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

我们已经成功地开发了一种伪受体系统，它可以拯救和繁殖显示单链抗体片段(ScFv)的重定向病毒。因此，受体的选择不是靶向麻疹病毒的显著限制，使用单链抗体进行靶向可能使我们能够将病毒重定向到任何选择的细胞受体。在本研究中，我们试图利用肿瘤特异性单链抗体的鉴定技术来开发麻疹病毒展示的单链抗体文库(病毒体库)。以MOI为0的病毒体库感染人A549肺腺癌细胞或BxPC-3胰腺癌细胞。01~1，1h后用Opti-MEM(Invitgen)液冲洗细胞4次。感染后2~5d，通过反复冻融循环从GFP阳性感染细胞中获取增殖病毒。经过多次筛选，最终从GFP阳性的感染细胞中克隆出肿瘤靶向的麻疹病毒。用RT-PCR方法从病毒RNA基因组中扩增出单链抗体的编码区，并进行序列分析。然而，已鉴定的单链抗体并不具有肿瘤特异性抗体的功能。我们假设麻疹病毒显示的单链抗体文库的多样性不足以识别肿瘤特异性单链抗体。要解决这个问题，不是传统的结扎法，而是Gateway[0！采用基于技术的克隆方法(Invitgen)构建质粒文库。在埃德蒙斯顿麻疹病毒的全长cDNA克隆中，将不同类型的单链抗体插入受体失活的H基因的C末端。此外，利用表达T7 RNA聚合酶的辅助痘苗病毒从全长cDNA克隆中解救出显示麻疹单链抗体的传染性病毒。这些修饰极大地增加了麻疹病毒展示的单链抗体文库的多样性。

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.