Ricin-like toxin variants for treatment of cancer, viral or parasitic infections

Abstract

The present invention provides a protein having an A chain of a ricin-like toxin, a B chain of a ricin-like toxin and a heterologous linker amino acid sequence, linking the A and B chains. The linker sequence contains a cleavage recognition site for a disease specific protease such as a cancer, fungal, viral or parasitic protease. The invention also relates to a nucleic acid molecule encoding the protein and to expression vectors incorporating the nucleic acid molecule. Also provided is a method of inhibiting or destroying mammalian cancer cells, cells infected with a virus, a fungus, or parasite, or parasites utilizing the nucleic acid molecules and proteins of the invention and pharmaceutical compositions for treating human cancer, viral infection, fungal infection, or parasitic infection.

1 - 128 . (canceled) 
     
     
         129 : A method of treating a disease comprising administering a recombinant protein comprising an A chain of a ricin-like toxin, a B chain of a ricin-like toxin and a heterologous linker amino acid sequence linking the A and B chains, the heterologous linker sequence containing a cleavage recognition site for a protease localized in cells or tissues affected by a specific disease to an animal in need thereof. 
     
     
         130 : The method according to  claim 129  for treating a mammal with cancer or infected with a fungus, virus or parasite comprising administering a recombinant protein comprising an A chain of a ricin-like toxin, a B chain of a ricin-like toxin and a heterologous linker amino acid sequence, linking the A and B chains, wherein the linker sequence contains a cleavage recognition site for a disease-specific protease selected from the group consisting of: a cancer associated protease, a viral protease, a fungal protease, and a parasitic protease. 
     
     
         131 : The method of  claim 130  wherein in the recombinant protein the A chain is ricin A chain, abrin toxin B chain, diphtheria toxin A chain, or Domain II/III of  Pseudomonas  exotoxin. 
     
     
         132 . The method of  claim 130  wherein in the recombinant protein the A chain is volkensin toxin A chain, cholera toxin A chain, modeccin toxin A chain or shiga toxin A chain. 
     
     
         133 : The method of  claim 130  wherein in the recombinant protein the B chain is ricin B chain, abrin toxin B chain, diphtheria toxin B chain, or Domain I of  Pseudomonas  exotoxin. 
     
     
         134 : The method of  claim 130  wherein in the recombinant protein the B chain is volkensin toxin B chain, cholera toxin B chain, modeccin toxin B chain or shiga toxin B chain. 
     
     
         135 : The method of  claim 130  wherein the cancer-associated protease is selected from the group consisting of: cathepsin B, an Epstein-Barr virus specific protease, a matrix metalloproteinase, cathespin L, cathespin D, urokinase-type plasminogen activator, tissue-type plasminogen activator, human prostate-specific antigen, kallikrein, neutrophil elastase, and calpain. 
     
     
         136 : The method of  claim 135  wherein in the recombinant protein the linker comprises the amino acid sequence according to SEQ ID NO: 40; SEQ ID NO: 41; SEQ ID NO: 42; SEQ ID NO: 43; SEQ ID NO: 44; SEQ ID NO: 45; SEQ ID NO: 46; SEQ ID NO: 87; SEQ ID NO: 90; SEQ ID NO: 93; SEQ ID NO: 96; SEQ ID NO: 99; SEQ ID NO: 102; SEQ ID NO: 105; SEQ ID NO: 108; SEQ ID NO: 111; SEQ ID NO: 114; SEQ ID NO: 117; SEQ ID NO: 120; SEQ ID NO: 123; or SEQ ID NO: 126. 
     
     
         137 : The method of  claim 130  wherein the parasitic protease is a  Plasmodium falciparum  protease. 
     
     
         138 : The method of  claim 137  wherein in the recombinant protein the linker comprises the amino acid sequence according to SEQ ID NO: 55; SEQ ID NO: 56; or SEQ ID NO: 57; SEQ ID NO: 58; or SEQ ID NO: 59. 
     
