Isolation Of Single Domain Antibodies Against Human EpCAM By Phage Display And Their Preliminary Charaeterization

Purposes: Epithelial cellular adhension molecule(Ep CAM) is a type Ⅰ transmembrane protein(40 k Da). In this study, phage display technology was used to isolate single domain antibodies(VH) against human Ep CAM. In the future, these antibodies will be studied for their anti-tumor activities. These antibodies may be used for the treatment of human cancers. Methods:(1) Human single domain antibody library was screened by phage display technology against a peptide coded by the exon 2 of human Ep CAM gene as an antigen.(2) Monoclonal phage ELISA was performed for the identification of the positive antibody clones. HER2 was used as an unrelated negative antigen control, and monoclonal phage antibody derived from the screening of the library was used as the primary antibody for ELISA.(3) The other six unrelated antigens were used to further verify the antibodies by ELISA.(4) The verified positive antibody clones were sequenced, and the homology among these sequences of different antibodies was analyzed.(5) ELISA was performed to examine the binding of sequenced antibodies with the purchased antigen containing the full extracellular domain of human Ep CAM protein. Results:(1) After six round of screening of the single domain antibody library with human EPCAM as an antigen, polyclonal phage ELISA analysis showed the significant enrichment of Ep CAM-specific antibody clones.(2) Monoclonal phage ELSIA analysis showed that 17 antibody clones could bind to Ep CAM with high affinity and specificity.(3) The DNA sequences analysis showed that some clones are the same antibody with the same DNA sequences. A total of 8 different antibodies against Ep CAM were identified.(4) The ELISA analysis showed that a EP1, a EP149, a EP198 and a EP269 had a strong binding ability with the full extracellular domain of purchased Ep CAM. These antibodies can be used in subsequent experiments for the study of their anti-cancer activity.Conclusions: After the human single domain antibody library was screened by phage display technology. Four single domain antibodies(a EP1, a EP149, a EP198 and a EP269) were isolated against the full extracellular domain of Ep CAM. These antibodies showed a high binding ability and a strong specificity against the human Ep CAM. This study laid a solid foundation for the further study of these antibodies for their anti-tumor activity in vitro and in vivo.