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  • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
  • C12Y304/24—Metalloendopeptidases (3.4.24)
  • C12Y304/24069—Bontoxilysin (3.4.24.69), i.e. botulinum neurotoxin
• • A—HUMAN NECESSITIES
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  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
  • A61K2039/6031—Proteins
  • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
  • A61K2039/6093—Synthetic polymers, e.g. polyethyleneglycol [PEG], Polymers or copolymers of (D) glutamate and (D) lysine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • C12Y—ENZYMES
  • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
  • C12Y304/24—Metalloendopeptidases (3.4.24)
  • C12Y304/24068—Tentoxilysin (3.4.24.68), i.e. tetanus neurotoxin
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• TeNT neurotoxin conjugated to polyethylene glycol (PEG) and a second TeNT.
• PEG polyethylene glycol
• the invention also relates to a method of treating hypotonia using the composition.
• Tetanus neurotoxin is produced by Clostridium tetani . TeNT acts at the spinal cord and blocks release at the spinal inhibitory interneurons of g-aminobutyric acid (GABA) and glycine, which are inhibitory neurotransmitters. As such, TeNT causes spastic paralysis. TeNT does not occur in multiple serotypes.
• US 2002/0197278 A1 discloses use of PEGylated botulinum toxins for treating disorders of inappropriate muscle contraction.
• WO 2016/001762 Al discloses use of a PEGylated TeNT fragment c (c) for increasing muscle mass.
• Fragment c 50 kDa is generated when TeNT is enzymatically cleaved by papain and corresponds to the 451 amino acids at the C-terminus of the TeNT heavy chain. Fragment c retains the binding, internalization and trans-synaptic transport capabilities of undigested TeNT, but does not disrupt any neuronal processes, and is therefore nontoxic.
• the inventors have produced a family of modified TeNTs and a treatment regimen that address these problems.
• the invention provides a family of PEGylated TeNTs (PEG-TeNTs) that each evade the immune system, and a tiered treatment regimen in which a different, replacement PEG-TeNT is used for treatment when efficacy of a previously administered PEG-TeNT decreases or when a patient' s immunological profile precludes the use of another PEG-TeNT.
• PEG-TeNTs PEGylated TeNTs
• a first aspect provides a tetanus neurotoxin (TeNT) or ragment thereof, comprising one or more surface serine to cysteine amino acid substitutions relative ID NO: 1.
• PEG polyethylene glycol
• a second aspe t provides a TeNT or fragment thereof
• a third aspect provides a composition comprising: (i) a first PEGylated tetanus neurotoxin (PEG-TeNT) comprising tetanus
• PEG-TeNT PEGylated tetanus neurotoxin
• TeNT neurotoxin conjugated to polyethylene glycol (PEG); and (ii) a second TeNT, wherein the first PEG-TeNT is not conjugated to the second TeNT.
• a fourth aspect provides a method for treating hypotonia, the method comprising administering to a subject: (i) a first PEGylated tetanus neurotoxin (PEG-TeNT) comprising tetanus neurotoxin (TeNT) conjugated to polyethylene glycol (PEG) ; and (ii) a second TeNT.
• PEG-TeNT PEGylated tetanus neurotoxin
• TeNT tetanus neurotoxin conjugated to polyethylene glycol
• the fourth aspec :;t also provides use of a first PEGylated tetanus neurotoxin ( PEG-TeNT) comprising a tetanus neurotoxin (TeNT) con ugated to polyethylene glycol (PEG) in the manufacture of a medicament for treating hypotonia in a subject administered a second PEGylated tetanus neurotoxin (PEG-TeNT) comprising a tetanus neurotoxin (TeNT) con ugated to polyethylene glycol (PEG) in the manufacture of a medicament for treating hypotonia in a subject administered a second PEGylated tetanus neurotoxin (PEG)
• PEG polyethylene glycol
• the fourth aspect also provides use of a second tetanus neurotoxin (TeNT) in the manufacture of a medicament for treating hypotonia in a subject administered a first PEGylated tetanus neurotoxin (PEG-TeNT) comprising a tetanus neurotoxin (TeNT) conjugated to polyethylene glycol (PEG) .
• PEG-TeNT PEGylated tetanus neurotoxin
• TeNT tetanus neurotoxin conjugated to polyethylene glycol
• the fourth aspect also provides a second tetanus neurotoxin (TeNT) for use in a method for treating hypotonia in a subj ect administered a first PEGylated tetanus neurotoxin (PEG—TeNT) comprising a tetanus neurotoxin (TeNT) conjugated to polyethylene glycol (PEG) .
• PEG—TeNT PEGylated tetanus neurotoxin
• TeNT polyethylene glycol
• the fourth aspect also provides: (i) a first PEGylated tetanus neurotoxin (PEG-TeNT) comprising a tetanus neurotoxin (TeNT) conjugated to polyethylene glycol (PEG); and (ii) a second TeNT for use in a method for treating hypotonia.
• a composition may comprise the first PEG-TeNT and the second TeNT.
• the fourth aspect also provides a composition comprising:
• PEG-TeNT PEGylated tetanus neurotoxin
• TeNT tetanus neurotoxin conjugated to polyethylene glycol
• first PEG-TeNT or the second TeNT is PEG-TeNT-HC comprising a PEGylated HC.
• LC is not PEGylated.
• first PEG-TeNT or the second TeNT is PEG-TeNT-LC-c comprising a PEGylated LC and a
• HN is not PEGylated.
• the first PEG-TeNT is PEG-TeNT-HC
• the second TeNT is PEG-TeNT-LC-c.
• the second TeNT comprises an inactivated TeNT.
• the inactivated TeNT may comprise: R1225K; R1225E; W1288A; W1288Y; W1288F; W1288L; R1225K and W1288A; R1225E and W1288A; R1225K and W1288Y; R1225E and W1288Y; R1225K and W1288F; R1225E and W1288F; R1225K and W1288L; R1225E and W1288L; R1225del; W1288del; R1225del or W1288del; E270A; Y374A; E270A and U374 ⁇ ; G270del; Y374del; or a combination thereof.
• the hypotonia is obstructive sleep apnoea.
• the composition is a therapeutic composition. In another embodiment, the composition is a cosmetic composition .
• LC-HN TeNT HN
• a fifth aspect provides a PEGylated tetanus neurotoxin (PEG- TeNT), comprising tetanus neurotoxin (TeNT) conjugated to
• PEG polyethylene glycol
• PEG is conjugated to a lysine residue of TeNT. In another embodiment, PEG is conjugated to a cysteine residue of TeNT. In one embodiment, PEG is conjugated to a cysteine residue of TeNT, wherein the cysteine residue is either native or a substitution for a serine residue relative to SEQ ID NO: 1.
• the PEG has a molecular weight of about 2 kDa, about 5 kDa, about 10 kDa, or about 20 kDa, or about 30 kDa .
• a sixth aspect provides a method for treating hypotonia, the method comprising administering to a subject the PEG-TeNT of the fifth aspect, wherein the PEG-TeNT is not conjugated to a second TeNT .
• the sixth aspect also provides use of the PEG-TeNT of the fifth aspect in the manufacture of a medicament for treating hypotonia, wherein the PEG-TeNT is not conjugated to a second TeNT.
