The Preliminary Studies Of Hu3D3/cSA Directed Pretargeting Therapy Of Lung Cancer With Multiple Anti-tumor Drugs

Antibody-directed targeting therapy of tumor has become one of the hot points in anti-tumor research and clinical application, but the traditional antibody-directed targeting therapy are based on anti-tumor monoclonal antibodies directly conjugated with the effectors, the immunoconjugate can only carry a kind of drug together with its large molecular weight and bad penetration capacity, so it couldn't receive good effects and its clinical applications was restricted in some degree. In order to overcome these problems, this study is intended to construct a fusion protein that composed of humanized single-domain antibody hu3D3 and core steptavidin as a universal carrier targeting to lung cancer, which can simultaneously carry several kinds of biotinylated drugs to tumor areas to realize the pretargeting therapy of lung cancer with multiple anti-tumor drugs via the streptavidin and biotin system.Core streptavidin (cSA) gene was acquired by PCR, and inserted into plasmid hu3D3/pET-22b(+) to construct a recombinant plasmid hu3D3/cSA/pET-22b(+). The recombinant plasmid was transformed into E.coli BL21 (DE3) and expressed with high yield under optimal conditions. The fusion protein was purified through Nickel-affinity chromatography column and renatured using dialysis and gel filtration chromatography. The tetrameric hu3D3/cSA complexes were analyzed by SDS-PAGE and Western blot. The fusion proteins hu3D3/cSA were labeled with FITC, then their antigen-binding activity was analyzed using fluorescence microscope, and the avidity of hu3D3/cSA and hu3D3 were analyzed and compared by Flow Cytometry. The biotin-binding activity of hu3D3/cSA was identified by ELISA and Western blot. After lung cancer cells A549 were treated by all combinations of hu3D3/cSA and various anti-tumor drugs (biotinylated or non-biotinylated), cells growth curve were described via Trypan Blue Staining, and the cytotoxicities to A549 cells in vitro were analyzed and compared by means of MTT assay. The results indicated that the recombinant plasmid hu3D3/cSA/pET-22b(+) with correct sequence was obtained. The fusion protein was found after expression in E.coli BL21(DE3) mainly in the form of inclusion bodies. After purified and refolded, tetrameric complexes were formed. The purified tetrameric hu3D3/cSA complexes retained both antigen-binding activity of hu3D3 moiety and biotin-binding activity of cSA moiety; furthermore, the avidity of hu3D3/cSA to its target antigen was increased by about six to nine times as compared with that of monomeric hu3D3. The cells growth curves illustrated that the groups which were treated by the combinations of hu3D3/cSA and biotinylated anti-tumor drugs had the lowest cell livability. In vitro cytotoxicity assays displayed that the killing rates of the combinations of hu3D3/cSA and biotinylated anti-tumor drugs were increased by 25 percent to 35 percent compared with biotinylated anti-tumor drugs alone.In conclusion, the fusion proteins hu3D3/cSA with both antigen- and biotin-binding activity were successfully prepared through genetic engineering, and the avidity of hu3D3 moiety to 3D3 antigen was enhanced. In vitro cytotoxicity tests indicated that hu3D3/cSA could be used as a universal carrier directing several biotinylated anti-tumor drugs to kill target cells more selectively and effectively, and could realize the pretargeting therapy of lung cancer with multiple anti-tumor drugs. Theses research results would contribute to the further studies of hu3D3/cSA directed pretargeting therapy of lung cancer in vivo.