Nianxi Zhao, Yimin Ge, Paul J Chiao and Youli Zu
Epithelial cellular adhesion molecule (EpCAM) is a biomarker highly expressed on the surface of epithelial carcinoma cells. Consequently, it has been widely used for detection of carcinoma tumors and isolation of individual circulating cancer cells and EpCAM-targeting therapeutics are undergoing pre-clinical and clinical testing against several types of carcinomas. Aptamers are a new class of small oligonucleotide ligands that bind to their targets with high affinity and specificity, and are thus termed “chemical antibodies.” In comparison to protein antibodies, aptamers are smaller in size, which improves their tissue penetration potential, and elicit few to no immunogenic responses. Furthermore, our previous studies revealed that ssDNA, compared to RNA aptamers are significantly more stable in human serum and thus, are suitable for in vivo use. In this study, by using a hybrid cell-biomarker selection approach, we developed ssDNA-based aptamers specific for EpCAM. We identified a dominant aptamer sequence (58.7% of >0.5 million sequence reads) that contained both loop and stem structures, and specifically targeted EpCAM-expressing carcinoma cells with high binding affinity (Kd=12 nM). Validation studies indicated that the newly synthesized aptamer targeted a variety of cultured carcinoma cells, including pancreatic, breast, ovarian, prostate, and colon cancer cells, but did not react with cultured EpCAM-negative leukemia and lymphoma cells. In addition, the aptamer recognized EpCAM-expressing, patient-derived primary normal and carcinoma cells. Finally, the aptamer selectively targeted carcinoma cells in complex cell mixtures, and showed no reactivity to mononucleated or red blood cells, demonstrating its suitability for therapeutic in vivo use.
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