• the sixth aspect provides the PEG-TeNT of the fifth aspect for use in a method for treating hypotonia, the method comprising administering to a subject the PEG-TeNT, wherein the PEG- TeNT is not conjugated to a second TeNT .
• the subject is administered: a PEG- TeNT comprising a PEGylated c (PEG-TeNT-c) ; and/or a first PEG-TeNT comprising a PEGylated HC ( PEG—TeNT-HC) and a second PEG-TeNT comprising a PEGylated LC-c (PEG-TeNT-LC-c) ; and/or a PEG-TeNT comprising a PEGylated HC and a PEGylated LC (PEG-TeNT-LC-HC) .
• treating comprises administering to the subject: a PEG-TeNT comprising PEGylated c (PEG-TeNT-c) until efficacy decreases; then a PEG-TeNT-HC and a PEG-TeNT-LC-c until efficacy decreases; then a PEG-TeNT comprising a PEGylated LC and a PEGylated HC (PEG-TeNT-LC-HC) .
• PEG-TeNT-c PEGylated c
• treating comprises administering to the subject: a PEG-TeNT comprising PEGylated c (PEG-TeNT-c) comprising PEG having a molecular weight of about 5 kDa until efficacy
• treating may comprise
• a PEG-TeNT-HC and a PEG-TeNT-LC-c either or both of which comprise PEG having a molecular weight of 5 kDa, until efficacy decreases
• a PEG-TeNT-HC and a PEG-TeNT- LC-c either or both of which comprise PEG having a molecular weight of 10 kDa, until efficacy decreases
• a PEG-TeNT-HC and a PEG- TeNT-LC-c either or both of which comprise PEG having a molecular weight of 20 kDa, until efficacy decreases.
• treating may comprise administering to the subject: a PEG-TeNT comprising a PEGylated LC and a PEGylated HC ( PEG-TeNT-LC-HC) , either or both of which comprise PEG having a molecular weight of 5 kDa, until efficacy decreases; then a PEG-TeNT comprising a PEGylated LC and a PEGylated HC (PEG-TeNT-LC-HC), either or both of which comprise PEG having a molecular weight of 10 kDa, until efficacy decreases; then a PEG-TeNT comprising a PEGylated LC and a PEGylated HC ( PEG-TeNT- LC-HC) , either or both of which comprise PEG having a molecular weight of 20 kDa.
• PEG-TeNT-LC-HC PEG-TeNT-LC-HC
• treating comprises adminstering to the subject: a PEG-TeNT comprising PEGylated c (PEG-TeNT-c) ; then a composition comprising PEG-TeNT-HC and PEG-TeNT-LC-c; then a PEG- TeNT comprising a PEGylated LC and a PEGylated HC (PEG-TeNT-LC-HC), to determine the immunological profile of anti-TeNT antibodies of the subject and determine the effective composition of PEG-TeNTs based on that profile .
• PEG-TeNT-c PEGylated c
• treating with a TeNT further comprises administering an inactivated TeNT.
• the inactivated TeNT may comprise: R1225K; R1225E; W1228A; W1288Y;
• the hypotonia is obstructive sleep apnoea.
• a seventh aspect provides a kit comprising the TeNT of the first aspect, the composition of the second aspect or the PEG-TeNT of the fifth aspect.
• composition or PEG-TeNT is used according to the method of the third or sixth aspect, respectively.
• Figure 1 is a schematic representation of example PEG-TeNTs of the invention: A PEG-TeNT-c; B PEG-TeNT-HC, C PEG-TeNT-LC-c, D PEG-TeNT-LC-HC, where the TeNT is entire and active, i.e. the TeNT comprises both chains, and PEGylation is present on specific regions, i.e. c, HC, LC-c, or LC-HC, respectively.
• Figure 2 is the amino acid sequence (SEQ ID NO: 1) of mature TeNT comprising 1314 amino acids.
• Figure 3 is a nucleic acid sequence (SEQ ID NO: 2) of the vector pRSET-TeNT encoding TeNT.
• Figure 4 is a map of the vector pRSET-TeNT encoding TeNT .
• TeNT is expressed with an N-terminal His 6 -tag.
• the nucleic acid was inserted within the MCS of the pRSET-A vector and expressed under the control of a T7 promoter.
• Figure 5 is the amino acid sequence (SEQ ID NO: 3) of HC comprising amino acids 457 to 1314 of SEQ ID NO: 1.
• Figure 6 is the amino acid sequence (SEQ ID NO: 4) of c comprising amino acids 864 to 1314 of SEQ ID NO: 1.
• Figure 7 is a nucleic acid sequence (SEQ ID NO: 5) of the vector pRSET-TeNT-c encoding c.
• Figure 8 is a map of the vector pRSET-TeNT-c encoding c. c is expressed with an N-terminal His 6 -tag. The nucleic acid was inserted within the MCS of the pRSET-A vector and expressed under the control of a T7 promoter.
• Figure 9 is the amino acid sequence (SEQ ID NO: 6) of LC-HN comprising amino acids 1 to 863 of SEQ ID NO: 1.
• Figure 10 is a nucleic acid sequence (SEQ ID NO: 7) of the vector pRSET-TeNT-LC-HN encoding LC-HN.
• Figure 12 is an amino acid sequence (SEQ ID NO: 8) of a non functional c comprising amino acid substitutions W1288A and R1225E relative to SEQ ID NO: 1.
• Figure 14 is a map of the vector pRSET-TeNT-c encoding the non-functional c of Figure 12 (SEQ ID NO: 8) comprising amino acid substitutions W1288A and R1225E relative to SEQ ID NO: 1.
• c comprising amino acid substitutions W1288A and R1225E relative to SEQ ID NO: 1, is expressed with an N-terminal His 6 -tag.
• the nucleic acid was inserted within the MCS of the pRSET-A vector and expressed under the control of a T7 promoter.
• Figure 15 is an amino acid sequence (SEQ ID NO: 10) of a non functional TeNT comprising amino acid substitutions W1288A and R1225E relative to SEQ ID NO: 1.
• Figure 16 is a nucleic acid sequence (SEQ ID NO: 11) of the vector pRSET-TeNT encoding the non-functional TeNT of Figure 15 (SEQ ID NO: 10) comprising amino acid substitutions W1288A and R1225E relative to SEQ ID NO: 1.
• Figure 17 is a map of the vector pRSET-TeNT encoding the non functional TeNT of Figure 15 (SEQ ID NO: 10) comprising amino acid substitutions W1288A and R1225E relative to SEQ ID NO: 1.
• TeNT comprising amino acid substitutions W1288A and R1225E relative to SEQ ID NO: 1, is expressed with an N-terminal His 6 -tag.
• the nucleic acid was inserted within the MCS of the pRSET-A vector and expressed under the control of a T7 promoter.
• Figure 19 is a nucleic acid sequence (SEQ ID NO: 13) of the vector pRSET-TeNT encoding the surface serine to cysteine
• Figure 20 is a map of the vector pRSET-TeNT of Figure 19 (SEQ ID NO: 13) encoding the surface serine to cysteine substituted mature TeNT of Figure 18 (SEQ ID NO: 12) .
