Compositions and methods for treatment of pain

Abstract

Embodied herein are engineered fusion proteins that bind and target nociceptor neurons, compositions comprising these engineered fusion proteins, and methods for treatment of pain using these engineered fusion proteins or compositions containing the engineered fusion proteins. The engineered fusion proteins contain domains derived from protein toxins such as the anthrax toxin, clostridial botulinum family of toxins, disulphide-containing toxins, and AB component type toxins.

1 . A fusion protein comprising:
 a) a  botulinum  neurotoxin (BTx) or a tetanus neurotoxin (TTx), and   b) an anthrax toxin protective antigen (PA), or a C-terminal receptor-binding domain of PA,
 wherein part (a) and (b) are linked or fused together. 
   
     
     
         2 . The fusion protein of  claim 1 , wherein the BTx or TTx comprises a BTx or TTx enzymatic moiety and the translocation signal. 
     
     
         3 . The fusion protein of  claim 2 , wherein the BTx enzymatic moiety or translocation signal is selected from the BTx light chain and heavy chain domains of any one of BTx/A, BTx/B, BTx/C, BTx/D, BTx/E, BTx/F, BTx/G, and a non-Clostridial  botulinum -like toxin. 
     
     
         4 . The fusion protein of  claim 2 , wherein the BTx or TTx enzymatic moiety or translocation signal is selected from the respective LC enzymatic moiety or H N  translocation peptide found in Table 1. 
     
     
         5 . The fusion protein of  claim 1 , wherein the part (a) and (b), or the enzymatic moiety and translocation peptide are linked by a linker peptide. 
     
     
         6 . The fusion protein of  claim 5 , wherein the linker peptide is 1-20 amino acids long. 
     
     
         7 . The fusion protein of  claim 6 , wherein the linker peptide is stable in human serum for at least 1 minute. 
     
     
         8 . The fusion protein of  claim 7 , wherein the linker peptide comprises at least one amino acid that is Gly or Ser. 
     
     
         9 . The fusion protein of  claim 6 , wherein the linker peptide does not comprise Lys and/or Arg. 
     
     
         10 . The fusion protein of  claim 1 , wherein the C-terminal receptor-binding domain of PA comprises a PAd4 domain, a PAd2 and a PAd4 domain, or PA63. 
     
     
         11 . The fusion protein of  claim 1 , wherein the PA or the C-terminal receptor-binding domain of PA is resistant to cleavage by a protease. 
     
     
         12 . The fusion protein of  claim 11 , wherein the the PA or the C-terminal receptor-binding domain of PA is resistant to furin cleavage, or Lys C cleavage or both. 
     
     
         13 . A fusion protein comprising:
 a) a non-cytotoxic protease, which protease is capable of cleaving a SNARE protein in a nociceptor neuron; and   b) a protein capable of binding to an anthrax toxin protective antigen (PA) or a fragment thereof,   wherein the PA or fragment binds a receptor expressed on the nociceptor neuron.   
     
     
         14 . The fusion protein of  claim 13 , wherein the non-cytotoxic protease comprises a clostridial neurotoxin L-chain. 
     
     
         15 . The fusion protein of  claim 14 , wherein the clostridial neurotoxin is  botulinum  neurotoxin (BTx) or tetanus neurotoxin (TTx). 
     
     
         16 . The fusion protein of  claim 14 , wherein the clostridial neurotoxin L-chain is selected from Table 1. 
     
     
         17 . The fusion protein of  claim 13 , wherein the PA-binding receptor expressed on the nociceptor neuron is ANTXR2 (CMG2). 
     
     
         18 . The fusion protein of  claim 13 , wherein the protein capable of binding to PA is:
 i) an anthrax toxin lethal factor (LF); or   ii) an anthrax toxin edema factor (EF).   
     
     
         19 . The fusion protein of  claim 18 , wherein PA binding domain of LF is the N-terminal domain of LF, (abbreviated as LFPABD or LFn). 
     
