Abstract
L-arginine (L-Arg) depletion induced by randomly PEGylated arginine deiminase (ADI-PEG20) can treat arginosuccinate synthase (ASS)-negative cancers, and ADI-PEG20 is undergoing phase III clinical trials. Unfortunately, ASS-positive cancers are resistant to ADI-PEG20. Moreover, the yield of ADI production is low because of the formation of inclusion bodies. Here, we report a thermostable arginine-depleting enzyme, Bacillus caldovelox arginase mutant (BCA-M: Ser161->Cys161). An abundant amount of BCA-M was easily obtained via high cell-density fermentation and heat treatment purification. Subsequently, we prepared BCA-M-PEG20, by conjugating a single 20 kDa PEG monomer onto the Cys161 residue via thio-chemistry. Unlike ADI-PEG20, BCA-M-PEG20 significantly inhibited ASS-positive lung cancer cell growth. Pharmacodynamic studies showed that a single intraperitoneal injection (i.p). administration of 250 U/mouse of BCA-M-PEG20 induced low L-Arg level over 168 h. The mono-PEGylation of BCA-M prolonged its elimination half-life from 6.4 to 91.4 h (a 14-fold increase). In an A549 lung cancer xenograft model, a weekly administration of 250 U/mouse of BCA-M-PEG20 suppressed tumor growth significantly. We also observed that BCA-M-PEG20 did not cause any significant safety issue in mouse models. Overall, BCA-M-PEG20 showed excellent results in drug production, potency, and stability. Thereby, it has great potential to become a promising candidate for lung cancer therapy.
Highlights
Lung carcinoma is one of the top-ranking killer cancers worldwide, with poor survival rates
Our results revealed that the injection of 250 U/mouse of Bacillus caldovelox arginase mutant (BCA-M)-PEG20 provided long-lasting serum arginine depletion for 168 h, as compared with only 6 h with the BCA-M (Figure 6A)
For in vivo anti-tumor efficacy tests of BCA-M-PEG20 and cisplatin on nude mice bearing A549 tumor xenografts, weekly administration of 250 U/mouse of BCA-M-PEG20 and weekly administration of 1.5 mg of cisplatin exhibited ~41% and ~47% tumor suppression, respectively. These results show that BCA-M-PEG20 is a potential candidate as good as cisplatin for treating argininosuccinate synthetase (ASS)-positive lung cancers
Summary
Lung carcinoma is one of the top-ranking killer cancers worldwide, with poor survival rates. Conventional treatment options for lung cancer, surgery, radiotherapy, and chemotherapy, are far from satisfactory in both aspects of efficacy and adverse side effects. L-arginine (L-Arg) depletion is a new, promising, and safe strategy to treat several cancer types, including, leukemia, melanoma, colorectal cancer, brain cancer, and hepatocellular carcinoma. They have been reported to be arginine-auxotrophic cancers because these cells do not express argininosuccinate synthetase (ASS) or ornithine transcarbamylase (OTC) in the urea cycle [4,5,6,7,8]. Thereby, arginine-depleting enzymes may be an effective and safe way of treating lung cancers
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