Pegylated tetanus neurotoxin and treatment of hypotension

Abstract

The present invention relates to a composition comprising: a first pegylated tetanus neurotoxin (PEG-TeNT) and a second tetanus neurotoxin (TeNT), the PEG-TeNT comprising a tetanus neurotoxin (TeNT) conjugated to polyethylene glycol (PEG). The invention also relates to a variety of PEG-TeNT. The invention also relates to a method for treating hypotension by using the composition or the various PEG-TeNTs, and a kit comprising the composition or the various PEG-TeNTs. In one embodiment, the hypotension is an obstructive sleep apnea.

Description

[0001] This application is a divisional application of PCT international patent application PCT / AU2019 / 050793 with an application date of July 30, 2019, which entered the Chinese national phase, with Chinese patent application number 201980057158.3 and the invention name being “PEGylated tetanus neurotoxin and treatment of hypotonia”. Technical Field

[0002] 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 a method of treating hypotonia using the composition. Background Art

[0003] Tetanus neurotoxin (TeNT) is produced by Clostridium 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. Consequently, TeNT causes spastic paralysis. TeNT does not have multiple serotypes.

[0004] Therapies have been proposed that exploit the biological properties of TeNT, or at least TeNT fragments. However, long-term treatment with protein therapeutics often results in a targeted immune response. Because only one serotype of TeNT is known, TeNT-based therapies do not offer the option of serotype switching to circumvent vaccination. Furthermore, many populations are vaccinated against TeNT, precluding TeNT-based therapies.

[0005] US2002 / 0197278 A1 discloses the use of pegylated botulinum toxin for treating inappropriate muscle contraction disorders. US2002 / 0197278 A1 also proposes the use of pegylated TeNT for treating inappropriate muscle contraction disorders, such as migratory headaches or strabismus. However, as described above, TeNT causes muscle contraction, thus precluding its use for treating inappropriate muscle contraction disorders, whether pegylated or not.

[0006] Wan et al., Process Biochemistry (2017) 52: 183-191 disclosed the effects of PEGylation on the anti-PEG immune response resulting from administration of PEGylated proteins, but did not utilize their findings for any therapeutic use.

[0007] WO 2016 / 001762 A1 discloses the use of PEGylated TeNT fragment c (c) for increasing muscle mass. When TeNT is enzymatically cleaved by papain, fragment c (50 kDa) is produced, and this fragment c corresponds to the 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.

[0008] There is a need for a TeNT-based therapy that avoids pre-existing anti-TeNT immune responses in subjects immunized with tetanus toxoid.

[0009] It should be appreciated that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms part of the common general knowledge in the art in Australia or any other country. Summary of the Invention

[0010] The inventors of the present application have recognized that the utilization of TeNT has not been fully realized due to the adaptive immune system, which, upon administration of a protein therapeutic, produces an antibody response that reduces the efficacy of the protein therapeutic. This adaptive immune response may be intentional, as a result of vaccination, as demonstrated by the large number of people who have been immunized against TeNT. Alternatively, the adaptive immune response may be unintentional, resulting from repeated exposure to the protein therapeutic.

[0011] The inventors of the present application have generated a family of modified TeNTs and therapeutic regimens that address these problems. Specifically, the present invention provides a family of PEGylated TeNTs (PEG-TeNTs) that each evade the immune system and a hierarchical therapeutic regimen, wherein when the efficacy of a previously administered PEG-TeNT decreases or when the patient's immunological profile precludes the use of another PEG-TeNT, a different alternative PEG-TeNT is used for treatment.

[0012] The first aspect provides a tetanus neurotoxin (TeNT) or a fragment thereof, which comprises one or more surface serine→cysteine ​​amino acid substitutions relative to SEQ ID NO: 1.

[0013] In one embodiment of the first aspect, the substituted cysteine ​​is conjugated to polyethylene glycol (PEG).

[0014] The second aspect provides a TeNT or a fragment thereof, which is 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; R1225K and W1288L; R1225E and W1288L; R1225del; W1288del; or R1225del and W1288del; E270A; Y374A; E270A and Y374; G270del; Y374del; or a combination thereof, wherein the TeNT or fragment is inactive.

[0015] 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) a second TeNT, wherein the first PEG-TeNT is not conjugated to the second TeNT.

[0016] 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.

[0017] A fourth aspect also provides use of a first pegylated tetanus neurotoxin (PEG-TeNT) comprising a tetanus neurotoxin (TeNT) conjugated to polyethylene glycol (PEG) in the manufacture of a medicament for treating hypotonia in a subject administered a second TeNT.

[0018] A fourth aspect also provides the use of a second tetanus neurotoxin (TeNT) in the manufacture of a medicament for treating hypotonia in a subject who has received a first pegylated tetanus neurotoxin (PEG-TeNT), wherein the first pegylated tetanus neurotoxin comprises a tetanus neurotoxin (TeNT) conjugated to polyethylene glycol (PEG).

[0019] A fourth aspect also provides the use of: (i) a first PEGylated tetanus neurotoxin (PEG-TeNT) comprising tetanus neurotoxin (TeNT) conjugated to polyethylene glycol (PEG); and (ii) a second TeNT in the manufacture of a medicament for treating hypotonia.

[0020] A fourth aspect also provides a first pegylated tetanus neurotoxin (PEG-TeNT) comprising a tetanus neurotoxin (TeNT) conjugated to polyethylene glycol (PEG) for use in a method of treating hypotonia in a subject administered a second TeNT.

[0021] The fourth aspect also provides a second tetanus neurotoxin (TeNT) for use in a method of treating hypotonia in a subject administered a first pegylated tetanus neurotoxin (PEG-TeNT), wherein the first pegylated tetanus neurotoxin comprises a tetanus neurotoxin (TeNT) conjugated to polyethylene glycol (PEG).

[0022] A 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; the composition being used in a method for treating hypotonia.

[0023] In embodiments of the fourth aspect, the composition may comprise the first PEG-TeNT and the second TeNT.

[0024] A fourth aspect also provides a composition comprising: (i) a first pegylated tetanus neurotoxin (PEG-TeNT), comprising tetanus neurotoxin (TeNT) conjugated to polyethylene glycol (PEG); and (ii) a second TeNT, wherein the composition is used in a method for treating hypotonia.

[0025] In one embodiment, the first PEG-TeNT or the 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 the second TeNT comprises PEG-TeNT-LC-HC.

[0026] In another embodiment, the first PEG-TeNT or the second TeNT is a PEG-TeNT-HC comprising a PEGylated HC. In this embodiment, the LC is not PEGylated. In another embodiment, the first PEG-TeNT or the second TeNT is a PEG-TeNT-LC-c comprising a PEGylated LC and a PEGylated c. In this embodiment, the HN is not PEGylated.

[0027] In another embodiment, the first PEG-TeNT is PEG-TeNT-HC and the second TeNT is PEG-TeNT-LC-c.

[0028] In one embodiment, the second TeNT comprises inactivated TeNT. Relative to SEQ ID NO: 1, 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 Y374A; G270del; Y374del; or a combination thereof. In one embodiment, the second TeNT comprises an inactivated TeNT comprising R1225E and W1288A.

[0029] In one embodiment, the subject is administered: a PEG-TeNT comprising PEGylated c (PEG-TeNT-c) until efficacy decreases; then a composition comprising PEG-TeNT-HC and PEG-TeNT-LC-c until efficacy decreases; then a PEG-TeNT comprising PEGylated LC and PEGylated HC (PEG-TeNT-LC-HC).

[0030] In one embodiment, the treatment comprises administering to the subject: PEG-TeNT comprising PEGylated c (PEG-TeNT-c); then administering a composition comprising PEG-TeNT-HC and PEG-TeNT-LC-c; then administering PEG-TeNT comprising PEGylated LC and PEGylated HC (PEG-TeNT-LC-HC), to determine the immunological profile of the subject's anti-TeNT antibodies and determining an effective composition of PEG-TeNT based on the profile.

[0031] In one embodiment, the hypotonia is obstructive sleep apnea.

[0032] In another embodiment, the composition is a therapeutic composition. In another embodiment, the composition is a cosmetic composition.

[0033] Also disclosed is a method for preparing a protein composed of TeNT LC and TeNT HN (LC-HN), the method comprising expressing a nucleic acid molecule encoding an amino acid sequence comprising SEQ ID NO: 6 in a host cell.

[0034] A fifth aspect provides a pegylated tetanus neurotoxin (PEG-TeNT), comprising tetanus neurotoxin (TeNT) conjugated to polyethylene glycol (PEG).

[0035] 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 the c is PEGylated (PEG-TeNT-LC-c).

[0036] In another embodiment, 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 native or a substitution for a serine residue relative to SEQ ID NO: 1.

[0037] In one embodiment, PEG has a molecular weight of about 2 kDa, about 5 kDa, about 10 kDa, or about 20 kDa, or about 30 kDa.

[0038] A sixth aspect provides a method for treating hypotonia, the method comprising administering the PEG-TeNT of the fifth aspect to a subject, wherein the PEG-TeNT is not conjugated to a second TeNT.

[0039] 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.

[0040] Alternatively, a sixth aspect provides the PEG-TeNT of the fifth aspect for use in a method of treating hypotonia, the method comprising administering the PEG-TeNT to a subject, wherein the PEG-TeNT is not conjugated to a second TeNT.

[0041] In one embodiment, a first PEG-TeNT comprising a PEGylated HC (PEG-TeNT-HC) and a second PEG-TeNT comprising a PEGylated LC and a PEGylated c (PEG-TeNT-LC-c) are administered to the subject.

