AIM:To create new diabodies with improved binding activity to antigen of the variable light-variable heavy (VH-VL)oriented single-chain Fv dimers genes(scFv). METHODS:The linker between VH and VL genes was shortened to 3-5 amino acid residues and cloned into the vector pCANTAB5E.The recombinant plasmids were transformed into TG1 cells and sequenced.The positive transformed cells were infected by M13K07 helper phage to form human recombinant phage antibodies.Expressed products were identified by SDS-PAGE,Western blotting,size exclusion gel chromatography(SEC),ELISA and immunohistochemistry. RESULTS:Three scFv(scFv-3,scFv-4,scFv-5)were constructed successfully with binding ability to hepato-cellular carcinoma 3.5-6 fold greater than their parental scFv.The single-chain Fv dimer(scFv-5,termed BDM3) with the best binding ability was successfully expressed in Yeast pichlia,as shown by SDS-PAGE and Western blotting.SEC results suggested the molecular weight of the expressed products was about 61 kDa.Expressed products showed significantly stronger binding to hepatocellular carcinoma cells than scFv,still having 50% binding activity even after 16 h incubation as 37℃.The purified dimers were bound specifically to the tumor antigen of HCC. CONCLUSION:we have generated scFv dimers by shortening a series of linkers to 3-5 amino acid residues in VH-linker-VL orientation,resulting in highly stable and affinity-improved dimeric molecules.These will become an attractive targeting moiety in immunotherapeutic and diagnostic applications for HCC. AIM: To create new diabodies with improved binding activity to antigen of the variable light-variable heavy (VH-VL) oriented single-chain Fv dimers genes (scFv). METHODS: The linker between VH and VL genes was shortened to 3-5 amino acid residues and cloned into the vector pCANTAB5E.The recombinant plasmids were transformed into TG1 cells and sequenced. The positive transformed cells were infected by M13K07 helper phage to form human recombinant phage antibodies. Expressed products were identified by SDS-PAGE, Western blotting, RESULTS: Three scFv (scFv-3, scFv-4, scFv-5) were constructed successfully with binding ability to hepato-cellular carcinoma 3.5-6 fold greater than their parental scFv. The single-chain Fv dimer (scFv-5, termed BDM3) with the best binding ability was successfully expressed in Yeast pichlia, as shown by SDS-PAGE and Western blotting. SEC results suggested the molecular weight of the expressed products was abou t 61 kDa.Expressed products showed significantly stronger binding to hepatocellular carcinoma cells than scFv, still having 50% binding activity even after 16 h incubation as 37 ° C. The purified dimers were bound specifically to the tumor antigen of HCC. CONCLUSION: we have generated scFv dimers by shortening a series of linkers to 3-5 amino acid residues in VH-linker-VL orientation, resulting in highly stable and affinity-improved dimeric molecules. These will become an attractive targeting moiety in immunotherapeutic and diagnostic applications for HCC.