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

全人源抗人TRAIL受体单克隆抗体对恶性胶质瘤的治疗活性研究

基本信息

批准号：
17591529
负责人：
NAGANE Motoo
金额：
$ 2.24万
依托单位：
Kyorin University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17591529/
关键词：
TRAIL glioma monoclonal antibody apoptosis TRAIL receptor DR5 c-FLIP_L Akt apoptosis DR4 caspase

项目摘要

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

目标.目的探讨抗人TRAIL受体单克隆抗体（mAb）对人脑胶质瘤细胞的治疗作用及其分子机制。本研究中使用了12个人胶质瘤细胞系。完全人抗人TRAIL受体mAb（B12：对DR 4特异，E11，H48和KMTR 2：对DR 5特异）由Kirin Brewery Co. Ltd提供。通过MTT测定评估细胞毒性。TUNEL法检测细胞凋亡。通过集落形成效率测定来评估克隆形成存活。Western blot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。将胶质瘤细胞接种到裸小鼠的大脑或大脑中，建立胶质瘤异种移植模型。结果。人脑胶质瘤细胞仅对抗DR 5单抗敏感，而对抗DR 4单抗完全不敏感。用抗DR 5单克隆抗体处理产生快速细胞毒性并导致细胞凋亡诱导。抗DR 5 ...更多信息 mAb处理导致引发剂胱天蛋白酶（胱天蛋白酶-8）的切割和活化，其继而切割效应物胱天蛋白酶（胱天蛋白酶-3）。Bid（Bc 1 -2家族成员的仅含BH 3的分子）的进一步裂解发生，从而激活涉及caspase-9裂解的线粒体凋亡途径。人脑胶质瘤细胞系的敏感性与细胞表面DR 5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DR 5 mAb的敏感性显著相关。使用siRNA下调c-FLIP_L蛋白表达导致人胶质瘤细胞对抗DR 5 mAb敏感。此外，通过转染c-FLIP_L表达载体而过表达c-FLIP_L的胶质瘤细胞变得对抗DR 5 mAb治疗具有抗性。与用对照非特异性抗体治疗的那些相比，用抗DR 5 mAb治疗裸鼠显著抑制皮下胶质瘤异种移植物的生长。同样，用抗DR 5单克隆抗体治疗荷脑胶质瘤异种移植物的裸鼠显著延长了寿命。DR 5是主要的TRAIL受体，其表达于细胞表面并介导人胶质瘤细胞中的凋亡信号。人脑胶质瘤细胞对抗DR 5单克隆抗体的敏感性可能至少部分由c-FLIPL的表达水平决定。抗DR 5 mAb在体外和体内均发挥抗肿瘤作用。我们的研究结果表明，特异性靶向死亡受体通路通过DR 5使用全人类单克隆抗体可能会提供一种新的治疗策略，难治性恶性胶质瘤。少