title: Strategies for optimizing the serum persistence of engineered human arginase I for cancer therapy.
abstract: Systemic L-arginine depletion following intravenous administration of l-arginine hydrolyzing enzymes has been shown to selectively impact tumors displaying urea cycle defects including a large fraction of hepatocellular carcinomas, metastatic melanomas and small cell lung carcinomas. However, the human arginases display poor serum stability (t(1/2)=4.8h) whereas a bacterial arginine deiminase evaluated in phase II clinical trials was reported to be immunogenic, eliciting strong neutralizing antibody responses. Recently, we showed that substitution of the Mn(2+) metal center in human Arginase I with Co(2+) (Co-hArgI) results in an enzyme that displays 10-fold higher catalytic efficiency for L-Arg hydrolysis, 12-15 fold reduction in the IC(50) towards a variety of malignant cell lines and, importantly a t(1/2)=22h in serum. To investigate the utility of Co-hArgI for L-Arg depletion therapy in cancer we systematically investigated three strategies for enhancing the persistence of the enzyme in circulation: (i) site specific conjugation of Co-hArgI engineered with an accessible N-terminal Cys residue to 20kDa PEG-maleimide (Co-hArgI-C(PEG-20K)); (ii) engineering of the homotrimeric Co-hArgI into a linked, monomeric 110kDa polypeptide (Co-hArgI x3) and (iii) lysyl conjugation of 5kDa PEG-N-hydroxysuccinimide (NHS) ester (Co-hArgI-K(PEG-5K)). Surprisingly, even though all three formulations resulted in proteins with a predicted hydrodynamic radius larger than the cut-off for renal filtration, only Co-hArgI amine conjugated to 5kDa PEG remained in circulation for sufficiently long durations. Using Co-hArgI-K(PEG-5K) labeled with an end-terminal fluorescein for easy detection, we demonstrated that following intraperitoneal administration at 6mg/kg weight, a well tolerated dose, the circulation t(1/2) of the protein in Balb/c mice is 63±10h. Very low levels of serum L-Arg (<5μM) could be sustained for over 75h after injection, representing a 9-fold increase in pharmacodynamic efficacy relative to similarly prepared Mn(2+)-containing hArgI conjugated to 5kDa PEG-NHS ester (Mn-hArgI-K(PEG-5K)). The favorable pharmacokinetic and pharmacodynamic properties of Co-hArgI-K(PEG-5K) reported here, coupled with its human origin which should reduce the likelihood of adverse immune responses, make it a promising candidate for cancer therapy.
authors: Department of Chemical Engineering, Center for Molecular, College of Pharmacy, University of Texas, Austin, TX 78712, USA.
authors: Everett
authors: E
authors: Stone
authors: Lynne
authors: L
authors: Chantranupong
authors: Candice
authors: C
authors: Gonzalez
authors: Jamye
authors: J
authors: O'Neal
authors: Mridula
authors: M
authors: Rani
authors: Carla
authors: C
authors: VanDenBerg
authors: George
authors: G
authors: Georgiou
mesh_terms: D000818:Animals; D001119:Arginase / Q000008:administration & dosage* / Q000737:chemistry / Q000235:genetics / Q000493:pharmacokinetics; D001120:Arginine / Q000097:blood; D003545:Cysteine / Q000008:administration & dosage* / Q000737:chemistry / Q000493:pharmacokinetics; D005260:Female; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D009369:Neoplasms / Q000188:drug therapy; D011092:Polyethylene Glycols / Q000008:administration & dosage* / Q000737:chemistry / Q000493:pharmacokinetics
chemical_list: D011092:Polyethylene Glycols; D001120:Arginine; D001119:Arginase; D003545:Cysteine