Intracellular Delivery of Recombinant Arginine Deiminase (rADI) by Heparin-Binding Hemagglutinin Adhesion Peptide Restores Sensitivity in rADI-Resistant Cancer Cells

Recombinant arginine deiminase (rADI) has been used in clinical trials for arginine-auxotrophic cancers. However, the emergence of rADI resistance, due to the overexpression of argininosuccinate synthetase (AS), has introduced an obstacle in its clinical application. Here, we have proposed a strategy for the intracellular delivery of rADI, which depletes both extracellular and intracellular arginine, to restore the sensitivity of rADI-resistant cancer cells. In this study, the C terminus of heparin-binding hemagglutinin adhesion protein from Mycobacterium tuberculosis (HBHAc), which contains 23 amino acids, was used to deliver rADI into rADI-resistant human breast adenocarcinoma cells (MCF-7). Chemical conjugates (L- and D-HBHAc-SPDP-rADI) and a recombinant fusion protein (rHBHAc-ADI) were produced. L- and D-HBHAc-SPDP-rADI showed a significantly higher cellular uptake of rADI by MCF-7 cells compared to that of rADI alone. Cell viability was significantly decreased in a dose-dependent manner in response to L- and D-HBHAc-SPDP-rADI treatments. In addition, the ratio of intracellular concentration of citrulline to arginine in cells treated with L- and D-HBHAc-SPDP-rADI was significantly increased by 1.4- and 1.7-fold, respectively, compared with that obtained in cells treated with rADI alone (p < 0.001). Similar results were obtained with the recombinant fusion protein rHBHAc-ADI. Our study demonstrates that the increased cellular uptake of rADI by HBHAc modification can restore the sensitivity of rADI treatment in MCF-7 cells. rHBHAc-ADI may represent a novel class of antitumor enzyme with an intracellular mechanism that is independent of AS expression.