Mono-PEGylation of a Thermostable Arginine-Depleting Enzyme for the Treatment of Lung Cancer

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.

Recombinant Bacillus caldovelox Arginase Mutant (BCA-M) Induces Apoptosis, Autophagy, Cell Cycle Arrest and Growth Inhibition in Human Cervical Cancer Cells

“…On the other hand, our results provided strong evidence that BCA-M is a promising broad spectrum arginine depletion-based anticancer drug candidate. In fact, we have previously demonstrated that a PEGylated form of BCA-M, with equivalent arginine depletion capability but more stable in vivo, had a remarkable anti-tumor effect on A549 lung tumor-bearing nude mice [ 10 ]. Our unpublished data also showed that repeated administrations of PEGylated BCA-M could maintain systemic arginine depletion for the whole testing period of a least a month in normal mice.…”

“…Our unpublished data also showed that repeated administrations of PEGylated BCA-M could maintain systemic arginine depletion for the whole testing period of a least a month in normal mice. With the in vitro, cancer-cell-suppressive IC 50 values of BCA-M on the tested cervical cancer cells being lower than that of the A549 lung cancer cells [ 10 ], we expect the PEGylated BCA-M to be a feasible and promising anti-cervical cancer drug candidate. When applied systemically, PEGylated BCA-M would certainly have the overwhelming advantage of in vivo stability over BCA-M and should allow the PEGylated drug to be effective even for cancer at the metastatic stage.…”

“…Recombinant Bacillus caldovelox arginase mutant (BCA-M) was prepared from E. coli harboring the expression vector for the his-tagged Bacillus caldovelox arginase and purified using nickel affinity column chromatography [ 10 , 48 ]. Recombinant L-arginine deiminase (ADI) were expressed and purified as described previously [ 49 ].…”

“…Despite the recent development of the HPV vaccine, cervical cancer is still the fourth most common and lethal cancer for women around the world [ 2 ]. With our recent success in developing an arginase-based anticancer drug against liver, lung, skin and colorectal cancers [ 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 ], we would like to explore the potential of a novel recombinant Bacillus caldovelox arginase mutant (BCA-M) [ 10 ] in the treatment of human cervical cancers.…”

From Enzyme Stability to Enzymatic Bioelectrode Stabilization Processes

“…In the "grafting to" strategy, the polymer is first synthetized; then, an end-group functionality is attached to one amino-acid on the protein surface. As an example, a mono-PEGylated arginase was constructed by linkage of PEG-maleimide to a cysteine residue on the enzyme surface [74]. The protein was protected against proteases thanks to the shielding effect of PEG, allowing the modified arginase to operate as a promising anticancer drug candidate.…”