新型抗ErbB2、抗CD16三价双特异性抗体的构建及功能鉴定

Construction and functional identification of a novel trivalent anti-ErbB2/anti-CD16 bispecific antibody

目的构建一种新型三价抗ErbB2、抗CD16 BsAb。方法构建重组载体pET22b(+)/BsAb;转化大肠杆菌BL21(DE3),获得重组蛋白的表达,通过包涵体复性纯化蛋白。应用悬浮培养系统扩增稳定表达抗人CD16 scFv-Fc融合蛋白的CHO细胞株(CG5细胞)和稳定表达抗人ErbB2 scFv-Fc融合蛋白的CHO细胞株(HG2细胞),通过rProtein A Sepharose Fast Flow亲和层析柱纯化融合蛋白,应用流式细胞术分析BsAb的结合能力。结果该BsAb可在大肠杆菌BL21(DE3)中以包涵体形式高效表达,通过对重组蛋白的复性可获得高纯度的蛋白;复性后的BsAb具有与其亲本抗体相似性的结合靶抗原的能力。结论新型三价抗ErbB2、抗CD16 BsAb能与高表达ErbB2的乳腺癌细胞系SKBR3细胞高亲和力结合和表达CD16的人外周血单个核细胞低亲和力结合。

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,CG 5 cells,CHO cells stably expressing anti-CD16 scFv-Fc fusion protein and HG 2 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 BsAb was efficiently expressed in E.coli BL21 strain（DE3） as inclusion bodies.High purity of recombinant protein could be obtained by refolding.The BsAb possessed the capability of binding to the target antigens as its parental antibodies. Conclusion The novel BsAb was able to bind human breast cancer cell line SK-BR-3 highly expressing ErbB2 and human peripheral blood mononuclear cells expressing CD16.