• the surface serine to cysteine substituted TeNT is expressed with an N-terminal His 6 -tag.
• the nucleic acid was inserted within the MCS of the pRSET-A vector and expressed under the control of a T7 promoter.
• Figure 21 is the amino acid sequence (SEQ ID NO: 14) of a mature 1314 amino acid TeNT comprising surface serine to cysteine amino acid substitutions S81C, S120C, S144C, S248C, S335C, S428C, S963C, S1041C, S1155C, and S1187C, relative to SEQ ID NO: 1, in the LC and c regions .
• Figure 22 is a nucleic acid sequence (SEQ ID NO: 15) of the vector pRSET-TeNT encoding the surface serine to cysteine
• Figure 23 is a map of the vector pRSET-TeNT of Figure 22 (SEQ ID NO: 15) encoding the surface serine to cysteine substituted mature TeNT of Figure 21 (SEQ ID NO: 14) .
• the surface serine to cysteine substituted TeNT is expressed with an N-terminal His 6 -tag.
• the nucleic acid was inserted within the MCS of the pRSET-A vector and expressed under the control of a T7 promoter.
• Figure 24 is the amino acid sequence (SEQ ID NO: 16) of a TeNT comprising HC surface serine to cysteine amino acid
• Figure 25 is a nucleic acid sequence (SEQ ID NO: 17) of the vector pRSET-TeNT encoding the surface serine to cysteine
• Figure 26 is a map of the vector pRSET-TeNT of Figure 25 (SEQ ID NO: 17) encoding the surface serine to cysteine substituted TeNT of Figure 24 (SEQ ID NO: 16) .
• the surface serine to cysteine substituted TeNT is expressed with an N-terminal His 6 -tag.
• the nucleic acid was inserted within the MCS of the pRSET-A vector and expressed under the control of a T7 promoter.
• Figure 27 is a 3-dimensional protein structure model of TeNT derived from crystallography data deposited in the protein data bank (accession ID PDB: 5N0B) mapping epitopes recognised by major human antibody clonotypes, as identified by da Silva Antunes et al (2017) and Palermo et al (2017), onto the model using Discovery Studio. Surface serine residues in or around the identified epitopes were selected for mutation to cysteine for subsequent PEGylation.
• Figure 28 comprises two photographs of SDS-PAGE analyses of PEG-TeNTs comprising PEGs of increasing molecular weight and detected (A) using Coomassie blue and (B) by Western blot using polyclonal anti-TeNT antibodies. (B) shows that immunogenicity is proportional to PEG molecular weight.
• Figure 29 comprises four line graphs representing competitive ELISA assays.
• TeNT was adsorbed to an ELISA plate and then probed with a polyclonal anti-TeNT antibody pre-incubated with each of four concentrations (10 pg/mL, 1 pg/mL, 0.1 pg/mL and 0.01 pg/mL) of four PEG-TeNT antigens (2 kDa, 5 kDa, 10 kDa and 20 kDa) .
• concentrations 10 pg/mL, 1 pg/mL, 0.1 pg/mL and 0.01 pg/mL
• PEG-TeNT antigens (2 kDa, 5 kDa, 10 kDa and 20 kDa
• Each PEG-TeNT (2 kDa, 5 kDa, 10 kDa and 20 kDa) was adsorbed to a separate ELISA plate and then probed with a polyclonal anti-TeNT antibody pre-incubated with each of four concentrations (10 pg/mL, 1 pg/mL, 0.1 pg/mL and 0.01 pg/mL) of TeNT antigen.
• TeNT-LC-c Serine mutant was adsorbed to an ELISA plate and then probed with polyclonal anti-TeNT antibody pre-incubated with each of four concentrations (10 pg/mL, 1 pg/mL, 0.1 pg/mL and 0.01 pg/mL) of four PEG-TeNT-LC-c Serine mutant antigens (2 kDa, 5 kDa, 10 kDa and 20 kDa) .
• Figure 30 comprises four photographs showing localised limb tetany in the hind limb of a female C57BL/6 mouse injected with PEG- TeNT-LC-HC.
• a and B show localised limb tetany in a naive mouse.
• C shows a lack of limb tetany after injection of 100 units of TeNT in a mouse vaccinated with tetanus toxoid
• D shows sustained limb tetany after injection of 80 units of PEG-TeNT LC-HC 20kDa in a mouse vaccinated with tetanus toxoid.
• Figure 31 comprises two dot plots showing the clinical tetanus level exhibited in the hind leg of female C57BL/6 mice after injection with a defined number of units of TeNT, PEG-TeNT-LC-HC 20 kDa, TeNT-LC-c Serine mutant, PEG-TeNT-LC-c Serine mutant 2 kDa, or PEG-TeNT-LC-c Serine mutant 20kDa.
• mice were injected with TeNT, TeNT-LC-c Serine mutant, PEG-TeNT-LC-c Serine mutant 2 kDa or PEG-TeNT-LC-c Serine mutant 20 kDa.
• One unit is the minimum amount of toxin required to produce stage four tetany within 24 hours in a naive mouse .
• Figure 32 comprises two line graphs showing the progression of clinical tetany in mice vaccinated with tetanus toxoid.
• A shows the progression of tetany after injection with 4000 units of TeNT with or an without deactivated TeNT decoy.
• B shows the progression of tetany after injection with 40 units of TeNT-PEG 20kDa with or without deactivated TeNT decoy. In both cases the progression is clearly enhanced by the presence of decoy.
• Figure 33 is a box plot showing the effect of increasing doses of tetanus toxin on the Respiratory Disturbance Index (RDI) of British Bulldogs treated according to example 23.
• the black bars represent the median RDI of the six studies at each dose of tetanus toxin.
• the shaded boxes represent the interquartile range and the brackets represent the minimum and maximum RDI sample at each dose.
• Placebo and 10 IU/kg medians are additionally represented by o and *, respectively.
• the present invention relates to immune-evading TeNT and PEG- TeNT molecules ( Figure 1), compositions thereof, and their
• the invention relates to treating hypotonia, optionally obstructive sleep apnoea.
• an active TeNT is modified by adding PEG, introducing specific mutations, or a combination thereof, for delivering a biologically active compound capable of increasing muscle tone in a tetanus-immune patient.
• the activity can be demonstrated by the administration of a unit defined dose of modified toxin, or formulation, where at the same unit dose TeNT would exhibit no activity in a vaccinated subject.
• TeNT modified toxin
• the combinations of PEGylation, site-directed mutation and decoy molecule formulations greatly increased the effect of the molecule on increasing muscle tone in vaccinated mammalian models, relative to the equivalent units administered of TeNT.
• US 2002/0197278 disclosed a series of PEGylated botulinum toxins for treating disorders of inappropriate muscle contraction and suggested that TeNT could be used as an alternative to botulium toxin. However, TeNT cannot be used to treat muscle contraction. Further, the alleged invention of US 2002/0197278 appears not to be enabled, because the methods disclosed do not include site directed masking of epitopes, and, to the best of the present inventors' knowledge, the three-dimensional structure and identification of epitopes required for deliberate masking of TeNT epitopes was not available at the priority date of US 2002/0197278.
