肝癌是全球第五大恶性肿瘤，目前对于中晚期肝癌还没有理想的治疗药物或者治疗方案。Glypican-3蛋白是一种在肝癌细胞表面特异性表达的肿瘤标志物。本课题组前期筛选鉴定了一个特异靶向GPC3的人源单域抗体HN3，能够在细胞水平和小鼠体内显著抑制肝癌细胞的增殖。为进一步加强HN3的活性，本项目以HN3抗体为基础，融入抗CD3蛋白的抗体结构，构建双特异性抗体。CD3是T-细胞的表面分化标志物，双特异性抗体通过HN3抗体靶向肝癌细胞，通过CD3抗体招募并激活人体自身的T细胞，攻击肿瘤细胞。本项目拟制备两种结构形式的双特异性抗体: 单一肽链的的BiTE和基于hFc异二聚化的BsFc (Bispecific scFv-Fc），并进行细胞和小鼠体内的活性实验及其作用机制研究、抗体特异性实验（细胞结合与杀伤）、稳定性实验、小鼠体内毒负作用检测等关键实验，确定其成药性的可能，为肝癌治疗性抗体的研制奠定基础。

Liver cancer is the fifth most common cancer. So far there is no curative treatment for advanced liver cancer. Glypican-3 is a GPI-anchored cell surface protein that highly expresses in liver cancer cells but not in normal adult tissues. Previously our lab identified a GPC3-specific human domain antibody HN3 (i.e. VH only), which can directly inhibit cell proliferation in vitro and suppress tumor growth in xenograft mouse model. To further boost HN3 potency, the current project aims to develop bispecific antibodies by incorporating anti-CD3 antibody with HN3. CD3 is a T cell differentiation marker. The bispecific antibody can bind liver cancer cells via HN3 antibody and recruit T cells via the anti-CD3 antibody. The recruited T cells will gather and proliferate in the tumor mass locations, and attack the tumor cells. The current project will make two forms of bispecific antibodies, the single polypeptide BiTE（Bi-specific T-cell Engager）and BsFc (Bispecific scFv-Fc）based on the hFc heterodimerization. Subsequent functional studies include cell killing and in vivo anti-tumor activity, the functional mechanism of the antibody, specificity of cell binding and killing, thermostability, and in vivo side effects in mice, which are closely related to the druggability of the antibodies. The results of the project will provide foundations for the development of therapeutic antibodies for liver cancer treatment.