     
         20 . The fusion protein of  claim 18 , wherein PA binding domain of EF is the N-terminal domain of EF, (abbreviated as EFPABD or EFn). 
     
     
         21 . A fusion protein comprising:
 a) a disulfide-containing peptide toxin (these include the channel blocking toxins having a cysteine-knot motif), which is capable of blocking ion channels in a nociceptor neuron; and   b) a targeting moiety (TM) that is capable of binding to a binding site on the nociceptor neuron, wherein the nociceptor neuron expresses said ion channels therein (e.g., sodium or calcium or both sodium and calcium);   
     
     
         22 . The fusion protein of  claim 21 , wherein the disulfide-containing peptide toxin comprises a cysteine knot motif. 
     
     
         23 . The fusion protein of  claim 21 , wherein the disulfide-containing peptide toxin is a conotoxin, an agatoxin, a delta-palutoxin, a huwentotoxin or a ProTx II toxin. 
     
     
         24 . The fusion protein according to  claim 21 , wherein the TM is selected from the group consisting of:
 i) an anthrax toxin protective antigen (PA);   ii) a C-terminal receptor-binding domain of PA;   iii) a nociceptor neuron-binding protein.   
     
     
         25 . The fusion protein according to  claim 24 , wherein the PA or C-terminal receptor-binding domain of PA interacts with and binds the ANTXR2 (CMG2) receptor expressed on the nociceptor neuron. 
     
     
         26 - 29 . (canceled) 
     
     
         30 . The fusion protein of  claim 24 , wherein the nociceptor neuron-binding protein is an antibody. 
     
     
         31 . The fusion protein of  claim 30 , wherein the antibody specifically binds to nerve growth factor receptor, the ANTXR2 receptor, or an ion-channel protein present on nociceptor neurons. 
     
     
         32 . The fusion protein of  claim 31 , wherein the ion-channel protein is selected from Nav1.7, Nav1.8 or Nav1.9. 
     
     
         33 . A fusion protein comprising:
 a) a disulfide-containing peptide toxin (this are channel blocking toxin having a cysteine-knot motif), which is capable of blocking sodium or calcium or both sodium and calcium channels in a nociceptor neuron; and   b) a protein capable of binding to an anthrax toxin protective antigen (PA) or a fragment thereof,   wherein the fragment binds a receptor expressed on the nociceptor neuron.   
     
     
         34 - 37 . (canceled) 
     
     
         38 . A fusion protein comprising:
 a) an AB toxin;   b) an anthrax toxin protective antigen (PA) or a fragment thereof, wherein the PA or fragment binds a receptor expressed on the nociceptor neuron; and   c) a Translocation domain (TL) that is capable of translocating the protease from within an endosome, across the endosomal membrane and into the cytosol of the nociceptor neuron.   
     
     
         39 . The fusion protein of  claim 38 , wherein the AB toxin is selected from
 a. Ricin toxin,   b. Cholera toxin A-part and B-part;   c.  Pseudomonas aeruginosa  Exotoxin A A-part and B-part;   d. Shiga toxin A-part and B-part; and   e. Diphtheria toxin A-part and B-part.   
     
     
         40 . (canceled) 
     
     
         41 . The fusion protein of  claim 38 , wherein the PA fragment is a C-terminal receptor-binding domain of PA. 
     
     
         42 . The fusion protein of  claim 38 , wherein the TL is
 i) a clostridial neurotoxin translocation domain;   ii) a holotoxin; or   iii) a variant form of the holotoxin that has the toxin receptor-binding function of the AB toxin negated.   
     
     
         43 - 49 . (canceled) 
     
     
         50 . A method for treatment of pain, the method comprising administering to a subject in need thereof the fusion protein of  claim 1 . 
     
     
         51 . A method of treating pain comprising administering to a subject in need thereof, native mature anthrax toxin protective antigen (PA) and anthrax toxin edema factor (EF), anthrax toxin lethal factor (LF) or any combination thereof. 
     
