Screening And Identification Of EpCAM Targeting Peptides

Background:Epithelial/endothelial cell adhesion molecule（EpCAM）is a glycoprotein expressed highly on the surface of epithelial-derived tumor cells and tumor stem cells.A large number of studies have found that CSC has the ability of tumor initiation,and it is related to tumor metastasis,recurrence,as well as the therapeutic effect of chemotherapy and radiotherapy.Targeting CSC has gradually become a research hotspot of targeted tumor therapy.The targeted peptides to EpCAM obtained by affinity screening,which has an important value for the tumor targeted therapy including cancer stem cells!Biopanning of phage display technology is the most effective technology for screening targeted peptides at present.Polypeptides have many advantages,such as small molecular weight,non-toxic,low immunogenicity,easy synthesis,high affinity and sensitivity,and can be used as targeted molecular probes for the early molecular imaging diagnosis of cancer,they also can be coupled with other biological materials,nanoparticles,anticancer drugs to form targeted therapeutic drugs,as well as improve drug efficacy significantly and reduced the side effects of drugs.It is of great significance for prevention and treatment of tumor.We can use biopanning from phage display random peptide library to screen all kinds of target molecules that we needIn this study,phage peptide library was used to screen polypeptides（12AA）with high affinity for EpCAM that were highly expressed in tumor stem cells,and the peptides were initially verified at the cellular level for specificity,as well as molecular simulation affinity verification with EpCAM.This study will contribute to the development of targeted anticancer drugs,especially targeted therapy drugs for cancer stem cells.Methods:In this study,the Ph.D.-12 phage peptide library produced by NEB,which is the most authorized at present,was used to conduct five rounds biopanning,Using EpCAM as the target,in which 47 phage clones were randomLy selected.Positive phage clones were identified using ELISA,sequenced,and we obtained the consensus sequence.Then,the affinity of positive clones to EpCAM in cell was detected by Immunofluorescence assay for obtaining the best clone.Finally,the best cloned peptide sequence was docked with EpCAM,the affinity of the peptide with EpCAM was confirm.Results:1.47 phage clones were randomLy selected from the library from last round,they were amplified and purified respectively.After ELISA assays,30 clones were defined as positive clones.And we get 5 consensus sequences after sequencing analysis of the 30 positive clones:NTXXXXXXXXQS、SHXXXXXXXXLP、GNXXXXXXXXKG、DSXXXXXXXXDT、VGXXXXXXXXEF,they were named ESP1、ESP2、ESP3、ESP4 and ESP5,among which ESP1 has the best targeted efficency.2.The clone with the strongest affinity for EpCAM was R8 by immunofluorescence analysis.It showed high specificity and sensitivity to MCF-7 cells,and non-binding with HepG2 and HEK293 cells which confirmed that R8 was the best positive clone.3.The homology and interaction between ESP1 peptide and some proteins were found by similarity retrieval of ESP1 and protein database.4.Confrimed ESP1 has a strong affinity with EpCAM through molecular docking（-6.7kcal/mol<-1.2kcal/mol）.ConclusionThe best peptide sequence targeting EpCAM was obtained by using phage display technique for biological panning.The identification at cell level and molecular Docking analysis suggest that the best peptide sequence obtained has good affinity specificity and sensitivity to EpCAM.The study may be of great value in the research and development of new tumor targeted therapies,especially targeting tumor stem cells.