Investigation of therapeutic activity of fully human anti-human TRAIL receptor monoclonal antibodies against malignant glioma

目标。目的探讨全人抗人TRAIL受体单抗(MAbs)对人脑胶质瘤细胞的治疗作用及其分子机制。本研究使用了12个人脑胶质瘤细胞系。全人抗人TRAIL受体单抗(B12：针对DR4、E11、H48和KMTR2：针对DR5)由麒麟啤酒有限公司提供。采用原位末端标记法检测细胞凋亡率。克隆形成效率法检测克隆存活率。Western印迹分析细胞蛋白表达。用流式细胞仪检测细胞表面TRAIL受体的表达。将胶质瘤细胞接种于裸鼠的大脑或大脑，建立脑胶质瘤移植瘤模型。人脑胶质瘤细胞仅对抗DR5单抗敏感，而对抗DR4单抗完全不敏感。抗DR5单抗可迅速发挥细胞毒作用，并诱导细胞凋亡。抗DR5…更多的单抗处理导致caspase-8的裂解和激活，而caspase-8又裂解了效应caspase-3。Bid是Bc1-2家族成员中只有BH3的分子，它被进一步切割，从而激活了涉及caspase-9切割的线粒体凋亡途径。人脑胶质瘤细胞株的敏感性与细胞表面DR5的表达水平密切相关。C-flip_L、Akt和Cyclin D1的蛋白表达水平与抗DR5mAb S的敏感性显著相关，小干扰RNA下调c-flip_L蛋白的表达导致人脑胶质瘤细胞对抗DR5mAb的增敏。此外，通过c-flip_L表达载体过表达c-flip_L的胶质瘤细胞对抗DR5mAb产生了抗性。与对照组相比，用抗DR5单抗治疗裸鼠明显抑制了皮下胶质瘤移植瘤的生长。同样，抗DR5单抗治疗裸鼠脑内胶质瘤移植瘤，可显著延长裸鼠的生存期。DR5是主要的TRAIL受体，表达于细胞表面，介导人脑胶质瘤细胞的凋亡信号。人脑胶质瘤细胞对抗DR5单抗的敏感性可能至少部分取决于c-FLIPL的表达水平。抗DR5单抗在体内和体外均具有抗肿瘤作用。我们的结果表明，利用全人单抗通过DR5特异性靶向死亡受体通路可能为难治性恶性胶质瘤提供一种新的治疗策略。较少

Objectives. To investigate therapeutic efficacy and molecular mechanisms of fully human anti-human TRAIL receptor monoclonal antibodies (mAbs) against human glioma cells.Methods. Twelve human glioma cell lines were used in this study. Fully human anti-human TRAIL receptor mAbs (B12 : specific to DR4, Ell, H48, and KMTR2 : specific to DR5) were provided by Kirin Brewery Co. Ltd. Cytotoxicity was assessed by MTT assay. Apoptosis was detected by TUNEL assay. Clonogenic survival was assessed by colony formation efficiency assay. Cellular protein expression was analyzed with Western blot. Cell surface expression of TRAIL receptors was quantified by flow cytometry. Glioma cells were inoculated into either the frank or cerebrum of nude mice to generate glioma xenografts models.Results. Human glioma cells were sensitive to only anti-DR5 mAbs, whereas they were totally insensitive to anti-DR4 mAb. Treatment with anti-DR5 mAbs exerted rapid cytotoxicity and lead to apoptosis induction. Anti-DR5 … More mAb treatment resulted in cleavage and activation of, an initiator caspase, caspase-8, which in turn cleaved an effector caspase, caspase-3. Further cleavage of Bid, a BH3-only molecule of the Bc1-2 family members, occurred, thereby activating mitochondrial apoptosis pathways involving cleavage of caspase-9. The sensitivity of human glioma cell lines was closely associated with the expression level of DR5 at the cell surface. Cellular protein expression levels of c-FLIP_L, Akt, and Cyclin D1 significantly correlated with sensitivity to anti-DR5 mAb s. Downregulation of c-FLIP_L protein expression using siRNA resulted in sensitization of human glioma cells to anti-DR5 mAbs. Furthermore, glioma cells overexpressing c-FLIP_L by transfecting the c-FLIP_L expression vector became resistant to anti-DR5 mAb treatment. Treatment of nude mice with anti-DR5 mAbs significantly suppressed growth of subcutaneous glioma xenografts compared with those treated with control non-specific antibodies. Similarly, treatment of nude mice bearing intracerebral glioma xenografts with anti-DR5 mAbs significantly elongated life span.Conclusions. DR5 is the predominant TRAIL receptor, which is expressed at the cell surface and mediates apoptotic signals in human glioma cells. Sensitivity of human glioma cells to anti-DR5 mAbs might be determined at least in part by expression level of c-FLIPL. Anti-DR5 mAbs exert anti-tumor effects both in vitro and in vivo. Our results suggest that specific targeting of death receptor pathway through DR5 using fully human mAbs might provide a novel therapeutic strategy for intractable malignant gliomas. Less