[0042] In another embodiment, 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).

[0043] In one embodiment, the treatment comprises administering to the subject: PEG-TeNT comprising PEGylated c (PEG-TeNT-c) until efficacy decreases. Thereafter, the treatment may comprise administering to the subject: PEG-TeNT-HC and PEG-TeNT-LC-c until efficacy decreases. Thereafter, the treatment may comprise administering to the subject: PEG-TeNT comprising PEGylated LC and PEGylated HC (PEG-TeNT-LC-HC).

[0044] In one embodiment, the treatment comprises administering to the subject: PEG-TeNT comprising PEGylated c (PEG-TeNT-c) until efficacy decreases; then administering PEG-TeNT-HC and PEG-TeNT-LC-c until efficacy decreases; then administering PEG-TeNT comprising PEGylated LC and PEGylated HC (PEG-TeNT-LC-HC).

[0045] In one embodiment, the treatment 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 the efficacy is reduced; then administering a PEG-TeNT comprising PEGylated c (PEG-TeNT-c) comprising PEG having a molecular weight of about 10 kDa, until the efficacy is reduced; then administering a PEG-TeNT comprising PEGylated c (PEG-TeNT-c) comprising PEG having a molecular weight of about 20 kDa, until the efficacy is reduced. Thereafter, treatment may comprise 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 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 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 comprise administering to the subject: PEG-TeNT comprising a PEGylated LC and a PEGylated HC (PEG-TeNT-LC-HC), either or both comprising a PEG having a molecular weight of 5 kDa, until efficacy decreases; then administering PEG-TeNT comprising a PEGylated LC and a PEGylated HC (PEG-TeNT-LC-HC), either or both comprising a PEG having a molecular weight of 10 kDa, until efficacy decreases; then administering PEG-TeNT comprising a PEGylated LC and a PEGylated HC (PEG-TeNT-LC-HC), either or both comprising a PEG having a molecular weight of 20 kDa.

[0046] In one embodiment, the treatment comprises administering to the subject: PEG-TeNT comprising PEGylated c (PEG-TeNT-c); then administering a composition comprising PEG-TeNT-HC and PEG-TeNT-LC-c; then administering PEG-TeNT comprising PEGylated LC and PEGylated HC (PEG-TeNT-LC-HC), to determine the immunological profile of the subject's anti-TeNT antibodies and determining an effective composition of PEG-TeNT based on the profile.

[0047] 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 an inactivated TeNT comprising R1225E and W1288A; E270A; Y374A; E270A and Y374A; G270del; Y374del; or a combination thereof.

[0048] In one embodiment, the hypotonia is obstructive sleep apnea.

[0049] The 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.

[0050] In one embodiment, the composition or PEG-TeNT is used according to the method of the third aspect or the sixth aspect, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 Schematic diagram of exemplary PEG-TeNT of the present invention: A is PEG-TeNT-c; B is PEG-TeNT-HC; C is PEG-TeNT-LC-c; D is PEG-TeNT-LC-HC, wherein the TeNT is intact and active, i.e., TeNT contains two chains, and PEGylation is present on specific regions, i.e., c, HC, LC-c or LC-HC, respectively.

[0052] Figure 2 is the amino acid sequence of mature TeNT comprising 1314 amino acids (SEQ ID NO: 1).

[0053] Figure 3 The nucleic acid sequence of the TeNT-encoding vector pRSET-TeNT (SEQ ID NO: 2) is shown.

[0054] Figure 4Figure 1 is a diagram of the TeNT-encoding vector pRSET-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.

[0055] Figure 5 The amino acid sequence of HC comprising amino acids 457 to 1314 of SEQ ID NO: 1 (SEQ ID NO: 3).

[0056] Figure 6 The amino acid sequence (SEQ ID NO: 4) is an amino acid sequence comprising c of amino acids 864 to 1314 of SEQ ID NO: 1.

[0057] Figure 7 The nucleic acid sequence of the vector pRSET-TeNT-c encoding c (SEQ ID NO: 5).

[0058] Figure 8 Figure 2 is a diagram 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.

[0059] Figure 9 is the amino acid sequence of LC-HN comprising amino acids 1 to 863 of SEQ ID NO: 1 (SEQ ID NO: 6).

[0060] Figure 10 The nucleic acid sequence of the vector pRSET-TeNT-LC-HN encoding LC-HN (SEQ ID NO: 7) is shown.

[0061] Figure 11 Figure 1 is a diagram of the LC-HN encoding vector pRSET-TeNT-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.

[0062] Figure 12 The amino acid sequence of a non-functional c comprising amino acid substitutions W1288A and R1225E relative to SEQ ID NO: 1 (SEQ ID NO: 8).

[0063] Figure 13 is a protein encoding a protein comprising amino acid substitutions W1288A and R1225E relative to SEQ ID NO: 1 Figure 12 The nucleic acid sequence of the non-functional c (SEQ ID NO: 8) vector pRSET-TeNT-c (SEQ ID NO: 9).

[0064] Figure 14 is a protein encoding a protein comprising amino acid substitutions W1288A and R1225E relative to SEQ ID NO: 1 Figure 12 Figure 1 shows a vector pRSET-TeNT-c containing a non-functional c (SEQ ID NO: 8). c containing amino acid substitutions W1288A and R1225E relative to SEQ ID NO: 1 is expressed with an N-terminal His6 tag. This nucleic acid is inserted into the MCS of the pRSET-A vector and expressed under the control of the T7 promoter.

[0065] Figure 15 is the amino acid sequence of a non-functional TeNT comprising amino acid substitutions W1288A and R1225E relative to SEQ ID NO: 1 (SEQ ID NO: 10).

[0066] Figure 16 is a protein encoding a protein comprising amino acid substitutions W1288A and R1225E relative to SEQ ID NO: 1 Figure 15 The nucleic acid sequence (SEQ ID NO: 11) of the vector pRSET-TeNT containing the non-functional TeNT (SEQ ID NO: 10) was detected.

[0067] Figure 17 is a protein encoding a protein comprising amino acid substitutions W1288A and R1225E relative to SEQ ID NO: 1 Figure 15 Figure 1 shows a non-functional TeNT vector (SEQ ID NO: 10) containing the amino acid substitutions W1288A and R1225E relative to SEQ ID NO: 1. TeNT was expressed with an N-terminal His6 tag. The nucleic acid was inserted into the MCS of the pRSET-A vector and expressed under the control of the T7 promoter.

[0068] Figure 18 is the amino acid sequence of a mature TeNT having 1314 amino acids (SEQ ID NO: 12), which comprises surface serine→cysteine ​​amino acid substitutions S81C, S120C, S144C, S248C, S335C, S428C, S600C, S963C, S1041C, S1155C, and S1187C relative to SEQ ID NO: 1.

[0069] Figure 19 It is encoding Figure 18 The nucleic acid sequence of the vector pRSET-TeNT (SEQ ID NO: 13) containing the mature TeNT (SEQ ID NO: 12) in which the surface serine is replaced by cysteine.

[0070] Figure 20 It is encoding Figure 18 The mature TeNT (SEQ ID NO: 12) in which the surface serine is replaced by cysteine Figure 19 Figure 1 shows a vector pRSET-TeNT (SEQ ID NO: 13). TeNT, in which surface serines are substituted with cysteine, 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.

[0071] Figure 21 is the amino acid sequence of a mature TeNT having 1314 amino acids (SEQ ID NO: 14), which comprises surface serine→cysteine ​​amino acid substitutions S81C, S120C, S144C, S248C, S335C, S428C, S963C, S1041C, S1155C, and S1187C in the LC region and c region relative to SEQ ID NO: 1.

[0072] Figure 22 It is encoding Figure 21 The nucleic acid sequence of the vector pRSET-TeNT (SEQ ID NO: 15) containing the mature TeNT (SEQ ID NO: 14) in which the surface serine is replaced by cysteine.

[0073] Figure 23 It is encoding Figure 21 The mature TeNT (SEQ ID NO: 14) in which the surface serine is replaced by cysteine Figure 22 Figure 1 shows a vector pRSET-TeNT (SEQ ID NO: 15). TeNT, in which surface serines are substituted with cysteine, 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.

[0074] Figure 24 The amino acid sequence of TeNT (SEQ ID NO: 16) comprising amino acid substitutions S600C, S963C, S1041C, S1155C, and S1187C on the HC surface serine→cysteine ​​relative to SEQ ID NO: 1.

[0075] Figure 25 It is encoding Figure 24 The nucleic acid sequence of the vector pRSET-TeNT (SEQ ID NO: 17) containing the mature TeNT (SEQ ID NO: 16) in which the surface serine is replaced by cysteine.

[0076] Figure 26 It is encoding Figure 24TeNT (SEQ ID NO: 16) in which the surface serine is replaced by cysteine Figure 25 Figure 1 shows a vector pRSET-TeNT (SEQ ID NO: 17). TeNT, in which surface serines are substituted with cysteine, 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.

[0077] Figure 27 A 3-dimensional protein structure model of TeNT derived from crystallographic data deposited in the Protein Data Bank (Accession ID PDB: 5N0B) was mapped onto the model using Discovery Studio to identify epitopes recognized by major human antibody clonotypes (as identified by da Silva Antunes et al. (2017) and Palermo et al. (2017)). Surface serine residues in or near the identified epitopes were selected for mutation to cysteine ​​for subsequent PEGylation.

