Abstract
Asparaginase (ASNase) is a biopharmaceutical for Acute Lymphoblastic Leukemia (ALL) treatment. However, it shows undesirable side effects such as short lifetimes, susceptibility to proteases, and immunogenicity. Here, ASNase encapsidation was genetically directed in bacteriophage P22-based virus-like particles (VLPs) (ASNase-P22 nanoreactors) as a strategy to overcome these challenges. ASNase-P22 was composed of 58.4 ± 7.9% of coat protein and 41.6 ± 8.1% of tetrameric ASNase. Km and Kcat values of ASNase-P22 were 15- and 2-fold higher than those obtained for the free enzyme, respectively. Resulting Kcat/Km value was 2.19 × 105 M−1 s−1. ASNase-P22 showed an aggregation of 60% of the volume sample when incubated at 37 °C for 12 days. In comparison, commercial asparaginase was completely aggregated under the same conditions. ASNase-P22 was stable for up to 24 h at 37 °C, independent of the presence of human blood serum (HBS) or whether ASNase-P22 nanoreactors were uncoated or PEGylated. Finally, we found that ASNase-P22 caused cytotoxicity in the leukemic cell line MOLT-4 in a concentration dependent manner. To our knowledge, this is the first work where ASNase is encapsulated inside of VLPs, as a promising alternative to fight ALL.
Highlights
Acute Lymphoblastic Leukemia (ALL) is a hematologic disorder in the bone marrow, blood, and extramedullary sites characterized by the uncontrolled proliferation of immature lymphocytes
E. coli BL21 DE3/pLysS cells were transformed with the plasmids pBAD/ASNaseSP and pRSFDuet1/P22-coat proteins (CP), and successful overexpression of both proteins, ASNase-scaffold proteins (SP)
Bacteriophage P22-based virus-like particles (VLPs) with ASNase activity were purified by ultracentrifugation and size exclusion chromatography (Figure 2a)
Summary
Acute Lymphoblastic Leukemia (ALL) is a hematologic disorder in the bone marrow, blood, and extramedullary sites characterized by the uncontrolled proliferation of immature lymphocytes. ALL accounts for approximately 25% of all childhood cancers and about 80% of all leukemias, representing the most common type of childhood cancer and the leading cause of death from disease in children [2]. The most common drugs used for ALL treatment are vincristine (an antineoplasic alkaloid), corticosteroids, and Asparaginase (ASNase; EC 3.5.1.1) [4,5]. ASNase is found in the three domains of life; it has a great value in the chemotherapeutic treatment of ALL and other types of leukemia such as lymphosarcoma, Hodgkin’s disease, acute myelogenous leukemia, chronic lymphocytic leukemia, reticulosarcoma, and melanosarcoma [6,7]
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