• Wan et al is directed to the effect of PEGylation on the anti-PEG immune response resulting from administration of PEGylated proteins, but does not exploit its findings for any therapy.
• Wan et al disclose that a PEGylated tetanus toxoid demonstrated reduced immunogenicity relative to non-PEGylated tetanus toxoid, Wan et al. do not present a therapeutically relevant molecule or formulation. Moreover, PEGylation of tetanus toxoid is not relevant to modification of active TeNT, because tetanus toxoid is a biologically inactive TeNT used for vaccination, which may be produced by formaldehyde cross-linking of TeNT. That is, tetanus toxoid, PEGylated or not, does not possess the combination of enzymatic, binding and translocational activity of active TeNT.
• WO 2016/001762 Al relates to a TeNT c-fragment alone, which is a molecule with no specific activity beyond binding
• Tetanus neuro oxin TeNT
• TeNT is approximately 150 kDa and is expressed from the tetX gene.
• a codon optimised nucleic acid sequence corresponding to the coding region of tetX, but lacking the initiator methionine codon, is provided in the vector sequence of Figure 3 (SEQ ID NO: 2) .
• TeNT is expressed as one protein that is post-translationally cleaved - first to remove the initiator methionine and then into two parts : a 50kDa light chain (LC or A-chain) derived from the N-terminus of the uncleaved protein and a 100 kDa heavy chain (HC or B-chain) derived from the C-terminus of the uncleaved protein. The two chains are connected by an interchain disulfide bond, which is essential for neurotoxicity.
• the 1314 amino acid sequence of mature TeNT is provided in Figure 2 (SEQ ID NO: 1) .
• LC has zinc endopeptidase activity and attacks the vesicle- associated membrane protein (VAMP) that is necessary for vesicle fusion to membranes, thereby preventing neurotransmitter release.
• VAMP vesicle-associated membrane protein
• HC Upon digestion with papain, HC can be cleaved into two domains, each of 50 kDa: an N-terminus translocation domain named HN; and a C-terminus ganglioside (membrane) binding domain named fragment c (c) .
• HN N-terminus translocation domain
• c C-terminus ganglioside binding domain
• TeNT lacking c is referred to herein as LC-HN.
• c harbours two polysialoganglioside binding sites and binds to polysialogangliosides (GD2 GDlb, and GTlb) on the neuronal membrane.
• c mediates binding of TeNT to the presynaptic membrane of peripheral motor axons and aids movement of TeNT across that membrane into the neuron.
• TeNT lacking the initiator methionine is provided in Figure 2 (SEQ ID NO: 1) ;
• HC is provided in Figure 5 (SEQ ID NO: 3);
• LC-HN is provided in Figure 9 (SEQ ID NO: 6) .
• LC-HN are provided in Figure 10 (SEQ ID NO: 7) and Figure 11.
• TeNT is used in reference to a full TeNT molecule, consisting of the heavy chain and light chain.
• Subdomains and fragments are referred to herein by their abbreviations : light chain “LC”; heavy chain “HC”; heavy chain N-terminus domain “HN”; heavy chain fragment c "c”; light chain plus heavy chain N-terminus domain "LC-HN” (i.e. TeNT molecule lacking c) .
• the prefix PEG is used: PEG-LC; PEG-HC; PEG-HN; PEG-c; PEG-LC-HN.
• TeNTs disclosed herein may be active or inactive.
• An active TeNT possesses the same biological activities of native TeNT.
• An inactive TeNT lacks one or more activities of native TeNT.
• an inactive TeNT does not block release of inhibitory neurotransmitters.
• An inactive TeNT of the disclosure may also be referred to as a "decoy".
• inactive TeNT includes tetanus toxoid.
• an inactive TeNT is an inactive TeNT as disclosed herein .
• two or more TeNTs may be conjugated.
• TeNTs are not conjugated.
• a first PEG—TeNT is not conjugated to a second TeNT.
• Immune-evading PEG-TeNTs disclosed herein and depicted in Figure 1 include :
• PEG-TeNT-c advantageously evades the pre-existing immune response of the adaptive immune system in vaccinated subjects.
• PEG-TeNT-c provides first tier treatment to be used until efficacy decreases.
• PEG-TeNT-HC and PEG-TeNT-LC-c advantageously evade the pre existing immune response of the adaptive immune system in vaccinated subjects and also evade the immune response of the adaptive immune system elicited by repeated exposure to PEG-TeNT-c.
• PEG-TeNT-HC and PEG-TeNT-LC-c together provide second tier treatment to be used until their efficacy decreases .
• PEG-TeNT-LC-HC advantageously evades the pre-existing immune response of the adaptive immune system in vaccinated subjects and also evades the immune response of the adaptive immune system elicited by repeated exposure to PEG-TeNT-c and to PEG-TeNT-HC plus PEG-TeNT-LC-c.
• PEG-TeNT-LC-HC provides third tier treatment to be used until its efficacy decreases.
• TeNT with PEGylated light chain PEG-TeNT-LC
• TeNT with PEGylated LC and HN PEG-TeNT-LC-HN
• TeNT with PEGylated HN PEG-TeNT-HN
• PEG may be conjugated, for example, to lysine (e.g. amino- PEGylation) , cysteine (e.g. thiol-PEGylation and bridging
• PEGylation PEGylation
• histidine arginine, aspartic acid, asparagine (e.g. N- glyco-PEGylation) , glutamic acid, glutamine (e.g. transglutaminase- mediated PEGylation), serine (e.g. O-glyco-PEGylation) , threonine (e.g. O-glyco-PEGylation), or tyrosine residues in TeNT.
• PEGylation also include N-terminus PEGylation and C-terminus
• PEGylation may be achieved by reacting PEG with a functional group that is hydroxyl-reactive, for example anhydrides, acid chlorides, chloroformates and carbonates.
• PEGylation may be achieved with functional groups such as aldehyde, ester, and amide .
• PEG may be linear or branched.
• PEG may be a modified PEG, for example, poly [oligo (ethylene glycol) methyl ether methacrylate] (POEGMA) .
• POEGMA poly [oligo (ethylene glycol) methyl ether methacrylate]
• PEGylation may be site-specific PEGylation.
• surface serine residues of TeNT or a TeNT fragment are mutated to surface cysteine residues to facilitate directed PEG conjugation at immunogenic epitopes.
• a mutation is synonymous with substitution, for instance, a serine to cysteine substitution.
• Such mutations, or substitutions include one or more, in any combination, of: S81C; S120C; S144C; S248C; S335C; S428C; S600C; S963C; S1041C; S1155C; and S1187C.
• Functional groups for heterobifunctional PEGs include maleimide, vinyl sulfone, pyridyl disulfide, amine, carboxylic acid, and NHS ester.
• PEG is conjugated to cal TeNT using
• the invention also contemplates a PEG-TeNT comprising different molecular weight PEGs conjugated to different subdomains or fragments of TeNT.
• PEG may be conjugated or attached to TeNTs of the disclosure between 4°C and 25 °C for between 2 and 6 hours, for example. In one embodiment, PEG was conjugated to TeNT at room temperature for 6 hours .
