Study Of Identfiying EGFRvⅢ Specific Single-chain Fragments Variable And Its Biological Activity

Objective: To identfiy an EGFRvⅢspecific single-chain Fv (scFv) by phage display library and detect its biological activity.Methods: BALB/c mice were immunized by PEP3-KLH. Genes of variable heavy (VH) and variable light (VL) chains of EGFRvⅢantibodies were amplificated by RT-PCR from the splenic cells of immunized mice and further spliced into scFv fragments, then cloned into the phagemid vector pCANTAB5E-Thrombin. And the recombinant phage which contains scFv was transformed into electrocompetent Hpd3cells to gain phage antibody library. A positive clone identified by ELISA was sequenced and re-cloned into pCANTAB5E-Thrombin-His vector and transfected into E.coli HB2151 cells to express. After induced by IPTG the precipitated supernatant was purificated with Ni Sepharose 4 Fast Flow to get the soluble scFv. The specific binding biological activity to EGFRvⅢof the scFv was studied by indirect immunofluorescence and in vivo immunofluorescence imaging.Results: A EGFRvⅢ-scFv phage library with a complexity of approximately 2.23×10~8 was successfully constructed and 16 EGFRvⅢ-scFv positive clones were identified by ELISA. One clone named EGFRvⅢ-scFv-2A1 was re-cloned into pCANTAB-Thrombin-His vector and soluble EGFRvⅢ-scFv-2A1 was successfully obtained. EGFRvⅢ-scFv-2A1 could specifically bind with HuH7-EGFRvⅢ, but not HuH7 cells in vitro. In vivo, fluorescence-labeled EGFRvⅢ-scFv-2A1 could only bind with U87MG-EGFRvⅢcell implanted tumor tissues, but not that of U87MG cells.Conclusions: EGFRvⅢ-scFv-2A1 will be applied in diagnostic imaging and target cancer therapy in the future.