     
         52 - 69 . (canceled) 
     
     
         70 . A fusion protein comprising:
 a) a  botulinum  neurotoxin N-terminal enzymatic domain of a  botulinum  neurotoxin (BTx) moiety (L chain of BTx), and   b) a N-terminal domain (LFn) of anthrax toxin lethal factor, which domain binds to oligomeric forms of PA63, the proteolytically activated form of anthrax PA; or the N-terminal domain (EFn) of anthrax toxin edema factor, which domain binds to oligomeric forms of PA63, the proteolytically activated form of anthrax PAthe enzymatic domain,   wherein part (a) is linked N-terminally or C-terminally or both N-terminally and C-terminally to part (b).   
     
     
         71 . The fusion protein of  claim 70  further comprising an amino acid sequence defining a belt corresponding to the N-terminal part of the BTx H N  domain which is located at C-terminal side of the BTx moiety, and the belt is linked to the L chain. 
     
     
         72 . The fusion protein of  claim 71 , wherein the residue corresponding to the L chain junction with the belt of the BTx H N  domain has been cleaved. 
     
     
         73 . The fusion protein of  claim 71 , wherein the Cys residues in the BTx L chain and the belt corresponding to the N-terminal part of the BTx H N  domain have been changed to Ala, Ser, or Thr. 
     
     
         74 . The fusion protein of  claim 70 , further comprising at least one D-amino acid at the N-terminus of the fusion protein. 
     
     
         75 . The fusion protein of  claim 70 , further comprising a linker peptide between the BTx L moiety and the LFn or EFn domain. 
     
     
         76 - 79 . (canceled) 
     
     
         80 . A composition comprising  claim 70 . 
     
     
         81 . (canceled) 
     
     
         82 . The composition of  claim 80  and a native anthrax toxin protective antigen (PA) protein. 
     
     
         83 - 85 . (canceled) 
     
     
         86 . A fusion protein comprising:
 a) anthrax toxin protective antigen (PA); an anthrax toxin protective antigen C-terminal receptor binding domain (PAd4); or a nociceptor neuron-binding protein linked to,   b) a disulfide-containing peptide toxin.   
     
     
         87 . The fusion protein of  claim 86 , wherein the disulfide-containing peptide toxin is an inhibitor cysteine knot toxin. 
     
     
         88 . (canceled) 
     
     
         89 . The fusion protein of  claim 86 , further comprising a linker peptide between the PA, PAd4 or nociceptor-binding protein and the inhibitor cysteine knot toxin. 
     
     
         90 - 104 . (canceled) 
     
     
         105 . A fusion protein comprising a disulfide-containing peptide toxin operably linked N-terminally or C-terminally or both N-terminally and C-terminally, or chemically crosslinked at one or more sites to the N-terminal domain (LFn) of anthrax toxin lethal factor, which domain binds to oligomeric forms of PA63, the proteolytically activated form of anthrax PA; or the N-terminal domain (EFn) of anthrax toxin edema factor, which domain binds to oligomeric forms of PA63, the proteolytically activated form of anthrax PA. 
     
     
         106 . The fusion protein of  claim 105 , further comprising a linker peptide between the LFn and the toxin or the EFn and the toxin. 
     
     
         107 - 115 . (canceled) 
     
     
         116 . A fusion protein comprising an AB toxin fused to a linker peptide operably linked to a C-terminal receptor-binding domain of anthrax toxin protective antigen (PAd4 domain), wherein the fusion protein further comprises a translocation domain, a holotoxin or a mutant form of the holotoxin that has the toxin receptor-binding function of the AB toxin negated. 
     
     
         117 - 121 . (canceled) 
     
     
         122 . The fusion protein of  claim 116 , wherein 1-60 consecutive amino acids from the N-terminal side adjacent to the native PAd4 domain are further incorporated between the AB toxin and the PAd4. 
     
     
         123 - 131 . (canceled) 
     
     
         132 . A fusion protein comprising an N-terminal enzymatic domain (Chain A) together with the translocation/pore-forming domain from a tetanus neurotoxin (TTx) operably linked to a C-terminal receptor-binding domain of anthrax toxin protective antigen (PAd4 domain). 
     
