Pegylated tetanus neurotoxin and treatment of hypotonia

Abstract

The present invention relates to a composition comprising a first PEGylated tetanus neurotoxin (PEG-TeNT) comprising tetanus neurotoxin (TeNT) conjugated to polyethylene glycol (PEG) and a second TeNT. The present invention also relates to various PEG-TeNTs. The invention also relates to methods of treating hypotonia using the compositions or various PEG-TeNTs, and kits comprising the compositions or various PEG-TeNTs. In one embodiment, the hypotonia is obstructive sleep apnea.SOLUTION: The present inventors have created a family of modified TeNTs and treatment regimens to address these issues.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a composition comprising a first PEGylated tetanus neurotoxin (PEG-TeNT) comprising a tetanus neurotoxin (TeNT) conjugated with polyethylene glycol (PEG), and a second TeNT. The present invention also relates to a method for treating hypotonia using the composition. [Background technology]

[0002] Tetanus neurotoxin (TeNT) is a neurotoxin produced by Clostridium tetani. TeNT is produced by the serogroup Tetani. TeNT acts in the spinal cord and blocks the release of the inhibitory neurotransmitters gamma-aminobutyric acid (GABA) and glycine from spinal inhibitory interneurons. As such, TeNT causes spastic paralysis. TeNT does not occur in multiple serotypes.

[0003] The biological properties of TeNT, or at least TeNT fragments, have been proposed for therapeutic use. However, long-term treatment with protein therapeutics tends to result in targeted immune responses. Because only one serotype of TeNT is known, serotype switching to evade immunity is not an option for TeNT-based therapy. Furthermore, many people are vaccinated against TeNT, hindering TeNT-based therapy.

[0004] Patent Document 1 discloses the use of PEGylated botulinum toxin for the treatment of diseases of inappropriate muscle contraction. Patent Document 1 also suggests the use of PEGylated TeNT for the treatment of diseases of inappropriate muscle contraction, such as migration headache or strabismus. However, as noted above, TeNT induces muscle contraction, which precludes its use for the treatment of diseases of inappropriate muscle contraction, whether PEGylated or not.

[0005] Non-Patent Document 1 discloses the effect of PEGylation on anti-PEG immune responses obtained from the administration of PEGylated proteins, but does not utilize this knowledge for any therapeutic purposes.

[0006] Patent Document 2 discloses the use of PEGylated TeNT fragment c (c) to increase muscle mass. When TeNT is enzymatically cleaved with papain, fragment c (50 kDa) is generated, corresponding to 451 amino acids at the C-terminus of the TeNT heavy chain. Fragment c retains the binding, internalization, and transsynaptic transport capabilities of undigested TeNT, but does not disrupt any neuronal processes and is therefore non-toxic.

[0007] There is a need for a TeNT-based therapy that circumvents a pre-existing anti-TeNT immune response in tetanus toxoid-immunized subjects.

[0008] Where a prior art publication is referred to herein, such reference is not an admission that the publication forms part of the common general knowledge in the art in Australia or any other country. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] U.S. Patent Application Publication No. 2002 / 0197278(A1) [Patent Document 2] International Publication No. 2016 / 001762(A1) [Non-patent literature]

[0010] [Non-Patent Document 1] Wan et al.Process Biochemistry(2017)52:183-191 Summary of the Invention [Problem to be solved by the invention]

[0011] The inventors recognize that the utility of TeNT has not been fully realized because, upon administration of a protein therapeutic, the adaptive immune system mounts an antibody response, reducing the effectiveness of the protein therapeutic. The adaptive immune response may be intentional, as a result of vaccination, as demonstrated by the large population that is immune to TeNT. Alternatively, the adaptive immune response may be unintentional and result from repeated exposure to the protein therapeutic. [Means for solving the problem]

[0012] The present inventors have created a family of modified TeNTs and treatment regimens to address these problems. Specifically, the present invention provides a family of PEGylated TeNTs (PEG-TeNTs), each of which evades the immune system, and a stepwise treatment regimen in which different alternative PEG-TeNTs are used for treatment when the effectiveness of a previously administered PEG-TeNT decreases or when a patient's immune profile precludes the use of another PEG-TeNT.

[0013] A first aspect provides a tetanus neurotoxin (TeNT) or fragment thereof comprising one or more surface serine to cysteine ​​amino acid substitutions relative to SEQ ID NO:1.

[0014] In one embodiment of the first aspect, the substituted cysteine ​​is conjugated to polyethylene glycol (PEG).

[0015] A second embodiment is TeNT or a fragment thereof, which, relative to SEQ ID NO: 1, comprises: R1225K; R1225E; W1288A; W1288Y; W1288F; W1288L; R1225K and W1288A; R1225E and W1288A; R1225K and W1288Y; R1225E and W1288Y; R1225K and W1288F; R1225E and W1288F or R1225del and W1288del; E270A; Y374A; E270A and Y374; G270del; Y374del; or combinations thereof, wherein the TeNT or fragment thereof is inactive.

[0016] A third aspect provides a composition comprising: (i) a first PEGylated tetanus neurotoxin (PEG-TeNT) comprising a tetanus neurotoxin (TeNT) conjugated to polyethylene glycol (PEG); and (ii) the first PEG-TeNT comprises a second TeNT that is not conjugated to the second TeNT.

[0017] 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 a tetanus neurotoxin (TeNT) conjugated with polyethylene glycol (PEG); and (ii) a second TeNT.

[0018] A fourth aspect also provides the use of a first PEGylated tetanus neurotoxin (PEG-TeNT) comprising a tetanus neurotoxin (TeNT) conjugated with polyethylene glycol (PEG) in a pharmaceutical formulation for the treatment of hypotonia in a subject receiving a second TeNT.

[0019] The fourth embodiment is a polyethylene glycol (PEG)-conjugated tetanus neurotoxin (TeNT). Also provided is the use of a second tetanus neurotoxin (TeNT) in a pharmaceutical formulation for the treatment of hypotonia in a subject receiving a first PEGylated tetanus neurotoxin (PEG-TeNT) comprising:

[0020] A fourth aspect also provides the use of (i) a first PEGylated tetanus neurotoxin (PEG-TeNT) comprising a tetanus neurotoxin (TeNT) conjugated with polyethylene glycol (PEG); and (ii) a second TeNT in a pharmaceutical formulation for the treatment of hypotonia.

[0021] The fourth aspect also provides a first PEGylated tetanus neurotoxin (PEG-TeNT) comprising a tetanus neurotoxin (TeNT) conjugated with polyethylene glycol (PEG) for use in a method for treating hypotonia in a subject administered a second TeNT.

[0022] A fourth aspect also provides the use of a second tetanus neurotoxin (TeNT) for use in a method for treating hypotonia in a subject receiving a first PEGylated tetanus neurotoxin (PEG-TeNT) comprising a tetanus neurotoxin (TeNT) conjugated with polyethylene glycol (PEG).

[0023] The fourth aspect also provides (i) a first PEGylated tetanus neurotoxin (PEG-TeNT) comprising a tetanus neurotoxin (TeNT) conjugated with polyethylene glycol (PEG); and (ii) a second TeNT, for use in a method for treating hypotonia.

[0024] In an embodiment of the fourth aspect, the composition may comprise a first PEG-TeNT and a second TeNT.

[0025] The fourth aspect also provides a composition comprising: (i) a first PEGylated tetanus neurotoxin (PEG-TeNT) comprising a tetanus neurotoxin (TeNT) conjugated with polyethylene glycol (PEG); and (ii) a second TeNT, for use in a method for treating hypotonia.

[0026] In one embodiment, the first PEG-TeNT or second TeNT comprises a PEGylated TeNT light chain (LC), a PEGylated TeNT heavy chain (HC), a PEGylated TeNT heavy chain (HC) and a PEGylated TeNT light chain (LC), or a PEGylated TeNT fragment c(c). In one embodiment, the first PEG-TeNT or second TeNT comprises PEG-TeNT-LC-HC.

[0027] In another embodiment, the first PEG-TeNT or the second TeNT is PEG-TeNT-HC, which comprises a PEGylated HC. In this embodiment, LC is not PEGylated. In another embodiment, the first PEG-TeNT or the second TeNT is PEG-TeNT-LC-c, which comprises a PEGylated LC and a PEGylated c. In this embodiment, HN is not PEGylated.

[0028] In a further embodiment, the first PEG-TeNT is PEG-TeNT-HC and the second TeNT is PEG-TeNT-LC-c.

[0029] In one embodiment, the second TeNT comprises an inactivated TeNT. Relative to SEQ ID NO: 1, an 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 Y374A; G270del; Y374del; or combinations thereof. In one embodiment, the second TeNT comprises an inactivated TeNT comprising R1225E and W1288A.

[0030] In one embodiment, a subject is administered PEG-TeNT containing PEGylated c (PEG-TeNT-c) until efficacy declines; then a composition containing PEG-TeNT-HC and PEG-TeNT-LC-c is administered until efficacy declines; then PEG-TeNT containing PEGylated LC and PEGylated HC (PEG-TeNT-LC-HC) is administered.

[0031] In one embodiment, the treatment involves administering PEG-TeNT containing PEGylated c (PEG-TeNT-c) to the subject; then administering a composition containing PEG-TeNT-HC and PEG-TeNT-LC-c to the subject; and then administering PEG-TeNT containing PEGylated LC and PEGylated HC (PEG-TeNT-LC-HC) to the subject, determining the subject's anti-TeNT antibody immune profile, and determining an effective composition of PEG-TeNT based on this profile.

[0032] In one embodiment, the hypotonia is obstructive sleep apnea.

[0033] In another embodiment, the composition is a therapeutic composition, hi another embodiment, the composition is a cosmetic composition.

[0034] Also disclosed is a method for producing a protein consisting of TeNT LC and TeNT HN (LC-HN), the method comprising expressing in a host cell a nucleic acid molecule encoding an amino acid sequence comprising SEQ ID NO:6.

