US2006110410A1PendingUtilityA1

Recombinant toxin fragments

62
Individually held — no corporate assignee on recordPriority: Sep 12, 2002Filed: Sep 12, 2003Published: May 25, 2006
Est. expirySep 12, 2022(expired)· nominal 20-yr term from priority
Inventors:Clifford ShoneKeith FosterJohn ChaddockPhilip MarksJ. Mark SuttonPatrick StancombeJonathan Wayne
C07K 2319/55A61K 2039/53C07K 2319/705A61K 38/00C07K 14/33A61K 39/08A61K 47/6415A61K 2039/505C07K 2319/00A61K 47/646C07K 2319/75C07K 2319/50C07K 2319/20C07K 2319/23C12N 9/52A61P 31/04Y10S530/825A61K 2039/627C12N 15/62A61P 43/00A61K 39/00
62
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Claims

Abstract

A single polypeptide is provided which comprises first and second domains. The first domain enables the polypeptide to cleave one or more vesicle or plasma-membrane associated proteins essential to exocytosis, and the second domain enables the polypeptide to be translocated into a target cell or increases the solubility of the polypeptide, or both. The polypeptide thus combines useful properties of a clostridial toxin, such as a botulinum or tetanus toxin, without the toxicity associated with the natural molecule. The polypeptide can also contain a third domain that targets it to a specific cell, rendering the polypeptide useful in inhibition of exocytosis in target cells. Fusion proteins comprising the polypeptide, nucleic acids encoding the polypeptide and methods of making the polypeptide are also provided. Controlled activation of the polypeptide, is possible and the polypeptide can be incorporated into vaccines and toxin assays.

Claims

exact text as granted — not AI-modified
1 . A single chain polypeptide comprising first and second domains, wherein:—said first domain is a clostridial neurotoxin light chain or a fragment or a variant thereof, wherein said first domain is capable of cleaving one or more vesicle or plasma membrane associated proteins essential to exocytosis; and said second domain is a clostridial neurotoxin heavy chain H N  portion or a fragment or a variant thereof, wherein said second domain is capable of (i) translocating the polypeptide into a cell or (ii) increasing the solubility of the polypeptide compared to the solubility of the first domain on its own or (iii) both translocating the polypeptide into a cell and increasing the solubility of the polypeptide compared to the solubility of the first domain on its own; and said second domain lacks a functional C-terminal part of a clostridial neurotoxin heavy chain designated H C  thereby rendering the polypeptide incapable of binding to cell surface receptors that are the natural cell surface receptors to which native clostridial neurotoxin binds; and wherein said single chain polypeptide comprises a sequence selected from the group consisting of:—
 (I) SEQ ID NO: 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 139, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, and 175; or    (II) a fragment or variant of (I) having a first domain that is capable of cleaving one or more vesicle or plasma membrane associated proteins essential to exocytosis.    
     
     
         2 . A polypeptide according to  claim 1  wherein said clostridial toxin heavy chain is a botulinum neurotoxin heavy chain.  
     
     
         3 . A polypeptide according to  claim 1  wherein said clostridial toxin heavy chain is a tetanus neurotoxin heavy chain.  
     
     
         4 . A polypeptide according to  claim 1 , wherein the first domain exhibits endopeptidase activity specific for a substrate selected from one or more of SNAP-25, synaptobrevin/VAMP and syntaxin.  
     
     
         5 . A polypeptide according to  claim 1 , wherein said second domain is a clostridial toxin heavy chain H N  portion.  
     
     
         6 . A polypeptide according to  claim 1 , wherein said clostridial neurotoxin heavy chain is a botulinum neurotoxin type A chain.  
     
     
         7 . A polypeptide according to  claim 1 , wherein the second domain comprises the 423 N-terminal amino acids of botulinum toxin type A heavy chain.  
     
     
         8 . A polypeptide according to  claim 1 , wherein said clostridial neurotoxin heavy chain is a botulinum neurotoxin type B chain.  
     
     
         9 . A polypeptide according to  claim 1 , wherein the second domain comprises the 107 N-terminal amino acids of a botulinum toxin type B heavy chain.  
     
     
         10 . A polypeptide according to  claim 1 , wherein the second domain comprises the 417 N-terminal amino acids of botulinum toxin type B heavy chain.  
     
     
         11 . A polypeptide according to  claim 1  wherein the second domain comprises the 422 N-terminal amino acids of tetanus heavy chain.  
     
     
         12 . A polypeptide according to  claim 1  wherein the second domain comprises the 100 N-terminal amino acids of a clostridial neurotoxin heavy chain.  
     