• hypotonia refers to any disorder comprising involuntary muscle weakness that may be treated by inhibiting inhibitory neurotransmitters , for instance GABA or glycine sue
• hypotonia includes reduced muscle tone secondary to reduced neurological drive or other causes and conditions of reduced or inadequate muscle tone, strength or neurological drive.
• hypotonia may be neurological hypotonia.
• hypotonia disorders that may be treated with a PEG-TeNT, a composition or method according to the disclosure include
• ALS amyotrophic lateral sclerosis
• disorders that may be treated with a PEG-TeNT, a composition or method according to the disclosure include muscular atrophy, muscular dystrophy, decrease in muscle mass, nasal
• a disorder to be treated according to the disclosure is not a hypotonia disorder per se
• increasing muscle tone by treating with a TeNT of the disclosure may alleviate a symptom of the disorder.
• Cosmetic applications of PEG-TeNT may include tightening of the abdominal muscles, tightening of the pectoral muscles,
• Smooth muscles, skeletal muscles, tissues or organs that may be treated with a PEG-TeNT, a composition or method according to the disclosure include upper oesophagus, oesophageal wall, oesophageal sphincter, lower oesophageal sphincter, anal sphincter, bladder, bladder sphincter, vaginal sphincter, pyloric sphincter, sphincter of Oddi, ileocaecal sphincter, pelvic floor muscles, vaginal wall muscles, prostate gland, submandibular gland, parotid gland, sublingual gland, minor salivary glands of the oral mucosa, vocal folds, facial muscles, mastication muscles, scalp muscles, chest muscles, back muscles, upper limb muscles, forearm muscles, lower limb muscles, hand muscles, foot muscles, stomach wall muscles, colon wall muscles, neck muscles, throat dilator muscles, masseter muscle, medial
• stylopharyngeus styloglossus, mylohyoid, stylohyoid, hyoglossus, diagastricus , sternocleidomastoid muscle, trapezius muscle,
• temporalis muscles cricopharyngeus muscle, uterine muscle and cervix, gastric nerve supply, intranasal mucosa, pulmonary mucosa, skin, thymus, bone, coronary artery, pulmonary smooth muscle, and cardiac muscle.
• compositions and administration are provided.
• composition of the disclosure may be a therapeutic composition or a cosmetic composition. That is, the composition may be used for therapy or cosmetic purposes.
• composition As used herein, the term "therapeutic composition” or
• composition refers to a composition comprising a TeNT that inhibits or treats hypotonia in the subject as described herein.
• the composition has been formulated for administration to a subject.
• the composition is sterile.
• the composition is pyrogen-free.
• the composition may comprise a pharmaceutically acceptable carrier.
• the composition is manufactured according to Good Laboratory Practice (GLP) or Good Manufacturing Practice (GMP) .
• a TeNT of the disclosure may be administered at up to
• a TeNT of the disclosure may be administered at about 1 fg/kg, about 5 fg/kg, about 10 fg/kg, about 50 fg/kg, about 100 fg/kg, about 500 fg/kg, about 1 pg/kg, about 5 pg/kg, about 10 pg/kg, about 50 pg/kg, about 100 pg/kg, about 500 pg/kg, about 1 ng/kg, about 2 ng/kg, about 3 ng/kg, about 4 ng/kg, about 5 ng/kg, about 6 ng/kg, about 7 ng/kg, about 8 ng/kg, about 9 ng/kg,
• a TeNT of the disclosure may be administered at up to
• a TeNT of the disclosure may be administered at about 0.1 IU/kg, about 0.2 IU/kg, about 0.3 IU/kg, about 0.4 IU/kg, about 0.5 IU/kg, about 0.6 IU/kg, about 0.7 IU/kg, about 0.8 IU/kg, about 0.9 IU/kg, about 1 IU/kg, about 2 IU/kg, about 3 IU/kg, about 4 IU/kg, about 5 IU/kg, about 6 IU/kg, about 7 IU/kg,
• IU/kg about 8 IU/kg, about 9 IU/kg, about 10 IU/kg, about 11 IU/kg, about 12 IU/kg, about 13 IU/kg, about 14 IU/kg, about 15 IU/kg, about 16 IU/kg, about 17 IU/kg, about 18 IU/kg, about 19 IU/kg, about 20 IU/kg, about 30 IU/kg, about 40 IU/kg, about 50 IU/kg, about 60 IU/kg, about 70 IU/kg, about 80 IU/kg, about 90 IU/kg, about 100 IU/kg, about 200 IU/kg, about 300 IU/kg, about 400 IU/kg, about 500 IU/kg, about 600 IU/kg, about 700 IU/kg, about 800 IU/kg, about 900 IU/kg, about 1 000 IU/kg.
• a TeNT of the disclosure may be administered within any range of any of the doses listed above.
• composition comprising two PEG-TeNTs, for example a composition comprising a first PEG-TeNT, wherein TeNT-HC is
• the ratio of the first PEG-TeNT to the second PEG-TeNT may be varied.
• the ratio of first PEG—TeNT to second PEG-TeNT may be about 1000:1, about 500:1, about 100:1, about 50:1, about 10:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1; about 1:2, about 1:3, about 1:4, about 1:5, about. 1:10, about 1:50, about 1:100, about 1:500, or about 1:1000.
• a composition may comprise any combination of TeNTs and is not to be limited to a combination of PEG-TeNT-HC and PEG-TeNT-LC-c.
• a composition may comprise: PEG-TeNT-c and PEG-TeNT-HC; PEG-TeNT-c and PEG-TeNT-LC-c; PEG-TeNT-c and PEG-TeNT-LC-HC; PEG-TeNT-HC and
• PEG-TeNT-LC-HC Als dosed is a composition comprising: PEG-TeNT-c, PEG-TeNT-HC and PEG-TeNT- LC-c; PEG-TeNT-c, PEG-TeNT-HC and PEG-TeNT-LC-HC; PEG-TeNT-c, PEG- TeNT-LC-c and PEG-TeNT-LC-HC; PEG-TeNT-HC, PEG-TeNT-LC-c and PEG- TeNT-LC-HC; and PEG-TeNT-c, PEG-TeNT-HC, PEG-TeNT-LC-c and PEG-Tel
• any TeNT may be substituted for, and any composition may further comprise, PEG-TeNT-LC, PEG-TeNT-LC-HN, and/or PEG-TeNT-HN.
• the composition further comprises an inactivated TeNT that acts as a decoy for anti-TeNT antibodies produced by prior exposure to TeNT, for example by vaccination
• the inactivated TeNT may, relative to SEQ ID NO: 1, comprise: R1225K; R1225E; W1228A; W1288Y; W1288F; W1288L; R1225K and W1288A; R1225K and W1288Y; R1225E and W1288Y; R1225K and W1288F; R1225E and W1288F; R1225K and W1288L; R1225E and W1288L; R1225del; W1288del; or
• the second TeNT comprises an inactivated TeNT comprising R1225E, W1288A, E270A and Y374A.
• the decoy TeNT will be in molar excess relative to the PEG- TeNT.