[0035] A fifth aspect provides a PEGylated tetanus neurotoxin (PEG-TeNT), which comprises tetanus neurotoxin (TeNT) conjugated with polyethylene glycol (PEG).

[0036] In one embodiment, the TeNT light chain (LC) is PEGylated, the TeNT heavy chain (HC) is PEGylated, or the TeNT fragment c (c) is PEGylated. In one embodiment, the LC is PEGylated and the HC is PEGylated (PEG-TeNT-LC-HC), or the LC is PEGylated and c is PEGylated (PEG-TeNT-LC-c).

[0037] In another embodiment, PEG is attached to a lysine residue of TeNT. In another embodiment, PEG is attached to a cysteine ​​residue of TeNT. In one embodiment, PEG is attached to a cysteine ​​residue of TeNT, which is either native to SEQ ID NO: 1 or substituted for a serine residue.

[0038] In one embodiment, the PEG has a molecular weight of about 2 kDa, about 5 kDa, about 10 kDa, or about 20 kDa, or about 30 kDa.

[0039] 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.

[0040] A sixth aspect also provides the use of the PEG-TeNT of the fifth aspect in a pharmaceutical formulation for treating hypotonia, wherein the PEG-TeNT is not conjugated to a second TeNT.

[0041] Alternatively, a sixth aspect provides the PEG-TeNT of the fifth aspect for use in a method for treating hypotonia, the method comprising administering the PEG-TeNT to a subject, wherein the PEG-TeNT is not conjugated to a second TeNT.

[0042] In one embodiment, a subject is administered a first PEG-TeNT containing a PEGylated HC (PEG-TeNT-HC) and a second PEG-TeNT containing a PEGylated LC and a PEGylated c (PEG-TeNT-LC-c).

[0043] In another embodiment, the subject is administered PEG-TeNT comprising PEGylated c (PEG-TeNT-c); and / or a first PEG-TeNT comprising PEGylated HC (PEG-TeNT-HC) and a second PEG-TeNT comprising PEGylated c (PEG-TeNT-LC-c); and / or a PEG-TeNT comprising PEGylated HC and PEGylated LC (PEG-TeNT-LC-HC).

[0044] In one embodiment, treatment involves administering PEG-TeNT containing PEGylated c (PEG-TeNT-c) to the subject until efficacy is reduced. Thereafter, treatment may involve administering PEG-TeNT-HC and PEG-TeNT-LC-c to the subject until efficacy is reduced. Thereafter, treatment may involve administering PEG-TeNT containing PEGylated LC and PEGylated HC (PEG-TeNT-LC-HC) to the subject.

[0045] In one embodiment, treatment involves administering PEG-TeNT containing PEGylated c (PEG-TeNT-c) to the subject until efficacy decreases; then administering PEG-TeNT-HC and PEG-TeNT-LC-c to the subject until efficacy decreases; and then administering PEG-TeNT containing PEGylated LC and PEGylated HC (PEG-TeNT-LC-HC) to the subject.

[0046] In one embodiment, treatment comprises administering to the subject PEG-TeNT (PEG-TeNT-c) comprising PEGylated c comprising PEG with a molecular weight of about 5 kDa until efficacy is reduced; then administering to the subject PEG-TeNT (PEG-TeNT-c) comprising PEG with a molecular weight of about 10 kDa until efficacy is reduced; then administering to the subject PEG-TeNT (PEG-TeNT-c) comprising PEG with a molecular weight of about 20 kDa until efficacy is reduced. Thereafter, treatment may include administering to the subject PEG-TeNT-HC and PEG-TeNT-LC-c, either or both of which comprise PEG having a molecular weight of 5 kDa, until efficacy decreases; then administering to the subject PEG-TeNT-HC and PEG-TeNT-LC-c, either or both of which comprise PEG having a molecular weight of 10 kDa, until efficacy decreases; then administering to the subject PEG-TeNT-HC and PEG-TeNT-LC-c, either or both of which comprise PEG having a molecular weight of 20 kDa, until efficacy decreases. Thereafter, treatment may include administering to the subject PEG-TeNT comprising a PEGylated LC and a PEGylated HC, either or both of which comprise PEG having a molecular weight of 5 kDa (PEG-TeNT-LC-HC), until efficacy decreases; then administering to the subject PEG-TeNT comprising a PEGylated LC and a PEGylated HC, either or both of which comprise PEG having a molecular weight of 10 kDa (PEG-TeNT-LC-HC), until efficacy decreases; then administering to the subject PEG-TeNT comprising a PEGylated LC and a PEGylated HC, either or both of which comprise PEG having a molecular weight of 20 kDa (PEG-TeNT-LC-HC), until efficacy decreases.

[0047] In one embodiment, treatment involves administering to the subject PEG-TeNT containing PEGylated c (PEG-TeNT-c); then administering to the subject a composition containing PEG-TeNT-HC and PEG-TeNT-LC-c; then administering to the subject a composition containing PEG-TeNT containing PEGylated LC and PEGylated HC. The method includes administering PEG-TeNT (PEG-TeNT-LC-HC) to a subject, determining the subject's anti-TeNT antibody immune profile, and determining an effective composition of PEG-TeNT based on this profile.

[0048] In another embodiment, treatment with TeNT further comprises administering inactivated TeNT. Relative to SEQ ID NO: 1, the inactivated TeNT may comprise: R1225K; R1225E; W1228A; 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. In one embodiment, the second TeNT comprises R1225E and W1288A; E270A; Y374A; E270A and Y374A; G270del; Y374del; or a combination thereof.

[0049] In one embodiment, the hypotonia is obstructive sleep apnea.

[0050] 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.

[0051] In one embodiment, the composition or PEG-TeNT is used according to the method of the third or sixth aspect, respectively. [Brief explanation of the drawings]

