Modified microbial toxin receptor for delivering agents into cells

Abstract

We described a novel system of targeted cell therapy with a protein toxin, such as anthrax toxin, that has been modified to re-direct it to a desired cell target instead of its natural cell target. The system can be used for, e.g., targeted killing of undesired cells in a population of cells, such as cancer or overly active immune system cells.

We claim: 
     
         1 . An altered binary toxin system for delivery of an active molecule to a target cell comprising:
 a fusion protein comprising a receptor-ablated pore-forming AB toxin unit fused to a non-toxin-associated receptor-binding ligand specific for a target cell, and   a complementary toxin unit capable of associating with the pore-forming toxin unit for delivery of a therapeutic protein to the cytosol of the target cell.   
     
     
         2 . The altered binary toxin system of  claim 1 , wherein the AB toxin is  botulinum  neurotoxin, anthrax toxin, diphtheria toxin, ricin, shiga toxin, shiga like toxin, exotoxin A, or cholera toxin. 
     
     
         3 . The altered binary toxin system of  claim 2 , wherein the receptor-ablated pore-forming unit is derived from the B unit of  botulinum  neurotoxin, diphtheria toxin, ricin, shiga toxin, shiga like toxin, exotoxin A, or cholera toxin. 
     
     
         4 . The altered binary toxin system of  claim 3 , wherein the complementary toxin unit is derived from the A unit of  Botulinum  neurotoxin, diphtheria toxin, ricin, shiga toxin, shiga like toxin, exotoxin A, or cholera toxin. 
     
     
         5 . The altered binary toxin system of  claim 1 , wherein the therapeutic protein is the catalytic domain of diphtheria toxin (DTA), ricin, shiga toxin, or  Pseudomonas  exotoxin A. 
     
     
         6 . The altered binary toxin system of  claim 1 , wherein the therapeutic protein is the cytotoxic domain of shiga toxin, shiga-like toxin 1 and 2, ricin, ricin toxin A chain, abrin, gelonin, pokeweed antiviral protein, saporin, trichsanthin, pepcin, maize RIP, alpha-sarcin,  Clostridium perfringens  epsilon toxin,  Botulinum  neurotoxins,  Staphylococcus  enterotoxins,  Clostridium difficile  toxins, pertussis toxins, or  Pseudomonas  exotoxin. 
     
     
         7 . The altered binary toxin system of  claim 3 , wherein the binary toxin is  Clostridium perfringens  toxins alpha, beta, epsilon or iota;  Clostridium botulinum  C2 toxin; or  Clostridium spiroforme  Iota-like toxin. 
     
     
         8 . The altered binary toxin system of  claim 1 , wherein the non-toxin associated specific target receptor-binding ligand is an epidermal growth factor-1 or epidermal growth factor 2. 
     
     
         9 . The altered binary toxin system of  claim 1 , wherein the receptor-binding ligand binds to a receptor selected from epidermal growth factor receptors HER1, HER2, HER3 or HER4; vascular endothelial growth factor receptors VEGFR-1, VEGFR-2 or VEGFR-3; insulin-like growth factor 1 receptor; fibroblast growth factor receptors; thrombospondin 1 receptor; estrogen receptors; urokinase receptors; progesterone receptors; testosterone receptors; carcinoembryonic antigens; prostate-specific antigens; farnesoid X receptors; transforming growth factor receptors; transferrin receptors; hepatocyte growth factor receptors; or vasoactive intestinal polypeptide receptors 1 and 2. 
     
     
         10 . The altered binary toxin system of  claim 1 , wherein the receptor-binding ligand is selected from an antibody or an affibody. 
     
     
         11 . The altered binary toxin system of  claim 10 , wherein the affibody is a HER2 affibody. 
     
     
         12 . The altered binary toxin system of  claim 11 , wherein the HER2 affibody is ZHER2. 
     
     
         13 . A kit for delivering bioactive molecules to a eukaryotic cell, comprising an altered binary toxin system of  claim 1 . 
     
     
         14 . A method for treating cancer comprising administering to a subject diagnosed with cancer the altered binary toxin system of  claim 1 . 
     
     
         15 . The method of  claim 14 , wherein the altered binary toxin system comprises a receptor-redirected B unit of  botulinum  neurotoxin, diphtheria toxin, ricin, shiga toxin, shiga like toxin, exotoxin A, or cholera toxin. 
     
     
         16 . The method of  claim 15 , wherein the cancer is a HER2 positive cancer and the receptor-redirected B unit of  botulinum  neurotoxin, diphtheria toxin, ricin, shiga toxin, shiga like toxin, exotoxin A, or cholera toxin is fused with a HER2 binding ligand. 
     
     
         17 . The method of  claim 16 , wherein the HER2 binding ligand is an antibody or an affibody. 
     
     
         18 . The method of  claim 16 , wherein the HER2 affibody is ZHER2. 
     
     
         19 . The method of  claim 14 , wherein the altered binary toxin system comprises:
 a therapeutic protein that is the cytotoxic domain of shiga toxin, shiga-like toxin 1 and 2, ricin, ricin toxin A chain, abrin, gelonin, pokeweed antiviral protein, saporin, trichsanthin, pepcin, maize RIP, alpha-sarcin,  Clostridium perfringens  epsilon toxin,  Botulinum  neurotoxins,  Staphylococcus  enterotoxins,  Clostridium difficile  toxins, pertussis toxins, or  Pseudomonas  exotoxin, or   a therapeutic protein that is the catalytic domain of diphtheria toxin (DTA), ricin, shiga toxin, or  Pseudomonas  exotoxin A.   
     
     
         20 . The method of  claim 15 , wherein the altered binary toxin system comprises a complementary toxin unit is derived from the A unit of  botulinum  neurotoxin, diphtheria toxin, ricin, shiga toxin, shiga like toxin, exotoxin A, or cholera toxin.