Expression of a recombinant FLT3 ligand and its emtansine conjugate as a therapeutic candidate against acute myeloid leukemia cells with FLT3 expression.
Expression of a recombinant FLT3 ligand and its emtansine conjugate as a therapeutic candidate against acute myeloid leukemia cells with FLT3 expression.
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DOI：
10.1186/s12934-021-01559-6
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发表时间：
2021-03-10
影响因子：
6.4
通讯作者：
Chen Y
中科院分区：
文献类型：
--
作者：
Zhang D;Guo Y;Zhao Y;Yu L;Chang Z;Pei H;Huang J;Chen C;Xue H;Xu X;Pan Y;Li N;Zhu C;Zhao ZJ;Yu J;Chen Y
大多数急性髓细胞白血病（AML）患者仍然无法治愈，需要新的治疗方法。功能获得性FMS样酪氨酸激酶3（FLT 3）突变存在于30-40%的AML患者中，并作为有吸引力的治疗靶点。此外，FLT 3在> 90%的AML患者的原始细胞上异常表达，使得基于FLT 3配体的药物缀合物成为治疗AML患者的有希望的治疗策略。这里，E。以大肠杆菌为宿主表达重组人FLT 3配体（rhFL），并将其作为特异性载体，将细胞毒药物递送至FLT 3 + AML细胞。重组hFL在大肠杆菌BL 21（DE 3）中得到表达和纯化。杆菌纯化的rhFL和emtansine（DM 1）通过N-琥珀酰亚胺基3-（2-吡啶基二硫代）丙酸酯（SPDP）接头缀合。我们通过检查活力、凋亡和细胞周期来评估缀合产物FL-DM 1对表达FLT 3的AML细胞的效力。通过免疫印迹检测与FLT 3信号通路和凋亡通路的活化相关的蛋白的活化。在我们独特的HCD-57细胞系中评估了FL-DM 1的选择性，该细胞系用FLT 3内部串联重复突变体（FLT 3-ITD）转化。可溶性rhFL在E.杆菌纯化的rhFL具有刺激AML细胞中FLT 3信号传导的生物活性，并且药物缀合物FL-DM 1在细胞信号传导和内化中显示活性。FL-DM 1可有效抑制表达FLT 3的THP-1和MV-4-11 AML细胞的存活，半数最大抑制浓度（IC 50）为12.9 nM和1.1 nM。此外，FL-DM 1诱导caspase-3依赖性凋亡，并将细胞周期阻滞在G2/M期。此外，FL-DM 1选择性地靶向由FLT 3-ITD转化的HCD-57细胞，而不是无FLT 3表达的亲本HCD-57细胞。FL-DM 1还可以在原代FLT 3阳性AML细胞中体外诱导明显的凋亡。我们的数据表明，可溶性rhFL可以在重组E.杆菌FL可用作将DM 1递送到表达FLT 3的AML细胞中的特异性载体。FL-DM 1在表达FLT 3的AML细胞系和原代AML细胞中表现出细胞毒性。FL-DM 1可能在治疗FLT 3阳性AML患者中具有潜在的临床应用。在线版本包含补充材料，可通过10.1186/s12934-021-01559-6获得。
Most patients with acute myeloid leukemia (AML) remain uncurable and require novel therapeutic methods. Gain-of-function FMS-like tyrosine kinase 3 (FLT3) mutations are present in 30–40% of AML patients and serve as an attractive therapeutic target. In addition, FLT3 is aberrantly expressed on blasts in > 90% of patients with AML, making the FLT3 ligand-based drug conjugate a promising therapeutic strategy for the treatment of patients with AML. Here, E. coli was used as a host to express recombinant human FLT3 ligand (rhFL), which was used as a specific vehicle to deliver cytotoxic drugs to FLT3 + AML cells. Recombinant hFL was expressed and purified from induced recombinant BL21 (DE3) E. coli. Purified rhFL and emtansine (DM1) were conjugated by an N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) linker. We evaluated the potency of the conjugation product FL-DM1 against FLT3-expressing AML cells by examining viability, apoptosis and the cell cycle. The activation of proteins related to the activation of FLT3 signaling and apoptosis pathways was detected by immunoblotting. The selectivity of FL-DM1 was assessed in our unique HCD-57 cell line, which was transformed with the FLT3 internal tandem duplication mutant (FLT3-ITD). Soluble rhFL was successfully expressed in the periplasm of recombinant E. coli. The purified rhFL was bioactive in stimulating FLT3 signaling in AML cells, and the drug conjugate FL-DM1 showed activity in cell signaling and internalization. FL-DM1 was effective in inhibiting the survival of FLT3-expressing THP-1 and MV-4-11 AML cells, with half maximal inhibitory concentration (IC50) of 12.9 nM and 1.1 nM. Additionally, FL-DM1 induced caspase-3-dependent apoptosis and arrested the cell cycle at the G2/M phase. Moreover, FL-DM1 selectively targeted HCD-57 cells transformed by FLT3-ITD but not parental HCD-57 cells without FLT3 expression. FL-DM1 can also induce obvious apoptosis in primary FLT3-positive AML cells ex vivo. Our data demonstrated that soluble rhFL can be produced in a bioactive form in the periplasm of recombinant E. coli. FL can be used as a specific vehicle to deliver DM1 into FLT3-expressing AML cells. FL-DM1 exhibited cytotoxicity in FLT3-expressing AML cell lines and primary AML cells. FL-DM1 may have potential clinical applications in treating patients with FLT3-positive AML. The online version contains supplementary material available at 10.1186/s12934-021-01559-6.
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DOI：
10.1186/s13046-017-0667-1
发表时间：
2018-02-06
期刊：
Journal of experimental & clinical cancer research : CR
影响因子：
--
作者：
Chalouni C;Doll S
通讯作者：
Doll S
影响因子：
12.8
作者：
Estey, Elihu H.
通讯作者：
Estey, Elihu H.
影响因子：
7.3
作者：
Lambert, John M.;Chari, Ravi V. J.
通讯作者：
Chari, Ravi V. J.
影响因子：
7.4
作者：
Le, Lan N.;Moore, Jamie M. R.;Galush, William J.
通讯作者：
Galush, William J.
DOI：
10.3390/molecules21101375
发表时间：
2016-10-15
期刊：
Molecules (Basel, Switzerland)
影响因子：
--
作者：
Dong M;Liu F;Zhou H;Zhai S;Yan B
通讯作者：
Yan B