Ricin-like toxins for treatment of cancer

Abstract

The present invention provides a protein having chain of a ricin-like toxin, a B chain of a ricin-like toxin and a novel heterologous linker amino acid sequence, linking the A and B chains. The linker sequence contains a cleavage recognition site for a specific protease such as those found in inflammatory cells and cancer cells. 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 cells having a specific protease, such as cancer cells or inflammatory cells utilizing the nucleic acid molecules and proteins of the invention and pharmaceutical compositions for treating human inflammation and cancer.

1 - 42 . (canceled) 
     
     
         43 . A purified and isolated nucleic acid molecule comprising (a) a nucleotide sequence encoding an A chain of a ricin-like toxin, (b) a nucleotide sequence encoding a B chain of a ricin-like toxin and (c) a nucleotide sequence encoding a heterologous linker amino acid sequence linking the A and B chains, the heterologous linker sequence containing a cleavage recognition site for a specific protease. 
     
     
         44 . A nucleic acid molecule of  claim 43  wherein the specific protease is an MMP or UPA. 
     
     
         45 . A nucleic acid molecule according to  claim 43  wherein the protease is associated with a cancer cell. 
     
     
         46 . A nucleic acid molecule according to  claim 45  wherein the cancer cell is one found in T- and B-cell lymphoproliferative diseases, ovarian cancer, pancreatic cancer, head and neck cancer, squamous cell carcinoma, gastrointestinal cancer, breast cancer, prostate, cancer or non small cell lung cancer. 
     
     
         47 . A nucleic acid molecule according to  claim 43  wherein the protease is associated with an inflammatory cell. 
     
     
         48 . A nucleic acid molecule according to  claim 47  wherein the cell is one found in rheumatoid arthritis, atherosclerotic cells, Crohn's disease, or central nervous system disease. 
     
     
         49 . A nucleic acid molecule of  claim 43  wherein the A chain is ricin A chain, abrin toxin A chain, diphtheria toxin A chain, Domain III of  Pseudomonas  exotoxin, volkensin toxin A chain, cholera toxin A chain, modeccin toxin A chain, viscumin toxin A chain or shiga toxin A chain. 
     
     
         50 . A nucleic acid molecule of  claim 43  wherein the B chain is ricin B chain, abrin toxin B chain, diphtheria toxin B chain, Domain I/II of  Pseudomonas  exotoxin, volkensin toxin B chain, cholera toxin B chain, modeccin toxin B chain, viscumin toxin B chain or shiga toxin B chain. 
     
     
         51 . A nucleic acid molecule according to  claim 43  having a nucleic acid sequence selected from the group consisting of the nucleic acid sequence of pAP301 as shown in  FIG. 1B  (SEQ ID NO:5); the nucleic acid sequence of pAP302 as shown in  FIG. 2B  (SEQ ID NO:12); the nucleic acid sequence of pAP303 as shown in  FIG. 3B  (SEQ ID NO:19); the nucleic acid sequence of pAP304 as shown in  FIG. 4B  (SEQ ID NO:26); the nucleic acid sequence of pAP305 as shown in  FIG. 5B  (SEQ ID NO:33); the nucleic acid 5 sequence of pAP308 as shown in  FIG. 6B  (SEQ ID NO:40); the nucleic acid sequence of pAP309 as shown in  FIG. 7B  (SEQ ID NO:47); the nucleic acid sequence of pAP313 as shown in  FIG. 8B  (SEQ ID NO:54); the nucleic acid sequence of pAP314 as shown in  FIG. 9B  (SEQ ID NO:61); the nucleic acid sequence of pAP315 as shown in  FIG. 10B  (SEQ ID NO:68); the nucleic acid sequence of pAP316 as shown in  FIG. 11B  (SEQ ID NO:75); the nucleic acid sequence of pAP318 as shown in  FIG. 12B  (SEQ ID NO:82); the nucleic acid sequence of pAP320 as shown in  FIG. 13B  (SEQ ID NO:89); the nucleic acid sequence of pAP321 as shown in  FIG. 14B  (SEQ ID NO:96); the nucleic acid sequence of pAP322 as shown in  FIG. 15B  (SEQ ID NO:103); the nucleic acid sequence of pAP323 as shown in  FIG. 16B  (SEQ ID NO:110); the nucleic acid sequence of pAP324 as shown in  FIG. 17B  (SEQ ID NO:117); and the nucleic acid sequence of pAP325 as shown in  FIG. 18B  (SEQ ID NO:124). 
     
