| Budget Amount *help |
¥3,220,000 (Direct Cost: ¥2,800,000、Indirect Cost: ¥420,000)
Fiscal Year 2011: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
Fiscal Year 2010: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Research Abstract |
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.
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