[0078] Figure 28 Included are two photographs of SDS-PAGE analysis of PEG-TeNT, which contains PEG with increasing molecular weight, where (A) detection was performed using Coomassie blue and (B) detection was performed by Western blot using a polyclonal anti-TeNT antibody. (B) Immunogenicity is shown to be proportional to the molecular weight of the PEG. The left edge has molecular weight markers in kDa, lane 1; TeNT lane 2; 2kDa PEG-TeNT-HC-LC lane 3; 5kDa PEG-TeNT-LC-HC lane 4; 10kDa PEG-TeNT-LC-HC lane 5; 20kDa PEG-TeNT-LC-HC lane 6.

[0079] Figure 29Included are four line graphs representing competitive ELISA assays. Four PEG-TeNT and four PEG-TeNT-LC-c serine mutants, each containing different molecular weight PEG (2 kDa, 5 kDa, 10 kDa, and 20 kDa), were assayed against TeNT using a polyclonal anti-TeNT antibody. (A) TeNT was adsorbed onto an ELISA plate and then probed using a polyclonal anti-TeNT antibody 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 a polyclonal anti-TeNT antibody preincubated with each of the four TeNT antigen concentrations (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 a polyclonal anti-TeNT antibody preincubated with each of the four PEG-TeNT-LC-c 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). (D) Each PEG-TeNT-LC-c serine mutant (2 kDa, 5 kDa, 10 kDa, and 20 kDa) was adsorbed onto a separate ELISA plate and then probed with a polyclonal anti-TeNT antibody preincubated with each of the four concentrations (10 μg / mL, 1 μg / mL, 0.1 μg / mL, and 0.01 μg / mL) of the TeNT-LC-c serine mutant antigen.

[0080] Figure 30 Included are four photographs showing localized limb twitching in the hind limbs of female C57BL / 6 mice injected with PEG-TeNT-LC-HC. A and B show localized limb twitching in naive mice. C shows the absence of limb twitching after injection of 100 units of TeNT in a tetanus toxoid-vaccinated mouse, while D shows persistent limb twitching after injection of 80 units of PEG-TeNT LC-HC 20 kDa in a tetanus toxoid-vaccinated mouse.

[0081] Figure 31Included are two dot plots showing clinical tetanus levels in the hind limbs of female C57BL / 6 mice following injection of defined numbers of units of TeNT, PEG-TeNT-LC-HC 20kDa, TeNT-LC-cSer, PEG-TeNT-LC-cSer 2kDa, or PEG-TeNT-LC-cSer 20kDa. A) Mice were injected with TeNT or PEG-TeNT-LC-HC 20kDa. B) Mice were injected with TeNT, TeNT-LC-cSer, PEG-TeNT-LC-cSer 2kDa, or PEG-TeNT-LC-cSer 20kDa. One unit is the minimum amount of toxin required to produce stage IV convulsions in naive mice within 24 hours.

[0082] Figure 32 Included are two line graphs showing the progression of clinical convulsions in mice vaccinated with tetanus toxoid vaccine. A shows the progression of convulsions after injection of 4000 units of TeNT in the presence or absence of inactivated TeNT bait. B shows the progression of convulsions after injection of 40 units of TeNT-PEG 20 kDa in the presence or absence of inactivated TeNT bait. In both cases, the presence of bait significantly accelerated progression.

[0083] Figure 33 23 is a box plot showing the effect of increasing the dose of tetanus toxin on the respiratory disturbance index (RDI) of English bulldogs treated according to Example 23. The black bars represent the median RDI of six studies conducted at each tetanus toxin dose. The shaded boxes represent the interquartile range and the brackets represent the minimum and maximum RDI samples at each dose. Compared to the RDI after placebo, increasing tetanus toxin after administration of 10 IU / kg was associated with a decrease in RDI (P=0.043; Wilcoxon Signed Ranks test). The median values ​​for placebo and 10 IU / kg are additionally represented by ○ and *, respectively. DETAILED DESCRIPTION

[0084] The present invention relates to TeNT and PEG-TeNT molecules that evade immunity ( Figure 1 ), compositions thereof and their therapeutic and cosmetic uses. In one embodiment, the present invention relates to the treatment of hypotonia, optionally obstructive sleep apnea.

[0085] Described herein are the uses of specifically modified TeNT and compositions of TeNT and decoy (inactive TeNT) for treating hypotonia in subjects with a protective immune response to tetanus toxoid. To achieve this goal, active TeNT is modified by adding PEG, introducing specific mutations, or a combination thereof, for use in delivering biologically active compounds capable of increasing muscle tone in tetanus-immunized patients.

[0086] The activity can be demonstrated by administering a unit dose of the modified toxin or formulation, where at the same unit dose, TeNT would not exhibit activity in a vaccinated subject. Based on analysis of the three-dimensional structure of TeNT, the introduction of specific surface mutations for the directed attachment of PEG molecules allows for the masking of specific TeNT epitopes known to be targeted by protective antibody responses in vaccinated subjects. The combination of PEGylation, site-directed mutagenesis, and decoy molecule formulations significantly enhances the ability of the molecule to increase muscle tone in a vaccinated mammalian model relative to an equivalent unit of TeNT administered.

[0087] US2002 / 0197278 discloses a series of pegylated botulinum toxins for treating inappropriate muscle contraction disorders and suggests that TeNT could be used as a replacement for botulinum toxin. However, TeNT cannot be used to treat muscle contraction. Furthermore, because the disclosed method does not include site-specific masking of epitopes, the purported invention of US2002 / 0197278 appears unrealizable, and to the best of the inventors' knowledge, the three-dimensional structure and epitope identity required for intentional masking of TeNT epitopes were unknown at the priority date of US2002 / 0197278.

[0088] Wan et al. addressed the effects of PEGylation on the anti-PEG immune response generated by administration of PEGylated proteins, but did not exploit their findings for any therapeutic application. Although Wan et al. disclosed that PEGylated tetanus toxoid exhibited reduced immunogenicity relative to non-PEGylated tetanus toxoid, Wan et al. did not propose therapeutically relevant molecules or formulations. Furthermore, PEGylation of tetanus toxoid is unrelated to the modification of active TeNT, as tetanus toxoid is a biologically inactive TeNT used for vaccination, which can be produced by formaldehyde cross-linking of TeNT. In other words, PEGylated or non-PEGylated tetanus toxoid does not possess the combination of enzymatic activity, binding activity, and translocation activity of active TeNT.

[0089] WO 2016 / 001762 A1 relates only to TeNTc fragments, which are molecules with no specific activity other than binding to neurotransmitters and entering neurons.

[0090] Therefore, there is a need for a TeNT-based therapy that avoids pre-existing anti-TeNT immunity in subjects immunized with tetanus toxoid.Disclosed herein is a solution to this problem, provided in part by masked active and therapeutically relevant PEGylated TeNT.

[0091] Tetanus neurotoxin (TeNT)

[0092] 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 initiator methionine codon, is provided in Figure 3 TeNT is expressed as a protein that is post-translationally cleaved, first by removing the initiating methionine, and then by cleaving into two parts: a 50 kDa 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. These two chains are linked by an interchain disulfide bond, which is crucial for neurotoxicity. The 1314 amino acid sequence of mature TeNT is provided in Figure 2 in (SEQ ID NO: 1).

[0093] LC has zinc endopeptidase activity and attacks vesicle-associated membrane protein (VAMP), which is necessary for vesicle fusion with the membrane, thereby preventing the release of neurotransmitters.

[0094] After digestion with papain, HC can be cleaved into two domains of 50 kDa each: an N-terminal translocation domain designated as HN; and a C-terminal ganglioside (membrane) binding domain designated as fragment c (c). TeNT lacking c is referred to herein as LC-HN.

[0095] c has two polysialoganglioside binding sites and binds to polysialogangliosides (GD2, GD1b, and GT1b) on neuronal membranes. Thus, c mediates the binding of TeNT to the presynaptic membrane of peripheral motor axons and helps TeNT move across the membrane into neurons.

[0096] Amino acid sequence:

[0097] The amino acid sequence of TeNT lacking the initiating methionine is provided in Figure 2 in (SEQ ID NO:1);

[0098] The amino acid sequence of HC is provided in Figure 5 Medium (SEQ ID NO:3);

[0099] The amino acid sequence of c is provided in Figure 6(SEQ ID NO: 4); and

[0100] The amino acid sequence of LC-HN is provided in Figure 9 in (SEQ ID NO:6).

[0101] Codon-optimized nucleic acid sequences and vector maps of vectors encoding:

[0102] cProvided on Figure 7 (SEQ ID NO: 5) and Figure 8 in; and

[0103] LC-HN is provided in Figure 10 (SEQ ID NO: 7) and Figure 11 middle.

[0104] As used herein, "TeNT" is used to refer to the complete TeNT molecule consisting of a heavy chain and a light chain. Subdomains and fragments are referred to herein by their abbreviations: light chain "LC"; heavy chain "HC"; heavy chain N-terminal domain "HN"; heavy chain fragment c "c"; light chain plus heavy chain N-terminal domain "LC-HN" (i.e., a TeNT molecule lacking c). Where any subdomain or fragment is PEGylated, the prefix PEG is used: PEG-LC; PEG-HC; PEG-HN; PEG-c; PEG-LC-HN. In the complete TeNT molecule comprising a PEGylated fragment or subdomain, the prefix PEG is used and indicates 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.

[0105] It should be understood that because subdomains and fragments have specific functions, subdomains and fragments are not interchangeable with respect to intact TeNT.

