Enzyme prodrug cancer therapy selectively targeted to tumor cells or tumor vasculature and methods of production and use thereof

Abstract

Conjugates for use in an enzyme prodrug cancer therapy treatment are provided.

1 - 9 . (canceled) 
     
     
         10 . A kit, comprising:
 a conjugate, comprising:
 a ligand having the ability to specifically and stably bind to at least one of an external receptor and a binding site on an outer surface of at least one of a tumor vasculature endothelial cell and a cancer cell, wherein the at least one of an external receptor and a binding site is specific to the at least one of a tumor vasculature endothelial cell and a cancer cell; 
 an enzyme operatively attached to the ligand, wherein the enzyme is able to convert a prodrug into an active drug; and 
 wherein when the conjugate binds to the at least one of a tumor vasculature endothelial cell and a cancer cell, the conjugate is maintained on the outer surface of said cell; and 
   a prodrug comprising a substrate for the enzyme of the conjugate, whereby the prodrug is convertible into an active anticancer drug by the enzyme of the conjugate.   
     
     
         11 . The kit of  claim 10 , wherein:
 (a) the ligand of the conjugate is selected from the group consisting of annexin V;   antibodies to a receptor or aminophospholipid that is uniquely expressed or overexpressed on a surface of a tumor vasculature endothelial cell or cancer cell; RGD-motif peptides; NGR-motif peptides; F3; HWGF-motif peptides; the peptide CTTHWGFTLC; amino-terminal fragment (ATF) of urokinase; a phosphatidylserine (PS)-binding protein; and fragments or variants thereof which substantially retain the ability to bind to the receptor or binding site; and   (b) the enzyme of the conjugate is selected from the group consisting of L-methioninase, nitroreductase, cytochrome P450, purine-nucleoside phosphorylase, thymidine kinase, alkaline phosphatase, β-β-glucuronidase, glycosidase, carboxypeptidase, carboxyesterase, penicillin amidase, β-lactamase, and cytosine deaminase.   
     
     
         12 . The kit of  claim 11 , wherein the ligand comprises annexin V and the enzyme comprises L-methioninase. 
     
     
         13 . The kit of  claim 10 , wherein the enzyme of the conjugate is of nonhuman origin or is absent or expressed at low concentrations in normal human tissues. 
     
     
         14 . The kit of  claim 10 , wherein the enzyme has an anticancer activity. 
     
     
         15 . The kit of  claim 10 , wherein the enzyme and ligand are directly coupled together. 
     
     
         16 . The kit of  claim 10 , wherein the enzyme and ligand are indirectly coupled together via a linker. 
     
     
         17 . The kit of  claim 10 , wherein at least one of:
 (a) the enzyme of the conjugate is conjugated to polyethylene glycol (PEG); and   (b) the conjugate is encapsulated in a liposome.   
     
     
         18 . The kit of  claim 10 , wherein the prodrug comprises a selenomethionine prodrug and the enzyme of the conjugate comprises L-methioninase. 
     
     
         19 . The kit of  claim 10 , further comprising an immunostimulant selected from the group consisting of glycated chitosan; muramyldipeptide derivatives; trehalose-dimycolates; BCG-cell wall skeleton; various cytokines; and combinations and/or derivatives thereof. 
     
     
         20 . The kit of  claim 10 , further comprising at least one chemotherapeutic agent. 
     
     
         21 . The kit of  claim 10 , wherein the conjugate is further defined as comprising at least one of:
 (a) the amino acid sequence as set forth in at least one of SEQ ID NOS:2 and 4;   (b) an amino acid sequence encoded by the nucleotide sequence of at least one of SEQ ID NOS:1 and 3;   (c) an amino acid sequence that is at least 90% identical to at least one of SEQ ID NOS:2 and 4; and   (d) an amino acid sequence encoded by a nucleotide sequence that is at least 90% identical to at least one of SEQ ID NOS:1 and 3.   
     
