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Insulin-like growth factor-I receptor-targeted cancer therapy

胰岛素样生长因子-I 受体靶向癌症治疗

基本信息

批准号：
17570120
负责人：
FUJITA Yoko
金额：
$ 2.4万
依托单位：
Tokai 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 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-17570120/
关键词：
Anti-IGF-I receptor single chain antibody cancer molecular thera downregulation Anti-tumor erowth 抗IGF-I 受容体ヒト単鎖抗体 Phage display法 downregulation

项目摘要

The aims of this study were to produce more effective therapeutic antihodies against the IGF-I receptor which can inhibit cancer growth, and to understand mechanisms by which anti-IGFIR induce down-regulation of IGFIR.1. Preparation of human anti-IGFIR with higher affinities than that of the 1H7 single chain antibody (scFv).We obtained 20 clones from a phage library displaying human scFvs. After extensive analyses of those clones, however, we found that they were cross-reactive to the insulin receptor as well as some other non-related proteins. In contrast, scFvs prepared from hybridoma producing 1H7 or 3B7 anti-IGFIR monoclonal antibody (mAb) showed strict specificity for IGFIR. These results suggested that rigorous screening is required to obtain scFvs of our interest in vitro antibody production using phage display technologies. Construction, expression, purification, and characterization of the two scFvs derived from hybridomas resulted in valuable information on their affinity and epitopes. The results have been published.2. Understanding mechanism by which anti-IGFIR antibodies induce down-regulation of IGFIRWe found that all five mAbs used, despite showing different effects on IGFIR signaling, down-regulated IGFIR. In contrast, IGF-I apparently internalized the receptor, but did not lead to its degradation as evidenced by intact IGFIR subunits detected by immunoblotting. Pretreatment of MCF-7 cells with methylamine substantially reduced the antibody-mediated IGFIR down-regulation whereas MG115 did not. No ubiquitination was detected with IGFIR in MCF-7 cells treated by IGFIR mAb nor IGF-I. These results suggest that anti-IGFIR antibodies with different epitope-specificities can cause IGFIR down-regulation via a lysosome-dependent pathway in an IGFIR activation-independent manner. The results were presented at the Annual Meeting of ASBMB, 2007 (manuscript in preparation).

本研究的目的是制备更有效的抗igf - 1受体的治疗性抗体，以抑制癌症的生长，并了解抗igfir诱导igfir - 1下调的机制。制备亲和力高于1H7单链抗体（scFv）的人抗igfir。我们从噬菌体文库中获得了20个克隆，显示人类scFvs。然而，经过对这些克隆的广泛分析，我们发现它们对胰岛素受体和其他一些不相关的蛋白质有交叉反应。相比之下，产生1H7或3B7抗IGFIR单克隆抗体（mAb）的杂交瘤制备的单克隆抗体对IGFIR具有严格的特异性。这些结果表明，使用噬菌体展示技术获得我们感兴趣的体外抗体生产的scFvs需要严格的筛选。从杂交瘤中提取的两种scfv的构建、表达、纯化和表征为它们的亲和力和表位提供了有价值的信息。结果已经发表了。我们发现，尽管抗IGFIR抗体对IGFIR信号的作用不同，但所使用的五种单克隆抗体都能下调IGFIR。相比之下，IGF-I明显内化了受体，但没有导致其降解，免疫印迹检测到完整的IGFIR亚基证明了这一点。用甲胺预处理MCF-7细胞可显著降低抗体介导的IGFIR下调，而MG115则没有。在IGFIR单抗和IGF-I处理的MCF-7细胞中未检测到IGFIR泛素化。这些结果表明，具有不同表位特异性的抗IGFIR抗体可以通过溶酶体依赖途径以不依赖IGFIR激活的方式导致IGFIR下调。研究结果在2007年ASBMB年会上发表（手稿正在准备中）。