L-asparaginase (L-ASNase) is a pivotal enzymatic drug in the treatment of acute lymphoblastic leukemia (ALL). Unfortunately, in ~15% of L-ASNase treated patients, immune reactions are observed that result in loss of enzyme and thus anti-cancer activity. In addition, these bacterial L-ASNases harbor L-glutaminase (L-GLNase) co-activity, which is responsible for most of the observed adverse toxicities, including hepatoxicity and pancreatitis. Importantly, premature termination of L-ASNase therapy is associated with poor relapse-free survival. Furthermore, the L-ASNase associated toxicities seriously impact the short- and long -term quality-of-life of patients. As such, there is a great need for less toxic and less immunogenic L-ASNase preparations. Via rational design, we recently generated a humanized mammalian-derived L-ASNase formulation with no L-GLNase co-activity. Our unpublished results in mouse leukemia models demonstrate that this alternative L-ASNase has equivalent anti-cancer properties but with significantly reduced toxicities. However, this variant has a short in vivo half-life. Therefore, we propose to evaluate different methods for stabilization and slow release of this humanized L-ASNase variant for the treatment of ALL. The end goal of this translational research project is to validate the safety/ immunogenicity and efficacy of our alternative L-ASNase formulation in lymphoma dogs, as a final step required to initiate clinical trials in human ALL patients.