Heparinase III HLGAG fragments and uses thereof

Abstract

The invention relates to heparinase III and mutants thereof. Modified forms of heparinase III having reduced enzymatic activity which are useful for a variety of purposes, including sequencing of heparin-like glycosaminoglycans (HLGAGs), removing active heparan sulfate from a solution, inhibition of angiogenesis, etc. have been discovered according to the invention. The invention in other aspects relates to methods of treating cancer and inhibiting tumor cell growth and/or metastasis using heparinase III, or products produced by enzymatic cleavage by heparinase III of HLGAGs.

1 . A method of identifying a therapeutic HLGAG fragment, comprising: 
 a) isolating at least a portion of a tumor,    b) contacting the portion of the tumor with heparinase III, and    c) identifying a HLGAG fragment with a therapeutic effect.    
     
     
         2 . The method of  claim 1 , wherein the method further comprises sequencing the HLGAG fragment with a therapeutic effect.  
     
     
         3 . The method of  claim 1 , wherein the therapeutic effect is the prevention of tumor cell proliferation or metastasis.  
     
     
         4 . The method of  claim 3 , wherein the tumor cell is of a different type of tumor than the isolated tumor.  
     
     
         5 . The method of  claim 1 , wherein the tumor cell is from a biliary tract cancer, brain cancer, breast cancer, cervical cancer, choriocarcinoma, colon cancer, endometrial cancer, esophageal cancer, gastric cancer, intraepithelial neoplasm, lymphoma, liver cancer, lung cancer, melanoma, neuroblastoma, oral cancer, ovarian cancer, pancreas cancer, prostate cancer, rectal cancer, sarcoma, skin cancer, testicular cancer, thyroid cancer, or renal cancer.  
     
     
         6 . The method of  claim 5 , wherein the tumor cell is a prostate cancer or melanoma cell.  
     
     
         7 . The method of  claim 1 , wherein the therapeutic effect is the inhibition of neovascularization.  
     
     
         8 . The method of  claim 1 , wherein the therapeutic effect is the prevention of coagulation.  
     
     
         9 . The method of  claim 1 , wherein the therapeutic effect is the prevention of psoriasis.  
     
     
         10 . A method of identifying a therapeutic HLGAG fragment, comprising: 
 a) isolating at least a portion of a tumor,    b) contacting the portion of the tumor with a modified heparinase III, and    c) identifying a HLGAG fragment with a therapeutic effect.    
     
     
         11 . The method of  claim 10 , wherein the method further comprises sequencing the HLGAG fragment with a therapeutic effect.  
     
     
         12 . The method of  claim 10 , wherein the therapeutic effect is the prevention of tumor cell proliferation or metastasis.  
     
     
         13 . The method of  claim 12 , wherein the tumor cell is of a different type of tumor than the isolated tumor.  
     
     
         14 . The method of  claim 10 , wherein the tumor cell is from a biliary tract cancer, brain cancer, breast cancer, cervical cancer, choriocarcinoma, colon cancer, endometrial cancer, esophageal cancer, gastric cancer, intraepithelial neoplasm, lymphoma, liver cancer, lung cancer, melanoma, neuroblastoma, oral cancer, ovarian cancer, pancreas cancer, prostate cancer, rectal cancer, sarcoma, skin cancer, testicular cancer, thyroid cancer, or renal cancer.  
     
     
         15 . The method of  claim 14 , wherein the tumor cell is a prostate cancer or melanoma cell.  
     
     
         16 . The method of  claim 10 , wherein the therapeutic effect is the inhibition of neovascularization.  
     
     
         17 . The method of  claim 10 , wherein the therapeutic effect is the prevention of coagulation.  
     
     
         18 . The method of  claim 10 , wherein the therapeutic effect is the prevention of psoriasis.  
     
     
         19 . The method of  claim 10 , wherein the modified heparinase III comprises a histidine residue selected from the group consisting of His36, His105, His110, His139, His152, His225, His234, His241, His424, His469 and His539 that has been substituted with alanine, serine, tyrosine, threonine or lysine.  
     
     
         20 . The method of  claim 19 , wherein the modified heparinase III comprises a substitution at histidine residue His110, His241 or His139.  
     
     
         21 . The method of  claim 20 , wherein the substitution is at His110.  
     
     
         22 . The method of  claim 20 , wherein the substitution is at His241.  
     
     
         23 . The method of  claim 20 , wherein the substitution is at His139.  
     
     
         24 . A method of identifying a therapeutic HLGAG fragment, comprising: 
 a) isolating at least a portion of a tumor,    b) contacting the portion of the tumor with a modified heparinase II with the functional activity of heparinase III, and    c) identifying a HLGAG fragment with a therapeutic effect.    
     
     
         25 . The method of  claim 24 , wherein the method further comprises sequencing the HLGAG fragment with a therapeutic effect.  
     
     
         26 . The method of  claim 24 , wherein the therapeutic effect is the prevention of tumor cell proliferation or metastasis.  
     
     
         27 . The method of  claim 26 , wherein the tumor cell is of a different type of tumor than the isolated tumor.  
     
     
         28 . The method of  claim 24 , wherein the tumor cell is from a biliary tract cancer, brain cancer, breast cancer, cervical cancer, choriocarcinoma, colon cancer, endometrial cancer, esophageal cancer, gastric cancer, intraepithelial neoplasm, lymphoma, liver cancer, lung cancer, melanoma, neuroblastoma, oral cancer, ovarian cancer, pancreas cancer, prostate cancer, rectal cancer, sarcoma, skin cancer, testicular cancer, thyroid cancer, or renal cancer.  
     
     
         29 . The method of  claim 24 , wherein the tumor cell is a prostate tumor or melanoma cell.  
     
     
         30 . The method of  claim 24 , wherein the therapeutic effect is the inhibition of neovascularization.  
     
     
         31 . The method of  claim 24 , wherein the therapeutic effect is the prevention of coagulation.  
     
     
         32 . The method of  claim 24 , wherein the therapeutic effect is the prevention of psoriasis.  
     
     
         33 . The method of  claim 24 , wherein the modified heparinase II comprises a substitution at Cys348 or His440.  
     
     
         34 . The method of  claim 33 , wherein the substitution is at Cys348.  
     
     
         35 . The method of  claim 33 , wherein the substitution is at His440.  
     
     
         36 . A method for preparing HLGAG fragments, comprising: 
 a) isolating at least a portion of a tumor,    b) contacting the portion of the tumor with heparinase III, modified heparinase III or a modified heparinase II to produce HLGAG fragments, and    c) isolating the HLGAG fragments.    
     
     
         37 . The method of  claim 36 , wherein the method further comprises determining the sequence of the HLGAG fragments.  
     
     
         38 . The method of  claim 36 , wherein the modified heparinase III comprises a histidine residue selected from the group consisting of His36, His105, His110, His139, His152, His225, His234, His241, His424, His469 and His539 that has been substituted with alanine, serine, tyrosine, threonine or lysine.  
     
     
         39 . The method of  claim 38 , wherein the modified heparinase III comprises a substitution at histidine residue His110, His139 or His241.  
     
     
         40 . The method of  claim 36 , wherein the modified heparinase II comprises a substitution at Cys348 or His440.