Mutations in Subunit Interface and B-cell Epitopes Improve Antileukemic Activities of Escherichia coli Asparaginase-II EVALUATION OF IMMUNOGENICITY IN MICE
Mutations in Subunit Interface and B-cell Epitopes Improve Antileukemic Activities of Escherichia coli Asparaginase-II EVALUATION OF IMMUNOGENICITY IN MICE
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DOI：
10.4331/wjbc.v5.i3.355
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发表时间：
2014-02-07
影响因子：
4.8
通讯作者：
Sonawane, Avinash
中科院分区：
文献类型：
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作者：
Mehta, Ranjit Kumar;Verma, Shikha;Sonawane, Avinash
背景：由于EcA的免疫原性和患者体内半衰期短，其治疗潜力有限。结果：构建了几种EcA变体，其对白血病淋巴母细胞的免疫原性和细胞毒性显着降低。结论：亚单位界面和B细胞表位的微小变化显著降低免疫原性，增强细胞毒性。重要性：大肠杆菌L-天冬酰胺酶II（EcA）是治疗急性淋巴细胞白血病（ALL）的主要成分。然而，由于免疫原性和患者体内半衰期短，EcA的治疗功效有限。在这里，我们进行了合理的诱变，以获得具有改善ALL治疗潜力的EcA变体。几种变体，特别是W 66 Y和Y176 F，比野生型EcA（WT-EcA）更有效地杀死ALL细胞，尽管非白血病外周血单核细胞不受影响。包括蛋白质印迹、膜联蛋白-V/碘化丙啶结合、彗星和微核试验在内的几项试验表明，白血病细胞活力的降低是由于半胱天冬酶-3增加、细胞色素c释放、聚（ADP-核糖）聚合酶激活、抗凋亡蛋白Bcl-XL下调、细胞周期停滞在G（0）/G（1）期以及最终凋亡。W 66 Y和Y176 F均诱导ALL患者淋巴细胞凋亡。此外，Y176 F和Y176 S表现出极大降低的转氨酶活性，而K288 S/Y176 F，一种在免疫显性表位中突变的变体，表现出降低的抗原性。进一步的小鼠体内免疫原性研究表明，与WT-EcA相比，K288 S/Y176 F的免疫原性低10倍。此外，从WT-EcA免疫的小鼠和给予天冬酰胺酶治疗数周的ALL患者获得的血清识别K288 S/Y176 F突变体显著低于WT-EcA。进一步的机制研究表明，与WT-EcA相比，W 66 Y，Y176 F和K288 S/Y176 F快速耗尽天冬酰胺，并下调天冬酰胺合成酶的转录。这些变异体的这些高度期望的属性可以在未来显著地推进白血病的天冬酰胺酶治疗。
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 G(0)/G(1) 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 with WT-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 asparagine and also 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.