Recombinant toxin fragments

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.

1 - 55 . (canceled)  
     
     
         56 . An antigenic composition comprising a single chain polypeptide consisting essentially of first and second domains, wherein: 
 said first domain is a clostridial neurotoxin light chain or a variant thereof or a fragment of said light chain or variant, wherein said variant or fragment has a common antigenic cross-reactivity to said clostridial neurotoxin light chain; and    said second domain is a clostridial neurotoxin heavy chain H N  portion or a variant thereof, or a fragment of said heavy chain H N  portion or variant, wherein said variant or fragment has a common antigenic cross-reactivity to said clostridial neurotoxin heavy chain H N  portion; and wherein said second domain (i) translocates the polypeptide into a cell or (ii) increases the solubility of the polypeptide compared to the solubility of the first domain on its own or (iii) both translocates the polypeptide into a cell and increases the solubility of the polypeptide compared to the solubility of the first domain on its own; and wherein the 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.    
     
     
         57 . The antigenic composition according to  claim 56  wherein said clostridial neurotoxin light chain is a botulinum neurotoxin light chain.  
     
     
         58 . The antigenic composition according to  claim 56  wherein said clostridial neurotoxin light chain is a tetanus neurotoxin light chain.  
     
     
         59 . The antigenic composition according to  claim 56  wherein said clostridial neurotoxin heavy chain is a botulinum neurotoxin heavy chain.  
     
     
         60 . The antigenic composition according to  claim 56  wherein said clostridial neurotoxin heavy chain is a tetanus neurotoxin heavy chain.  
     
     
         61 . The antigenic composition according to  claim 56  wherein said second domain is a clostridial neurotoxin heavy chain H N  portion.  
     
     
         62 . The antigenic composition according to  claim 56  wherein one or both of said clostridial neurotoxin light chain and said clostridial neurotoxin heavy chain is a botulinum neurotoxin type A chain.  
     
     
         63 . The antigenic composition according to  claim 62  wherein the botulinum neurotoxin type A light chain has at residue 2 a glutamate, at residue 26 a lysine and at residue 27 a tyrosine.  
     
     
         64 . The antigenic composition according to  claim 56  wherein the second domain comprises the 423 N-terminal amino acids of botulinum neurotoxin type A heavy chain.  
     
     
         65 . The antigenic composition according to  claim 56  wherein one or both of said clostridial neurotoxin light chain and said clostridial neurotoxin heavy chain is a botulinum neurotoxin type B chain.  
     
     
         66 . The antigenic composition according to  claim 56  wherein the second domain comprises the 107 N-terminal amino acids of a botulinum neurotoxin type B heavy chain.  
     
     
         67 . The antigenic composition according to  claim 56  wherein the second domain comprises the 417 N-terminal amino acids of botulinum neurotoxin type B heavy chain.  
     
     
         68 . The antigenic composition according to  claim 56  wherein the clostridial neurotoxin light chain is a botulinum neurotoxin type B light chain, and the second domain comprises the 417 N-terminal amino acids of a botulinum neurotoxin type B heavy chain.  
     
     
         69 . The antigenic composition according to  claim 56  wherein one or both of said clostridial neurotoxin light chain and said clostridial neurotoxin heavy chain is a tetanus toxin chain.  
     
     
         70 . The antigenic composition according to  claim 56  wherein the second domain comprises the 422 N-terminal amino acids of tetanus heavy chain.  
     
     
         71 . The antigenic composition according to  claim 56  wherein the second domain comprises the 100 N-terminal amino acids of a clostridial neurotoxin heavy chain.  
     
     
         72 . The antigenic composition according to  claim 70  lacking a C-terminal part of a clostridial neurotoxin heavy chain designated Hc.  
     
     
         73 . The antigenic composition according to  claim 71  lacking a C-terminal part of a clostridial neurotoxin heavy chain designated Hc.  
     
     
         74 . The antigenic composition according to  claim 56  comprising a site for cleavage by a proteolytic enzyme, wherein said cleavage site is located between said first domain and said second domain.  
     
     
         75 . The antigenic composition according to  claim 74 , wherein the cleavage site is not present in a native clostridial neurotoxin.  
     
     
         76 . The antigenic composition according to  claim 74 , wherein the site for cleavage allows proteolytic cleavage of the first and second domains.  
     
     
         77 . The antigenic composition according to  claim 74  produced by a process comprising (a) inserting a first nucleic acid sequence encoding said cleavage site into a second nucleic acid sequence encoding the polypeptide according to  claim 56 , and (b) expressing said first and second nucleic acid sequences to obtain said polypeptide.  
     