     
         139 : The method of  claim 130  wherein the viral protease is selected from the group consisting of: human cytomegalovirus, human herpes virus, varicella zoster virus, hepatitis A virus, hepatitis C virus and infectious laryngotracheitis virus. 
     
     
         140 : The method of  claim 139  wherein in the recombinant protein the linker comprises the amino acid sequence according to SEQ ID NO: 60; SEQ ID NO: 61; SEQ ID NO: 62; SEQ ID NO: 63; SEQ ID NO: 64; SEQ ID NO: 65; SEQ ID NO: 66; SEQ ID NO: 67; SEQ ID NO: 68; SEQ ID NO: 69; SEQ ID NO: 75; SEQ ID NO: 78; SEQ ID NO: 81; or SEQ ID NO: 84. 
     
     
         141 : The method of  claim 130  wherein the fungal protease is a  Candida  acid protease. 
     
     
         142 : The method of  claim 141  wherein in the recombinant protein the linker comprises the amino acid sequence according to SEQ ID NO: 70; SEQ ID NO: 71; or SEQ ID NO: 72. 
     
     
         143 : The method of  claim 135 , wherein in the recombinant protein the A chain is ricin A chain, the B chain is ricin B chain, and the heterologous linker contains a cleavage recognition site for a matrix metalloproteinase. 
     
     
         144 : The method of  claim 143 , wherein the heterologous linker contains a cleavage recognition site for matrix metalloproteinase-9. 
     
     
         145 : A method of treating a disease comprising administering a nucleic acid molecule having a nucleotide sequence encoding an A chain of a ricin-like toxin, a B chain of a ricin-like toxin and a heterologous linker amino acid sequence linking the A and B chains, the heterologous linker sequence containing a cleavage recognition site for a protease localized in cells or tissues affected by a specific disease to an animal in need thereof. 
     
     
         146 : A pharmaceutical composition for treating cancer or a fungal, or viral, or parasitic infection in an animal comprising a nucleic acid molecule having a nucleotide sequence encoding an A chain of a ricin-like toxin, a B chain of a ricin-like toxin and a heterologous linker amino acid sequence linking the A and B chains, the heterologous linker sequence containing a cleavage recognition site for a protease localized in cells or tissues affected by a specific disease to an animal in need thereof and a pharmaceutically acceptable carrier, diluent or excipient. 
     
     
         147 : A process for preparing a pharmaceutical for treating a mammal with cancer, fungal infection, viral infection or parasitic infection, comprising the steps of:
 (a) preparing a purified and isolated nucleic acid having a nucleotide sequence encoding an A chain of a ricin-like toxin, a B chain of a ricin-like toxin, and a heterologous linker amino acid sequence, linking the A and B chains, wherein the linker sequence contains a cleavage recognition site for a cancer, viral or parasitic protease;   (b) introducing the nucleic acid into a host cell and expressing the nucleic acid in the host cell to obtain a recombinant protein comprising an A chain of a ricin-like toxin, a B chain of a ricin-like toxin and a linker amino acid sequence;   (c) suspending the protein in a pharmaceutically acceptable carrier, diluent or excipient.   
     
     
         148 : A method of inhibiting or destroying cells affected by a disease, which cells are associated with a protease specific to the disease comprising the steps of:
 (a) preparing a purified and isolated nucleic acid having a nucleotide sequence encoding an A chain of a ricin-like toxin, a B chain of a ricin-like toxin, and a heterologous linker amino acid sequence, linking the A and B chains, wherein the linker sequence contains a cleavage recognition site for the protease;   (b) introducing the nucleic acid into a host cell and expressing the nucleic acid in the host cell to obtain a recombinant protein comprising an A chain of a ricin-like toxin, a B chain of a ricin-like toxin and a linker amino acid sequence;   (c) suspending the protein in a pharmaceutically acceptable carrier, diluent or excipient, and   (d) contacting the cells with the recombinant protein.   
     
     
         149 : The method of  claim 148  where the disease is one of cancer or cells infected with a fungus, virus or parasite.