诱导细胞凋亡的抗hDR5单抗的研究

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DR5的McAb；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FITC-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。

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

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-FTTC/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 cyto-toxicity named as mDRA-6. The epitope type is of confonnation and mDRA-6 specially identificates rDR5. The flow cytometiy 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 apoptosis-inducing McAb against hDR5 has been prepared successfully. mDRA-6 is a novel agonistic anti-human 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.