     
         52 . A nucleic acid molecule according to  claim 43  wherein the nucleotide sequence of the linker is selected from the group consisting of: the nucleic acid sequence of pAP301 as shown in  FIG. 1A  (SEQ ID NO:4); the nucleic acid sequence of pAP302 as shown in  FIG. 2A  (SEQ ID NO:11); the nucleic acid sequence of pAP303 as shown in  FIG. 3A  (SEQ ID NO:18); the nucleic acid sequence of pAP304 as shown in  FIG. 4A  (SEQ ID NO:25); the nucleic acid sequence of pAP305 as shown in  FIG. 5A  (SEQ ID NO:32); the nucleic acid sequence of pAP308 as shown in  FIG. 6A  (SEQ ID NO:39); the nucleic acid sequence of pAP309 as shown in  FIG. 7A  (SEQ ID NO:46); the nucleic acid sequence of pAP313 as shown in  FIG. 8A  (SEQ ID NO:53); the nucleic acid sequence of pAP314 as shown in  FIG. 9A  (SEQ ID NO:60); the nucleic acid sequence of pAP315 as shown in  FIG. 10A  (SEQ ID NO:67); the nucleic acid sequence of pAP316 as shown in  FIG. 11A  (SEQ ID NO:74); the nucleic acid sequence of pAP318 as shown in  FIG. 12A  (SEQ ID NO:81); the nucleic acid sequence of pAP320 as shown in  FIG. 13A  (SEQ ID NO:88); the nucleic acid sequence of pAP321 as shown in  FIG. 14A  (SEQ ID NO:95); the nucleic acid sequence of pAP322 as shown in  FIG. 15A  (SEQ ID NO:102); the nucleic acid sequence of pAP323 as shown in  FIG. 16A  (SEQ ID NO:109); the nucleic acid sequence of pAP324 as shown in  FIG. 17A  (SEQ ID NO:116); and the nucleic acid sequence of pAP325 as shown in  FIG. 18A  (SEQ ID NO:123). 
     
     
         53 . A plasmid incorporating the nucleic acid molecule of  claim 43 . 
     
     
         54 . A baculovirus transfer vector incorporating the nucleic acid molecule according to  claim 43 . 
     
     
         55 . 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 specific protease. 
     
     
         56 . A recombinant protein of  claim 55  wherein the specific protease is an MMP or UPA. 
     
     
         57 . A protein according to  claim 55  wherein the protease is associated with a cancer cell. 
     
     
         58 . A protein according to  claim 57  wherein the cancer cell is one found in T- and B cell lymphoproliferative diseases, ovarian cancer, pancreatic cancer, head and neck cancer, squamous cell carcinoma, gastrointestinal cancer, breast cancer, prostate, cancer or non small cell lung cancer. 
     
     
         59 . A protein according to  claim 55  wherein the protease is associated with an inflammatory cell. 
     
     
         60 . A protein according to  claim 59  wherein the cell is one found in rheumatoid arthritis, atherosclerotic cells, Crohn's disease, or central nervous system disease. 
     
     
         61 . A recombinant protein of  claim 55  wherein the A chain is ricin A chain, abrin toxin A chain, diphtheria toxin A chain, Domain III of  Pseudomonas  exotoxin, volkensin toxin A chain, cholera toxin A chain, modeccin toxin A chain, viscumin toxin A chain, or shiga toxin A chain. 
     
     
         62 . A recombinant protein of  claim 55  wherein the B chain is ricin B chain, abrin toxin B chain, diphtheria toxin B chain, Domain I/II of  Pseudomonas  exotoxin, volkensin toxin B chain, cholera toxin B chain, modeccin toxin B chain, viscumin toxin B chain, or shiga toxin B chain. 
     
     
         63 . A recombinant protein of  claim 55  wherein the linker amino acid sequence is selected from the group consisting of: the amino acid sequence of PAP301 as shown in  FIG. 1C  (SEQ ID NO:7); the amino acid sequence of PAP309 as shown in  FIG. 7C  (SEQ ID NO:49); the amino acid sequence of PAP314 as shown in  FIG. 9C  (SEQ ID NO:63); the amino acid sequence of PAP315 as shown in  FIG. 10C  (SEQ ID NO:70); the amino acid sequence of PAP318 as shown in  FIG. 12C  (SEQ ID NO:84); the amino acid sequence of PAP320 as shown in  FIG. 13C  (SEQ ID NO:91); the amino acid sequence of PAP321 as shown in  FIG. 14C  (SEQ ID NO:98); and the amino acid sequence of PAP322 as shown in  FIG. 15C  (SEQ ID NO:105. 
     
     
         64 . A method of inhibiting or destroying cells having a specific protease 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.   
     
     
         65 . A method according to  claim 64  wherein the protease is an MMP or UPA. 
     
     
         66 . A method according to  claim 64  wherein the protease is associated with a cancer cell. 
     
     
         67 . A method according to  claim 66  wherein the cancer cell is one found in T- and B cell lymphoproliferative diseases, ovarian cancer, pancreatic cancer, head and neck cancer, squamous cell carcinoma, gastrointestinal cancer, breast cancer, prostate, cancer or non small cell lung cancer. 
     
     
         68 . A method according to  claim 64  wherein the protease is associated with an inflammatory cell. 
     
     
         69 . A method according to  claim 68  wherein the cell is one found in rheumatoid arthritis, atherosclerotic cells, Crohn's disease, or central nervous system disease. 
     