     
         22 . A method of treating at least one of a cancer tumor and cancer cells wherein the cancer tumor/cancer cells is supplied by a tumor vasculature, comprising the step of:
 providing a conjugate comprising a ligand having an enzyme operatively attached thereto, wherein the ligand has the ability to specifically and stably bind to at least one of an external receptor and a binding site on an outer surface of at least one of a tumor vasculature endothelial cell and a cancer cell, wherein the at least one of an external receptor and a binding site is specific to the at least one of a tumor vasculature endothelial cell and a cancer cell, and wherein the enzyme is able to convert a prodrug into an active drug;   providing a prodrug comprising a substrate for the enzyme of the conjugate, whereby the prodrug is convertible into an active anticancer drug by the enzyme of the conjugate;   contacting at least one blood vessel supplying a tumor with a therapeutically effective amount of the conjugate, whereby the conjugate is maintained on the outer surface of the at least one tumor vasculature endothelial cell and cancer cell;   contacting the at least one blood vessel having the conjugate maintained thereon with a therapeutically effective amount of the prodrug, whereby the prodrug comes into contact with the conjugate and is converted into an active anticancer drug by the enzyme of the conjugate in close proximity to the at least one of a tumor vasculature endothelial cell and a cancer cell and is toxic thereto.   
     
     
         23 . The method of  claim 22  wherein, in the step of providing a conjugate:
 (a) the ligand of the conjugate is selected from the group consisting of annexin V; 
 antibodies to a receptor or aminophospholipid that is uniquely expressed or overexpressed on a surface of a tumor vasculature endothelial cell or cancer cell; RGD-motif peptides; NGR-motif peptides; F3; HWGF-motif peptides; the peptide CTTHWGFTLC; amino-terminal fragment (ATF) of urokinase; a phosphatidylserine (PS)-binding protein; and fragments or variants thereof which substantially retain the ability to bind to the receptor or binding site; and 
 (b) the enzyme of the conjugate is selected from the group consisting of L-methioninase, nitroreductase, cytochrome P450, purine-nucleoside phosphorylase, thymidine kinase, alkaline phosphatase, β-β-glucuronidase, glycosidase, carboxypeptidase, carboxyesterase, penicillin amidase, β-lactamase, and cytosine deaminase. 
 
     
     
         24 . The method of  claim 23 , wherein the ligand comprises annexin V and the enzyme comprises L-methioninase. 
     
     
         25 . The method of  claim 22  wherein, in the step of providing a conjugate, the enzyme of the conjugate is of nonhuman origin or is absent or expressed at low concentrations in normal human tissues. 
     
     
         26 . The method of  claim 22  wherein, in the step of providing a conjugate, the enzyme has an anticancer activity. 
     
     
         27 . The method of  claim 22  wherein, in the step of providing a conjugate, the enzyme and ligand are directly coupled together. 
     
     
         28 . The method of  claim 22  wherein, in the step of providing a conjugate, the enzyme and ligand are indirectly coupled together via a linker. 
     
     
         29 . The method of  claim 22  wherein, in the step of providing a conjugate, at least one of:
 (a) the enzyme is conjugated to polyethylene glycol (PEG); and 
 (b) the conjugate is encapsulated in a liposome. 
 
     
     
         30 . The method of  claim 22 , wherein the prodrug comprises a selenomethionine prodrug and the enzyme of the conjugate comprises L-methioninase. 
     
     
         31 . The method of  claim 22 , wherein the active anticancer drug is taken up by the at least one of a tumor vasculature endothelial cell and a cancer cell and is toxic thereto. 
     
     
         32 . The method of  claim 22  wherein, in the step of providing a conjugate, the conjugate is further defined as comprising at least one of:
 (a) the amino acid sequence as set forth in at least one of SEQ ID NOS:2 and 4; 
 (b) an amino acid sequence encoded by the nucleotide sequence of at least one of SEQ ID NOS:1 and 3; 
 (c) an amino acid sequence that is at least 90% identical to at least one of SEQ ID NOS:2 and 4; and 
 (d) an amino acid sequence encoded by a nucleotide sequence that is at least 90% identical to at least one of SEQ ID NOS:1 and 3. 
 
     
     
         33 - 47 . (canceled)