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目的 研制一种具有抑制肝癌生长和肿瘤肝转移作用的单克隆抗体Fab′片段与平阳霉素 (PYM)偶联物。方法 以胃蛋白酶酶解结合二巯基苏糖醇 (DTT)还原法制备单抗Fab′片段 ;以葡聚糖T 40 (dextranT 40 )为中介体制备Fab′片段和PYM偶联物Fab′ PYM ;用间接ELISA法、2 ,3 ,5 氯化三苯基四氮唑 (TTC)抑菌效价检测法 ,克隆形成法及动物移植性肿瘤模型测定Fab′ PYM偶联物在体外的生物学活性与在小鼠体内的抗肿瘤作用。结果 Fab′ PYM与BEL 740 2、肝癌 2 2 (H2 2 )及HT 2 9细胞呈免疫学阳性反应 ,但与KB细胞无反应 ;偶联物中PYM的抑菌活性为游离PYM的 15 % ;Fab′ PYM对体外培养的BEL 740 2细胞、HT 2 9细胞和KB细胞的 5 0 %抑制浓度 (IC50 )分别为 0 0 2、0 0 8及 0 5 0μmol/L ;静脉注射 10mg/kgFab′ PYM偶联物和PYM皮下接种对小鼠肝癌 2 2的抑瘤率分别为 86 %和6 2 % ;腹腔注射 ,10mg/kgFab′ PYM偶联物和PYM对小鼠脾内接种的结肠癌 2 6肝转移的抑制率分别为 91%和 6 2 %。结论 Fab′ PYM偶联物具有比游离平阳霉素更强的抑制肝癌生长与肿瘤肝转移作用。
Objective To develop a monoclonal antibody Fab′ fragment with pingyangmycin (PYM) conjugate that inhibits hepatoma growth and tumor liver metastasis. Methods Fab′ fragment was prepared by pepsin enzymatic hydrolysis combined with dithiothreitol (DTT) reduction method. Fab′ fragment and PYM conjugate Fab′ PYM were prepared by using dextran T 40 as the mediator. The biological activity of Fab′ PYM conjugates in vitro was determined by indirect ELISA, 2, 3, 5 triphenyltetrazolium chloride (TTC) bacteriostatic assay, cloning method and animal transplantable tumor model. With anti-tumor effects in mice. Results Immunologically positive reaction of Fab′ PYM with BEL 740 2, liver cancer 2 2 (H 2 2) and HT 2 9 cells, but no response with KB cells; antibacterial activity of PYM in conjugates was 15% of free PYM; The 50 % inhibitory concentration (IC50) of Fab’ PYM on BEL 740 2 cells, HT 2 9 cells and KB cells in vitro was 0 0 2, 0 0 8 and 0 50 μmol/L, respectively. Intravenous injection of 10 mg/kg of Fab’ PYM conjugates and PYM subcutaneously inoculated mice with hepatocellular carcinoma 2 2 with tumor inhibition rates of 86% and 6 2%, respectively; intraperitoneal injections, 10 mg/kg of Fab′ PYM conjugates and PYM against mice inoculated with spleen 2 6 The inhibitory rates of hepatic metastasis were 91% and 62%, respectively. Conclusion The Fab′ PYM conjugate has stronger inhibition of liver cancer growth and tumor liver metastasis than free pingyangmycin.