[0106] The TeNTs disclosed herein can be active or inactive. Active TeNTs have the same biological activity as natural TeNTs. Inactive TeNTs lack one or more activities of natural TeNTs. In one embodiment, inactive TeNTs do not block the release of inhibitory neurotransmitters. The inactive TeNTs disclosed herein can also be referred to as "bait." Inactive TeNTs include tetanus toxoid. In one embodiment, the inactive TeNTs are inactive TeNTs as disclosed herein.

[0107] In one embodiment, two or more TeNTs can be conjugated.

[0108] In another embodiment, the TeNT is not conjugated. In one embodiment of the compositions, methods, and uses disclosed herein, the first PEG-TeNT is not conjugated to the second TeNT.

[0109] This article discloses and Figure 1 The immune-evading PEG-TeNTs depicted in

[15] include:

[0110] TeNT with PEGylated fragment c (PEG-TeNT-c) ( Figure 1 A);

[0111] TeNT with PEGylated heavy chain (PEG-TeNT-HC) ( Figure 1 B);

[0112] TeNT with PEGylated light chain and PEGylated fragment c (PEG-TeNT-LC-c) ( Figure 1 C); and

[0113] Fully PEGylated TeNT (PEG-TeNT-LC-HC) Figure 1 D).

[0114] PEG-TeNT-c advantageously evades pre-existing immune responses of the adaptive immune system in vaccinated subjects. In one embodiment, PEG-TeNT-c provides a first-level therapy to be used until efficacy decreases.

[0115] PEG-TeNT-HC and PEG-TeNT-LC-c advantageously evade pre-existing immune responses of the adaptive immune system in vaccinated subjects and also evade immune responses of the adaptive immune system elicited by repeated exposure to PEG-TeNT-c.

[0116] In one embodiment, PEG-TeNT-HC and PEG-TeNT-LC-c together provide a second level of treatment to be used until their efficacy decreases.

[0117] PEG-TeNT-LC-HC advantageously evades pre-existing immune responses of the adaptive immune system in vaccinated subjects and also evades immune responses of the adaptive immune system elicited by repeated exposure to PEG-TeNT-c and exposure to PEG-TeNT-HC plus PEG-TeNT-LC-c.

[0118] In one embodiment, PEG-TeNT-LC-HC provides a tertiary treatment to be used until its efficacy decreases.

[0119] 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).

[0120] Those skilled in the art will appreciate that the specific combinations of PEG-TeNTs and the order in which those PEG-TeNT combinations are used for treatment can vary.

[0121] Polyethylene glycol (PEG)

[0122] PEG can be conjugated to the following amino acid residues in TeNT, for example: lysine (e.g., amino-PEGylation), cysteine ​​(e.g., thiol-PEGylation and bridged PEGylation), histidine, arginine, aspartic acid, asparagine (e.g., N-glycosyl-PEGylation), glutamic acid, glutamine (e.g., transglutaminase-mediated PEGylation), serine (e.g., O-glycosyl-PEGylation), threonine (e.g., O-glycosyl-PEGylation), or tyrosine residues. Examples of PEGylation also include N-terminal PEGylation and C-terminal PEGylation.

[0123] PEGylation can be achieved by reacting PEG with hydroxyl-reactive functional groups such as anhydrides, acid chlorides, chloroformates, and carbonates. Alternatively, PEGylation can be achieved with functional groups such as aldehydes, esters, and amides.

[0124] PEG can be linear or branched.

[0125] The PEG can be a modified PEG, such as poly[oligo(ethylene glycol) methyl ether methacrylate] (POEGMA).

[0126] The PEGylation may be site-specific PEGylation.

[0127] In one embodiment, surface serine residues of TeNT or TeNT fragments are mutated to surface cysteine ​​residues to facilitate targeted PEG conjugation at immunogenic epitopes. In this context, mutation is synonymous with substitution, e.g., substitution of serine with cysteine. Such mutations or substitutions include any combination of one or more of the following: S81C; S120C; S144C; S248C; S335C; S428C; S600C; S963C; S1041C; S1155C; and S1187C.

[0128] Functional groups used in heterobifunctional PEGs include maleimide, vinyl sulfone, pyridyl disulfide, amine, carboxylic acid, and NHS ester.

[0129] In one embodiment, PEG is conjugated to TeNT using carbodiimide-EDC and sulfo-NHS with carboxyl-amine cross-linking.

[0130] The present invention also contemplates PEG-TeNT comprising PEG of varying molecular weight conjugated to different subdomains or fragments of TeNT.

[0131] For example, PEG can be conjugated or linked to the TeNT of the present disclosure for 2 to 6 hours at 4° C. to 25° C. In one embodiment, PEG is conjugated to TeNT at room temperature for 6 hours.

[0132] Indications

[0133] As used herein, "hypotonia" refers to any condition involving involuntary muscle weakness that can be treated by inhibiting inhibitory neurotransmitters such as GABA or glycine. Thus, "hypotonia" includes decreased muscle tone secondary to decreased nervous system drive or other causes and conditions of decreased or insufficient muscle tone, strength, or nervous system drive. Thus, in one embodiment, the hypotonia can be neurogenic hypotonia.

[0134] Hypotonic conditions that can be treated with the PEG-TeNT, compositions, or methods according to the present disclosure include obstructive sleep apnea, apnea, snoring, ptosis, Horner's syndrome, muscle atrophy, impaired muscle nerve function, amyotrophic lateral sclerosis (ALS), motor neuron disease, any myopathy, multiple sclerosis, myasthenia gravis, facial hypotonia, optionally ectropion, flaccid paralysis or weakness of any skeletal or smooth muscle of any etiology, respiratory muscle weakness of any etiology, including post-ventilator weakness; muscle weakness caused by trauma or poor posture due to muscle relaxation, pelvic floor muscle relaxation or weakness, or nasal or upper airway relaxation.

[0135] Other conditions that can be treated with PEG-TeNT, compositions, or methods according to the present disclosure include muscle wasting, muscular dystrophy, or loss of muscle mass.

[0136] In cases where the condition to be treated according to the present disclosure is not itself a hypotonic condition, increasing muscle tone through treatment with the TeNTs of the present disclosure may alleviate the symptoms of the condition.

[0137] Cosmetic applications of PEG-TeNT may include tightening abdominal muscles, tightening pectoral muscles, tightening gluteus maximus muscles, tightening skeletal muscles, or treating facial sagging caused by muscle laxity.

[0138] Smooth muscles, skeletal muscles, tissues or organs that can be treated with PEG-TeNT, compositions or methods according to the present disclosure include the 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 cords, facial muscles, masticatory 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, pharyngeal dilator muscles, masseter muscles, medial pterygoid muscles, lateral pterygoid muscles, geniohyoid muscles, genioglossus muscles, tensor veli palatini, levator veli palatini, stylophodon muscles, styloglossus muscles, mylohyoid muscles, stylohyoid muscles, hyoglossus muscles, digastric muscles, sternocleidomastoid muscles, trapezius muscles, temporalis muscles, cricopharyngeus muscles, uterine muscles and cervix, gastric nerve supply, nasal mucosa, lung mucosa, skin, thymus gland, bones, coronary arteries, pulmonary smooth muscle and myocardium.

[0139] Composition and administration

[0140] The compositions of the present disclosure can be therapeutic compositions or cosmetic compositions. That is, the compositions can be used for therapeutic or cosmetic purposes.

[0141] As used herein, the term "therapeutic composition" or "cosmetic composition" refers to a composition comprising TeNT as described herein for inhibiting or treating hypotonia in a subject. The composition has been formulated for administration to a subject. In one embodiment, the composition is sterile. In one embodiment, the composition is pyrogen-free. The composition may comprise a pharmaceutically acceptable carrier. Preferably, the composition is manufactured according to Good Laboratory Practice (GLP) or Good Manufacturing Practice (GMP).

[0142] The TeNTs of the present disclosure can be administered at doses up to 10 mg / kg or higher. The TeNTs of the present disclosure can be administered at doses of 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, The TeNTs of the present disclosure can be administered within any range of any of the doses listed above.

[0143] The TeNT of the present disclosure can be administered at a dose of up to 1000 IU / kg or more. The TeNT of the present disclosure can be administered at a dose of 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

[0014] The TeNTs of the present disclosure can be administered within any range of any of the doses listed above.

[0144] In a composition comprising two PEG-TeNTs, for example, a first PEG-TeNT in which TeNT-HC is PEGylated (PEG-TeNT-HC) and a second PEG-TeNT in which TeNT-LC is PEGylated and TeNT-c is 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.

[0145] The composition can include any combination of TeNTs and is not limited to the combination of PEG-TeNT-HC and PEG-TeNT-LC-c. The composition can 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. Also disclosed are compositions 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-TeNT-LC-HC. Any TeNT can be substituted in the composition, and any composition can also comprise PEG-TeNT-LC, PEG-TeNT-LC-HN, and / or PEG-TeNT-HN.

[0146] In one embodiment, the composition further comprises inactivated TeNT, which serves as a bait for anti-TeNT antibodies generated by prior exposure to TeNT, such as by vaccination. Relative to SEQ ID NO: 1, the inactivated TeNT may 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 R1225del and W1288del; E270A; Y374A; E270A and Y374A; E270del; Y374del or a combination thereof. In one embodiment, the second TeNT comprises an inactivated TeNT comprising R1225E, W1288A, E270A, and Y374A.