     
         13 . A polypeptide according to  claim 1  comprising a site for cleavage by a proteolytic enzyme.  
     
     
         14 . A polypeptide according to  claim 13 , wherein the cleavage site is not present in a native clostridial neurotoxin.  
     
     
         15 . A polypeptide according to  claim 13 , wherein the cleavage site allows proteolytic cleavage of the first and second domains.  
     
     
         16 . A polypeptide according to  claim 13 , wherein the cleavage site allows proteolytic cleavage of the first and second domains, and when so cleaved said first domain exhibits greater enzyme activity in cleaving said one or more vesicle or plasma membrane associated protein than does the polypeptide prior to said proteolytic cleavage.  
     
     
         17 . A polypeptide according to  claim 13  obtainable by providing a first nucleic acid sequence encoding said cleavage site within a second nucleic acid sequence encoding said single chain polypeptide.  
     
     
         18 . A polypeptide according to  claim 1 , wherein the second domain lacks a C-terminal part of a clostridial neurotoxin heavy chain designated H C .  
     
     
         19 . A polypeptide according to  claim 1 , further comprising a third domain that binds the polypeptide to a cell, by binding of the third domain directly to a cell or by binding of the third domain to a ligand or to ligands that bind to a cell.  
     
     
         20 . A polypeptide according to  claim 19 , wherein said third domain is for binding the polypeptide to an immunoglobulin.  
     
     
         21 . A polypeptide according to  claim 20 , wherein said third domain is a tandem repeat synthetic IgG binding domain derived from domain b of Staphylococcal protein A.  
     
     
         22 . A polypeptide according to  claim 19 , wherein said third domain comprises an amino acid sequence that binds to a cell surface receptor.  
     
     
         23 . A polypeptide according to  claim 22 , wherein said third domain is insulin-like growth factor-1 (IGF-1).  
     
     
         24 . A polypeptide according to  claim 1  including a spacer molecule between the first and second domains.  
     
     
         25 . A polypeptide according to  claim 19  including a spacer molecule between the second and third domains.  
     
     
         26 . A polypeptide according to  claim 1 , further comprising a purification tag that binds to an affinity matrix thereby facilitating purification of the polypeptide using said matrix.  
     
     
         27 . A polypeptide according to  claim 26  including a spacer molecule between the purification tag and the polypeptide.  
     
     
         28 . A polypeptide according to  claim 26 , wherein said purification tag binds to an affinity matrix of glutathione sepharose.  
     
     
         29 . A polypeptide according to  claim 26 , wherein a first protease cleavage site is incorporated between said single chain polypeptide and the purification tag, said protease cleavage site enabling proteolytic separation of said polypeptide from said purification tag.  
     
     
         30 . A polypeptide according to  claim 26 , wherein a second proteolytic cleavage site is incorporated between the first and second domains of said single chain polypeptide, said protease cleavage site enabling proteolytic cleavage of the first and second domains.  
     
     
         31 . A nucleic acid encoding a polypeptide according to  claim 1 .  
     
     
         32 . A nucleic acid according to  claim 31 , wherein said nucleic acid lacks nucleotides encoding a portion designated H C  of a clostridial neurotoxin.  
     
     
         33 . A nucleic acid according to  claim 31 , comprising nucleotides encoding residues 1-423 of a botulinum toxin type A heavy chain H N  domain.  
     
     
         34 . A nucleic acid according to  claim 31 , comprising nucleotides encoding residues 1-417 of a botulinum toxin type B heavy chain H N  domain.  
     
     
         35 . A nucleic acid according to  claim 31 , comprising nucleotides encoding a proteolytic cleavage site.  
     
     
         36 . A nucleic acid according to  claim 35 , wherein the proteolytic cleavage site is not present in a native clostridial neurotoxin.  
     
     
         37 . A nucleic acid according to  claim 36 , wherein said proteolytic cleavage site is located between the first and second domains of the polypeptide.  
     
     
         38 . (canceled)  
     
     
         39 . A nucleic acid sequence selected from the group consisting of:—SEQ ID 69, 71, 73, 75, 77, 113, 134, or a fragment or variant thereof.  
     
     
         40 . A single chain polypeptide selected from the group consisting of:—SEQ ID 70, 72, 74, 76, 78, 114, or a fragment or variant thereof.  
     
     
         41 . A nucleic acid sequence encoding a polypeptide according to  claim 29 , wherein said nucleic acid sequence comprises nucleotides encoding a proteolytic cleavage site which is not present in a native clostridial neurotoxin, and wherein said nucleic acid sequence is obtainable by providing said nucleotides encoding a proteolytic cleavage site within said nucleic acid sequence.

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