Development of tumor-targeted therapy using measles virus-displayed antibody library

利用麻疹病毒展示抗体库开发肿瘤靶向治疗

• 批准号: 22659227
• 负责人: NAKAMURA Takafumi
• 金额: $ 2.06万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Challenging Exploratory Research
• 财政年份: 2010
• 资助国家: 日本
• 起止时间: 2010 至 2011
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-22659227/
• 关键词: 実験外科学; 癌; ウイルス; 抗体; バイオテクノロジー; 生体機能利用

We have successfully developed a pseudoreceptor system which allows rescue and propagation of retargeted viruses displaying single chain antibody fragments(scFvs). Thus, receptor choice is not a significant limitation for targeting measles viruses and the use of single chain antibodies for targeting potentially allows us to redirect the virus against any chosen cellular receptor.In this study, we try to develop measles virus-displayed scFv library(virobody library) using the technology for identification of tumor-specific scFv. Human A549 lung, or BxPC-3 pancreatic carcinoma cells were infected with virobody library at an MOI of 0. 01 to 1, one hour later the cells were washed with Opti-MEM medium(Invitrogen) four times. 2 to 5 days after infection, propagated viruses were harvested by repeated freeze-thaw cycles from the GFP-positive infected cells. The biopanning was repeated several times, and finally the tumor-targeted measles virus was cloned from the GFP-positive infected cells. The cDNA encoding scFv from the viral RNA genome was amplified by RT-PCR for sequence analysis. However, the identified scFvs were not functional as a tumor-specific antibody. We have assumed that the diversity of measles virus-displayed scFv library is not sufficient enough to identify tumor-specific scFvs. To address this question, not traditional ligation method but Gateway[○! R] Technology-based cloning method(Invitrogen) was used for generation of plasmid library. Various kind of scFvs were incorporated into the C terminus of a receptor-blind H gene in a full-length cDNA clone of Edmonston measles virus. Furthermore, infectious scFv-displaying measles viruses were rescued from the full-length cDNA clones using helper vaccinia virus expressing T7 RNA polymerase. These modifications dramatically increased the diversity of measles virus-displayed scFv library.

我们已经成功地开发了一种假受体系统，该系统允许拯救和繁殖展示单链抗体片段（scFv）的重靶向病毒。因此，受体的选择并不是麻疹病毒靶向治疗的一个重要限制因素，而单链抗体的靶向作用使我们能够将麻疹病毒重新导向任何选择的细胞受体。人A549肺或BxPC-3胰腺癌细胞用MOI为0的病毒体文库感染。01至1，1小时后，用Opti-MEM培养基（Invitrogen）洗涤细胞四次。感染后2至5天，通过反复冻融循环从GFP阳性感染细胞收获增殖的病毒。重复多次生物淘选，最终从GFP阳性感染细胞中克隆出肿瘤靶向麻疹病毒。通过RT-PCR从病毒RNA基因组中扩增编码scFv的cDNA用于序列分析。然而，鉴定的scFv不具有肿瘤特异性抗体的功能。我们假设麻疹病毒展示的scFv文库的多样性不足以鉴定肿瘤特异性scFv。为了解决这个问题，不是传统的结扎方法，而是Gateway[○！使用基于技术的克隆方法（Invitrogen）来产生质粒文库。将不同种类的scFv整合到Edmonston麻疹病毒全长cDNA克隆的受体盲H基因的C末端。此外，感染性的scFv展示麻疹病毒获救的全长cDNA克隆使用辅助痘苗病毒表达T7 RNA聚合酶。这些修饰极大地增加了麻疹病毒展示的单链抗体库的多样性。

项目成果

Exploitation of the interaction of measles virus fusogenic envelope proteins with the surface receptor CD46 on human cells for microcell-mediated chromosome transfer.

• DOI: 10.1186/1472-6750-10-37
• 发表时间: 2010-05-06
• 期刊: BMC biotechnology
• 影响因子: 3.5
• 作者: Katoh M;Kazuki Y;Kazuki K;Kajitani N;Takiguchi M;Nakayama Y;Nakamura T;Oshimura M
• 通讯作者: Oshimura M

マイクロRNAによって制御されるウイルスの開発とその応用

microRNA控制病毒的研制及其应用

• 发表时间: 2010
• 作者: Sawada Y;Sugita K;Kabashima R;Hino R;Nakamura M;Koga C;Tokura Y;高橋昭久;中村貴史
• 通讯作者: 中村貴史

MicroRNA Targeting of Oncolytic Viruses for cancer therapy

用于癌症治疗的溶瘤病毒的 MicroRNA 靶向

• 发表时间: 2011
• 作者: Takafumi Nakamura.

Highly Attenuated Vaccinia Virus as a Potential Oncolytic Agent for Cancer Virotherapy

高度减毒痘苗病毒作为癌症病毒治疗的潜在溶瘤剂

• 发表时间: 2010
• 作者: Fusasaki T;Narita R;Hiura M;Abe S;Tabaru A;Hino R;Matsuyama A;Shimajiri S;Tokura Y;Sasaguri Y;Harada M;Nakamura T
• 通讯作者: Nakamura T

MicroRNA-regulated oncolytic vaccinia virus not only enhances the oncolytic activity but also reduces the viral pathogenicity

MicroRNA调控的溶瘤痘苗病毒不仅增强溶瘤活性还降低病毒致病性

• 发表时间: 2010
• 作者: Nakamura M;Sugita K;Tokura Y;大西武雄;Nakamura T
• 通讯作者: Nakamura T