[0147] In a composition comprising at least one PEG-TeNT and a decoy TeNT, the decoy TeNT will be in molar excess relative to the PEG-TeNT. For example, the ratio of decoy TeNT to PEG-TeNT can 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.

[0148] The PEG-TeNT of the present disclosure can be administered once, twice, or three times a week; once, twice, or three times a month; once, twice, or three times a quarter; once, twice, or three times every 6 months; or once, twice, or three times a year.

[0149] PEG-TeNT can be administered in a single dose, divided doses, or multiple doses. Where muscles are paired, PEG-TeNT can be administered unilaterally to one muscle in the pair or bilaterally to both muscles in the pair.

[0150] As an alternative to a combination comprising two or more PEG-TeNTs of the present disclosure, the two or more PEG-TeNTs can be administered in combination, either sequentially or simultaneously.

[0151] PEG-TeNT can be administered topically to a subject by any suitable method, such as by injection, surgical implantation, topical administration, or intranasal administration. In one embodiment, TeNT is administered intramuscularly by injection into the affected muscle.

[0152] PEG-TeNT will be formulated, dosed, and applied in a manner consistent with good medical practice. Factors to consider in this context include the specific type of hypotonia being treated, the specific subject being treated, the subject's clinical condition, the site of administration, the method of administration, the timing of administration, and other factors known to physicians, including dentists. The therapeutically effective amount of PEG-TeNT to be administered will depend on these considerations.

[0153] Pharmaceutically acceptable carriers include water, buffered water, saline solutions, such as physiological saline or balanced saline solutions, such as Hank's balanced solution or Earle's balanced solution, glycine and hyaluronic acid.

[0154] The composition can be formulated for intramuscular administration. The composition for intramuscular administration can comprise pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions and sterile powders for reconstitution into sterile injectable solutions or dispersions. The example of suitable aqueous and non-aqueous carriers, solvents, diluents or vehicles comprises water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol), carboxymethyl cellulose and mixtures thereof, vegetable oils (such as olive oil), injectable organic esters (such as ethyl oleate).

[0155] The compositions may include a penetration enhancer to facilitate their TeNT delivery. 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, 1-monocaprin, monoglycerides and diglycerides, and physiologically acceptable salts thereof.

[0156] The composition may also include a chelating agent, such as ethylenediaminetetraacetic acid (EDTA), citric acid, salicylates (eg, sodium salicylate, 5-methoxysalicylate, homovanillate).

[0157] Also provided is an article and / or kit comprising a container comprising PEG-TeNT or a composition comprising PEG-TeNT. The container can be a bottle, vial, or syringe containing PEG-TeNT or a composition (optionally in unit dosage form). For example, the PEG-TeNT or composition can be in the form of an injectable solution in a disposable container, optionally a syringe. The article and / or kit can also include printed instructions and / or labels, etc., indicating that a subject is to be treated according to the methods disclosed herein.

[0158] The term "therapeutically effective amount" refers to an amount of PEG-TeNT effective to treat hypotonia in a subject.

[0159] The terms "treat," "treating," or "treatment" refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent, reduce, or ameliorate hypotonia in a subject or to slow (lessen) 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.

[0160] The terms "preventing," "prevention," "prophylactic," or "preventative" refer to avoiding the occurrence of hypotonia or hindering, guarding against, or preventing the occurrence of hypotonia. A subject in need of prevention may be susceptible to developing hypotonia.

[0161] The terms "ameliorate" or "amelioration" refer to lessen, reduce, or eliminate hypotonia.

[0162] Hypotonia can be quantified. Hypotonia can be quantified on a semi-quantitative scale, for example, 0 to 5, where 0 represents absence, 1 to 4 represent a discernible increase in severity, and 5 represents the maximum severity. Alternatively, hypotonia can be quantified as a binary event, i.e., present or absent (0 or 1). Other semi-quantitative scales will be apparent to those skilled in the art. In another embodiment, hypotonia can be quantified on a quantitative scale, for example, using a dynamometer.

[0163] Any quantification of hypotonia can be compared to a control, such as a healthy control subject who did not receive PEG-TeNT, a diseased control subject who received treatment to reduce hypotonia but was not treated with PEG-TeNT, or a human population.

[0164] Treatment of hypotonia by administration of PEG-TeNT can be a decrease in hypotonia by about 1%, by about 2%, by about 3%, by about 4%, by about 5%, by about 6%, by about 7%, by about 8%, by about 9%, by about 10%, by about 20%, by about 30%, by about 40%, by about 50%, by about 60%, by about 70%, by about 80%, by about 90%, or by about 100%.

[0165] As used herein, the term "subject" can refer to a mammal. The mammal can be a primate, particularly a human, or can be a livestock animal, zoo animal, or companion animal. While the PEG-TeNT, compositions, and methods disclosed herein are specifically contemplated for use in the medical treatment of humans, they are also suitable for veterinary treatment, including the treatment of livestock, such as horses, cattle, and sheep; companion animals, such as dogs and cats; or zoo animals, such as felines, canines, bovines, and ungulates.

[0166] Unless otherwise defined in this specification, technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which the present invention belongs by referring to the disclosure.

[0167] It should be noted that the term "a / an" refers to one or more, for example, "a TeNT" should be understood to mean one or more TeNTs. Thus, the terms "a / an," "one or more," and "at least one" are used interchangeably herein.

[0168] In the appended claims and description of the present invention, unless the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" are used in an inclusive sense, i.e. specifying the presence of stated features but not excluding the presence or addition of further features in various embodiments of the invention.

[0169] As used herein, the term "about" encompasses a range of values ​​of a given quantity that vary from the magnitude of that quantity by ±25%. In other embodiments, the term "about" encompasses a range of values ​​of a given quantity that vary from the magnitude of that quantity by ±20%, ±15%, ±10%, ±5%, ±4%, ±3%, ±2%, or ±1%. For example, in one embodiment, "about 3 grams" means a value of 2.7 grams to 3.3 grams (i.e., 3 grams ±10%), etc.

[0170] Similarly, the time or duration of an event can vary by at least 25%. For example, although a specific event can be disclosed as lasting one day in one embodiment, the event can last more than one day or less than one day. For example, "one day" can include a time period of approximately 18 hours to approximately 30 hours. In other embodiments, the time period can vary by ± 20%, ± 15%, ± 10%, ± 5%, ± 4%, ± 3%, ± 2% or ± 1% of the time period.

[0171] This application includes the following embodiments.

[0172] 1. A tetanus neurotoxin (TeNT) or a fragment thereof comprising one or more surface serine→cysteine ​​amino acid substitutions relative to SEQ ID NO: 1.

[0173] 2. The TeNT or fragment thereof according to item 1, wherein relative to SEQ ID NO: 1, the substituted serine comprises one or more of S81C, S120C, S144C, S248C, S335C, S428C, S600C, S963C, S1041C, S1155C and S1187C.

[0174] 3. TeNT or a fragment thereof according to item 1 or 2, wherein the substituted cysteine ​​is conjugated to polyethylene glycol (PEG).

[0175] 4. TeNT or a fragment thereof according to item 3, wherein the PEG is about 2 kDa, 5 kDa, about 10 kDa or about 20 kDa.

[0176] 5. TeNT or a fragment thereof according to item 3 or 4, wherein the PEG is linear or branched.

[0177] 6. A TeNT or a fragment thereof, relative to SEQ ID NO: 1, comprising: 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 and W1288del; E270A; E270del; or a combination thereof, wherein the TeNT or fragment is inactive.

[0178] 7. A composition comprising: (i) a first pegylated tetanus neurotoxin (PEG-TeNT) comprising a tetanus neurotoxin (TeNT) conjugated to polyethylene glycol (PEG); and (ii) a second TeNT.

[0179] 8. The composition of item 7, wherein the first PEG-TeNT comprises the TeNT of any one of items 3 to 5.

[0180] 9. The composition according to item 7 or 8, wherein the second TeNT is the TeNT according to any one of items 1 to 6.

[0181] 10. 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 to polyethylene glycol (PEG); and (ii) a second TeNT.

[0182] 11. Use of a first pegylated tetanus neurotoxin (PEG-TeNT) comprising a tetanus neurotoxin (TeNT) conjugated to polyethylene glycol (PEG) in the manufacture of a medicament for treating hypotonia in a subject to which a second TeNT is administered.

[0183] 12. Use of a second TeNT in the manufacture of a medicament for treating hypotonia in a subject administered a first PEGylated tetanus neurotoxin (PEG-TeNT), said first PEGylated tetanus neurotoxin comprising tetanus neurotoxin (TeNT) conjugated to polyethylene glycol (PEG).

[0184] 13. Use of: (i) a first PEGylated tetanus neurotoxin (PEG-TeNT) comprising tetanus neurotoxin (TeNT) conjugated to polyethylene glycol (PEG); and (ii) a second TeNT in the manufacture of a medicament for treating hypotonia.

[0185] 14. The composition of any one of items 7 to 9, the method of item 10, or the use of any one of items 11 to 13, wherein the first PEG-TeNT or the second TeNT comprises a PEGylated TeNT light chain (LC), a PEGylated TeNT heavy chain (HC), or a PEGylated TeNT fragment c(c).

[0186] 15. The composition, method or use of any one of items 7 to 14, wherein the first PEG-TeNT or the second TeNT is PEG-TeNT-HC comprising a PEGylated HC, or is PEG-TeNT-LC-c comprising a PEGylated LC and a PEGylated c.