• the ratio of decoy TeNT to the PEG-TeNT may be about 10 6 : 1; 10 b :l, 1Q 4 :1 , 1000:1, about 500:1, about 400:1, about 300:1, about 200:1, about 100:1, about 90:1, about 80:1, about 70:1, about 60:1, about 50:1, about 40:1, about 30:1, about 20:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1.
• a PEG-TeNT of the disclosure may be administered once, twice or three times per week, once, twice or three times per month, once, twice or three times per quarter, once, twice or three times per 6 months, or once, twice or three times per year.
• the PEG-TeNT may be administered in a single dose, a split dose, or in multiple doses. Where a muscle exists as a pair, the PEG-TeNT may be administered unilaterally to one muscle of the pair or bilaterally to both muscles of the pair.
• such two or more PEG-TeNTs may be administered in combination sequentially or simultaneously.
• the PEG-TeNT may be administered to a subject locally by any suitable method, for example by injection, surgical implantation, topical application, or intranasal administration.
• any suitable method for example by injection, surgical implantation, topical application, or intranasal administration.
• the TeNT is administered intramuscularly by injection to the affected muscle.
• the PEG-TeNT will be formulated, dosed, and administered in a fashion consistent with good medical practice.
• the therapeutically effective amount of the PEG-TeNT to be administered will be governed by such considerations.
• Pharmaceutically acceptable carriers include water, buffered water, saline solutions such as, for example, normal saline or balanced saline solutions such as Hank' s or Earle' s balanced solutions, glycine, and hyaluronic acid.
• saline solutions such as, for example, normal saline or balanced saline solutions such as Hank' s or Earle' s balanced solutions, glycine, and hyaluronic acid.
• composition may be formulated for intramuscular
• compositions for intramuscular administration may comprise pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions.
• suitable aqueous and non-aqueous carriers, solvents, diluents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol),
• carboxymethylcellulose and mixtures thereof vegetable oils (such as olive oil), injectable organic esters (e.g. ethyl oleate) .
• the composition may comprise penetration enhancers to enhance their delivery of TeNT .
• Penetration enhancers may include fatty acids such as oleic acid, lauric acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid,
• dicaprate reclineate, monoolein, dilaurin, caprylic acid
• arachidonic acid glyceryl 1-monocaprate, mono and di-glycerides and physiologically acceptable salts thereof.
• composition may further include chelating agents such as, for example, ethylenediaminetetraacetic acid (EDTA) , citric acid, salicylates (e.g. sodium salycilate, 5-methoxysalicylate,
• chelating agents such as, for example, ethylenediaminetetraacetic acid (EDTA) , citric acid, salicylates (e.g. sodium salycilate, 5-methoxysalicylate,
• an article of manufacture and/or a kit comprising a container comprising the PEG-TeNT or composition comprising the PEG-TeNT.
• the container may be a bottle, vial or syringe comprising the PEG-TeNT or composition, optionally in unit dosage form.
• the PEG-TeNT or composition may be in the form of an injectable solution in a disposable container, optionally a syringe.
• the article of manufacture and/or kit may further comprise printed instructions and/or a label or the like, indicating treatment of a subject according to the method disclosed herein.
• terapéuticaally effective amount refers to an amount of PEG-TeNT effective to treat hypotonia in a subject.
• treat refers to both therapeutic treatment and prophylactic or preventative measures, wherein the aim is to prevent, reduce, or ameliorate hypotonia in a subject or slow down (lessen) progression of hypotonia in a subject.
• Subjects in need of treatment include those already with hypotonia as well as those in which hypotonia is to be prevented or
• prophylactic refers to keeping from occurring, or to hinder, defend from, or protect from the occurrence of hypotonia. A subject in need of prevention may be prone to develop hypotonia.
• ameliorate or “amelioration” refers to a decrease, reduction or elimination of hypotonia.
• Hypotonia may be quantified. Hypotonia may be quantified on a semi-quantitative scale, for example 0 to 5 , where 0 represents absence, 1 to 4 represent identifiable increases in severity, and 5 represents maximum severity. Alternatively, hypotonia may be quantified as a binary event, i.e. presence or absence, 0 or 1.
• hypotonia may be quantified on a quantitative scale, for instance using a force gauge .
• Any quantification of hypotonia may be compared to a control, for example a healthy control subject not receiving a PEG-TeNT, an affected control subject receiving treatment for hypotonia, but not treated with a PEG-TeNT, or a population.
• Treating hypotonia by administering a PEG-TeNT may be about a 1% decrease, about a 2% decrease, about a 3% decrease, about a 4% decrease, about a 5% decrease, about a 6% decrease, about a 7% decrease, about an 8% decrease, about a 9% decrease, about a 10% decrease, about a 20% decrease, about a 30% decrease, about a 40% decrease, about a 50% decrease, about a 60% decrease, about a 70% decrease, about an 80% decrease, about a 90% decrease, or about a
• the term "subject" may refer to a mammal.
• the mammal may be a primate, particularly a human, or may be a domestic, zoo, or companion animal.
• the PEG-TeNTs, compositions and method disclosed herein are suitable for medical treatment of humans, it is also applicable to veterinary treatment, including treatment of domestic animals such as horses, cattle and sheep, companion animals such as dogs and cats, or zoo animals such as felids, canids, bovids and ungulates.
• the term "about” as used herein contemplates a range of values for a given number of +25% the magnitude of that number. In other embodiments, the term “about” contemplates a range of values for a given number of +20%, ⁇ 15%, ⁇ 10%, ⁇ 5%, ⁇ 4%, ⁇ 3%, ⁇ 2%, or ⁇ 1% the magnitude of that number. For example, in one embodiment, “about 3 grams” indicates a value of 2.7 grams to 3.3 grams (i.e. 3 grams ⁇ 10%), and the like.
• the timing or duration of events may be varied by at least 25%.
• a particular event may be disclosed in one embodiment as lasting one day, the event may last for more or less than one day.
• "one day” may include a period of about 18 hours to about 30 hours.
• periods of time may vary by +20%, +15%, +10%, +5%, +4%, +3%, +2%, or +1% of that period of time.
• surface serine residues of TeNT-c will be mutated to surface cysteine residues to facilitate directed PEG conjugation at immunogenic epitopes to produce the molecule of Figure 1A.
• the mutations will be: S963C, S1041C, S1155C, and S1187C.
• substitutions S963C, S1041C, S1155C, and S1187C will be synthesised by a commercial provider, for example Integrated DNA Technologies.
• the gene will be sub-cloned into the pRSET-A expression vector by restriction digestion so that 6x Histidine tag from the vector is added to the N-terminus of the mutant protein.
• TeNT comprising S963C, S1041C, S1155C, and S1187C will be expressed, PEGylated and purified according to Example 5 to produce PEG-TeNT-c.
• surface serine residues of TeNT-HC will be mutated to surface cysteine residues to facilitate directed PEG conjugation at immunogenic epitopes to produce the molecule of Figure IB.
• the mutations will be: S600C, S963C, S1041C, S1155C, and S1187C ( Figure 24 SEQ ID NO: 16) .
• substitutions S600C, S963C, S1041C, S1155C, and S1187C will be synthesised by a commercial provider, for example Integrated DNA Technologies .
