Site-directed pegylation of arginases and the use thereof as anti-cancer and anti-viral agents
The present invention provides a site-specific pegylated arginase conjugate and method for producing thereof. The site-specific pegylated arginase is homogeneous in molecular weight and shows therapeutic effect for treating cancers and viral infections. The method for producing the arginase conjugate comprises genetically modifying the gene encoding an arginase so that the PEG moiety can be attached to the enzyme at a predetermined, specific intended sites. This is achieved by removing the PEG-attaching amino acid residue(s) at undesirable site(s) while keeping or adding cysteine(s) at the desirable site(s) of the enzyme. Two exemplary embodiments of the pegylated arginase conjugate are directed to human arginase I (HAI) where a polyethylene glycol (PEG) moiety is site-specific covalently bonded to Cys 45 of the enzyme and Bacillus caldovelox arginase (BCA) where a polyethylene glycol (PEG) moiety is site-specific covalently bonded to Cys 161 of the enzyme.
1. A pharmaceutical composition for treating an arginine-dependent disease comprising a polyethylene glycol-arginase conjugate having a polyethylene glycol moiety covalently attached to a genetically-modified Sus scrofa arginase, wherein said genetically-modified Sus scrofa arginase has a single cysteine residue for covalently attaching to the polyethylene glycol moiety, wherein said single cysteine residue is located at a distance away from the active binding site of the genetically-modified Sus scrofa arginase such that the polyethylene glycol attachment does not interfere with the active site, wherein the polyethylene glycol moiety has a molecular weight of 10,000-30,000 Da,
wherein the genetically-modified Sus scrofa arginase comprises all of SEQ ID No: 26 except for substitutions at positions 54, 119, 168 and 303 of SEQ ID NO: 26 and the single cysteine for the attachment of the polyethylene glycol moiety is at a position corresponding to position 45 of SEQ ID NO: 26.
2. The pharmaceutical composition of claim 1 further comprising a therapeutic agent for treating an arginine-dependent disease.
3. The pharmaceutical composition of claim 2 wherein the therapeutic agent is for treating cancer.
4. The pharmaceutical composition of claim 1 wherein the polyethylene glycol is a single chain or branched chain polyethylene glycol.
5. A method for treating an arginine-dependent disease comprising administering the pharmaceutical composition of claim 1 in combination with an anti-neoplastic compound to a subject in needs thereof, wherein said subject comprises human and other organisms.