[0187] 16. The composition, method or use of any one of items 7 to 15, wherein the first PEG-TeNT is PEG-TeNT-HC and the second TeNT is PEG-TeNT-LC-c.

[0188] 17. The composition, method or use of any one of items 7 to 16, wherein the second TeNT comprises an inactivated TeNT.

[0189] 18. The composition, method or use according to item 17, wherein relative to SEQ ID NO:1, the inactivated TeNT comprises: 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 and W1288del; E270A; Y374A; E270A and Y374A; E270del; Y374del; or a combination thereof.

[0190] 19. The method or use according to any one of items 9 to 18, wherein the subject is administered:

[0191] PEG-TeNT comprising PEGylated c (PEG-TeNT-c) until efficacy decreases; then

[0192] a composition comprising PEG-TeNT-HC and PEG-TeNT-LC-c until the efficacy decreases; and then

[0193] PEG-TeNT comprising PEGylated LC and PEGylated HC (PEG-TeNT-LC-HC).

[0194] 20. The method or use according to any one of items 9 to 19, wherein the hypotonia is obstructive sleep apnea.

[0195] 21. The composition according to any one of items 7 or 14 to 18, wherein the composition is a therapeutic composition.

[0196] 22. A pegylated tetanus neurotoxin (PEG-TeNT), comprising a tetanus neurotoxin (TeNT) conjugated to polyethylene glycol (PEG), wherein the PEG is conjugated to a cysteine ​​residue, wherein the cysteine ​​residue is a surface serine→cysteine ​​substitution relative to SEQ ID NO: 1.

[0197] 23. The PEG-TeNT according to item 22, wherein the TeNT light chain (LC) is PEGylated, the TeNT heavy chain (HC) is PEGylated, or the TeNT fragment c(c) is PEGylated.

[0198] 24. The PEG-TeNT according to item 22 or 23, wherein LC is PEGylated and HC is PEGylated (PEG-TeNT-LC-HC), or LC is PEGylated and c is PEGylated (PEG-TeNT-LC-c).

[0199] 25. The PEG-TeNT of any one of items 22 to 24, wherein the PEG has a molecular weight of about 5 kDa, about 10 kDa, or about 20 kDa. 26. A method for treating hypotonia, the method comprising administering to a subject a PEGylated tetanus neurotoxin (PEG-TeNT) comprising a tetanus neurotoxin (TeNT) conjugated to polyethylene glycol (PEG), optionally wherein the PEG-TeNT is the PEG-TeNT of any one of items 22 to 25.

[0200] 27. Use of a pegylated tetanus neurotoxin (PEG-TeNT) comprising tetanus neurotoxin (TeNT) conjugated to polyethylene glycol (PEG), optionally the PEG-TeNT according to any one of items 22 to 25, in the manufacture of a medicament for the treatment of hypotonia.

[0201] 28. The method of item 26 or the use of item 27, wherein a first PEG-TeNT comprising PEGylated HC (PEG-TeNT-HC) and a second PEG-TeNT comprising PEGylated LC and PEGylated c (PEG-TeNT-LC-c) are administered to the subject.

[0202] 29. The method or use according to any one of items 26 to 28, wherein the subject is administered:

[0203] PEG-TeNT comprising PEGylated c (PEG-TeNT-c); and / or

[0204] 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

[0205] PEG-TeNT comprising PEGylated HC and PEGylated LC (PEG-TeNT-LC-HC).

[0206] 30. The method or use according to any one of items 26 to 29, wherein the treatment comprises administering to the subject:

[0207] administering PEG-TeNT comprising PEGylated c (PEG-TeNT-c) until efficacy decreases; then

[0208] PEG-TeNT-HC and PEG-TeNT-LC-c were administered until efficacy decreased; then

[0209] PEG-TeNT comprising PEGylated LC and PEGylated HC (PEG-TeNT-LC-HC) was administered.

[0210] 31. The method or use according to any one of items 26 to 30, wherein the treatment further comprises administering inactivated TeNT.

[0211] 32. The method or use according to item 31, wherein relative to SEQ ID NO:1, the inactivated TeNT 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; R1225K and W1288L; R1225E and W1288L; R1225del; W1288del; R1225del and W1288del; E270A; Y374A; E270A and Y374A; E270del; Y374del; or a combination thereof.

[0212] 33. The method or use according to any one of items 26 to 32, wherein the hypotonia is obstructive sleep apnea.

[0213] 34. A kit comprising the TeNT according to any one of items 1 to 6, the composition according to any one of items 7, 14 to 18, or 21, or the PEG-TeNT according to any one of items 22 to 25.

[0214] Example

[0215] Example 1: Preparation of PEG-TeNT-c ( Figure 1 A)

[0216] In this example, surface serine residues of TeNT-c were mutated to surface cysteine ​​residues to facilitate targeted PEG conjugation at immunogenic epitopes. Figure 1 Molecule of A. The mutations would be: S963C, S1041C, S1155C and S1187C.

[0217] The gene for TeNT with surface serine→cysteine ​​substitutions S963C, S1041C, S1155C, and S1187C will be synthesized by a commercial supplier (e.g., Integrated DNA Technologies, Inc.) The gene will be subcloned into the pRSET-A expression vector by restriction enzyme digestion to add a 6× histidine tag from the vector to the N-terminus of the mutant protein.

[0218] TeNT containing S963C, S1041C, S1155C, and S1187C will be expressed, PEGylated, and purified according to Example 5 to produce PEG-TeNT-c.

[0219] Example 2: Preparation of PEG-TeNT-HC ( Figure 1 B)

[0220] In this example, surface serine residues of TeNT-HC were mutated to surface cysteine ​​residues to facilitate targeted PEG conjugation at immunogenic epitopes. Figure 1 B. The mutations would be: S600C, S963C, S1041C, S1155C and S1187C ( Figure 24 , SEQ ID NO: 16).

[0221] The genes for TeNT with surface serine→cysteine ​​substitutions S600C, S963C, S1041C, S1155C, and S1187C will be synthesized by a commercial supplier (e.g., Integrated DNA Technologies). The genes will be subcloned into the pRSET-A expression vector ( Figure 25 , SEQ ID NO: 17, Figure 26 ) to add a 6×histidine tag from the vector to the N-terminus of the mutant protein.

[0222] TeNT containing S600C, S963C, S1041C, S1155C, and S1187C was expressed, PEGylated, and purified according to Example 5 to produce PEG-TeNT-HC.

[0223] Example 3: Preparation of PEG-TeNT-LC-c ( Figure 1 C)

[0224] In this example, surface serine residues of LC and c were mutated to surface cysteine ​​residues to facilitate targeted PEG conjugation at immunogenic epitopes. Figure 1C. The mutations are: S81C, S120C, S144C, S248C, S335C, S428C, S963C, S1041C, S1155C and S1187C ( Figure 21 , SEQ ID NO: 14).

[0225] The genes for TeNTs with surface serine→cysteine ​​substitutions S81C, S120C, S144C, S248C, S335C, S428C, S963C, S1041C, S1155C, and S1187C in LC and c were synthesized by a commercial supplier (Integrated DNA Technologies). The genes were subcloned into the pRSET-A expression vector ( Figure 22 , SEQ ID NO: 15, Figure 23 ) to add a 6×histidine tag from the vector to the N-terminus of the mutant protein.

[0226] TeNT containing 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.

[0227] Endotoxin removal

[0228] Endotoxin was removed from TeNT-LC-c serine mutant according to Example 4.

[0229] Attachment of PEG to cysteine ​​residues.

[0230] 1. A molar excess of PEG-maleimide, wherein the PEG has about 2 kDa, about 5 kDa, about 10 kDa, or about 20 kDa, is combined with serine mutant TeNT-LC-c (0.5 mg / mL-2 mg / mL) in PBS at pH 6.5-7.5.

[0231] 2. Ligation was performed at room temperature for 6 hours.

[0232] 3. Remove excess PEG by size exclusion chromatography.

[0233] Trypsin digestion activation of proteins

[0234] Trypsin digestion was performed according to Example 4 to activate the TeNT-LC-c serine mutant.

[0235] Example 4: Preparation of PEG-TeNT-LC-HC ( Figure 1 D) (Method 1)

[0236] Preparation of TeNT

[0237] 1. Use pRSET-TeNT vector ( Figure 3 and Figure 4 ) E. coli BL21 DE3 pLysS strain was electrotransformed and grown on LB agar with selective antibiotics (ampicillin and chloramphenicol) at 37°C overnight.

[0238] 2. Inoculate 200 mL of pre-induction broth with one colony from the selection plate. Pre-induction broth, pH 7.2-7.4, contains: 1.2% tryptone; 2.4% yeast extract; 2% glucose; 0.4% glycerol; 17 mM KH2PO4; 72 mM K2HPO4; and selection antibiotics (ampicillin and chloramphenicol).

[0239] 3. Incubate the culture at 30°C overnight with rapid shaking.

[0240] 4. Harvest the overnight culture by centrifugation at 4000 g for 10 minutes.

[0241] 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.

[0242] 6. Protein was expressed at 30°C with rapid shaking for 6 hours.

[0243] 7. Harvest cells by centrifugation at 4500 g for 15 minutes and resuspend the pellet in 30 mL of TBS (pH 8) containing 20 mM imidazole.

[0244] 8. Lyse the cells by sonication.

[0245] 9. Clarify the cell lysate by centrifugation at 4500 g for 20 minutes and filter through a 0.45 μm filter.