[0052] [Figure 1]Figure 1 shows a schematic diagram of an example PEG-TeNT of the present invention. Figure 1A shows PEG-TeNT-c, Figure 1B shows PEG-TeNT-HC, Figure 1C shows PEG-TeNT-LC-c, and Figure 1D shows PEG-TeNT-LC-HC, where TeNT is intact and active, i.e., TeNT contains both chains, and PEGylation is present in specific regions, i.e., c, HC, LC-c, or LC-HC, respectively. [Figure 2] FIG. 2 is the amino acid sequence of mature TeNT (SEQ ID NO: 1), which contains 1314 amino acids. [Figure 3] FIG. 3 is the nucleic acid sequence of the vector pRSET-TeNT (SEQ ID NO: 2), which encodes TeNT. [Figure 4] Figure 4 shows a map of the vector pRSET-TeNT encoding TeNT. TeNT is expressed with an N-terminal His6 tag. The nucleic acid is inserted into the MCS of the pRSET-A vector and expressed under the control of the T7 promoter. [Figure 5] FIG. 5 shows the amino acid sequence of HC (SEQ ID NO: 3) containing amino acids 457 to 1314 of SEQ ID NO: 1. [Figure 6] FIG. 6 shows the amino acid sequence of c (SEQ ID NO: 4) containing amino acids 864 to 1314 of SEQ ID NO: 1. [Figure 7] FIG. 7 is the nucleic acid sequence of vector pRSET-TeNT-c encoding c (SEQ ID NO:5). [Figure 8] Figure 8 shows a map of the vector pRSET-TeNT-c encoding c. c is expressed with an N-terminal His6 tag. The nucleic acid is inserted into the MCS of the pRSET-A vector and expressed under the control of the T7 promoter. [Figure 9] FIG. 9 shows the amino acid sequence of LC-HN (SEQ ID NO: 6) containing amino acids 1 to 863 of SEQ ID NO: 1. [Figure 10] FIG. 10 is the nucleic acid sequence of the vector pRSET-TeNT-LC-HN encoding LC-HN (SEQ ID NO: 7). [Figure 11]Figure 11 shows a map of the vector pRSET-TeNT-LC-HN encoding LC-HN. LC-HN is expressed with an N-terminal His6 tag. The nucleic acid is inserted into the MCS of the pRSET-A vector and expressed under the control of the T7 promoter. [Figure 12] FIG. 12 is the amino acid sequence of non-functional c (SEQ ID NO: 8) containing the amino acid substitutions W1288A and R1225E relative to SEQ ID NO: 1. [Figure 13] FIG. 13 is the nucleic acid sequence (SEQ ID NO: 9) of the vector pRSET-TeNT-c encoding the non-functional c of FIG. 12 (SEQ ID NO: 8) containing the amino acid substitutions W1288A and R1225E relative to SEQ ID NO: 1. [Figure 14] Figure 14 is a map of vector pRSET-TeNT-c encoding the non-functional c of Figure 12 (SEQ ID NO: 8) containing the amino acid substitutions W1288A and R1225E relative to SEQ ID NO: 1. c containing the amino acid substitutions W1288A and R1225E relative to SEQ ID NO: 1 is expressed with an N-terminal His6 tag. The nucleic acid is inserted into the MCS of the pRSET-A vector and expressed under the control of the T7 promoter. [Figure 15] FIG. 15 is the amino acid sequence of non-functional TeNT (SEQ ID NO: 10) containing the amino acid substitutions W1288A and R1225E relative to SEQ ID NO: 1. [Figure 16] FIG. 16 is the nucleic acid sequence (SEQ ID NO: 11) of vector pRSET-TeNT encoding the non-functional TeNT of FIG. 15 (SEQ ID NO: 10) containing the amino acid substitutions W1288A and R1225E relative to SEQ ID NO: 1. [Figure 17] Figure 17 is a map of vector pRSET-TeNT, which encodes the non-functional TeNT of Figure 15 (SEQ ID NO: 10) containing the amino acid substitutions W1288A and R1225E relative to SEQ ID NO: 1. TeNT containing the amino acid substitutions W1288A and R1225E relative to SEQ ID NO: 1 is expressed with an N-terminal His6 tag. The nucleic acid is inserted into the MCS of the pRSET-A vector and expressed under the control of the T7 promoter. [Figure 18]FIG. 18 is the amino acid sequence of the mature 1,314 amino acid TeNT (SEQ ID NO: 12), which contains the surface serine-to-cysteine ​​amino acid substitutions S81C, S120C, S144C, S248C, S335C, S428C, S600C, S963C, S1041C, S1155C, and S1187C relative to SEQ ID NO: 1. [Figure 19] FIG. 19 is the nucleic acid sequence (SEQ ID NO: 13) of the vector pRSET-TeNT encoding mature TeNT with a surface serine-to-cysteine ​​substitution of FIG. 18 (SEQ ID NO: 12). [Figure 20] Figure 20 is a map of the vector pRSET-TeNT of Figure 19 (SEQ ID NO: 13), which encodes mature TeNT with a surface serine-to-cysteine ​​substitution of Figure 18 (SEQ ID NO: 12). TeNT with a surface serine-to-cysteine ​​substitution is expressed with an N-terminal His6 tag. The nucleic acid is inserted into the MCS of the pRSET-A vector and expressed under the control of the T7 promoter. [Figure 21] Figure 21 is the amino acid sequence of the mature 1314 amino acid TeNT (SEQ ID NO: 14) containing the 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] FIG. 22 is the nucleic acid sequence (SEQ ID NO: 15) of the vector pRSET-TeNT encoding mature TeNT with a surface serine-to-cysteine ​​substitution of FIG. 21 (SEQ ID NO: 14). [Figure 23] Figure 23 is a map of the vector pRSET-TeNT of Figure 22 (SEQ ID NO: 15), which encodes mature TeNT with a surface serine-to-cysteine ​​substitution of Figure 21 (SEQ ID NO: 14). TeNT with a surface serine-to-cysteine ​​substitution is expressed with an N-terminal His6 tag. The nucleic acid is inserted into the MCS of the pRSET-A vector and expressed under the control of the T7 promoter. [Figure 24]FIG. 24 is the amino acid sequence of TeNT (SEQ ID NO: 16) containing the HC surface serine-to-cysteine ​​amino acid substitutions S600C, S963C, S1041C, S1155C, and S1187C relative to SEQ ID NO: 1. [Figure 25] FIG. 25 is the nucleic acid sequence (SEQ ID NO: 17) of the vector pRSET-TeNT encoding mature TeNT with a surface serine-to-cysteine ​​substitution of FIG. 24 (SEQ ID NO: 16). [Figure 26] Figure 26 is a map of the vector pRSET-TeNT of Figure 25 (SEQ ID NO: 17), which encodes 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 His6 tag. The nucleic acid is inserted into the MCS of the pRSET-A vector and expressed under the control of the T7 promoter. [Figure 27] Figure 27 shows a three-dimensional protein structure model of TeNT derived from crystallography data deposited in the Protein Data Bank (accession ID PDB:5N0B) mapping epitopes recognized by the major human antibody clonotypes identified by da Silva Antunes et al. (2017) and Palermo et al. (2017) to a model using Discovery Studio. Surface serine residues in or surrounding the identified epitopes were selected for mutation to cysteines for subsequent PEGylation. [Figure 28] Figure 28 shows two photographs of SDS-PAGE analysis of PEG-TeNT containing increasing molecular weight PEGs and detected by Western blot using (A) Coomassie Blue and (B) a polyclonal anti-TeNT antibody. (B) shows that immunogenicity is proportional to the molecular weight of the PEG. Molecular weight markers in kDa are on the left: Lane 1, TeNT; Lane 2, 2 kDa PEG-TeNT-HC-LC; Lane 3, 5 kDa PEG-TeNT-LC-HC; Lane 4, 10 kDa PEG-TeNT-LC-HC; Lane 5, 20 kDa PEG-TeNT-LC-HC; Lane 6. [Figure 29]Figure 29 shows four line graphs representing competitive ELISA assays. Four PEG-TeNTs and four PEG-TeNT-LC-c serine mutants, each containing a different molecular weight PEG (2 kDa, 5 kDa, 10 kDa, and 20 kDa), were assayed for TeNT using a polyclonal anti-TeNT antibody. (A) TeNT was adsorbed to an ELISA plate and then probed with polyclonal anti-TeNT antibodies preincubated with each of the four PEG-TeNT antigens (2 kDa, 5 kDa, 10 kDa, and 20 kDa) at four concentrations (10 μg / ml, 1 μg / ml, 0.1 μg / ml, and 0.01 μg / ml). (B) Each PEG-TeNT (2 kDa, 5 kDa, 10 kDa, and 20 kDa) was adsorbed onto a separate ELISA plate and then probed with polyclonal anti-TeNT antibodies preincubated with each of four concentrations of TeNT antigen (10 μg / ml, 1 μg / ml, 0.1 μg / ml, and 0.01 μg / ml). (C) TeNT-LC-c serine mutants were adsorbed onto an ELISA plate and then probed with polyclonal anti-TeNT antibodies preincubated with each of four concentrations of PEG-TeNT-LC-c serine mutant antigens (2 kDa, 5 kDa, 10 kDa, and 20 kDa) (10 μg / ml, 1 μg / ml, 0.1 μg / ml, and 0.01 μg / ml). (D) Each PEG-TeNT-LC-c serine mutant (2 kDa, 5 kDa, 10 kDa, and 20 kDa) was adsorbed to a separate ELISA plate and then probed with polyclonal anti-TeNT antibody preincubated with each of four concentrations of TeNT-LC-c serine mutant antigen (10 μg / ml, 1 μg / ml, 0.1 μg / ml, and 0.01 μg / ml). [Figure 30]Figure 30 shows four photographs showing hindlimb localized muscle tetanus in the hindlimbs of female C57BL / 6 mice injected with PEG-TeNT-LC-HC. Figures 30A and 30B show hindlimb localized muscle tetanus in naive mice. Figure 30C shows the absence of hindlimb muscle tetanus in mice vaccinated with tetanus toxoid after injection of 100 units of TeNT, while Figure 30D shows the persistence of hindlimb muscle tetanus in mice vaccinated with tetanus toxoid after injection of 80 units of PEG-TeNT LC-HC 20 kDa. [Figure 31] Figure 31 shows two dot plots showing the clinical levels of tetanus in the hind paws of female C57BL / 6 mice after injection of defined units of TeNT, PEG-TeNT-LC-HC 20 kDa, TeNT-LC-cSerine mutant, PEG-TeNT-LC-cSerine mutant 2 kDa, or PEG-TeNT-LC-cSerine mutant 20 kDa. Figure 31A) Mice were injected with TeNT or PEG-TeNT-LC-HC 20 kDa. Figure 31B) Mice were injected with TeNT, TeNT-LC-cSerine mutant, PEG-TeNT-LC-cSerine mutant 2 kDa, or PEG-TeNT-LC-cSerine mutant 20 kDa. One unit is the minimum dose of toxin required to induce stage 4 muscle tetanus in naive mice within 24 hours. [Figure 32] Figure 32 shows two line graphs showing the progression of clinical tetanus in mice vaccinated with tetanus toxoid. Figure 32A shows the progression of tetanus after injection of 4000 units of TeNT with or without an inactivated TeNT decoy. Figure 32B shows the progression of tetanus after injection of 40 units of TeNT-PEG 20 kDa with or without an inactivated TeNT decoy. In both cases, progression is clearly enhanced by the presence of the decoy. [Figure 33]Figure 33 is a boxplot showing the effect of increasing doses of tetanus toxin on the respiratory disturbance index (RDI) in British bulldogs treated according to Example 23. The black bars represent the median RDI of six trials at each dose of tetanus toxin. The shaded boxes represent the interquartile range, and the whiskers represent the minimum and maximum RDI samples at each dose. Increasing tetanus toxin was associated with a lower RDI after administration of 10 IU / kg compared to the RDI after placebo administration (P=0.043; Wilcoxon signed-rank test). In addition, the median values ​​for placebo and 10 IU / kg are represented by ◯ and *, respectively. DETAILED DESCRIPTION OF THE INVENTION

[0053] The present invention relates to immune-evasive TeNT and PEG-TeNT molecules (FIG. 1), compositions thereof, and their therapeutic and cosmetic uses. In one embodiment, the invention relates to the treatment of hypotonia, and optionally, obstructive sleep apnea.

[0054] Described herein are the uses of specifically modified TeNT and compositions of TeNT and decoy (inactivated TeNT) to treat muscle hypotonia in subjects with a protective immune response to tetanus toxoid. To achieve this goal, active TeNT is modified by the addition of PEG, the introduction of specific mutations, or a combination thereof, to deliver a bioactive compound capable of increasing muscle tone in tetanus-immune patients.

[0055] Activity can be demonstrated by administering a defined unit dose of the modified toxin or formulation; at the same unit dose, TeNT will not exhibit activity in vaccinated subjects. Based on the three-dimensional structural analysis of TeNT, the introduction of specific surface mutations for specific binding of PEG molecules enabled masking of specific TeNT epitopes known to be targeted by protective antibody responses in vaccinated subjects. The combination of PEGylation, site-specific mutations, and decoy molecule formulation significantly enhanced the effect of the molecule on increasing muscle tone in vaccinated mammalian models compared to the administration of an equivalent unit of TeNT.

[0056] Patent Document 1 discloses a series of PEGylated botulinum toxins for the treatment of disorders of inappropriate muscle contraction and suggests that TeNT can be used as an alternative to botulinum toxin. However, TeNT cannot be used to treat muscle contraction. Furthermore, the claimed invention of Patent Document 1 does not appear to be effective because the disclosed method does not involve site-specific masking of epitopes, and to the best of the inventors' knowledge, the three-dimensional structure and identification of the epitopes required for the deliberate masking of TeNT epitopes were not available at the priority date of Patent Document 1.

