New anti-ErbB2 anti-CD16 bispecific antibodies trivalent Construction and identification of.doc

 PAGE \* MERGEFORMAT 21 New anti-ErbB2 anti-CD16 bispecific antibodies trivalent Construction and identification of Of: Miss Han, Xie Zhigang, WANG Ming, Shi Ming, Hu Meiru, in Ming, Ma distance, Bei-fen Shen, Ning Guo [Abstract] Objective To construct a new trivalent anti-ErbB2, anti-CD16 BsAb. Methods recombinant vector pET22b (+) / BsAb, transformed into E. coli BL21 (DE3), recombinant protein expression, protein purification by inclusion body refolding. Application of stable suspension culture amplified expression of anti human CD16 scFv-Fc fusion protein in CHO cell line (CG5 cells) and stable expression of anti-human ErbB2 scFv-Fc fusion protein in CHO cells (HG2 cells), by rProtein A Sepharose Fast Flow affinity chromatography purified fusion protein was analyzed by flow cytometry the binding capacity of BsAb. Results of the BsAb in E. coli BL21 (DE3) was highly expressed in the form of inclusion bodies by refolding of recombinant protein high purity protein, refolded BsAb with their parents a combination of the similarity of the antibody’s ability to target antigen. Conclusion The new trivalent anti-ErbB2, anti-CD16 BsAb with high expression of ErbB2 in breast cancer cell lines SKBR3 cells and expression of high affinity binding of CD16 human peripheral blood mononuclear cells with low affinity. [Keywords:] ErbB2 CD16 bispecific antibody tumor immunotherapy ABSTRACT: Objective To construct a novel trivalent anti-ErbB2/CD16 bispecific antibody. Methods The recombinant plasmid pET22b (+) / BsAb was constructed. The recombinant protein was expressed in E.coli BL21 strain (DE3) and purified by refolding. By using Spinner System, CG5 cells, CHO cells stably expressing anti-CD16 scFv-Fc fusion protein and HG2 cells, CHO cells stably expressing anti-ErbB2 scFv-Fc fusion protein were expanded. The fusion proteins were purified over rProtein A Sepharose Fast Flow Column. The binding activity of the BsAb was analyzed by flow cytometry. Results The BsA