• the gene will be sub-cloned into the pRSET-A
• TeNT comprising S600C, S963C, S1041C, S1155C, and S1187C will be expressed, PEGylated and purified according to Example 5 to produce PEG-TeNT-HC.
• surface serine residues of LC and c were mutated to surface cysteine residues to facilitate directed PEG conjugation at immunogenic epitopes to produce the molecule of Figure 1C.
• the mutations were: S81C, S120C, S144C, S248C, S335C, S428C, S963C, S1041C, S1155C, and S1187C ( Figure 21 SEQ ID NO: 14) .
• the gene was sub cloned into the pRSET-A expression vector ( Figure 22 SEQ ID NO: 15, Figure 23) by restriction digestion so that 6x Histidine tag from the vector was added to the N-terminus of the mutant protein.
• TeNT comprising S81C, S120C, S144C, S248C, S335C, S428C, S963C, S1041C, S1155C, and S1187C was expressed, PEGylated and purified according to Example 5 to produce PEG-TeNT-LC-c.
• TeNT 1 E. coli BL21 DE3 pLysS strain was electrotransformed with pRSET-TeNT vector ( Figures 3 and 4 ⁇ and grown overnight on LB agar with selection antibiotic (ampicillin and chloramphenicol ) at 37 °C.
• the pre-induction broth pH 7.2- 7.4 comprised: 1.2% Tryptone; 2.4% Yeast extract; 2% Glucose; 0.4% Glycerol; 17 mM KH2PO4; 72 mM K2HPO4; and selection antibiotics (ampicillin and chloramphenicol).
• the culture was incubated overnight at 30 °C with fast shaking .
• the overnight culture was harvested by centrifugation at 4000 g for 10 minutes.
• the pellet was resuspended in 200 mL expression broth pH 7.2-7.4, comprising: 1.2% Tryptone; 2.4% Yeast extract; 0.4% glycerol; 1 mM IPTG; 17 mM KH2PO4; 72 mM K2HPO4; 100 pg/mL
• the protein was expressed for 6 hours at 3Q°C with fast shaking .
• the cells were harvested by centrifugation at 4500 g for 15 min and the pellet resuspended in 30 mL of TBS with 20 mM imidazole at pH 8.
• the cells were lysed by sonication.
• the cell lysate was cleared by centrifugation at 4500 c; r for 20 min and filtered through a 0.45 pm filter.
• the protein was purified by His-tag affinity
• the purified protein underwent buffer exchange to PBS using size exclusion chromatography followed by a second stage purification by gel filtration using AKTA pure 25 FPLC with Superdex 200 increase 10/300 GL column.
• the column was incubated with end-over-end mixing at 4°C for at least 1 h.
• the gel was resuspended in about 0.2 mL of the digestion buffer .
• TeNT-LC-HC surface serine residues of TeNT-LC-HC were mutated to surface cysteine residues (S to C mutant) to facilitate directed PEG conjugation at immunogenic epitopes.
• the TeNT mutations were: S81C; S120C; S144C; S248C; S335C; S428C; S600C; S963C; S1041C; S1155C; and S1187C, relative to SEQ ID NO: 1.
• TeNT-LC-HC comprising the S to C mutations was expressed and purified according to Example 4 with the addition of treatment by 0.5 mM DTT for 15 minutes between step 10 and 11.
• Endotoxin was removed from the TeNT-LC-HC Serine mutant according to Example 4.PEG attachment to cysteine residues.
• inactivating R1225E and W1288A amino acid substitutions, relative to SEQ ID NO: 1, in the ganglioside binding region was produced.
• the inactivated c was combined with an equimolar amount of non-PEGylated LC-HN to produce the inactive decoy TeNT.
• the resulting inactive TeNT will be used as a decoy for the antibody-based neutralising response in subjects vaccinated against tetanus toxoid.
• the gene for c with R1225E and W1288A was synthesised by a commercial provider, Integrated DNA Technologies. The gene was sub cloned into the pRSET-A expression vector by restriction digestion so that 6x Histidine tag from the vector is added to the N-terminus of the mutant protein.
• E. coli BL21 (DE3) pLysS strain was electrotransformed with pRSET-TeNT-c vector ( Figure 13 (SEQ ID NO: 9) and Figure 14) encoding c comprising R1225E W1288A ( Figure 12, SEQ ID NO: 8) and grown overnight on LB agar with selection antibiotics (ampicillin, chloramphenicol) at 37 ° C .
• c comprising R1225E W1288A was expressed and purified according to Example 4.
• Endotoxin was removed from the purified protein according to Example 4.
• E. coli BL21 (DE3) pLysS strain was electrotransformed with pRSET-TeNT-LC-HN vector ( Figures 10 and 11) and grown overnight on LB agar with selection antibiotics (ampicillin, chloramphenicol) at 37 °C.
• LC-HN was expressed and purified according to Example 4.
• Endotoxin was removed from the purified protein according to Example 4.
• E. coli BL21 (DE3) pLysS strain was electrotransformed with pRSET-TeNT-LC-HN vector encoding LC-HN comprising E270A Y374A and grown overnight on LB agar with selection antibiotics (ampicillin, chloramphenicol) at 37°C.
• LC-HN comprising E270A Y374A was expressed and purified according to Example 4.
• Endotoxin was removed from the purified protein according to Example 4.
• the inactivated TeNT comprising R1225E and W1288A was expressed and purified according to Example 4.
• TeNT was prepared and PEGylated according to Example 4, then analysed by SDS-PAGE ( Figure 28) and detected (A) using Coomassie blue and (B) by Western blot using polyclonal anti-TeNT antibodies. (B) snows that immunogenicity is proportional to PEG molecular weight .
• PEG-TeNTs each comprising a different molecular weight PEG (2 kDa, 5 kDa, 10 kDa and 20 kDa) were prepared and PEGylated according to Example 4. The PEG-TeNTs were then assayed by
• TeNT was adsorbed to an ELISA plate. Adsorbed TeNT was then probed with a polyclonal anti- TeNT antibody pre-incubated with each of four concentrations
• each PEG-TeNT was adsorbed to a separate ELISA plate.
• Each adsorbed PEG-TeNT (2 kDa, 5 kDa, 10 kDa and 20 kDa) was then probed with a polyclonal anti-TeNT antibody pre-incubated with each of four concentrations (10 pg . /mL, 1 ag/mL, 0.1 pg/mL and 0.01 pg/mL) of TeNT antigen.
• concentrations 10 pg . /mL, 1 ag/mL, 0.1 pg/mL and 0.01 pg/mL
• TeNT preferentially to TeNT and that PEGylated TeNTs have reduced immunogenicity relative to TeNT (i.e. non-PEGylated) .
• TeNT-LC-c Serine mutant was prepared according to Example 5.
• Four samples of the TeNT-LC-c Serine mutant were PEGylated according to Example 5, each sample comprising a different molecular weight PEG (2 kDa, 5 kDa, 10 kDa and 20 kDa) .
• the PEG-TeNT-LC-c Serine mutants were then assayed by competitive ELISA against TeNT-LC-c Serine mutant.
