诱导细胞凋亡的抗hDR5单抗的研究

Preparation and characterization of a novel agonistic anti-human death receptor 5 monoclonal antibody with apoptosis activity

目的研制能诱导肿瘤细胞凋亡的抗人DR5单克隆抗体(McAb)。方法以可溶性人死亡受体(death receptor,DR)5胞外段免疫小鼠,采用杂交瘤技术制备抗人DIL5的MeAb;MTT方法筛选分泌有细胞毒活性McAb的杂交瘤细胞;亲和层析方法纯化McAb;夹心ELISA法测定McAb亚型; Western blot和斑点ELISA法检测McAb的抗原表位类型;间接ELISA法检测McAb的特异性;流式细胞仪检测FTTC-annexinⅤ／PI双色标记的Jurkat细胞的凋亡率;DNA琼脂糖凝胶电泳测定凋亡细胞的DNA片段化。结果获得1株杂交瘤细胞,其分泌的McAb命名为mDRA-6,为IgG1;其抗原表位类型为构象表位;其特异性识别hDR5,与hFas、hDR4等无交叉反应。mDRA-6对Jurkat细胞具有细胞毒作用;经McAb mDRA-6处理后,Jurkat细胞膜表面高表达丝氨酸磷脂,并导致Jurkat细胞中的DNA片段化。结论mDRA-6是一个具有诱导细胞凋亡活性的新的抗人DR5功能性抗体,在以TRAIL／DR5系统进行肿瘤治疗和探讨DR5的功能结构域研究方面具有广泛应用前景。

Objective To prepare anti-hDR5 monoclonal antibody（McAb） with apoptosis activity. Methods To immunize BALB/c mice with rhDR5, and to prepare McAb by hybridoma technique. Tumoricidal anti-human DR5 McAb was screened by MTT assay. McAb was purified by protein G affinity chromatography. IgG subclass was analysed by Sandwich-ELISA. The antigen epitope type of McAb was identified by Western blot and Dot-ELISA. McAb speciality was analysed by indirect-ELISA. The apoptosis of Jurkat cells was detected by flow cytometry with annexin V-FITC/PI staining. The DNA fragmentation in Jurkat cells was analysed by agarose gel electrophoresis. Results A hybridoma was obtained, which secrets an IgG1 anti-hDR5 McAb with intrinsic cytotoxicity named as mDRA-6. The epitope type is of conformation and mDRA-6 specially identificates rDR5. The flow cytometry analysis showed that phosphatidylserine（PS） highly expressed in Jurkat cell surfaces treated with mDRA-6, and agarose gel electrophoresis exhibited DNA fragmentation in that cells. Conclusion An apoptosisinducing McAb against hDR5 has been prepared successfully, mDRA-6 is a novel agonistic anti-hmnan DR5 McAb, and it may be a useful tool in tumor therapy by using DR5 as target molecule and in exploring the functional domain of DR5.