     
         133 . The fusion protein of  claim 132  further comprising a linker peptide between the TTx moiety and the PAd4 domain. 
     
     
         134 - 138 . (canceled) 
     
     
         139 . The fusion protein of  claim 132 , wherein 1-60 consecutive amino acids from the N-terminal side adjacent to the native PAd4 domain are further incorporated between the BTx moiety and the PAd4. 
     
     
         140 - 148 . (canceled) 
     
     
         149 . A nucleic acid encoding the fusion protein of  claim 132 . 
     
     
         150 . A vector comprising the nucleic acid of  claim 149 . 
     
     
         151 . The vector of  claim 150 , wherein the vector is a plasmid, a bacteriophage, a phagmid, a cosmid, a viral particle, or viral vector. 
     
     
         152 . A cell comprising the nucleic acid of  claim 149 . 
     
     
         153 . A method of producing fusion protein comprising:
 a) culturing the cell of  claim 152  in conditions such that the fusion protein is expressed; and   b) recovering the fusion protein.   
     
     
         154 . The fusion protein produced by the method of  claim 153 . 
     
     
         155 . The fusion protein of  claim 1 , which is glycosylated. 
     
     
         156 - 164 . (canceled) 
     
     
         165 . A use of fusion protein of  claim 132  for the treatment of pain. 
     
     
         166 . A use of fusion protein of  claim 132  for the manufacture of a medicament for the treatment of pain. 
     
     
         167 . A method for treatment of pain, the method comprising administering to a subject in need thereof the composition from  claim 80 . 
     
     
         168 . The method of  claim 167 , wherein the administering is performed by intrathecal infusion or intra-cerebroventricular infusion or by an epidural injection into the central nervous system, or by peripheral administration using intradermal injection, subcutaneous injection, intramuscular injection, intraneural injection, or intra-articular injection. 
     
     
         169 . The method of  claim 168 , wherein the pain is selected from diabetic neuropathic pain, cancer pain, fibromyalgia and other systemic pain disorders. 
     
     
         170 . A method for treatment of nerve, joint, skin, visceral, bladder, or muscle pain comprising administering peripherally by intradermal injection, subcutaneous injection, intramuscular injection, intraneural injection, or intra-articular injection to a subject in need thereof the composition from  claim 80 . 
     
     
         171 . A method for treatment of diabetic neuropathic pain, cancer pain, fibromyalgia or other systemic pain disorders comprising administering by epidural injection, intrathecal infusion or intra-cerebroventricular infusion into the central nervous system of a subject in need thereof the composition from  claim 80 . 
     
     
         172 . The method of  claim 167 , wherein the composition is administered separately before, simultaneously or after administering a composition comprising an anthrax protective antigen (PA) in a pharmaceutically acceptable carrier, excipient or diluent. 
     
     
         173 . (canceled) 
     
     
         174 . A method of manufacture of a pharmaceutical composition of  claim 80  for the treatment of pain, wherein the pharmaceutical composition comprising fusion protein and a pharmaceutically acceptable carrier or excipient. 
     
     
         175 . A fusion protein comprising:
 a. a  botulinum  neurotoxin (BTx) moiety, wherein the BTx moiety comprises an enzymatic moiety and a translocation domain and   b. a targeting moiety comprising a C-terminal receptor-binding domain of an anthrax toxin protective antigen (PA), wherein the C-terminal receptor-binding domain of PA comprises a PAd4 domain, wherein part (a) and (b) are linked or fused together.   
     
     
         176 . A nucleic acid encoding a fusion protein comprising:
 a. a  botulinum  neurotoxin (BTx) moiety that comprises an enzymatic moiety and a translocation domain; and   b. a targeting moiety comprising a C-terminal receptor-binding domain of anthrax toxin protective antigen (PA), wherein the C-terminal receptor-binding domain of PA comprises a PAd4 domain,   wherein part (a) and (b) are linked or fused together.