
  • the present invention therefore provides a di-chain clostridial neurotoxin obtainable by a method of the invention.
  • a therapeutic or cosmetic clostridial neurotoxin composition obtainable by a method of the invention, optionally wherein the therapeutic or cosmetic clostridial neurotoxin composition is packaged.
  • the term “obtainable” as used herein also the term “obtained”.
  • the invention provides an isolated binding substrate of the present invention.
  • the isolated binding substrate is bound to a clostridial neurotoxin polypeptide.
  • the invention provides an isolated complex comprising a clostridial neurotoxin bound to a binding substrate.
  • the binding substrate is immobilised to a support (e.g., a surface for performing the analysis of binding events and kinetics).
  • amino acids are indicated by the standard one-letter codes; preferably this method is used to align a sequence with a SEQ ID NO described herein to define amino acid position numbering, as described herein.
  • the "percent sequence identity" between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by the sequences. Thus, % identity may be calculated as the number of identical nucleotides / amino acids divided by the total number of nucleotides / amino acids, multiplied by 100.
  • a limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids may be substituted for polypeptide amino acid residues.
  • the polypeptides of the present invention can also comprise non-naturally occurring amino acid residues.
  • an in vitro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs.
  • Methods for synthesizing amino acids and aminoacylating tRNA are known in the art. Transcription and translation of plasmids containing nonsense mutations is carried out in a cell free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson et al., J. Am. Chem.
  • coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine).
  • a natural amino acid that is to be replaced e.g., phenylalanine
  • the desired non-naturally occurring amino acid(s) e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine.
  • the non-naturally occurring amino acid is incorporated into the polypeptide in place of its natural counterpart. See, Koide et al., Biochem.33:7470-6, 1994.
  • Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification.
  • Sites of biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labelling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett.309:59-64, 1992.
  • the identities of essential amino acids can also be inferred from analysis of homologies with related components (e.g. the translocation or protease components) of the polypeptides of the present invention.
  • any nucleic acid sequences are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.
  • the headings provided herein are not limitations of the various aspects or embodiments of this disclosure.
  • Amino acids are referred to herein using the name of the amino acid, the three letter abbreviation or the single letter abbreviation.
  • the term “protein”, as used herein, includes proteins, polypeptides, and peptides.
  • amino acid sequence is synonymous with the term “polypeptide” and/or the term “protein”. In some instances, the term “amino acid sequence” is synonymous with the term “peptide”.
  • a clostridial neurotoxin includes a plurality of such candidate agents and reference to “the clostridial neurotoxin” includes reference to one or more clostridial neurotoxins and equivalents thereof known to those skilled in the art, and so forth.
  • the publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. None herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto. SEQUENCE LISTING Where an initial Met amino acid residue or a corresponding initial codon is indicated in any of the following SEQ ID NOs, said residue/codon is optional.
  • Figure 1 shows an exemplary expression construct.
  • Figure 2 shows that FRET occurs in the absence of binding of BoNT/A to an exemplary binding substrate.
  • Figure 3 shows the expression of binding substrates comprising camelid antibodies.
  • Figure 4 is a schematic showing the immobilisation of proteins to an AR2G support.
  • Figure 5 shows the binding kinetics for glycosylated SV2c.
  • Figure 6 shows the binding kinetics for anti-BoNT/A (rabbit polyclonal antibody).
  • Figure 7 shows the binding kinetics for mClover3-ciA-C2-mRuby3.
  • Figure 8 shows the binding kinetics for mClover3-ciA-D12-ciA-B5-mRuby3.
  • Figure 9 shows the binding kinetics for mClover3-ciA-H7-mRuby3.
  • Figure 10 shows the binding kinetics for mClover3-SV2C 529-566-mRuby3.
  • Figure 11 shows the binding kinetics for mClover3-SV2C 454-580-mRuby3.
  • Figure 12 shows the binding kinetics for mClover3-SV2C 566-580-mRuby3.
  • Example 1 SV2 linker variants library for BoNT/A detection Materials & Methods Expression constructs encoding a binding substrate comprising a donor fluorophore (mClover3), a binding region and an acceptor fluorophore (mRuby3) were designed and cloned into pRSETa plasmids using Gibson Assembly (see, Fig. 1). The expression constructs comprise a binding region were selected from the following table.
  • Table 2 SV2 binding region library Residues included (relative to full length SV2c, Design SEQ ID NO: 21) Length (AA) 1 454-580 126 2 454-519 65 3 454-529 75 4 454-566 112 5 519-566 47 6 519-580 61 7 519-529 10 0 (Original) 529-566 37 9 529-580 51 10 566-580 14
  • the resulting plasmids were used to transform DH5 ⁇ cells. Colonies were picked and the presence of the plasmid confirmed by PCR and sequencing. Expression of the expression constructs was performed in BL21 cells and the resulting binding substrates were isolated.
  • Design 0 (i.e., comprising amino acids 529-566 of full length SV2c, SEQ ID NO: 21) was selected for further analysis.
  • the mClover3-SV2c-mRuby3 binding substrate was assessed to determine whether fluorescent energy transfer occurs 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, Fig.2). were selected based on their ability to bind to BoNT/A.
  • 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 1ng/ ml, preferably less than 0.1ng/ ml.
  • a method for real-time monitoring of clostridial neurotoxin production during a bacterial fermentation process comprising: a. culturing a bacterial host cell capable of producing a clostridial neurotoxin in a liquid medium; b. contacting said liquid medium with a biosensor according to any one or more of the preceding aspects; c. exciting said donor fluorophore; and d.
  • a method for real-time monitoring of clostridial neurotoxin production during a bacterial fermentation process comprising: a.
  • step d comprises detecting donor fluorescence intensity at said biosensor, wherein increased donor fluorescence intensity of at said biosensor as compared to said control is indicative of the presence of clostridial neurotoxin.
  • step d comprises detecting acceptor fluorescence intensity at said biosensor, wherein decreased acceptor fluorescence intensity at said biosensor as compared to said control is indicative of the presence of clostridial neurotoxin. 19. The method of any one of aspects 12 to 16, wherein step d comprises detecting an acceptor emission maximum and a donor fluorophore emission maximum at said biosensor, wherein a shift in emission maxima from near said acceptor emission maximum to near said donor fluorophore emission maximum is indicative of the presence of clostridial neurotoxin. 20.
  • step d comprises detecting the ratio of fluorescence amplitudes near an acceptor emission maximum to the fluorescence amplitudes near a donor fluorophore emission maximum, wherein a decreased ratio at said biosensor as compared to the control is indicative of the presence of clostridial neurotoxin.

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Abstract

La présente invention concerne un biocapteur pour le suivi en temps réel de la production de neurotoxine clostridiale pendant un processus de fermentation bactérienne. Le biocapteur comprend une surface pour effectuer l'analyse d'événements de liaison et de cinétique, et sur laquelle est immobilisé un substrat de liaison comprenant : un fluorophore donneur ; un accepteur présentant un spectre d'absorbance chevauchant le spectre d'émission du fluorophore donneur ; et une région de liaison qui se lie plus particulièrement à la neurotoxine clostridiale, ladite région de liaison étant positionnée entre le fluorophore donneur et l'accepteur de sorte que, à la suite de l'activation du fluorophore donneur, un transfert d'énergie de résonance est présenté entre ledit fluorophore donneur et ledit accepteur.
PCT/GB2024/053207 2023-12-28 2024-12-27 Biocapteur pour le suivi en temps réel de la production de neurotoxine clostridiale Pending WO2025141289A1 (fr)

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