Strategies for optimizing the serum persistence of engineered human arginase I for cancer therapy

摘要: Systemic l-Arginine depletion following intravenous administration of hydrolyzing enzymes has been shown to selectively impact tumors displaying urea-cycle defects including a large fraction hepatocellular carcinomas, metastatic melanomas and small cell lung carcinomas. However, the human arginases display poor serum stability (t1/2 =4.8 hrs) whereas bacterial arginine deiminase evaluated in phase II clinical trials was reported be immunogenic, eliciting strong neutralizing antibody responses. Recently, we showed that substitution Mn2+ metal center Arginase I with Co2+ (Co-hArgI) results an enzyme displays 10-fold higher catalytic efficiency for l-Arg hydrolysis, 12–15 fold reduction IC50 towards variety malignant lines and, importantly t1/2= 22 hrs serum. To investigate utility Co-hArgI therapy cancer systematically investigated three strategies enhancing persistence circulation: (i) site specific conjugation engineered accessible N-terminal Cys residue 20 KDa PEG-maleimide (Co-hArgI-CPEG-20K); (ii) engineering homotrimeric into linked, monomeric 110 polypeptide (Co-hArgI ×3) (iii) lysyl 5 PEG-N-hydroxysuccinimide (NHS) ester (Co-hArgI-KPEG-5K). Surprisingly, even though all formulations resulted proteins predicted hydrodynamic radius larger than cut-off renal filtration, only CohArgI amine conjugated PEG remained circulation sufficiently long durations. Using CohArgI-KPEG-5K labeled end-terminal fluorescein easy detection, demonstrated intraperitoneal at 6 mg/kg weight, well tolerated dose, t1/2 protein Balb/c mice is 63 ± 10 hrs. Very low levels (<5 μM) could sustained over 75 after injection, representing 9-fold increase pharmacodynamic efficacy relative similarly prepared Mn2+-containing hArgI PEG-NHS (Mn-hArgI-KPEG-5K). The favorable pharmacokinetic properties Co-hArgI-KPEG-5K here, coupled its origin which should reduce likelihood adverse immune responses, make it promising candidate therapy.