Mutations in subunit interface and B-cell Epitopes improve Antileukemic activities of Escherichia Coli asparaginase-II:Evaluation of Immunogenicity in Mice

Abstract

Background: The therapeutic potential of EcA is limited due to immunogenicity and short half-life in patients. Results: Several EcA variants were constructed that showed markedly reduced immunogenicity and cytotoxicity against leukemic lymphoblasts. Conclusion: Small changes in subunit interface and B-cell epitope significantly reduced immunogenicity and enhanced cytotoxicity. Significance: These variants have promising potential in the advanced asparaginase therapy of leukemia. L-Asparaginase-II from Escherichia coli (EcA) is a central component in the treatment of acute lymphoblastic leukemia (ALL). However, the therapeutic efficacy of EcA is limited due to immunogenicity and a short half-life in the patient. Here, we performed rational mutagenesis to obtain EcA variants with a potential to improve ALL treatment. Several variants, especially W66Y and Y176F, killed the ALL cells more efficiently than did wild-type EcA (WT-EcA), although nonleukemic peripheral blood monocytes were not affected. Several assays, including Western blotting, annexin-V/propidium iodide binding, comet, and micronuclei assays, showed that the reduction in viability of leukemic cells is due to the increase in caspase-3, cytochrome c release, poly(ADP-ribose) polymerase activation, down-regulation of anti-Apoptotic protein Bcl-XL, an arrest of the cell cycle at the G0/G1 phase, and eventually apoptosis. Both W66Y and Y176F induced significantly more apoptosis in lymphocytes derived from ALL patients. In addition, Y176F and Y176S exhibited greatly decreased glutaminase activity, whereas K288S/Y176F, a variant mutated in one of the immunodominant epitopes, showed reduced antigenicity. Further in vivo immunogenicity studies in mice showed that K288S/Y176F was 10-fold less immunogenic as compared withWT-EcA.Moreover, sera obtained from WT-EcA immunized mice and ALL patients who were given asparaginase therapy for several weeks recognized the K288S/ Y176F mutant significantly less than the WT-EcA. Further mechanistic studies revealed that W66Y, Y176F, and K288S/Y176F rapidly depleted asparagineandalso down-regulated the transcription of asparagine synthetase as compared with WT-EcA. These highly desirable attributes of these variants could significantly advance asparaginase therapy of leukemia in the future.© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.