Edmond H. Fischer Translational Medical Research Laboratory, Scientific Research Center, The Seventh Affiliated Hospital, Sun Yat-Sen University, Shenzhen, 518107, Guangdong, China.
Department of Pediatrics, The Seventh Affiliated Hospital, Sun Yat-Sen University, Shenzhen, 518107, Guangdong, China.
Microb Cell Fact. 2021 Mar 10;20(1):67. doi: 10.1186/s12934-021-01559-6.
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 (IC) 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.
大多数急性髓系白血病（AML）患者无法治愈，需要新的治疗方法。FMS 样酪氨酸激酶 3（FLT3）的功能获得性突变存在于 30-40%的 AML 患者中，是一个有吸引力的治疗靶点。此外，超过 90%的 AML 患者的白血病细胞中存在异常表达的 FLT3，这使得基于 FLT3 配体的药物偶联物成为治疗 AML 患者的有前途的治疗策略。在这里，我们使用大肠杆菌作为宿主来表达重组人 FLT3 配体（rhFL），rhFL 可作为一种特殊载体将细胞毒性药物递送至 FLT3+AML 细胞。
从诱导的重组 BL21（DE3）大肠杆菌中表达和纯化重组 hFL。纯化的 rhFL 和emtansine（DM1）通过 N-琥珀酰亚胺基 3-(2-吡啶基二硫代)丙酸酯（SPDP）接头连接。我们通过检查细胞活力、细胞凋亡和细胞周期来评估针对表达 FLT3 的 AML 细胞的缀合物 FL-DM1 的效力。通过免疫印迹检测与 FLT3 信号通路和细胞凋亡通路激活相关的蛋白的激活。我们在独特的 HCD-57 细胞系中评估了 FL-DM1 的选择性，该细胞系转化为 FLT3 内部串联重复突变（FLT3-ITD）。
可溶性 rhFL 成功在重组大肠杆菌的周质中表达。纯化的 rhFL 可刺激 AML 细胞中的 FLT3 信号，药物缀合物 FL-DM1 可在细胞信号转导和内化中发挥作用。FL-DM1 可有效抑制表达 FLT3 的 THP-1 和 MV-4-11 AML 细胞的存活，半数最大抑制浓度（IC）分别为 12.9 nM 和 1.1 nM。此外，FL-DM1 诱导 caspase-3 依赖性细胞凋亡并将细胞周期阻滞在 G2/M 期。此外，FL-DM1 选择性靶向由 FLT3-ITD 转化的 HCD-57 细胞，而不是没有 FLT3 表达的亲本 HCD-57 细胞。FL-DM1 还可以在体外诱导明显的原发性 FLT3 阳性 AML 细胞凋亡。
我们的数据表明，可溶性 rhFL 可以在重组大肠杆菌的周质中以生物活性形式产生。FL 可用作将 DM1 递送至表达 FLT3 的 AML 细胞的特异性载体。FL-DM1 在表达 FLT3 的 AML 细胞系和原发性 AML 细胞中表现出细胞毒性。FL-DM1 可能在治疗 FLT3 阳性 AML 患者方面具有潜在的临床应用价值。