[0057] Wan et al. addressed the effect of PEGylation on the anti-PEG immune response resulting from the administration of PEGylated proteins, but did not develop any therapeutic insights. Although Wan et al. demonstrated reduced immunogenicity of PEGylated tetanus toxoid relative to non-PEGylated tetanus toxoid, they did not present any therapeutically relevant molecules or formulations. Furthermore, because tetanus toxoid is a bioinactive form of TeNT used for vaccination that can be produced by formaldehyde crosslinking of TeNT, PEGylation of tetanus toxoid does not involve modification of active TeNT. That is, tetanus toxoid, whether PEGylated or not, does not possess the combined enzymatic, binding, and translocation activities of active TeNT.

[0058] Patent Document 2 relates to the TeNT c fragment alone, which is a molecule that has no specific activity beyond binding to neurotransmitters and entering neurons.

[0059] Thus, there is a need for a TeNT-based therapy that circumvents pre-existing anti-TeNT immunity in tetanus toxoid-immunized subjects. Disclosed herein is a solution to this problem, provided in part by a masked, active, and therapeutically suitable PEGylated TeNT.

[0060] Tetanus neurotoxin (TeNT) TeNT is approximately 150 kDa and is expressed from the tetX gene. A codon-optimized nucleic acid sequence corresponding to the coding region of tetX but lacking the initiating methionine codon is provided in the vector sequence (SEQ ID NO: 2) in Figure 3. TeNT is expressed as a single protein that is post-translationally cleaved to remove first the initiating methionine and then two portions: a 50 kDa light chain (LC or A chain) from the N-terminus of the uncleaved protein and a 100 kDa heavy chain (HC or B chain) 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).

[0061] LC has zinc endopeptidase activity and attacks vesicle-associated membrane proteins (VAMPs) that are required for vesicle fusion with the membrane, thereby preventing neurotransmitter release.

[0062] Upon digestion with papain, HC can be cleaved into two domains, each 50 kDa: an N-terminal translocation domain, designated HN; and a C-terminal ganglioside (membrane) binding domain, designated fragment c (c). c-deficient TeNT is designated herein as LC-HN.

[0063] c has two polysialoganglioside-binding sites and binds to polysialogangliosides (GD2, GD1b, and GT1b) on the neuronal membrane, thus mediating the binding of TeNT to the presynaptic membrane of peripheral motor axons and facilitating the translocation of TeNT across this membrane into neurons.

[0064] Regarding the amino acid sequence: The amino acid sequence of TeNT lacking the initiating methionine is provided in Figure 2 (SEQ ID NO: 1); The amino acid sequence of HC is provided in Figure 5 (SEQ ID NO:3); The amino acid sequence of c is provided in Figure 6 (SEQ ID NO:4); and The amino acid sequence of LC-HN is provided in Figure 9 (SEQ ID NO: 6).

[0065] For codon-optimized nucleic acid sequences encoding vectors and vector maps: The sequence of c is provided in Figure 7 (SEQ ID NO: 5) and Figure 8; and The sequence of LC-HN is provided in FIG. 10 (SEQ ID NO: 7) and FIG.

[0066] As used herein, "TeNT" refers to the complete TeNT molecule consisting of a heavy chain and a light chain. Subdomains and fragments are represented herein by these abbreviations: light chain "LC"; heavy chain "HC"; heavy chain N-terminal domain "HN"; heavy chain fragment c "c"; and light chain + heavy chain N-terminal domain "LC-HN" (i.e., a TeNT molecule without c). If any subdomain or fragment is PEGylated, the prefix PEG is used: PEG-LC; PEG-HC; PEG-HN; PEG-c; PEG-LC-HN. For a complete TeNT molecule containing a PEGylated fragment or subdomain, the prefix PEG is used to indicate the PEGylated subdomain or fragment: PEG-TeNT-LC; PEG-TeNT-HC; PEG-TeNT-LC-HC; PEG-TeNT-HN; PEG-TeNT-c; PEG-TeNT-LC-c; PEG-TeNT-LC-HN, etc.

[0067] It will be appreciated that subdomains and fragments are not interchangeable with respect to the complete TeNT, as they have unique functions.

[0068] The TeNT disclosed herein may be active or inactive. Active TeNT has the same biological activity as native TeNT. Inactive TeNT lacks one or more native TeNT activities. In one embodiment, inactive TeNT does not block the release of inhibitory neurotransmitters. The inactive TeNT of the present disclosure may also be referred to as a "decoy." Examples of inactive TeNT include tetanus toxoid. In one embodiment, the inactive TeNT is the inactive TeNT disclosed herein.

[0069] In one embodiment, two or more TeNTs may be linked.

[0070] In another embodiment, the TeNT is unconjugated. In one embodiment of the compositions, methods, and uses disclosed herein, the first PEG-TeNT is unconjugated to the second TeNT.

[0071] The immune evasive PEG-TeNTs disclosed herein and illustrated in FIG. 1 include: TeNT with PEGylated fragment c (PEG-TeNT-c) ( Figure 1 A); TeNT with PEGylated heavy chain (PEG-TeNT-HC) ( Figure 1 B); TeNT with PEGylated light chain and PEGylated fragment c (PEG-TeNT-LC-c) (Figure 1C); and Fully PEGylated TeNT (PEG-TeNT-LC-HC) (Figure 1D) Examples include:

[0072] PEG-TeNT-c advantageously circumvents pre-existing immune responses of the adaptive immune system in vaccinated subjects. In one embodiment, PEG-TeNT-c provides a first-line treatment that is used until its effectiveness diminishes.

[0073] PEG-TeNT-HC and PEG-TeNT-LC-c advantageously circumvent pre-existing immune responses of the adaptive immune system in vaccinated subjects, as well as immune responses of the adaptive immune system induced by repeated exposure to PEG-TeNT-c.

[0074] In one embodiment, PEG-TeNT-HC and PEG-TeNT-LC-c together provide a second line treatment that is used until their effectiveness diminishes.

[0075] PEG-TeNT-LC-HC advantageously evades pre-existing immune responses of the adaptive immune system in vaccinated subjects, and PEG-TeNT-c and PEG-TeNT-HC+PEG -It also avoids the immune response of the adaptive immune system induced by repeated exposure to TeNT-LC-c.

[0076] In one embodiment, PEG-TeNT-LC-HC provides a third line treatment that is used until its effectiveness diminishes.

[0077] Also disclosed are TeNT with a PEGylated light chain (PEG-TeNT-LC), TeNT with a PEGylated LC and HN (PEG-TeNT-LC-HN), and TeNT with a PEGylated HN (PEG-TeNT-HN).

[0078] Those skilled in the art will appreciate that the particular combinations of PEG-TeNT and the sequence of treatment with these PEG-TeNT combinations may be varied.

[0079] Polyethylene glycol (PEG) PEG can be attached to, for example, lysine (e.g., amino-PEGylation), cysteine ​​(e.g., thiol-PEGylation and cross-linking 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. Examples of PEGylation also include N-terminal PEGylation and C-terminal PEGylation.

[0080] PEGylation may be carried out by reacting PEG with hydroxyl-labile functional groups such as anhydrides, acid chlorides, chloroformates, and carbonates. Alternatively, PEGylation may be carried out using functional groups such as aldehydes, esters, and amides.

[0081] PEG can be linear or branched.

[0082] The PEG can be a modified PEG, for example, poly[oligo(ethylene glycol) methyl ether methacrylate] (POEGMA).

[0083] The PEGylation may be site-specific PEGylation.

[0084] In one embodiment, surface serine residues of TeNT or a TeNT fragment are mutated to surface cysteine ​​residues to facilitate specific PEG conjugation at immunogenic epitopes. In this context, mutation is synonymous with substitution, e.g., serine to cysteine ​​substitution. Such mutations or substitutions include any combination of one or more of S81C; S120C; S144C; S248C; S335C; S428C; S600C; S963C; S1041C; S1155C; and S1187C.

[0085] Functional groups of heterobifunctional PEGs include maleimide, vinyl sulfone, pyridyl disulfide, amine, carboxylic acid, and NHS ester.

[0086] In one embodiment, PEG is conjugated to TeNT using a carboxyl-amine crosslink using carbodiimide-EDC and sulfo-NHS.

[0087] The present invention also contemplates PEG-TeNT, which comprises PEG of different molecular weights attached to different subdomains or fragments of TeNT.

[0088] PEG may be conjugated or attached to the disclosed TeNT for, for example, 2 to 6 hours at 4° C. to 25° C. In one embodiment, PEG was conjugated to TeNT for 6 hours at room temperature.

[0089] efficacy As used herein, "hypotonia" refers to any disorder involving involuntary muscle weakness that can be treated by inhibiting inhibitory neurotransmitters, such as GABA or glycine. As such, "hypotonia" includes hypotonia secondary to neurological drive weakness or other causes, as well as conditions of reduced or insufficient muscle tone, strength, or neurological drive. Thus, in one embodiment, the hypotonia can be neurogenic hypotonia.

[0090] Hypotonic disorders that may be treated with a PEG-TeNT, composition, or method according to the present disclosure include obstructive sleep apnea, apnea, snoring, scoliosis, strabismus due to muscle atonia, ptosis, Horner's syndrome, muscle atrophy, neuropathic muscles, amyotrophic lateral sclerosis (ALS), motor neuron disease, any muscle disease, multiple sclerosis, Parkinson's disease, myasthenia gravis, facial muscle tone, possibly ectropion, flaccid paralysis or weakness due to any skeletal or smooth muscle cause, respiratory muscle weakness due to any cause including post-ventilation weakness, poor posture due to trauma-induced muscle weakness or atonia, pelvic floor muscle atonia or weakness, or nasal or upper airway relaxation.

[0091] Other diseases that may be treated with PEG-TeNT, compositions, or methods according to the present disclosure include muscle atrophy, muscular dystrophy, muscle mass loss, nasal congestion, impotence, alopecia, hypotension, temporomandibular syndrome, torticollis, neck pain, intramuscular nerve regeneration, migraine, headache, achalasia, obesity, irritated colon, anal fissures, tissues or organs affected by gastric acid, prostatic hyperplasia, rhinorrhea, salivation, irritation of the lung mucosa, psoriasis, immune tolerance, immune response.