     
         78 . An antigenic composition comprising a fusion protein consisting essentially of a fusion of (a) a single chain polypeptide consisting essentially of first and second domains and (b) a purification tag that binds to an affinity matrix thereby facilitating purification of the fusion protein using said matrix; 
 wherein said first domain is a clostridial neurotoxin light chain or a variant thereof or a fragment of said light chain or variant, wherein said variant or fragment has a common antigenic cross-reactivity to said clostridial neurotoxin light chain; and    said second domain is a clostridial neurotoxin heavy chain H N  portion or a variant thereof or a fragment of said heavy chain H N  portion or variant, wherein said variant or fragment has a common antigenic cross-reactivity to said clostridial neurotoxin heavy chain H N  portion; and wherein said second domain (i) translocates the polypeptide into a cell or (ii) increases the solubility of the polypeptide compared to the solubility of the first domain on its own or (iii) both translocates the polypeptide into a cell and increases the solubility of the polypeptide compared to the solubility of the first domain on its own; and wherein the 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.    
     
     
         79 . The antigenic composition fusion protein according to  claim 78  wherein said purification tag binds to an affinity matrix of glutathione sepharose.  
     
     
         80 . The antigenic composition according to  claim 78  wherein a first protease cleavage site is incorporated between the polypeptide and purification tag, said first protease cleavage site enabling proteolytic separation of the polypeptide from the purification tag.  
     
     
         81 . An antigenic composition comprising a single chain polypeptide consisting essentially of first and second domains, and a spacer molecule, wherein: 
 said first domain is a clostridial neurotoxin light chain or a variant thereof or a fragment of said light chain or variant, wherein said variant or fragment has a common antigenic cross-reactivity to said clostridial neurotoxin light chain; and    said second domain is a clostridial neurotoxin heavy chain H N  portion or a variant thereof or a fragment of said heavy chain H N  portion or variant, wherein said variant or fragment has a common antigenic cross-reactivity to said clostridial neurotoxin heavy chain H N  portion, and wherein said second domain (i) translocates the polypeptide into a cell or (ii) increases the solubility of the polypeptide compared to the solubility of the first domain on its own or (iii) both translocates the polypeptide into a cell and increases the solubility of the polypeptide compared to the solubility of the first domain on its own; and wherein the 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 the spacer molecule is between the first and second domains.    
     
     
         82 . The antigenic composition according to  claim 78  including a spacer molecule between the purification tag and the polypeptide.  
     
     
         83 . An antigenic composition comprising a single chain polypeptide selected from the group consisting of: SEQ ID: 32, 34, 68, 84, 145, 147, 151, 153, 161, and 173; wherein said single chain polypeptide 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.  
     
     
         84 . An antigenic composition comprising a single chain polypeptide consisting of first and second domains, wherein: 
 said first domain is a clostridial neurotoxin light chain or a variant thereof or a fragment of said light chain or variant, wherein said variant or fragment has a common antigenic cross-reactivity to said clostridial neurotoxin light chain; and    said second domain is a clostridial neurotoxin heavy chain H N  portion or a variant thereof or a fragment of said heavy chain H N  portion or variant, wherein said variant or fragment has a common antigenic cross-reactivity to said clostridial neurotoxin heavy chain H N  portion; and wherein said second domain (i) translocates the polypeptide into a cell or (ii) increases the solubility of the polypeptide compared to the solubility of the first domain on its own or (iii) both translocates the polypeptide into a cell and increases the solubility of the polypeptide compared to the solubility of the first domain on its own; and wherein the 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.    
     
     
         85 . An antigenic composition comprising a single chain polypeptide comprising first, second and third domains, wherein: 
 said first domain is a clostridial neurotoxin light chain or a variant thereof or a fragment of said light chain or variant, wherein said variant or fragment has a common antigenic cross-reactivity to said clostridial neurotoxin light chain; and    said second domain is a clostridial neurotoxin heavy chain H N  portion or a variant thereof or a fragment of said heavy chain H N  portion or variant, wherein said variant or fragment has a common antigenic cross-reactivity to said clostridial neurotoxin heavy chain H N  portion; and wherein said second domain (i) translocates the polypeptide into a cell or (ii) increases the solubility of the polypeptide compared to the solubility of the first domain on its own or (iii) both translocates the polypeptide into a cell and increases the solubility of the polypeptide compared to the solubility of the first domain on its own; wherein the 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    said third domain is a tandem repeat synthetic IgG binding domain derived from domain b of Staphylococcal protein A.    
     
     
         86 . An antigenic composition comprising a single chain polypeptide comprising first, second and third domains, wherein:—
 said first domain is a clostridial neurotoxin light chain or a variant thereof or a fragment of said light chain or variant, wherein said variant or fragment has a common antigenic cross-reactivity to said clostridial neurotoxin light chain;    said second domain is a clostridial neurotoxin heavy chain H N  portion or a variant thereof or a fragment of said heavy chain H N  portion or variant, wherein said variant or fragment has a common antigenic cross-reactivity to said clostridial neurotoxin heavy chain H N  portion; and wherein said second domain (i) translocates the polypeptide into a cell or (ii) increases the solubility of the polypeptide compared to the solubility of the first domain on its own or (iii) both translocates the polypeptide into a cell and increases the solubility of the polypeptide compared to the solubility of the first domain on its own; wherein the 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    said third domain is insulin-like growth factor-1 (IGF-1).    
     