• TeNT-LC-c Serine mutant was adsorbed to an ELISA plate. Adsorbed TeNT Serine mutant was then probed with a polyclonal anti-TeNT antibody pre-incubated with each of four concentrations (10 pg/mL, 1 pg/mL, 0.1 pg/mL and 0.01 pg/mL) of four PEG-TeNT Serine mutant antigens (2 kDa, 5 kDa, 10 kDa and 20 kDa) . In this assay, higher responses (OD 450 nm) indicated greater affinity for TeNT Serine mutant and therefore reduced
• each PEG-TeNT-LC-c Serine mutant was adsorbed to a separate ELISA plate.
• Each adsorbed PEG- TeNT Serine mutant (2 kDa, 5 kDa, 10 kDa and 20 kDa) was then probed with a polyclonal anti-TeNT antibody pre-incubated with each of four concentrations (10 pg/mL, 1 pg/mL, 0.1 pg/mL and 0.01 pg/mL) of TeNT Serine mutant antigen.
• lower responses indicated greater affinity for TeNT Serine mutant and therefore reduced immunogenicity for the PEG-TeNT Serine mutant.
• TeNT-LC-c Serine mutant preferentially to TeNT-LC-c Serine mutant and that PEGylated TeNT- LC-c Serine mutants have reduced immunogenicity relative to
• TeNT -LC-c Serine mutant ( i . e . non-PEGy1ated) .
• a competitive ELISA assay will be conducted in accordance with Example 9, except that the polyclonal antibody will be replaced by human serum collected from one or more subjects who have received a booster tetanus toxoid vaccination within the previous 12 months . Antibodies in the serum will show greater affinity for TeNT
• Example 12 In vivo model
• PEG-TeNT-LC-HC were prepared by attaching PEG (about 5 kDa, about 10 kDa or about 20 kDa) to the surface exposed lysine residues of recombinant TeNT accordng to Example 4.
• One or more units of PEG-TeNT-LC-HC in 15 pL of PBS was injected into the hind limb of female C57BL/6 mice. Each animal exhibited localised limb tetany within 48 hours of injection
• Example 13 In vivo model
• PEG-TeNT-LC-HC with 5 kDa PEG attached to surface lysine residues of TeNT according to Example 4, was combined with c decoy, i.e. c inactivated by R1225E W1288A, at a molar ratio of 1:10, Fifteen microlitres of the composition in PBS, containing 5 ng of the PEG-TeNT, was in ected into the muscle of the hind limb of female C57BL/6 mice. Each animal exhibited localised limb tetany within 48 hours, symptom development was indistinguishable from animals treated with the PEG-TeNT-LC-HC without the decoy present.
• c decoy i.e. c inactivated by R1225E W1288A
• PEG-TeNT-LC-HC comprising 5 kDa, 10 kDa or 20 kDa PEG was administered at 50 - 500 000 ng/kg intramuscularly to the hind leg muscle of mice previously immunized with tetanus toxoid. Increased muscle contraction was observed in the injected muscle for up to 3 days, and was greater than the effect observed in mice
• mice administered at 50 - 500 000 ng/kg intramuscularly to the hind leg muscle of mice previously immunized with tetanus toxoid. Increased muscle contraction was observed in the injected muscle for up to three days, and was greater than the effect observed in mice administered TeNT ( Figure 32A) .
• a composition comprising PEG-TeNT-LC-HC comprising 5 kDa, 10 kDa or 20 kDa PEG and a 10:1 or 100:1 molar excess of a decoy TeNT produced according to Example 6 was administered at 50 - 500 000 ng/kg intramuscularly to the hind leg muscle of mice previously immunized with tetanus toxoid. Increased muscle contraction was observed in the injected muscle for up to three days, and was greater than the effect observed in mice administered TeNT, and greater than the effect observed in mice administered PEG-TeNT only ( Figure 32B) .
• PEG-TeNT-LC-HC comprising 20 kDa PEG will be administered at 0.01 - 50 000 ng/kg intramuscularly to the left geniohyoid of a human subject previously vaccinated with tetanus toxoid. Increased muscle contraction will be observed in the injected muscle for up to 2 weeks, and will be greater than the effect observed in the right geniohyoid administered vehicle only of the same subject.
• a composition comprising PEG-TeNT-LC-HC comprising 20 kDa PEG and a 10:1 - 1000:1 molar excess of a decoy TeNT produced according to Example 6 will be administered at 50 ng/kg intramuscularly to the left geniohyoid of a human subject previously vaccinated with tetanus toxoid.
• PEG-TeNT-LC-HC comprising 20 kDa PEG will be administered at 50 ng/kg intramuscularly to the right geniohyoid of the same human subject.
• Increased muscle contraction will be observed in both the left and right geniohyoids for up to 2 weeks, but will be greater in the left geniohyoid treated with the composition comprising PEG- TeNT-LC-HC and the decoy TeNT compared to the PEG-TeNT-LC-HC alone.
• Bulldogs of approximately 30 kg will be administered 25 - 50 000 ng/kg PEG-TeNT-c comprising 20 kDa PEG intramuscularly with the dose divided bilaterally to the left and right geniohyoid.
• OSA obstructive sleep apnoea
• the bulldogs will be observed weekly for OSA and the PEG- TeNT-c dose will be repeated as needed until efficacy decreases, as determined by a return of OSA comparable to animals treated with vehic1e a1one.
• the bulldogs will be administered 25 - 50 000 ng/kg of PEG-TeNT-HC or 25 - 50 000 ng/kg PEG-TeNT-LC-c, each comprising 20 kDa PEG, divided bilaterally to the left and right geniohyoid.
• OSA will decrease in PEG-TeNT treated animals compared with animals treated with vehicle alone.
• the bulldogs will be observed weekly for OSA and the PEG-TeNT-HC and PEG-TeNT-LC-c dose will be repeated as needed until efficacy decreases, as determined by a return of OSA comparable to animals treated with vehicle alone.
• the bulldogs will be administered 25 - 50 000 ng/kg PEG-TeNT-LC-HC, comprising 20 kDa PEG, divided
• OSA will decrease in PEG-TeNT treated animals compared with animals treated with vehicle alone.
• the bulldogs will be observed weekly for OSA and the PEG-TeNT-LC-HC dose will be repeated as needed until efficacy decreases, as determined by a return of OSA comparable to animaIs treated with vehic1e alone.
• decoy TeNT (5 kDa, 10 kDa, or 20 kDa branched or linear PEG attached to surface lysine or cysteine residues) and a 10-1000 molar excess of decoy TeNT will be injected intramuscularly into the geniohyoid of a human vaccinated against tetanus toxoid. Localised tetani is observed for a period of weeks to months, greater than the effect observed in a human injected with native TeNT.
• TeNT administered 0.001 - 10 IU/Kg of TeNT intramuscularly, with the dose divided bilaterally to the left and right geniohyoid.
• obstructive sleep apnoea was observed to decrease significantly at the highest dose.
• the baseline respiratory disturbance index (RDI ) score was 19.9 (interquartile range 5.45) decreasing to 13.2 (interquartile range 4.45) after treatment with 10 IU/Kg TeNT, determined to be significant compared to

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