Yu.V. Krasotkina, N.N. Sokolov

Up to date microbial L-asparaginase is a single example of enzymes application in oncotherapy. For more that 30 years microbial asparaginase is using as a component in chemotherapy courses for treatment of acute lymphoblastic leukosis, lymphoblastomas and reticuloblastomas. Russian government incorporated asparaginase into the list of basic vital drugs in 1994. At the present time only two types of asparaginase are using in clinical practice: asparaginase from Escherichia coli (EcA) and asparaginase from Erwinia chrysanthemi (ErA). Undoubtedly, E. coli asparaginase is the most widely used enzyme. Several large pharmaceutical corporations such as Merck (Germany) and Enzon (USA) produced this enzyme, and E. coli asparaginase is an only asparaginase that is imported and is approved for clinical use in Russia. One more asparaginase, which is available on the market, is Erwinase. Ipsen Pharma (Great Britain) produces this enzyme from Erwinia sp. in limited quantity. In many cases, asparaginase treatment leads to development of hypersensitivity, up to anaphylaxis shock, to enzyme protein, which is alien to human organism. Use of pegylated form of asparaginase, i.e. conjugate of asparaginase and some polyethylenglycol, that produced under name Oncospar does not prevent development of allergic reactions. The basic approaches for leukemia treatment have been developed in the USA in late sixties of previous century. Since then drugs, which form so called «base therapy of ALL (acute lymphoblastic leukemia)», are in use. Optimization of chemotherapy courses leaded to further progress in leukemia treatment and recovery of leukemia patients reached 90-95%. Now it is widely accepted that main cause of asparaginase toxicity is the enzyme ability to catalyze L-glutamine hydrolysis, which leads to glutamine depletion in cells. There are two approaches to decrease asparaginase toxicity. The first way is to decrease enzyme glutaminase activity by directed mutagenesis and the second one is a search of new asparaginases with low glutaminase activity. Now the only asparaginase without glutaminase activity is enzyme isolated from Wolinella succinogenes (WsA). We have founded that asparaginase of Helicobacter pylori (NP_223379) (НрА) is one of ten the most close horologes of WsA. We amplified gene of H. pylori asparaginase and cloned it into E.coli cells. Glutaminase activity of enzyme, which we isolated from these cells, was ~0.01% of asparaginase activity. Studies in vitro demonstrated high cytotoxic activity of H. pylori asparaginase for acute lymphoblast leukosis cells MOLT-4, Burkitt's lymphoma Raji cells and chronic myelogenous leukaemia cells K-562. We expect that drug, which is based on HpA, would have the following advantages over existing asparaginase preparations: (a) low toxicity, which would allow to avoid numerous side-effects common during asparaginase therapy (b) low production cost of recombinant enzyme would decrease expenses of treatment (c) due to different antigen properties HpA may be used as substitute in EcA therapy We suppose that recombinant Н. pylori asparaginase would great interest as a base for creation of domestic drug for treatment of different leukosis forms.