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BACKGROUND: The treatment of acute lymphoblastic leukemia (ALL) uses the biopharmaceutical L-asparaginase (ASNase) as the main medication. This drug, from bacterial origin (Escherichia coli or Erwinia chrysanthemi), depletes L-asparagine (Asn) and secondarily L-glutamine (Gln -GLNase activity) from the bloodstream, leading leukemic cells to die by deprivation of Asn. The use of ASNase is limited by the high incidence of adverse effects, which collectively can specifically impair quality of life of patients. Its high toxicity caused by the product of the hydrolysis of amino acids and the formation of anti-ASNase antibodies often required treatment interruption, thus reducing the chances of cure and increasing the rates of disease relapse. RESULTS:In order to improve enzymatic activity, while reducing toxicity, we developed through directed evolution a doublemutant ASNase from Erwinia chrysanthemi (Erw_DM), which has specific activity for Asn 46% higher than the wild-type enzyme (Erw_WT). This makes it possible to reduce the amount of protein necessary for depletion of this amino acid and, consequently, the reduction of GLNase activity, considered toxic. In silico analysis showed that a lower number of epitopes was exposed, resulting in reduced recognition of the recombinant protein by antibody anti-ASNase observed in vitro assay. Furthermore, we observed the same cytotoxic profile for the MOLT-4 and REH ALL cell lines using 40% lower protein mass of Erw_DM to achieve the minimum enzyme activity required in the bloodstream during treatment.CONCLUSION: Altogether, our findings describe a potent and less immunogenic ASNase, an improvement that may alleviate treatment adverse effects developed in anti-ALL therapy.

Production of IL-12 is an important indicator of the macrophage's ability to regulate immune responses. In this study, we investigated the IL-12 production by macrophages in different developmental stages. To this end, macrophages were generated in vitro from precursors stimulated with M-CSF, GM-CSF or IL-3. Density separation yielded populations enriched in different maturation stages. Invariably, only cells banding at the 40-50% Percoll interface produced large amounts of IL-12p40 when stimulated with LPS, whereas only low levels of IL-12p70 were produced. These cells represented immature macrophages, as indicated by the absence of precursor markers CD31/ER-MP12, Ly-6C/ER-MP20 and ER-MP58, and by the low level of expression of mature-cell markers like ER-HR3, scavenger receptor and CD11b/Mac-1. Upon further maturation, the macrophages' ability to produce IL-12p40 decreased, coinciding with increased nitric oxide production upon LPS stimulation. These results show that immature macrophages produce high levels of IL-12p40 and thus may either contribute to IL-12p70 production or regulate it.