[0246] 10. The protein was purified by His-tag affinity chromatography using an AKTA pure 25 FPLC system (GE).

[0247] 11. The purified protein was buffer exchanged with PBS using size exclusion chromatography, followed by a second stage of purification by gel filtration using an AKTA pure 25 FPLC and a Superdex 200 increase 10 / 300 GL column.

[0248] Endotoxin removal

[0249] 1. Equilibrate a 0.5 mL endotoxin removal spin column to room temperature.

[0250] 2. Remove the plug from the bottom of the column, 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.

[0251] 3. Replace the column plug, remove the column cap, add 0.2N 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 hour to 2 hours.

[0252] 4. Remove the plug from the column, 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.

[0253] 5. Replace the column bottom plug, remove the column cap, add endotoxin-free 2M NaCl to the resin, replace the column cap, and invert the column several times to resuspend the resin.

[0254] 6. Remove the plug from the bottom of the column, 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.

[0255] 7. Replace the column bottom 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.

[0256] 8. Remove the plug from the bottom of the column, 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.

[0257] 9. Replace the column 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.

[0258] 10. Remove the plug from the bottom of the column, 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.

[0259] 11. Rinse the column two more times with phosphate buffer and discard the eluate.

[0260] 12. Replace the column bottom plug, remove the column cap, apply the sample to the resin, replace the column cap, and invert the column several times to resuspend the resin.

[0261] 13. Incubate the column at 4°C for at least 1 hour with inversion mixing.

[0262] 14. Remove the plug from the bottom of the column, 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. Reserve the sample.

[0263] 15. Repeat the endotoxin removal procedure using the regenerated spin column until endotoxin levels in the samples are at equivalent or lower levels so that all doses will contain less than 5 EU units of endotoxin per kg of subject.

[0264] Preparation of PEG-TeNT-LC-HC

[0265] 1. In a total volume of 500 μL of PBS (pH 7.4), 3 μmol of purified TeNT and 0.5 mmol of mpeg-NHS(SC) (Nanocs) were combined with one of 2 kDa, 5 kDa, 10 kDa, 20 kDa, or 30 kDa PEG.

[0266] 2. Mix the sample at room temperature for 3 hours.

[0267] 3. Remove excess PEG by size exclusion chromatography.

[0268] Trypsin digests proteins into their active form

[0269] 1. Dissolve 1 mg of protein in 0.5 mL of digestion buffer containing 0.1 M NH4HCO3 buffer (pH 8.0) or 0.1 M Tris buffer (pH 8.5).

[0270] 2. Wash 0.10 mL to 0.25 mL of immobilized TPCK trypsin with 3 x 500 μL of digestion buffer. Separate the gel from the buffer by centrifugation after each wash.

[0271] 3. Resuspend the gel in approximately 0.2 mL of digestion buffer.

[0272] 4. Add immobilized TPCK trypsin to the protein sample.

[0273] 5. Incubate the reaction mixture at 37°C in a rapid shaking incubator for 2 hours to 18 hours.

[0274] 6. Isolate the immobilized TPCK trypsin by centrifugation.

[0275] Example 5: Preparation of PEG-TeNT-LC-HC ( Figure 1 D) (Method 2)

[0276] In this example, surface serine residues of TeNT-LC-HC were mutated to surface cysteine ​​residues (S→C mutants) to facilitate targeted PEG conjugation at immunogenic epitopes. Relative to SEQ ID NO: 1, the TeNT mutations are: S81C; S120C; S144C; S248C; S335C; S428C; S600C; S963C; S1041C; S1155C; and S1187C.

[0277] Preparation of serine mutant TeNT-LC-HC

[0278] Encoding a gene containing an S→C mutation Figure 18 The vector pRSET-TeNT ( Figure 19 and Figure 20 ) The E. coli BL21(DE3) pLysS strain was electrotransformed. TeNT-LC-HC containing the S→C mutation was expressed and purified according to Example 4, except that a 15-minute treatment with 0.5 mM DTT was added between steps 10 and 11.

[0279] Endotoxin removal

[0280] Endotoxin was removed from TeNT-LC-HC serine mutants according to Example 4. Attachment of PEG to cysteine ​​residues.

[0281] 1. A molar excess of PEG-maleimide, wherein the PEG has about 2 kDa, about 5 kDa, about 10 kDa, or about 20 kDa, is combined with serine mutant TeNT-LC-HC (0.5 mg / mL-2 mg / mL) in PBS at pH 6.5-7.5.

[0282] 2. Ligation was performed at room temperature for 6 hours.

[0283] 3. Remove excess PEG by size exclusion chromatography.

[0284] Trypsin digestion activation of proteins

[0285] Trypsin digestion was performed according to Example 4 to activate the TeNT-LC-HC serine mutant.

[0286] Example 6: Inactive Bait TeNT

[0287] In this example, a non-PEGylated recombinant c was generated with inactivating R1225E and W1288A amino acid substitutions in the ganglioside binding region relative to SEQ ID NO: 1. The inactivated c was combined with an equimolar amount of non-PEGylated LC-HN to generate inactive decoy TeNT. The resulting inactive TeNT will be used as a decoy for antibody-based neutralization responses in subjects vaccinated with tetanus toxoid vaccine.

[0288] Preparation of c containing R1225E and W1288A

[0289] The c gene with R1225E and W1288A was synthesized by a commercial supplier (Integrated DNA Technologies) and subcloned into the pRSET-A expression vector by restriction enzyme digestion to add a 6×histidine tag from the vector to the N-terminus of the mutant protein.

[0290] c( Figure 12 , SEQ ID NO: 8) of the pRSET-TeNT-c vector ( Figure 13 (SEQ ID NO:9) and Figure 14 ) E. coli BL21 (DE3) pLysS strain was electroporated and grown overnight on LB agar containing selective antibiotics (ampicillin, chloramphenicol) at 37° C. c containing R1225E, W1288A was expressed and purified according to Example 4.

[0291] Endotoxin removal

[0292] Endotoxin was removed from the purified protein according to Example 4.

[0293] Preparation of LC-HN

[0294] Using pRSET-TeNT-LC-HN vector ( Figure 10 and Figure 11 ) E. coli BL21 (DE3) pLysS strain was electroporated and grown overnight on LB agar containing selective antibiotics (ampicillin, chloramphenicol) at 37° C. LC-HN was expressed and purified according to Example 4.

[0295] Endotoxin removal

[0296] Endotoxin was removed from the purified protein according to Example 4.

[0297] Preparation of LC-HN containing E270A and Y374A

[0298] E. coli BL21 (DE3) pLysS strain was electroporated with the pRSET-TeNT-LC-HN vector encoding LC-HN containing E270A, Y374A and grown overnight on LB agar containing selective antibiotics (ampicillin, chloramphenicol) at 37° C. LC-HN containing E270A, Y374A was expressed and purified according to Example 4.

[0299] Endotoxin removal

[0300] Endotoxin was removed from the purified protein according to Example 4.

[0301] Example 7: Inactive Bait TeNT

[0302] The gene for TeNT containing R1225E and W1288A was synthesized by a commercial supplier (Integrated DNA Technologies). The gene was subcloned into the pRSET-A expression vector by restriction enzyme digestion to add a 6× histidine tag from the vector to the N-terminus of the mutant protein ( Figure 15 (SEQ ID NO: 10), Figure 16 (SEQ ID NO: 11) and Figure 17 ).

[0303] Inactivated TeNT containing R1225E and W1288A was expressed and purified according to Example 4.

[0304] Endotoxin removal

[0305] Endotoxin was removed from the purified protein according to Example 4.

[0306] Example 8: PEG-TeNT Analysis

[0307] TeNT was prepared according to Example 4 and PEGylated, and then analyzed by SDS-PAGE ( Figure 28 ) and detected by Western blot (A) using Coomassie blue and (B) using polyclonal anti-TeNT antibodies. (B) Shows that immunogenicity is proportional to PEG molecular weight.

[0308] Example 9: Reduced immunogenicity of PEG-TeNT compared to TeNT

[0309] Four PEG-TeNTs, each containing PEG of a different molecular weight (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.

[0310] In the first determination ( Figure 29 In A), TeNT was adsorbed onto an ELISA plate. The adsorbed TeNT was then probed with a polyclonal anti-TeNT antibody pre-incubated with each of 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). In this assay, a higher response (OD 450 nm) indicates a greater affinity for TeNT and, therefore, reduced immunogenicity of PEG-TeNT.

[0311] In the second determination ( Figure 29 In B), each PEG-TeNT was adsorbed onto 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 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 a greater affinity for TeNT and, therefore, reduced immunogenicity of the PEG-TeNT.

[0312] This example demonstrates that anti-TeNT antibodies preferentially bind TeNT and that PEGylated TeNT has reduced immunogenicity relative to TeNT (ie, non-PEGylated).

[0313] Example 10: Reduced Immunogenicity of PEG-TeNT-LC-c Serine Mutant Compared to TeNT-LC-c Serine Mutant

[0314] TeNT-LC-c serine mutants were prepared according to Example 5. Four samples of TeNT-LC-c serine mutants were PEGylated according to Example 5, each sample containing PEG of different molecular weights (2 kDa, 5 kDa, 10 kDa, and 20 kDa). PEG-TeNT-LC-c serine mutants were then assayed against TeNT-LC-c serine mutants by competitive ELISA.

[0315] In the first determination ( Figure 29C), TeNT-LC-c serine mutants were adsorbed onto ELISA plates. The adsorbed TeNT serine mutants were then probed with polyclonal anti-TeNT antibodies pre-incubated with each of 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) indicated a greater affinity for the TeNT serine mutant and, therefore, reduced immunogenicity of the PEG-TeNT serine mutant.