[0092] In cases where the disorder being treated by the present disclosure is not a hypotonic disorder per se, increasing muscle tone by treatment with TeNT of the present disclosure may alleviate the symptoms of the disorder.

[0093] Cosmetic applications of PEG-TeNT can include abdominal muscle strengthening, pectoral muscle strengthening, gluteus maximus muscle strengthening, skeletal muscle strengthening, or treatment of facial ptosis caused by muscle relaxation.

[0094] Smooth muscle, skeletal muscle, tissue or organ that may be treated with PEG-TeNT, compositions or methods according to the present disclosure include upper esophagus, esophageal wall, esophageal sphincter, lower esophageal sphincter, anal sphincter, bladder, bladder sphincter, vaginal sphincter, pyloric sphincter, sphincter of Oddi, ileocecal sphincter, pelvic floor muscles, vaginal wall muscles, prostate, submandibular gland, parotid gland, sublingual gland, minor salivary glands of the oral mucosa, vocal folds, facial muscles, muscles of mastication, superficial cranial muscles, chest muscles, back muscles, upper limb muscles, These include muscles of the forearm, lower leg, hand, foot, stomach wall, colon wall, neck, dilator pharyngeal muscles, masseter, medial pterygoid, lateral pterygoid, geniohyoid, genioglossus, tensor veli palatini, levator veli palatini, stylopharyngeus, styloglossus, mylohyoid, stylohyoid, hyoglossus, digastric, sternocleidomastoid, trapezius, temporalis, cricopharyngeal, uterine muscles and cervix, gastric nerve distribution, nasal mucosa, pulmonary mucosa, skin, thymus, bone, coronary arteries, pulmonary smooth muscle, and cardiac muscle.

[0095] Compositions and Administration The compositions of the present disclosure may be therapeutic or cosmetic compositions, i.e., the compositions may be used for therapeutic or cosmetic purposes.

[0096] As used herein, the term "therapeutic composition" or "cosmetic composition" refers to a composition comprising TeNT that inhibits or treats hypotonia in a subject as described herein. The composition is formulated for administration to a subject. In one embodiment, the composition is sterile. In one embodiment, the composition is pyrogen-free. The composition may include a pharmaceutically acceptable carrier. Preferably, the composition is formulated according to Good Laboratory Practice (GLP) or pharmaceutical industry standards. It is manufactured in accordance with the Good Manufacturing Practice (GMP) regulations for quasi-drugs.

[0097] The TeNT of the present disclosure may be administered up to 10 mg / kg or more. The TeNT of the present 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, about 10 ng / kg, about 11 ng / kg, about 12 ng / kg, about 13 ng / kg, about 14 ng / kg, about 15 ng / kg, about 16 ng / kg, about 17 ng / kg, about 18 ng / kg, about 19 ng / kg, about 20 ng / kg, about 30 ng / kg, about 40 ng / kg, about 50 ng / kg, about 60 ng / kg, about 70 ng / kg, about 80 ng / kg, about 90 ng / kg, about 100 ng / kg, about 200 ng / kg, about 300 ng / kg, about 400 ng / kg, about 500 ng / kg, about 600 ng / kg, about 700 ng / kg, about 800 ng / kg, about 900 ng / kg, about 1 μg / kg, about 5 μg / kg, about 10 μg / kg, about 50 μg / kg, about 100 μg / kg, about 500 μg / kg, about 1 mg / kg, or about 10 mg / kg. The TeNT of the present disclosure may be administered within any range of any of the above dosages.

[0098] The TeNT of the present disclosure may be administered at up to 1000 IU / kg or more. The TeNT of the present 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, 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 21 IU / kg, about 22 IU / kg, about 23 IU / kg, about 24 IU / kg, about 25 IU / kg, about 26 IU / kg, about 27 IU / kg, about 28 IU / kg, about 29 IU / kg, about 30 IU / kg, about 31 IU / kg, about 32 IU / kg, about 33 IU / kg, about 34 IU / kg, about 35 IU / kg, about 36 IU / kg, about 37 IU / kg, about 38 IU / kg, about 39 IU / kg, about 40 IU / kg, about 41 IU / kg, about 42 IU / kg, about 43 IU / kg, about 44 IU / U / 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 1000 IU / kg. The TeNT of the present disclosure may be administered within any of the ranges of any of the above dosages.

[0099] In a composition comprising two PEG-TeNTs, e.g., a first PEG-TeNT in which TeNT-HC is PEGylated (PEG-TeNT-HC) and a second PEG-TeNT in which TeNT-LC and TeNT-c are PEGylated (PEG-TeNT-LC-c), the ratio of the first PEG-TeNT to the second PEG-TeNT can vary. For example, the ratio of the first PEG-TeNT to the second PEG-TeNT can 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.

[0100] The composition may include any combination of TeNTs, including but not limited to PEG-TeNT-HC and PEG-TeNT-LC-c. The composition may include: 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; and PEG-TeNT-LC-c and PEG-TeNT-LC-HC. 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 Also disclosed are compositions comprising TeNT-LC-c and PEG-TeNT-LC-HC; and PEG-TeNT-c, PEG-TeNT-HC, PEG-TeNT-LC-c, and PEG-TeNT-LC-HC. Any of the compositions may be substituted for TeNT, and any of the compositions may further comprise PEG-TeNT-LC, PEG-TeNT-LC-HN, and / or PEG-TeNT-HN.

[0101] In one embodiment, the composition further comprises inactivated TeNT to act as a decoy for anti-TeNT antibodies generated by prior exposure to TeNT, for example, by vaccination. The inactivated TeNT may comprise, relative to SEQ ID NO: 1, 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 R1225del and W1288del; E270A; Y374A; E270A and Y374A; E270del; Y374del, or combinations thereof. In one embodiment, the second TeNT comprises an inactivated TeNT comprising R1225E, W1288A, E270A and Y374A.

[0102] In a composition comprising at least one PEG-TeNT and decoy TeNT, the decoy TeNT may be in molar excess relative to the PEG-TeNT. For example, the ratio of decoy TeNT to PEG-TeNT may be about 10 6 :1;10 5 :1, 10 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.

[0103] PEG-TeNT of the present disclosure may be administered once, twice, or three times weekly, once, twice, or three times monthly, once, twice, or three times quarterly, once, twice, or three times every six months, or once, twice, or three times yearly.

[0104] PEG-TeNT may be administered in a single dose, divided doses, or multiple doses. When muscles are paired, PEG-TeNT may be administered unilaterally to one muscle of the pair or bilaterally to both muscles of the pair.

[0105] As an alternative to combinations of the present disclosure that include two or more PEG-TeNTs, such two or more PEG-TeNTs may be administered in combination, either sequentially or simultaneously.

[0106] PEG-TeNT may be administered locally to a subject by any suitable method, for example, injection, surgical implantation, topical application, or intranasal administration. In one embodiment, TeNT is administered intramuscularly by injection into the affected muscle.

[0107] PEG-TeNT will be formulated, dosed, and administered in a manner consistent with principles of good medical practice. Factors to consider in this regard include the particular type of hypotonia being treated, the particular subject being treated, the subject's clinical condition, the site of administration, the method of administration, the administration schedule, and other factors known to medical practitioners, including dentists. The therapeutically effective amount of PEG-TeNT to be administered will be governed by such considerations.

[0108] Pharmaceutically acceptable carriers include water, buffered water, saline solutions such as physiological saline or balanced saline solutions such as Hank's or Earle's balanced solution, glycine, and hyaluronic acid.

[0109] Compositions can be formulated for intramuscular administration. Compositions for intramuscular administration can include pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for preparation into sterile injectable solutions or dispersions. Examples of suitable aqueous or non-aqueous carriers, solvents, diluents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol), carboxymethylcellulose and their mixtures, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate).

[0110] The composition may include a penetration enhancer to enhance delivery of TeNT, which may include oleic acid, lauric acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprylate, reclineate, monoolein, dilaurin, caprylic acid, arachidonic acid, glyceryl 1-monocaprate, mono- and diglycerides, and physiologically acceptable salts thereof.

[0111] The compositions may further include chelating agents such as, for example, ethylenediaminetetraacetic acid (EDTA), citric acid, salicylates (eg, sodium salicylate, 5-methoxysalicylate, homovanilate).

[0112] Also provided are articles of manufacture and / or kits comprising a container housing PEG-TeNT or a composition comprising PEG-TeNT. The container may be a bottle, vial, or syringe containing PEG-TeNT or the composition, optionally in unit dosage form. For example, the PEG-TeNT or composition may be in the form of an injectable solution in a disposable container, optionally a syringe. The articles of manufacture and / or kits may further comprise printed instructions and / or labels, etc., indicating treatment of a subject with the methods disclosed herein.

[0113] The term "therapeutically effective amount" refers to an amount of PEG-TeNT effective to treat hypotonia in a subject.

[0114] The terms "treat," "treating," or "treatment" refer to both therapeutic treatments and prophylactic or preventative measures aimed at preventing, reducing, or ameliorating hypotonia in a subject, or slowing (reducing) the progression of hypotonia in a subject. Subjects in need of treatment include those already suffering from hypotonia as well as those in whom hypotonia is to be prevented or ameliorated.

[0115] The terms "preventing," "prevention," "preventative," or "prophylactic" refer to avoiding or hindering the onset of hypotonia, guarding against or protecting against the onset of hypotonia. A subject in need of prevention may be prone to suffering from hypotonia.

[0116] The term "ameliorate" or "amelioration" refers to decreasing, reducing or eliminating hypotonia.

[0117] 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 distinct 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. Other semi-quantitative scales will be readily apparent to those skilled in the art. In another embodiment, hypotonia may be quantified on a quantitative scale, for example, using a force gauge.