     
         70 . A method of inhibiting or destroying cells having a specific protease comprising contacting the cells with an effective amount a recombinant protein according to  claim 55 . 
     
     
         71 . A method of treating a cell having a specific protease comprising administering an effective amount of a recombinant protein according to  claim 55  to an animal in need thereof. 
     
     
         72 . A method of treating a cell having a specific protease comprising administering an effective amount of a nucleic acid molecule according to  claim 43  to an animal in need thereof. 
     
     
         73 . A process for preparing a pharmaceutical for treating a cell having a specific protease 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 specific 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.   
     
     
         74 . A process according to  claim 73  wherein the protease is an MMP or UPA. 
     
     
         75 . A process according to  claim 73  wherein the protease is associated with a cancer cell. 
     
     
         76 . A process according to  claim 75  wherein the cancer cell is one found in T- and B cell lymphoproliferative diseases, ovarian cancer, pancreatic cancer, head and neck cancer, squamous cell carcinoma, gastrointestinal cancer, breast cancer, prostate, cancer or non small cell lung cancer. 
     
     
         77 . A process according to  claim 73  wherein the protease is associated with an inflammatory cell. 
     
     
         78 . A process according to  claim 77  wherein the cell is one found in rheumatoid arthritis, atherosclerotic cells, Crohn's disease, or central nervous system disease. 
     
     
         79 . A pharmaceutical composition for treating cancer comprising a recombinant protein of  claim 55  and a pharmaceutically acceptable carrier, diluent or excipient. 
     
     
         80 . A pharmaceutical composition for treating inflammation comprising a recombinant protein of  claim 55  and a pharmaceutically acceptable carrier, diluent or excipient. 
     
     
         81 . A pharmaceutical composition for treating a cell having a specific protease comprising a nucleic acid molecule of  claim 43  and a pharmaceutically acceptable carrier, diluent or excipient. 
     
     
         82 . A pharmaceutical composition for treating a cell having a specific protease comprising an amino acid molecule of  claim 43  and a pharmaceutically acceptable carrier, diluent or excipient. 
     
     
         83 . A purified and isolated nucleic acid molecule having a nucleic acid sequence selected from the group consisting of: the nucleic acid sequence of pAP301 as shown in  FIG. 1A  (SEQ ID NO:4); the nucleic acid sequence of pAP302 as shown in  FIG. 2A  (SEQ ID NO:11); the nucleic acid sequence of pAP303 as shown in  FIG. 3A  (SEQ ID NO:18); the nucleic acid sequence of pAP304 as shown in  FIG. 4A  (SEQ ID NO:25); the nucleic acid sequence of pAP305 as shown in  FIG. 5A  (SEQ ID NO:32); the nucleic acid sequence of pAP308 as shown in  FIG. 6A  (SEQ ID NO:39); the nucleic acid sequence of pAP309 as shown in  FIG. 7A  (SEQ ID NO:46); the nucleic acid sequence of pAP313 as shown in  FIG. 8A  (SEQ ID NO:53); the nucleic acid sequence of pAP314 as shown in  FIG. 9A  (SEQ ID NO:60); the nucleic acid sequence of pAP315 as shown in  FIG. 10A  (SEQ ID NO:67); the nucleic acid sequence of pAP316 as shown in  FIG. 11A  (SEQ ID NO:74); the nucleic acid sequence of pAP318 as shown in  FIG. 12A  (SEQ ID NO:81); the nucleic acid sequence of pAP320 as shown in  FIG. 13A  (SEQ ID NO:88); the nucleic acid sequence of pAP321 as shown in  FIG. 14A  (SEQ ID NO:95); the nucleic acid sequence of pAP322 as shown in  FIG. 15A  (SEQ ID NO:102); the nucleic acid sequence of pAP323 as shown in  FIG. 16A  (SEQ ID NO:109); the nucleic acid sequence of pAP324 as shown in  FIG. 17A  (SEQ ID NO:116); and the nucleic acid sequence of pAP325 as shown in  FIG. 18A  (SEQ ID NO:123). 
     
     
         84 . A linker protein having an amino acid sequence selected from the group consisting of: the amino acid sequence of PAP301 as shown in  FIG. 1C  (SEQ ID NO:7); the amino acid sequence of PAP309 as shown in  FIG. 7C  (SEQ ID NO:49); the amino acid sequence of PAP314 as shown in  FIG. 9C  (SEQ ID NO:63); the amino acid sequence of PAP315 as shown in  FIG. 10C  (SEQ ID NO:70); the amino acid sequence of PAP318 as shown in  FIG. 12C  (SEQ ID NO:84); the amino acid sequence of PAP320 as shown in  FIG. 13C  (SEQ ID NO:91); the amino acid sequence of PAP321 as shown in  FIG. 14C  (SEQ ID NO:98); and the amino acid sequence of PAP322 as shown in  FIG. 15C  (SEQ ID NO:105).