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

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• 作者：Everett Stone^a ; ^b ; ^c ; Lynne Chantranupong^a ; ^b ; Candice Gonzalez^a ; ^b ; Jamye O'Neal^e ; Mridula Rani^a ; ^b ; Carla VanDenBerg^e ; George Georgiou^a ; ^b ; ^c ; ^d ; ^{gg@che.utexas.edu}
• 关键词：Serum persistence ; L-Arg depletion ; Systemic therapeutic ; Antineoplastic
• 刊名：Journal of Controlled Release
• 出版年：2012
• 期刊代码：25_01683659
• 类别：pha
• 出版时间：28 February, 2012
• 卷：158
• 期：1
• 页码：171-179
• 文件大小：520 K

摘要

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 = 4.8 h) 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 Mn²⁺ metal center in human Arginase I with Co²⁺ (Co-hArgI) results in an enzyme that displays 10-fold higher catalytic efficiency for L-Arg hydrolysis, 12-15 fold reduction in the IC₅₀ towards a variety of malignant cell lines and, importantly a t_1/2 = 22 h 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 20 kDa PEG-maleimide (Co-hArgI-C^PEG-20K); (ii) engineering of the homotrimeric Co-hArgI into a linked, monomeric 110 kDa polypeptide (Co-hArgI x3) and (iii) lysyl conjugation of 5 kDa 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 5 kDa 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 6 mg/kg weight, a well tolerated dose, the circulation t_1/2 of the protein in Balb/c mice is 63 卤 10 h. Very low levels of serum L-Arg (< 5 渭M) could be sustained for over 75 h after injection, representing a 9-fold increase in pharmacodynamic efficacy relative to similarly prepared Mn²⁺-containing hArgI conjugated to 5 kDa 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.