上皮细胞黏附分子（EpCAM）参与调节细胞粘附、增殖、分化等过程，在多种实体瘤表面高表达，是肿瘤靶向治疗的热门靶点。抗体分子和毒素分子组成的免疫毒素在治疗恶性肿瘤表现出良好的疗效。驼源纳米抗体（Nanobody, Nb）具有分子量小、穿透性强、免疫原性低等优点，在用于免疫毒素靶向治疗肿瘤方面比单克隆抗体更具优势。本项目前期对骆驼免疫了EpCAM蛋白，构建了高质量的噬菌体展示纳米抗体文库，成功淘选了12株CDR3序列不同的EpCAM Nbs；流式细胞鉴定出3株能够特异识别EpCAM阳性细胞的Nbs；并且成功地对其进行了人源化改造。结果显示人源化的纳米抗体（hNb）能特异结合并内吞进入EpCAM阳性细胞。本项目将继续构建新型免疫毒素EpCAM hNb-PE24分子；体外检测其对肿瘤细胞生长及凋亡的影响；评估该免疫毒素在小鼠体内抗肿瘤活性。本项目的开展将为EpCAM阳性肿瘤的靶向治疗开辟新途径。

Epithelial cell adhesion molecule (EpCAM) is a key regulator of adhesion, cell migration and proliferation. It was overexpressed in a variety of solid tumors that becoming a popular target for cancer therapy. Immunotoxins, consist of antibody and toxin molecular, have gained remarkable achievements in malignant tumor therapy. Nanobody (Nb), derived from camelid animals, is the smallest naturally antigen-binding fragment. Compared with conventional antibody, Nanobody has lower molecular weight, higher stability, more excellent penetrability and lower immunogenicity. It has been regarded as an alternative approach for antibody cancer therapy, and has more advantages in forming immunotoxins than other types of antibodies. In our preliminary experiments, we have immunized a camel with EpCAM antigen and constructed a high quality phage-display library. 12 nanobodies with different CDR3 sequences have been screened and 3 of them recognized the EpCAM based on FACS analysis. These nanobodies could enter the cell by endocytosis. Furthermore, these 3 candidate nanobodies have been successfully humanized and the humanized Nbs can combine and enter the EpCAM positive cells. In this proposal, we will continue to construct the novel immunotoxins, which consists of EpCAM hNb and Pseudomonas exotoxin A (PE24). Moreover, we will detect the function of immunotoxins in the process of cell growth and apoptosis. By using immunodeficient mice model, we will test the in vivo anti-tumor activity of immunotoxins. Overall, this proposal will create a new way to the EpCAM-targeted therapy.