Utilization of Wolinella succinogenes asparaginase to treat diseases associated with asparagine dependence

Abstract

Described herein are methods for producing recombinant forms of asparaginase derived from Wollinella succinogenes . In addition, methods for covalent modification of proteins, including asparaginases, by acylation are also provided. Certain embodiments provide for epitopic-labeling of the amino terminus of W. succinogenes asparaginase. Additional embodiments concern methods for the therapeutic utilization of the native, homotetrameric form of W. succinogenes asparaginase, as well as the use of epitopically-labeled or non-epitopically-labeled recombinant W. succinogenes asparaginase (or a covalently modified analog thereof) in the therapeutic treatment of malignant and non-malignant hematological disease and other diseases where asparagine depletion or deprivation would be efficacious or which respond to asparagine depletion or deprivation, as well as their potential utilization in the therapeutic treatment of autoimmune diseases such as rheumatoid arthritis, AIDS, and SLE.

I claim:  
     
         1 . Method of treating a disease which responds to asparaginase depletion, the method comprising the step of administering to a patient having a disease which responds to asparagine depletion a therapeutically effective amount of a  Wollinella succinogenes  asparaginase.  
     
     
         2 . A method according to  claim 1  wherein the disease is a malignant disease.  
     
     
         3 . A method according to  claim 2  wherein the malignant disease is a malignant hematologic disease.  
     
     
         4 . A method according to  claim 3  wherein the malignant disease is selected from the group consisting of a lymphoma, a leukemia, and a myeloma.  
     
     
         5 . A method according to  claim 4  wherein the malignant hematologic disease is a chronic disease.  
     
     
         6 . A method according to  claim 5  wherein the chronic malignant hematologic disease is in an acute phase.  
     
     
         7 . A method according to  claim 1  wherein the disease is a non-malignant disease.  
     
     
         8 . A method according to  claim 7  wherein the non-malignant disease is an autoimmune disease.  
     
     
         9 . A method according to  claim 8  wherein the autoimmune disease is selected from the group consisting of a rheumatoid arthritis, SLE, and AIDS.  
     
     
         10 . A method according to  claim 1  wherein the patient is a mammal selected from the group consisting of bovine, canine, equine, feline, bovine, porcine, and primate animals.  
     
     
         11 . A method according to  claim 1  wherein the patient is human.  
     
     
         12 . A method according to  claim 1  wherein  Wolinella Succinogenes  asparaginase is a native enzyme.  
     
     
         13 . A method according to  claim 1  wherein  Wolinella Succinogenes  asparaginase is a recombinant enzyme.  
     
     
         14 . A method according to  claim 12  or  13  wherein the enzyme is an analog of asparaginase.  
     
     
         15 . A method according to  claim 14  wherein the analog comprises at least one covalent modification.  
     
     
         16 . A method according to  claim 15  wherein the covalent modification is selected from the group consisting of pegylation and acetylation.  
     
     
         17 . A pharmaceutical composition comprising a purified  Wollinella succinogenes  asparaginase and a pharmaceutically acceptable carrier.  
     
     
         18 . A pharmaceutical composition according to  claim 17  wherein the asparaginase is a native enzyme.  
     
     
         19 . A pharmaceutical composition according to  claim 17  wherein the enzyme is a recombinant enzyme.  
     
     
         20 . A pharmaceutical composition according to  claim 17  or 18 wherein the enzyme is an analog of asparaginase.  
     
     
         21 . A pharmaceutical composition according to  claim 20  wherein the analog comprises at least one covalent modification.  
     
     
         22 . A pharmaceutical composition according to  claim 21  wherein the covalent modification is selected from the group consisting of pegylation and acetylation.  
     
     
         23 . A method of producing a recombinant form of  Wollinella succinogenes  asparaginase analog, the method comprising the steps of: 
 (a) obtaining a nucleic acid molecule encoding a polypeptide comprising a unique contiguous amino acid sequence of  Wollinella succinogenes  asparaginase, wherein the unique amino acid sequence comprises at least nine amino acids;    (b) cloning the nucleic acid sequence into an expression vector;    (c) introducing the expression vector into a suitable host cell or cells;    (d) culturing the host cell(s) under conditions which allow expression of the polypeptide from the expression vector in biologically active form or in a form from which biological activity can be reconstituted.    
     
     
         24 . A method according to  claim 23  further comprising the step of isolating the expressed polypeptide.  
     
     
         25 . A method according to  claim 23  performed in vivo.  
     
     
         26 . A method according to  claim 25  wherein the expression vector is carried in a gene delivery vehicle.  
     
     
         27 . A method according to  claim 26  wherein the gene delivery vehicle is selected from the group consisting of a recombinant virus, and a non-viral gene delivery system.  
     
     
         28 . A method according to  claim 23  wherein the expressed polypeptide is an analog of  Wollinella succinogenes  asparaginase.  
     
     
         29 . A method according to  claim 28  wherein the polypeptide comprises an N-terminal epitope. tag.  
     
     
         30 . A method according to  claim 28  wherein the polypeptide comprise one or more amino acid residue insertions, deletions, and/or substitutions, as compared to the amino acid sequence of the native form of  Wollinella succinogenes  asparaginase.  
     
     
         31 . A method according to  claim 25  wherein the isolated polypeptide is covalently modified by a process selected from the group consisting of acylation and pegylation.  
     
     
         32 . A method according to  claim 29  wherein the nucleic acid molecule encoding the polypeptide to be expressed comprises a nucleotide sequence according to SEQ ID NO:3.  
     
     
         33 . Nucleic acid molecule encoding an analog of  Wollinella succinogenes  asparaginase.  
     
     
         34 . A nucleic acid molecule according to  claim 33  wherein the analog comprises at least one amino acid residue substitution, deletion, and/or insertion as compared to the amino acid sequence of the native form of  Wollinella succinogenes  asparaginase.  
     
     
         35 . A nucleic acid molecule according to  claim 34  functionally inserted into an expression vector.  
     
     
         36 . A nucleic acid molecule encoding  Wollinella succinogenes  asparaginase or an analog thereof functionally inserted into an expression vector of a gene delivery vehicle.  
     
     
         37 . Recombinant host cell having a nucleic acid molecule according to  claim 35  contained therein.  
     
     
         38 . Method of covalently modifying a biologically active protein, the method comprising the step of acylating the protein.  
     
     
         39 . A method according to  claim 38  wherein the protein is  Wolinella succinogenes  asparaginase or an analog thereof.  
     
     
         40 . Method of altering a pharmacokinetic property of a protein by modifying the protein according to  claim 38 .  
     
     
         41 . A method of reducing immunogenecity of a therapeutic protein by modifying the protein according to claim  38 .