[0118] Any quantification of hypotonia may be compared to a control, e.g., a healthy control subject not receiving PEG-teNT, or a diseased control patient receiving treatment for hypotonia but not treated with PEG-TeNT.

[0119] Treatment of hypotonia by administering PEG-TeNT resulted in approximately 1% decrease in muscle tone, approximately 2% decrease, approximately 3% decrease, approximately 4% decrease, approximately 5% decrease, approximately 6% decrease, approximately 7% decrease, approximately 8% decrease, and approximately 9 % reduction, about 10% reduction, about 20% reduction, about 30% reduction, about 40% reduction, about 50% reduction, about 60% reduction, about 70% reduction, about 80% reduction, about 90% reduction, or about 100% reduction.

[0120] As used herein, the term "subject" may refer to a mammal. The mammal may be a primate, particularly a human, or may be a domestic animal, a zoo animal, or a companion animal. While the PEG-TeNT compositions and methods disclosed herein are particularly contemplated as being suitable for the medical treatment of humans, they are also applicable to veterinary treatment, including the treatment of domestic animals such as horses, cattle, and sheep, companion animals such as dogs and cats, or zoo animals such as felines, canines, bovines, and ungulates.

[0121] Unless otherwise defined herein, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art and in the published literature to which this invention belongs.

[0122] It should be noted that the terms "a" or "an" refer to one or more, e.g., "a TeNT" is understood to refer to one or more TeNT. Thus, the terms "a" or "an," "one or more," and "at least one" may be used interchangeably herein.

[0123] In the following claims and in the specification of the present invention, unless the context requires otherwise by language or implication, the words "comprise" or "comprises" or "comprising" are used in a non-exclusive sense, i.e., to specify the presence of stated features in various embodiments of the invention, but not to preclude the presence or addition of further features.

[0124] As used herein, the term "about" refers to a range of values ​​for a given number of ±25% of the magnitude of that number. In other embodiments, the term "about" refers to a range of values ​​for a given number of ±20%, ±15%, ±10%, ±5%, ±4%, ±3%, ±2%, or ±1% of the magnitude of that number. For example, in one embodiment, about 3 grams refers to 2.7 grams to 3.3 grams (i.e., 3 grams ±10%), etc.

[0125] Similarly, the timing or duration of an event may vary by at least 25%. For example, in one embodiment, a particular event may be disclosed that lasts one day, although the event may last longer or shorter than one day. For example, "one day" may include a period of about 18 hours to about 30 hours. In other embodiments, the time may vary by ±20%, ±15%, ±10%, ±5%, ±4%, ±3%, ±2%, or ±1% of that time. [Example]

[0126] Example 1 - Preparation of PEG-TeNT-c (Figure 1A) In this example, surface serine residues of TeNT-c are mutated to surface cysteine ​​residues to facilitate specific PEG attachment at immunogenic epitopes, producing the molecule shown in Figure 1A. The mutations are S963C, S1041C, S1155C, and S1187C.

[0127] Surface serine-to-cysteine ​​substitutions S963C, S1041C, S1155C, and The gene for TeNT with S1187C is synthesized by a commercial provider, such as Integrated DNA Technologies, and subcloned into the pRSET-A expression vector by restriction digestion, adding a 6x histidine tag from the vector to the N-terminus of the mutant protein.

[0128] TeNT containing S963C, S1041C, S1155C, and S1187C is expressed, PEGylated, and purified according to Example 5 to obtain PEG-TeNT-c.

[0129] Example 2 - Preparation of PEG-TeNT-HC (Figure 1B) In this example, surface serine residues of TeNT-HC are mutated to surface cysteine ​​residues to facilitate specific PEG attachment at immunogenic epitopes, producing the molecule of Figure 1B. The mutations are S600C, S963C, S1041C, S1155C, and S1187C (Figure 24, SEQ ID NO: 16).

[0130] The gene for TeNT with surface serine-to-cysteine ​​substitutions S600C, S963C, S1041C, S1155C, and S1187C is synthesized by a commercial provider, such as Integrated DNA Technologies, and subcloned into the pRSET-A expression vector (Figure 25, SEQ ID NO: 17, Figure 26) by restriction digestion, adding a 6x histidine tag from the vector to the N-terminus of the mutant protein.

[0131] TeNT containing S600C, S963C, S1041C, S1155C, and S1187C is expressed, PEGylated, and purified according to Example 5 to obtain PEG-TeNT-HC.

[0132] Example 3 - Preparation of PEG-TeNT-LC-c (Figure 1C) In this example, surface serine residues in LC and c were mutated to surface cysteine ​​residues to facilitate specific PEG attachment at the immunogenic epitope, producing 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).

[0133] The gene for TeNT with surface serine-to-cysteine ​​substitutions S81C, S120C, S144C, S248C, S335C, S428C, S963C, S1041C, S1155C, and S1187C in LC and c was synthesized by a commercial provider, Integrated DNA Technologies. The gene was subcloned into the pRSET-A expression vector (Figure 22, SEQ ID NO: 15, Figure 23) by restriction digestion, adding a 6x histidine tag from the vector to the N-terminus of the mutant protein.

[0134] TeNT containing S81C, S120C, S144C, S248C, S335C, S428C, S963C, S1041C, S1155C, and S1187C was expressed, PEGylated, and purified according to Example 5 to obtain PEG-TeNT-LC-c.

[0135] Endotoxin removal Endotoxin was removed from the TeNT-LC-cSerine mutant according to Example 4.

[0136] Conjugation of PEG to cysteine ​​residues 1. Molar excess of PEG - about 2 kDa, about 5 kDa, about 10 kDa, or about 20 kDa The maleimide bearing PEG was mixed with the serine mutant TeNT-LC-c (0.5–2 mg / mL) in PBS, pH 6.5–7.5. 2. Binding was carried out at room temperature for 6 hours. 3. Excess PEG was removed by size exclusion chromatography.

[0137] Protein trypsin digestion activity Trypsin digestion for activation of TeNT-LC-cSerine mutant was performed according to Example 4.

[0138] Example 4 - Preparation of PEG-TeNT-LC-HC (FIG. 1D) (Method 1)

[0139] TeNT production 1. E. coli BL21 DE3 pLysS strain was electrotransformed with the pRSET-TeNT vector (FIGS. 3 and 4) and grown overnight on LB agar with selection antibiotics (ampicillin and chloramphenicol) at 37°C. 2,200 mL of pre-induction broth was inoculated with one colony from the selection plate. 1.2% tryptone; 2.4% yeast extract; 2% glucose; 0.4% glycerol; 17 mM Pre-induction broth pH 7.2–7.4 containing: 72 mM KHPO; 72 mM KHPO; and selective antibiotics (ampicillin and chloramphenicol). 3. The culture was incubated overnight at 30°C with rapid shaking. 4. The overnight culture was harvested by centrifugation at 4000 g for 10 min. 5. Resuspend the pellet in 200 mL of expression broth pH 7.2-7.4 containing 1.2% tryptone; 2.4% yeast extract; 0.4% glycerol; 1 mM IPTG; 17 mM KH2PO4; 72 mM K2HPO4; 100 μg / mL ampicillin; and 10 μM ZnCl2. 6. The protein was expressed for 6 hours at 30°C with high speed shaking. 7. Cells were harvested by centrifugation at 4500 g for 15 minutes and the pellet was resuspended in 30 mL of TBS at pH 8 containing 20 mM imidazole. 8. Cells were lysed by sonication. 9. The cell lysate was clarified by centrifugation at 4500 g for 20 minutes and filtered through a 0.45 μm filter. 10. The protein was purified by His-tag affinity chromatography using an AKTA pure 25 FPLC system (GE). 11. The purified protein was buffer exchanged into PBS using size exclusion chromatography, followed by a second stage of purification by gel filtration using an AKTA Pure 25 FPLC system equipped with a Superdex200 increase 10 / 300GL column.

[0140] Endotoxin removal 1. Equilibrate a 0.5 mL endotoxin removal spin column to room temperature. 2. Remove the column button plug, loosen the column cap, place the column in a 15 mL tube, and centrifuge at 500 g for 1 minute to remove the solution from the column. Discard the solution. 3. Replace the column button plug, remove the column cap, add 0.2 N NaOH in 95% ethanol to the resin, replace the column cap, invert the column several times to resuspend the resin, and then incubate at room temperature for 1-2 hours. 4. Remove the column button plug, loosen the column cap, place the column in a 15 mL tube, and centrifuge at 500 g for 1 minute to remove the solution from the column. Discard the solution. 5. Replace the column button plug, remove the column cap, and Add 2M NaCl to the resin, replace the column cap, and invert the column several times to resuspend the resin. 6. Remove the column button plug, loosen the column cap, place the column in a 15 mL tube, and centrifuge at 500 g for 1 minute to remove the solution from the column. Discard the solution. 7. Replace the column button plug, remove the column cap, add endotoxin-free ultrapure water to the resin, replace the column cap, and invert the column several times to resuspend the resin. 8. Remove the column button plug, loosen the column cap, place the column in a 15 mL tube, and centrifuge at 500 g for 1 minute to remove the solution from the column. Discard the solution. 9. Replace the column button plug, remove the column cap, add endotoxin-free phosphate buffer to the resin, replace the column cap, and invert the column several times to resuspend the resin. 10. Remove the column button plug, loosen the column cap, place the column in a 15 mL tube, and centrifuge at 500 g for 1 minute to remove the solution from the column. Discard the solution. 11. The column was rinsed twice more with phosphate buffer and the eluent was discarded. 12. The column button plug was replaced, the column cap was removed, the sample was applied to the resin, the column cap was replaced, and the column was inverted several times to resuspend the resin. 13. The column was incubated at 4°C for at least 1 hour with end-over-end mixing. 14. Remove the column button plug, loosen the column cap, place the column in an endotoxin-free 15 mL tube, and centrifuge at 500 g for 1 minute to remove the solution from the column. Retain the sample. 15. The endotoxin removal procedure was repeated using the regenerated spin column until the samples had similar or lower levels of endotoxin and all doses contained less than 5 EU units of endotoxin per kilogram of target.

