内吞型EpCAM纳米抗体的筛选及鉴定

The screening and identification of internalized nanobody against EpCAM

上皮细胞黏附分子(epithelial cell adhesion molecule,EpCAM)是肿瘤靶向治疗的热门靶点,本研究开发了3株高特异性针对EpCAM的胞吞型纳米抗体,为基于此靶点的免疫毒素研究提供基础。研究前期对骆驼免疫EpCAM-Fc蛋白,构建了高质量的噬菌体展示纳米抗体文库,运用噬菌体展示技术通过3轮淘选成功获得了17株互补决定区(complementarity determining region,CDR)3序列不同的EpCAM纳米抗体,原核纯化获得高纯度的抗体蛋白后,流式细胞术确定其中7株具有细胞水平高结合活性。动物实验经复旦大学药学院实验动物伦理委员会(IACUC)批准。通过流式细胞术和激光共聚焦显微术最终鉴定出3株具有较高胞吞活性的EpCAM纳米抗体,且ForteBio测定具高亲和力。本研究以期为EpCAM阳性肿瘤的靶向治疗开辟新的途径,并为其他靶点的纳米抗体免疫毒素研究提供指导。

Epithelial cell adhesion molecule(EpCAM)is a popular target for cancer therapy.In this research,3 nanobodies with high specificity and endocytosis activity against EpCAM were developed,which provides a basis for the study of immunotoxin based on EpCAM.In our preliminary experiments,we have immunized a camel with EpCAM-Fc antigen and constructed a high-quality phage display library.Seventeen nanobodies with different complementarity determining region(CDR)3 sequences have been screened after 3 rounds of biopanning by phage display technology.The animal procedures were approved by the Institutional Animal Care and Use Committee(IACUC)of Fudan University School of Pharmacy.After purification,7 nanobodies showed high cell binding activity by fluorescent activated cell sorting(FACS)identification.Furthermore,3 nanobodies presented high endocytosis activity based on FACS and laser confocal microscopy,which also showed high affinity to EpCAM measured by ForteBio.According to this study,we aimed to provide a novel alternative approach to the EpCAMtargeted therapy and to provide guidance for the study of nanobody based immunotoxins for other targets.