     
         87 . An antigenic composition comprising a fusion of (a) the polypeptide of  claim 85  with (b) a purification tag that binds to an affinity matrix thereby facilitating purification of the fusion protein using said matrix.  
     
     
         88 . The antigenic composition of  claim 87  wherein a first protease cleavage site is incorporated between the polypeptide and purification tag, said first protease cleavage site enabling proteolytic separation of the polypeptide from the purification tag.  
     
     
         89 . The antigenic composition of  claim 88 , wherein a second proteolytic cleavage site is incorporated between the first and second domains of the polypeptide, said protease cleavage site enabling proteolytic cleavage of the first and second domains.  
     
     
         90 . The antigenic composition of  claim 85  including a spacer molecule between the second and third domains.  
     
     
         91 . The antigenic composition of  claim 87  including a spacer molecule between the purification tag and the polypeptide.  
     
     
         92 . An antigenic composition comprising a single chain polypeptide consisting essentially of first and second domains and a site for cleavage by a proteolytic agent, wherein: 
 said first domain is a clostridial neurotoxin light chain or a variant thereof or a fragment of said light chain or variant, wherein said variant or fragment has a common antigenic cross-reactivity to said clostridial neurotoxin light chain; and    said second domain is a clostridial neurotoxin heavy chain H N  portion or a variant thereof or a fragment of said heavy chain H N  portion or variant, wherein said variant or fragment has a common antigenic cross-reactivity to said clostridial neurotoxin heavy chain H N  portion; and wherein said second domain (i) translocates the polypeptide into a cell or (ii) increases the solubility of the polypeptide compared to the solubility of the first domain on its own or (iii) both translocates the polypeptide into a cell and increases the solubility of the polypeptide compared to the solubility of the first domain on its own; and wherein the 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 cleavage site is located between said first domain and said second domain.    
     
     
         93 . An antigenic composition comprising a single chain polypeptide consisting of first and second domains and a site for cleavage by a proteolytic agent, wherein:—
 said first domain is a clostridial neurotoxin light chain or a variant thereof, or a fragment of said light chain or variant, wherein said variant or fragment has a common antigenic cross-reactivity to said clostridial neurotoxin light chain; and    said second domain is a clostridial neurotoxin heavy chain H N  portion or a variant thereof or a fragment of said heavy chain H N  portion or variant, wherein said variant or fragment has a common antigenic cross-reactivity to said clostridial neurotoxin heavy chain H N  portion; wherein said second domain (i) translocates the polypeptide into a cell or (ii) increases the solubility of the polypeptide compared to the solubility of the first domain on its own or (iii) both translocates the polypeptide into a cell and increases the solubility of the polypeptide compared to the solubility of the first domain on its own; and wherein the 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 cleavage site is located between said first domain and said second domain.    
     
     
         94 . An antigenic composition comprising a fusion protein consisting of a fusion of (a) a single chain polypeptide consisting of a first and second domains and (b) a purification tag that binds to an affinity matrix thereby facilitating purification of the fusion protein using said matrix; 
 wherein said first domain is a clostridial neurotoxin light chain or a variant thereof or a fragment of said light chain or variant, wherein said variant or fragment has a common antigenic cross-reactivity to said clostridial neurotoxin light chain; and    said second domain is a clostridial neurotoxin heavy chain H N  portion or a variant thereof, or a fragment of said heavy chain H N  portion or variant, wherein said variant or fragment has a common antigenic cross-reactivity to said clostridial neurotoxin heavy chain H N  portion; and wherein said second domain (i) translocates the polypeptide into a cell or (ii) increases the solubility of the polypeptide compared to the_solubility of the first domain on its own or (iii) both translocates the polypeptide into a cell and increases the solubility of the polypeptide compared to the solubility of the first domain on its own; and wherein the 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.    
     
     
         95 . An antigenic composition comprising a single chain polypeptide consisting of first and second domains, and a spacer molecule wherein:—
 said first domain is a clostridial neurotoxin light chain or a variant thereof or a fragment of said light chain or variant, wherein said variant or fragment has a common antigenic cross-reactivity to said clostridial neurotoxin light chain; and    said second domain is a clostridial neurotoxin heavy chain H N  portion or a variant thereof or a fragment of said heavy chain H N  portion or variant, wherein said variant or fragment has a common antigenic cross-reactivity to said clostridial neurotoxin heavy chain H N  portion; and wherein said second domain (i) translocates the polypeptide into a cell or (ii) increases the solubility of the polypeptide compared to the solubility of the first domain on its own or (iii) both translocates the polypeptide into a cell and increases the solubility of the polypeptide compared to the solubility of the first domain on its own; and wherein the 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 the spacer molecule is between the first and second domains.