[0141] Preparation of PEG-TeNT-LC-HC In a total volume of 1.500 μL of PBS pH 7.4, 3 μmol of purified TeNT and 0.5 mmol of mpeg-NHS(SC) (Nanocs) were conjugated with 2 kDa, 5 kDa, 10 kDa, 20 kDa, or 30 kDa PEG. 2. The samples were mixed for 3 hours at room temperature. 3. Excess PEG was removed by size exclusion chromatography.

[0142] Trypsin digestion of proteins into active forms 1. 1 mg of protein was dissolved in 0.5 mL digestion buffer containing 0.1 M NH4HCO3 buffer, pH 8.0 or 0.1 M Tris buffer, pH 8.5. 2. 0.10 mL to 0.25 mL of immobilized TPCK trypsin was washed with 3 x 500 µL of digestion buffer. After each wash, the gel was separated from the buffer by centrifugation. 3. The gel was resuspended in approximately 0.2 mL of digestion buffer. 4. Immobilized TPCK trypsin was added to the protein sample. 5. The reaction mixture was incubated at 37°C in a high speed shaking incubator for 2-18 hours. 6. The immobilized TPCK trypsin was separated by centrifugation.

[0143] Example 5 - Preparation of PEG-TeNT-LC-HC (Figure 1D) (Method 2) In this example, surface serine residues in TeNT-LC-HC were mutated to surface cysteine ​​residues (S to C mutations) to facilitate specific PEG conjugation at the immunogenic epitope. The TeNT mutations were S81C; S120C; S144C; S248C, S335C; S428C; S600C; S963C; S1041C; S1155C; and S1187C relative to SEQ ID NO: 1.

[0144] Preparation of serine mutant TeNT-LC-HC E. coli strain BL21(DE3)pLysS was electrotransformed with the vector pRSET-TeNT (Figures 19 and 20), which encodes the amino acid sequence of Figure 18 (SEQ ID NO: 12) containing an S to C mutation. TeNT-LC-HC containing an S to C mutation was expressed and purified according to Example 4, with the addition of a 15-minute 0.5 mM DTT treatment between steps 10 and 11.

[0145] Endotoxin removal Endotoxin was removed from the TeNT-LC-HC serine mutant according to Example 4.

[0146] Conjugation of PEG to cysteine ​​residues 1. A molar excess of PEG-maleimide with PEG of approximately 2 kDa, approximately 5 kDa, approximately 10 kDa, or approximately 20 kDa was mixed with serine mutant TeNT-LC-HC (0.5-2 mg / mL) in PBS, pH 6.5-7.5. 2. Binding was carried out at room temperature for 6 hours. 3. Excess PEG was removed by size exclusion chromatography.

[0147] Protein trypsin digestion activity Trypsin digestion for activation of TeNT-LC-HC serine mutants was performed according to Example 4.

[0148] Example 6 - Inactivated Decoy TeNT In this example, a non-PEGylated recombinant c was produced containing R1225E and W1288A amino acid substitutions relative to SEQ ID NO: 1 in the ganglioside-binding region. The inactivated c was conjugated with an equimolar amount of non-PEGylated LC-HN to produce inactivated decoy TeNT. The resulting inactivated TeNT is used as a decoy for antibody-based neutralizing responses in subjects vaccinated with tetanus toxoid.

[0149] Manufacture of c containing R1225E W1288A The c gene containing R1225E and W1288A was synthesized by Integrated DNA Technologies, a commercial provider, and subcloned into the pRSET-A expression vector by restriction digestion, adding a 6x histidine tag from the vector to the N-terminus of the mutant protein.

[0150] E. coli strain BL21(DE3)pLysS was electrotransformed with the pRSET-TeNT-c vector (FIG. 13 (SEQ ID NO:9) and FIG. 14) encoding c containing R1225E W1288A (FIG. 12, SEQ ID NO:8) and grown overnight at 37°C on LB agar containing selection antibiotics (ampicillin and chloramphenicol). c containing R1225E W1288A was expressed and purified as described in Example 4.

[0151] Endotoxin removal Endotoxin was removed from the purified protein according to Example 4.

[0152] LC-HN production E. coli BL21(DE3)pLysS strain was electrotransformed with the pRSET-TeNT-LC-HN vector (Figs. 10 and 11) and incubated at 37°C with a selection antibiotic (Ant). The cells were grown overnight on LB agar with 5% ethanol (picillin, chloramphenicol). LC-HN was expressed and purified as described in Example 4.

[0153] Endotoxin removal Endotoxin was removed from the purified protein according to Example 4.

[0154] Preparation of LC-HN containing E270A Y374A E. coli BL21(DE3)pLysS strain was electrotransformed with the pRSET-TeNT-LC-HN vector encoding LC-HN containing E270A Y374A and grown overnight on LB agar with selection antibiotics (ampicillin, chloramphenicol) at 37°C. LC-HN containing E270A Y374A was expressed and purified as described in Example 4.

[0155] Endotoxin removal Endotoxin was removed from the purified protein according to Example 4.

[0156] Example 7 - Inert Decoy TeNT The gene for TeNT containing R1225E and W1288A was synthesized by a commercial provider, Integrated DNA Technologies. The gene was inserted into the vector to insert a 6x histidine tag into the mutant protein (Figure 15, SEQ ID NO: 10, Figure 16). The resulting fragment was subcloned into the pRSET-A expression vector by restriction digestion so as to be appended to the N-terminus of SEQ ID NO: 11 (FIG. 17).

[0157] Inactivated TeNT containing R1225E and W1288A was expressed and purified according to Example 4.

[0158] Endotoxin removal Endotoxin was removed from the purified protein according to Example 4.

[0159] Example 8 - PEG-TeNT Analysis TeNT was prepared and PEGylated according to Example 4, then analyzed by SDS-PAGE (FIG. 28) and detected by (A) Coomassie blue and (B) Western blot using a polyclonal anti-TeNT antibody. (B) shows that immunogenicity is proportional to PEG molecular weight.

[0160] Example 9 - Reduced immunogenicity of PEG-TeNT relative to TeNT Four PEG-TeNTs, each containing a different molecular weight of PEG (2 kDa, 5 kDa, 10 kDa, and 20 kDa), were prepared and PEGylated according to Example 4. The PEG-TeNTs were then assayed against TeNT by competitive ELISA.

[0161] In the first assay (Figure 29A), TeNT was adsorbed to an ELISA plate. The adsorbed TeNT was then probed with polyclonal anti-TeNT antibodies preincubated with each of four concentrations (10 μg / ml, 1 μg / ml, 0.1 μg / ml, and 0.01 μg / ml) of four PEG-TeNT antigens (2 kDa, 5 kDa, 10 kDa, and 20 kDa). In this assay, a higher response (OD 450 nm) indicates greater affinity for TeNT and, therefore, less immunogenicity to PEG-TeNT.

[0162] In the second assay (Figure 29B), each PEG-TeNT was adsorbed to a separate ELISA plate. Then, each adsorbed PEG-TeNT (2 kDa, 5 kDa, 10 kDa, and The PEG-TeNT antibodies (10 kDa and 20 kDa) were probed with polyclonal anti-TeNT antibodies preincubated with each of four concentrations of TeNT antigen (10 μg / ml, 1 μg / ml, 0.1 μg / ml, and 0.01 μg / ml). In this assay, a lower response (OD 450 nm) indicates greater affinity for TeNT and, therefore, less immunogenicity for PEG-TeNT.

[0163] This example demonstrated that anti-TeNT antibodies selectively bind TeNT and that PEGylated TeNT has reduced immunogenicity compared to TeNT (ie, non-PEGylated).

[0164] Example 10 - Reduced immunogenicity of PEG-TeNT-LC-cSerine variants relative to TeNT-LC-cSerine variants The 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 containing a different molecular weight of PEG (2 kDa, 5 kDa, 10 kDa, and 20 kDa). The PEG-TeNT-LC-c serine mutant was then assayed against the TeNT-LC-c serine mutant by competitive ELISA.

[0165] In the first assay (Figure 29C), TeNT-LC-c serine mutants were adsorbed to an ELISA plate. The adsorbed TeNT serine mutants were then probed with polyclonal anti-TeNT antibodies preincubated with four PEG-TeNT serine mutant antigens (2 kDa, 5 kDa, 10 kDa, and 20 kDa) at four concentrations (10 μg / ml, 1 μg / ml, 0.1 μg / ml, and 0.01 μg / ml). In this assay, a higher response (OD 450 nm) indicates greater affinity for the TeNT serine mutant and, therefore, less immunogenicity of the PEG-TeNT serine mutant.

[0166] In the second assay (Figure 29D), 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 polyclonal anti-TeNT antibodies preincubated with each of four concentrations of TeNT serine mutant antigen (10 μg / ml, 1 μg / ml, 0.1 μg / ml, and 0.01 μg / ml). In this assay, a lower response (OD 450 nm) indicates greater affinity for the TeNT serine mutant and, therefore, less immunogenicity for the PEG-TeNT serine mutant.

[0167] This example demonstrated that anti-TeNT antibodies selectively bound to the TeNT-LC-cSerine mutant, and that the PEGylated TeNT-LC-cSerine mutant had reduced immunogenicity compared to the TeNT-LC-cSerine mutant (i.e., non-PEGylated).

[0168] Example 11 - Reduced immunogenicity of PEG-TeNT relative to TeNT A competitive ELISA assay is performed according to Example 9, except that the polyclonal antibodies are replaced with human sera collected from one or more subjects who have received a booster tetanus toxoid vaccination within the previous 12 months. Antibodies in the sera will exhibit greater affinity for TeNT (i.e., non-PEGylated TeNT) versus PEG-TeNT.

[0169] Example 12 - In vivo model PEG-TeNT-LC-HC was prepared according to Example 4 by conjugating PEG (about 5 kDa, about 10 kDa, or about 20 kDa) to the surface-exposed lysine residues of recombinant TeNT.

