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

目标。探讨全人源抗人TRAIL受体单克隆抗体（mAb）对人胶质瘤细胞的治疗效果和分子机制。方法。本研究使用了十二种人类神经胶质瘤细胞系。全人抗人TRAIL受体单克隆抗体（B12：针对DR4、Ell、H48和KMTR2：针对DR5特异性）由Kirin Brewery Co. Ltd提供。通过MTT测定评估细胞毒性。通过TUNEL法检测细胞凋亡。通过集落形成效率测定评估克隆形成存活率。用蛋白质印迹分析细胞蛋白表达。通过流式细胞术定量 TRAIL 受体的细胞表面表达。将神经胶质瘤细胞接种到裸鼠的大脑或大脑中以产生神经胶质瘤异种移植模型。结果。人神经胶质瘤细胞仅对抗 DR5 mAb 敏感，而对抗 DR4 mAb 完全不敏感。用抗 DR5 mAb 治疗可发挥快速细胞毒性并诱导细胞凋亡。抗 DR5 … More mAb 处理导致起始 caspase caspase-8 的裂解和激活，进而裂解效应 caspase caspase-3。 Bid（Bc1-2 家族成员的仅 BH3 分子）发生进一步裂解，从而激活涉及 caspase-9 裂解的线粒体凋亡途径。人胶质瘤细胞系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 的细胞蛋白表达水平与抗 DR5 mAb 的敏感性显着相关。使用 siRNA 下调 c-FLIP_L 蛋白表达导致人神经胶质瘤细胞对抗 DR5 mAb 敏感。此外，通过转染c-FLIP_L表达载体而过度表达c-FLIP_L的神经胶质瘤细胞对抗DR5 mAb治疗产生抗性。与用对照非特异性抗体治疗的裸鼠相比，用抗 DR5 mAb 治疗的裸鼠显着抑制了皮下神经胶质瘤异种移植物的生长。同样，用抗 DR5 mAb 治疗携带脑内神经胶质瘤异种移植物的裸鼠可显着延长寿命。结论。 DR5 是主要的 TRAIL 受体，在细胞表面表达并介导人神经胶质瘤细胞的凋亡信号。人神经胶质瘤细胞对抗 DR5 mAb 的敏感性可能至少部分由 c-FLIPL 的表达水平决定。抗 DR5 mAb 在体外和体内均发挥抗肿瘤作用。我们的结果表明，使用全人单克隆抗体通过 DR5 特异性靶向死亡受体途径可能为难治性恶性神经胶质瘤提供一种新的治疗策略。较少的

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