[0316] In the second determination ( Figure 29 D), each PEG-TeNT-LC-c serine mutant was adsorbed onto 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 μg / mL, 1 μg / mL, 0.1 μg / mL, and 0.01 μg / mL) of TeNT serine mutant antigen. In this assay, a lower response (OD 450 nm) indicates a greater affinity for the TeNT serine mutant and, therefore, reduced immunogenicity of the PEG-TeNT serine mutant.

[0317] This example demonstrates that anti-TeNT antibodies preferentially bind to TeNT-LC-c serine mutants and that PEGylated TeNT-LC-c serine mutants have reduced immunogenicity relative to TeNT-LC-c serine mutants (ie, non-PEGylated).

[0318] Example 11: Reduced immunogenicity of PEG-TeNT compared to TeNT

[0319] A competitive ELISA assay will be performed according to Example 9, except that the polyclonal antibodies will be replaced with 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 (i.e., non-PEGylated TeNT) than for PEG-TeNT.

[0320] Example 12: In vivo model

[0321] According to Example 4, PEG-TeNT-LC-HC was prepared by attaching PEG (about 5 kDa, about 10 kDa, or about 20 kDa) to surface-exposed lysine residues of recombinant TeNT.

[0322] One or more units of PEG-TeNT-LC-HC in 15 μL PBS were injected into the hind limbs of female C57BL / 6 mice. Each animal showed localized limb twitching within 48 hours of injection ( Figure 30 A and Figure 30 B).

[0323] Example 13: In vivo model

[0324] PEG-TeNT-LC-HC, with 5 kDa PEG attached to surface lysine residues of TeNT according to Example 4, was combined with c bait (i.e., c inactivated by R1225E and W1288A) at a 1:10 molar ratio. 15 μl of the composition containing 5 ng of PEG-TeNT in PBS was injected into the hind limb muscles of female C57BL / 6 mice. Each animal exhibited localized limb twitching within 48 hours, with symptom progression indistinguishable from animals treated with PEG-TeNT-LC-HC in the absence of bait.

[0325] Example 14

[0326] PEG-TeNT-LC-HC containing 5 kDa, 10 kDa, or 20 kDa PEG was administered intramuscularly at 50 ng / kg to 500,000 ng / kg to the hind leg muscles of mice previously immunized with tetanus toxoid. Increased muscle contraction was observed in the injected muscles, lasting up to 3 days, and was greater than the effect observed in mice that received the same unit of TeNT. Figure 31 A reports the results for PEG-TeNT-LC-HC 20 kDa.

[0327] Example 15

[0328] Compositions comprising TeNT-LC-HC and a 10:1 or 100:1 molar excess of the decoy TeNT produced according to Example 6 were administered intramuscularly at 50 ng / kg to 500,000 ng / kg to the hind leg muscles of mice previously immunized with tetanus toxoid. Increased muscle contraction was observed in the injected muscles, lasting up to 3 days, and was greater than the effect observed in mice receiving TeNT administration ( Figure 32 A).

[0329] Example 16

[0330] 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 the decoy TeNT produced according to Example 6 were administered intramuscularly at 50 ng / kg to 500,000 ng / kg to the hind leg muscles of mice previously immunized with tetanus toxoid. Increased muscle contraction was observed in the injected muscles for up to 3 days, and the increase in muscle contraction was greater than the effect observed in mice that received TeNT administration and greater than the effect observed in mice that received PEG-TeNT administration alone ( Figure 32 B).

[0331] Example 17

[0332] PEG-TeNT-LC-HC containing 20 kDa PEG is administered intramuscularly at 0.01 ng / kg to 50,000 ng / kg to the left geniohyoid muscle of human subjects previously vaccinated with tetanus toxoid vaccine. 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 muscle of the same subject who received only vehicle administration.

[0333] Example 18

[0334] A composition containing PEG-TeNT-LC-HC containing 20 kDa PEG and a 10:1-1000:1 molar excess of decoy TeNT produced according to Example 6 was administered intramuscularly at 50 ng / kg to the left geniohyoid muscle of a human subject previously vaccinated with tetanus toxoid vaccine. PEG-TeNT-LC-HC containing 20 kDa PEG was administered intramuscularly at 50 ng / kg to the right geniohyoid muscle of the same human subject.

[0335] Increased muscle contraction will be observed in both the left and right geniohyoid muscles for up to 2 weeks, but will be greater in the left geniohyoid muscle treated with the composition comprising PEG-TeNT-LC-HC and decoy TeNT compared to PEG-TeNT-LC-HC alone.

[0336] Example 19

[0337] Approximately 30 kg bulldogs will be administered intramuscularly 25 ng / kg to 50,000 ng / kg of PEG-TeNT-c containing 20 kDa PEG, with the dose being administered bilaterally, split into the left and right geniohyoid muscles. Following administration, animals treated with PEG-TeNT will experience a reduction in obstructive sleep apnea (OSA) compared to animals treated with vehicle alone. Bulldogs will be observed weekly for OSA and PEG-TeNT-c doses will be repeated as needed until efficacy decreases, as determined by recovery of OSA comparable to animals treated with vehicle alone.

[0338] Thereafter, the bulldogs will be 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, administered bilaterally to the left and right geniohyoid muscles. Following administration, animals treated with PEG-TeNT will experience a reduction in OSA compared to animals treated with vehicle alone. Bulldogs will be observed weekly for OSA and PEG-TeNT-HC and PEG-TeNT-LC-c doses will be repeated as needed until efficacy is reduced, as determined by recovery of OSA comparable to animals treated with vehicle alone.

[0339] The bulldogs will then be administered 25-50,000 ng / kg of PEG-TeNT-LC-HC containing 20 kDa PEG, administered bilaterally to the left and right geniohyoid muscles. Following administration, animals treated with PEG-TeNT will experience a reduction in OSA compared to animals treated with vehicle alone. Bulldogs will be observed weekly for OSA and PEG-TeNT-LC-HC doses will be repeated as needed until efficacy is reduced, as determined by recovery of OSA comparable to animals treated with vehicle alone.

[0340] Example 20

[0341] The TeNT epitope recognized by the major human antibody clonotypes identified by da Silva Antunes et al. (2017) PloS One, 12(1), e0169086 and Palermo et al. (2017) Biotechnology Journal, 12(10), 1700197 was mapped onto a 3-dimensional model of TeNT derived from crystallographic data deposited in the Protein Data Bank (Accession ID PDB: 5N0B) using Discovery Studio. Surface serine residues in or around the identified epitope were identified and mutated to cysteines for subsequent PEGylation as described in Examples 1 to 3 and 5.

[0342] Example 21

[0343] A mixture of 1 nanogram to 64 micrograms of PEG-TeNT (5 kDa, 10 kDa, or 20 kDa branched or linear PEG attached to surface lysine or cysteine ​​residues) and a 10-1000-fold molar excess of decoy TeNT was injected intramuscularly into the hind leg muscles of mice vaccinated with tetanus toxoid. Localized convulsions were observed, lasting for a period of several hours to several months, which were greater than the effects observed in mice injected with native TeNT.

[0344] Example 22

[0345] A mixture of 1 ng to 64 μg of PEG-TeNT (5 kDa, 10 kDa, or 20 kDa branched or linear PEG attached to surface lysine or cysteine ​​residues) and a 10-1000-fold molar excess of decoy TeNT was injected intramuscularly into the geniohyoid muscle of humans vaccinated with tetanus toxoid. Localized twitching was observed, lasting for weeks to months, and was greater than the effect observed in humans injected with native TeNT.

[0346] Example 23

[0347] English bulldogs with sleep-disordered breathing were administered intramuscularly at 0.001-10 IU / Kg of TeNT, with the doses being administered bilaterally, split into the left and right geniohyoid muscles. Following administration, a significant reduction in obstructive sleep apnea (OSA) was observed at the highest dose. The baseline respiratory disturbance index (RDI) score was 19.9 (interquartile range 5.45), which decreased to 13.2 (interquartile range 4.45) after treatment with 10 IU / Kg TeNT, which was significant compared to placebo administration (P=0.043) as determined by the Wilcoxon signed-rank test. The bulldogs maintained the reduction in RDI four months after the trial; median RDI=13.4, P=0.043; Wilcoxon signed-rank test.

Claims

1. A composition comprising: (i) a first active tetanus neurotoxin (TeNT) and (ii) a second inactive TeNT, wherein the inactive TeNT contains amino acid substitutions R1225E and W1288A relative to the tetanus neurotoxin of SEQ ID NO:

1.

2. The composition of claim 1, wherein the composition is a therapeutic composition.

3. Use of a tetanus neurotoxin (TeNT) composition in the manufacture of a medicament for treating hypotonia, wherein the TeNT composition comprises: (i) a first active TeNT and (ii) a second inactive TeNT, wherein the inactive TeNT contains amino acid substitutions R1225E and W1288A relative to the tetanus neurotoxin of SEQ ID NO:

1.

4. The composition according to claim 1 or 2 or the use according to claim 3, wherein relative to SEQ ID NO: 1, the second inactive TeNT further comprises E270A; Y374A; E270A and Y374A; E270del; Y374del; or a combination thereof.

5. Use according to claim 3 or 4, wherein the hypotonia is obstructive sleep apnea. A kit comprising the composition according to claim 1 or 2.

Images