[0170] One or more units of PEG-TeNT-LC-HC in 15 μL of PBS were injected into the hind limbs of female C57BL / 6 mice, and each animal exhibited focal limb muscle tetanus within 48 hours of injection (FIGS. 30A and 30B).

[0171] Example 13 - In vivo model According to Example 4, PEG-TeNT-LC-HC, which contains 5 kDa PEG conjugated to surface lysine residues of TeNT, was conjugated to c decoy, i.e., c inactivated with R1225E W1288A, at a molar ratio of 1:10. A composition containing 5 ng of PEG-TeNT in 15 milliliters of PBS was injected intramuscularly into the hind limbs of female C57BL / 6 mice. Each animal developed focal limb muscle tetanus within 48 hours, with no discernible disease symptoms from animals treated with PEG-TeNT-LC-HC in the absence of the decoy.

[0172] Example 14. PEG-TeNT-LC-HC containing 5 kDa, 10 kDa, or 20 kDa PEG was administered intramuscularly at 50 to 500,000 ng / kg into the hindlimb muscles of mice pre-immunized with tetanus toxoid. Enhanced muscle contractions were observed in the injected muscles by day 3 and were greater than the effect observed in mice administered the same unit of TeNT. Figure 31A reports the results for PEG-TeNT-LC-HC 20 kDa.

[0173] Example 15. A composition containing TeNT-LC-HC prepared according to Example 6 and a 10:1 or 100:1 molar excess of decoy TeNT was administered intramuscularly at 50 to 500,000 ng / kg into the hind limb muscles of mice pre-immunized with tetanus toxoid. Increased muscle contractions were observed in the injected muscles for up to 3 days, an effect greater than that observed in mice administered TeNT (Figure 32A).

[0174] Example 16. Compositions containing PEG-TeNT-LC-HC containing 5 kDa, 10 kDa, or 20 kDa PEG and a 10:1 or 100:1 molar excess of decoy TeNT, prepared according to Example 6, were administered intramuscularly at 50 to 500,000 ng / kg into the hindlimb muscles of mice pre-immunized with tetanus toxoid. Increased muscle contractions were observed within the injected muscles for up to 3 days and were greater than the effects observed in mice administered TeNT and greater than the effects observed in mice administered PEG-TeNT alone (Figure 32B).

[0175] Example 17. PEG-TeNT-LC-HC containing 20 kDa PEG is administered intramuscularly at 0.01 to 50,000 ng / kg into the left geniohyoid muscle of human subjects previously vaccinated with tetanus toxoid. Increased muscle contraction is observed in the injected muscle for up to two weeks, and is greater than the effect observed in the right geniohyoid muscle of the same subjects receiving vehicle alone.

[0176] Example 18. A composition containing PEG-TeNT-LC-HC with 20 kDa PEG and a 10:1 or 1000:1 molar excess of decoy TeNT, prepared according to Example 6, is administered intramuscularly at 50 ng / kg into the left geniohyoid muscle of a human subject previously vaccinated with tetanus toxoid. PEG-TeNT-LC-HC with 20 kDa PEG is administered intramuscularly at 50 ng / kg into the right geniohyoid muscle of the same human subject.

[0177] Increased muscle contraction was observed in both the left and right geniohyoid muscles by 2 weeks, with PEG-TeNT-LC-HC and PEG-TeNT-LC-HC compared to PEG-TeNT-LC-HC alone. and the left geniohyoid muscle treated with a composition containing decoy TeNT.

[0178] Example 19. Approximately 30 kg bulldogs were intramuscularly administered 25-50,000 ng / kg of PEG-TeNT-c containing 20 kDa PEG in split doses bilaterally into the left and right geniohyoid muscles. Obstructive sleep apnea (OSA) was reduced in PEG-TeNT-treated animals compared to animals treated with vehicle alone. Bulldogs were monitored weekly for OSA, and PEG-TeNT-c administration was repeated as needed until efficacy was achieved, as determined by the resumption of OSA comparable to that in animals treated with vehicle alone.

[0179] Bulldogs were then administered 25-50,000 ng / kg of PEG-TeNT-HC or 25-50,000 ng / kg of PEG-TeNT-LC-c, each containing 20 kDa PEG, bilaterally into the left and right geniohyoid muscles. OSA was reduced in PEG-TeNT-treated animals compared to animals treated with vehicle alone. Bulldogs were monitored weekly for OSA, and PEG-TeNT-HC and PEG-TeNT-LC-c administration was repeated as needed until efficacy was achieved, as determined by the resumption of OSA comparable to that in animals treated with vehicle alone.

[0180] Bulldogs were then administered 25-50,000 ng / kg of PEG-TeNT-LC-HC, each containing 20 kDa of PEG, bilaterally into the left and right geniohyoid muscles. Upon administration, OSA was reduced in PEG-TeNT-treated animals compared to animals treated with vehicle alone. Bulldogs were monitored weekly for OSA, and PEG-TeNT-LC-HC administration was repeated as needed until efficacy was achieved, as determined by the resumption of OSA comparable to that in animals treated with vehicle alone.

[0181] Example 20. Discovery Studio was used to map TeNT recognized by the major human antibody clones identified by da Silva Antunes et al. (2017) PloS One, 12(1), e0169086 and Palermo et al. (2017) Biotechnology Journal, 12(10), 1700197 onto a three-dimensional model of TeNT derived from crystallographic data deposited in the Protein Data Bank. Surface serine residues in or surrounding the identified epitopes were identified for mutation to cysteine ​​for subsequent PEGylation as described in Examples 1-3 and 5.

[0182] Example 21. A mixture of 1 nanogram to 64 micrograms of PEG-TeNT (5 kDa, 10 kDa, or 20 kDa branched or linear PEG conjugated to surface lysine or cysteine ​​residues) and a 10-1000 molar excess of decoy TeNT is injected intramuscularly into the hind limbs of mice vaccinated with tetanus toxoid. Localized muscle tetanus contractions are observed for periods ranging from several hours to several months and are greater than those observed in mice injected with native TeNT.

[0183] Example 22. A mixture of 1 nanogram to 64 micrograms of PEG-TeNT (5 kDa, 10 kDa, or 20 kDa branched or linear PEG conjugated to surface lysine or cysteine ​​residues) and a 10-1000 molar excess of decoy TeNT is injected intramuscularly into the geniohyoid muscle of humans vaccinated with tetanus toxoid. Localized muscle tetanus contraction is observed for a period of weeks to months, greater than the effect observed in humans injected with native TeNT.

[0184] Example 23. British bulldogs with sleep-disordered breathing received intramuscular injections of 0.001 to 10 IU / kg TeNT in divided doses bilaterally into the left and right geniohyoid muscles. Obstructive sleep apnea (OSA) was significantly reduced at the highest dose. Baseline respiratory disorder index (RDI) scores were 19.9 (interquartile range 5.45) and reduced to 13.2 (interquartile range 4.45) after treatment with 10 IU / kg TeNT, a significant difference compared with placebo treatment determined by Wilcoxon signed-rank test; P = 0.043. Bulldogs were followed up for 4 months after treatment, and the reduction in RDI was maintained; median RDI = 13.4, P = 0.043; Wilcoxon signed-rank test.

Claims

1. 1. Use of tetanus neurotoxin (TeNT) in a pharmaceutical preparation for the treatment of hypotonia in a subject immunized with tetanus toxoid, comprising: The TeNT comprises: a first active tetanus neurotoxin (TeNT); and Second inactive TeNT Including, use.

2. The use of claim 1, wherein the first TeNT comprises a PEGylated tetanus neurotoxin (PEG-TeNT) comprising a TeNT conjugated with polyethylene glycol (PEG).

3. The use of claim 2, wherein the first PEG-TeNT comprises a PEGylated TeNT light chain (LC), a PEGylated TeNT heavy chain (HC), or a PEGylated TeNT fragment c(c).

4. The use according to claim 2 or 3, wherein the first PEG-TeNT is a PEG-TeNT-HC comprising a PEGylated HC, a PEG-TeNT-LC-HC comprising a PEGylated LC and a PEGylated HC, or a PEG-TeNT-LC-c comprising a PEGylated LC and a PEGylated c.

5. The use of any one of claims 2 to 4, wherein the first PEG-TeNT comprises one or more surface serine to cysteine ​​amino acid substitutions relative to SEQ ID NO:

1.

6. 6. The use of claim 5, wherein the substituted cysteine ​​is conjugated with polyethylene glycol (PEG).

7. The use of any one of claims 2 to 6, wherein the PEG is about 2 kDa, about 5 kDa, about 10 kDa, or about 20 kDa, and optionally the PEG is linear or branched.

8. The pharmaceutical PEG-TeNT containing 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 PEG-TeNT containing PEGylated HC and PEGylated LC (PEG-TeNT-LC-HC) The use according to any one of claims 2 to 7, wherein the composition is formulated for administration of

9. The use according to any one of claims 1 to 8, wherein the hypotonia is obstructive sleep apnea.

10. A composition comprising: (i) a first, active tetanus neurotoxin (TeNT); and (ii) a second, inactive TeNT.

11. 11. The composition of claim 10, wherein the first TeNT comprises (i) a first PEGylated tetanus neurotoxin (PEG-TeNT) comprising a tetanus neurotoxin (TeNT) conjugated with polyethylene glycol (PEG).

12. 12. The composition of claim 10 or 11, which is a therapeutic composition.

13. 13. The composition of claim 11 or 12, wherein the first PEG-TeNT comprises one or more surface serine-to-cysteine ​​amino acid substitutions relative to SEQ ID NO: 1, and the substituted cysteines are conjugated with polyethylene glycol (PEG).

14. 14. The composition of claim 13, wherein the PEG is about 2 kDa, about 5 kDa, about 10 kDa, or about 20 kDa, and optionally the PEG is linear or branched.

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