Compositions Of Engineered Human Arginases And Methods For Treating Cancer

Abstract

Compositions and methods for the treatment of cancer are described, and, more preferably, to the treatment of cancers that do not express, or are otherwise deficient in, argininosuccinate synthetase, with enzymes that deplete L-Arginine in serum. In one embodiment, the present invention contemplates an arginase protein, such as a human Arginase I protein, comprising at least one amino acid substitution and a metal cofactor, said protein comprising an increased catalytic activity when compared with a native human Arginase I.

What is claimed is: 
     
         1 . A protein comprising an amino acid sequence of human Arginase I or an amino acid sequence of human Arginase II and a non-native metal cofactor, wherein the non-native metal cofactor is cobalt. 
     
     
         2 . The protein of  claim 1 , wherein the amino acid sequence is further defined as Arginase I. 
     
     
         3 . The protein of  claim 2 , wherein the human Arginase I comprises SEQ ID NO:1. 
     
     
         4 . The protein of  claim 1 , wherein the amino acid sequence is further defined as Arginase II. 
     
     
         5 . The protein of  claim 4 , wherein the Arginase II comprises SEQ ID NO:2. 
     
     
         6 . The protein of  claim 1 , wherein the amino acid sequence comprises a truncated Arginase I or Arginase II sequence. 
     
     
         7 . The protein of  claim 6 , wherein the amino acid sequence lacks an N-terminal methionine. 
     
     
         8 . The protein of  claim 6 , wherein the amino acid sequence lacks the first 21 amino acids of the wild-type sequence. 
     
     
         9 . The protein of  claim 1 , wherein the protein displays a k cat /K m  between 400 mM −1  s −1  and 4,000 mM −1  s −1  at pH 7.4. 
     
     
         10 . The protein of  claim 9 , wherein the protein displays a k cat /K M  between 400 mM −1  s 4  and 2,500 mM −1  s −1  at pH 7.4. 
     
     
         11 . The protein of  claim 1 , further comprising at least one amino acid substitution at the metal binding site. 
     
     
         12 . The protein of  claim 11 , wherein the at least one amino acid substitution is at His101, Asp124, His126, Asp128, Asp232, Asp234, Trp122, Asp181, Ser230, His120, Asp143, His145, Asp147, Asp251, Asp253, Trp141, Asp200, Ser249, Cys303, or Glu256. 
     
     
         13 . The protein of  claim 12 , wherein the at least one amino acid substitution is Asp181Ser, Ser230Cys, Ser230Gly, Cys303Phe, Cys303Ile, Glu256Gln, Asp181Glu, or Ser230Ala. 
     
     
         14 . The protein of  claim 11 , further comprising at least two amino acid substitutions. 
     
     
         15 . A human Arginase I or Arginase II protein having a metal binding site and comprising at least one amino acid substitution at the metal binding site, wherein the protein displays an increase in the hydrolysis of Arginine that results in a k cat /K m  of at least two fold greater than that of a native human Arginase I having SEQ ID NO:1 or a native human Arginase II having SEQ ID NO:2. 
     
     
         16 . The protein of  claim 15 , wherein the protein displays a k cat /K m  between 400 mM −1  s −1  and 4,000 mM −1  s −1  at pH 7.4. 
     
     
         17 . The protein of  claim 16 , wherein the protein displays a k cat /K M  between 400 mM −1  s −1  and 2,500 mM −1  s −1  at pH 7.4. 
     
     
         18 . The protein of  claim 15 , wherein the arginase protein comprises one polypeptide chain. 
     
     
         19 . The protein of  claim 15 , wherein the at least one amino acid substitution is at His101, Asp124, His126, Asp128, Asp232, Asp234, Trp122, Asp181, Ser230, His120, Asp143, His145, Asp147, Asp251, Asp253, Trp141, Asp200, Ser249, Cys303, or Glu256. 
     
     
         20 . The protein of  claim 19 , wherein the at least one amino acid substitution is Asp181Ser, Ser230Cys, Ser230Gly, Cys303Phe, Cys303Ile, Glu256Gln, Asp181Glu, or Ser230Ala. 
     
     
         21 . The protein of  claim 15 , wherein the human Arginase comprises at least two amino acid substitutions. 
     
     
         22 . The protein of  claim 21 , wherein the amino acid substitutions are Asp181Glu and Ser230Ala. 
     
     
         23 . The protein of  claim 15 , further comprising a non-native metal cofactor. 
     
     
         24 . The protein of  claim 23 , wherein the non-native metal cofactor is cobalt. 
     
     
         25 . The protein of  claim 15 , wherein the amino acid sequence comprises a truncated Arginase I or Arginase II sequence. 
     
     
         26 . The protein of  claim 25 , wherein the amino acid sequence lacks an N-terminal methionine. 
     
     
         27 . The protein of  claim 25 , wherein the amino acid sequence lacks the first 21 amino acids of the wild-type sequence. 
     
     
         28 . The protein of  claim 15 , wherein the protein is covalently linked to polyethylene glycol. 
     
     
         29 . A nucleic acid encoding the protein of  claim 1 . 
     
     
         30 . The nucleic acid of  claim 29 , wherein the nucleic acid is codon optimized for expression in bacteria. 
     
     
         31 . A vector comprising the nucleic acid of  claim 29 . 
     
     
         32 . A host cell comprising the vector of  claim 31 . 
     
     
         33 . A fusion protein comprising the protein of  claim 1 , further comprising a heterologous peptide segment. 
     
     
         34 . The fusion protein of  claim 33 , wherein the heterologous peptide segment comprises the Fc region of an immunoglobulin or a portion thereof. 
     
     
         35 . The fusion protein of  claim 33 , wherein the amino acid sequence comprises a truncated Arginase I or Arginase II sequence. 
     
     
         36 . The fusion protein of  claim 35 , wherein the amino acid sequence lacks an N-terminal methionine. 
     
     
         37 . The fusion protein of  claim 35 , wherein the amino acid sequence lacks the first 21 amino acids of the wild-type sequence. 
     
     
         38 . A nucleic acid encoding the fusion protein of  claim 34 . 
     
     
         39 . The nucleic acid of  claim 38 , wherein the nucleic acid is codon optimized for expression in  E. coli.    
     
     
         40 . A vector comprising the nucleic acid of  claim 38 . 
     
     
         41 . A host cell comprising the vector of  claim 40 . 
     
     
         42 . A method of treating a human patient having an arginine auxotrophic cancer comprising administering to the patient a formulation comprising the protein of  claim 1 . 
     
     
         43 . The method of  claim 42 , wherein the patient has been diagnosed with a hepatocellular carcinoma, renal cell carcinoma, or melanoma. 
     
     
         44 . The method of  claim 42 , wherein the formulation is administered topically, intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intraocularly, intranasally, intravitreally, intravaginally, intrarectally, intramuscularly, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, orally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, via a catheter, or via a lavage. 
     
     
         45 . A pharmaceutical formulation comprising the protein of  claim 1  and a pharmaceutically acceptable excipient.