Despite significant progress in the treatment of hematological malignancies, in particular leukemias, a significant majority of patients do not achieve a remission, or relapse after a prior response. As a result intense research is being directed towards examining methods of improving outcome by increasing the efficacy) of the established agents. Nucleoside analogues, including arabinosylcytosine (AraC), fludarabine, cladribine, pentostatin, and more recently gemcitabine and AraG have been a major part of the arsenal for treating hematological malignancies and solid tumors. The cytotoxic activity of AraC as well as that of other nucleoside analogues, is dependent on their intracellular conversion to an active phosphorylated metabolite. This phosphorylation (activation) is catalyzed by intracellular kinases such as deoxycytidine kinase (dCK). This enzyme is the rate-limiting factor for the activation of prodrugs such as AraC, fludarabine, cladribine, and gemcitabine. We intend to investigate the three dimensional structure as well as the catalytic mechanism of dCK. to determine its substrate specificity to define the factors that limit the efficacy of prodrug phosphorylation by the enzyme. and to apply the information to design and test dCK mutants with enhanced enzymatic (phosphorylation) activity towards these compounds. In addition we will examine the allosteric properties of dCK with the aim of developing an allosteric activator of dCK. Selective delivery of a dCK variant with enhanced activity into leukemic blasts will be an integral part of this project. This will ensure the ultimate goal of increasing the efficacy/toxicity ratio of nucleoside analogues. We will examine the feasibility of using a monoclonal antibody-enzyme delivery system to target leukemic cells with our mutant enzymes. Such a system will have the theoretical advantage of delivering the enzyme selectively into the leukemic blasts, increasing the activity of the drug in these cells only. thereby sparing the normal cells. We will use humanized anti-CD33 antibody conjugated with the mutant dCK to increase the efficacy of AraC in the targeted cells. This Selective Enhanced Enzyme Delivery System (SEEDS) could be applicable in treating other malignancies using the appropriate antibodies, mutant enzymes and drugs.
