A novel splice variant of human Ep-CAM as a potential therapeutic antibody target for epithelial cell-based cancers

2706 Ep-CAM is a 40kDa transmembrane glycoprotein expressed on the basolateral surface of normal epithelia. Ep-CAM protein is significantly upregulated on the cell surface of a variety of carcinomas and consequently is being developed as a therapeutic target for both active and passive immunotherapy. Through internal efforts to find alternative transcripts associated with cancer, we identified a novel splice variant of Ep-CAM, named DD-O232v3. We validated the expression of this novel mRNA in human cancer tissues. The mRNA expression of DD-O232v3 is more cancer-specific than Ep-CAM, using real-time quantitative RT-PCR. For example, Ep-CAM mRNA exhibits significant expression in normal colon tissue, whereas DD-O232v3 mRNA is largely undetected in such samples. Aligning the coding region of Ep-CAM with that of DD-O232v3 shows that DD-O232v3 has 76 additional amino acids within the extracellular domain, a region thought to be important for the cell-cell adhesion function of Ep-CAM. In addition, there are potential integrin-binding and arginine-rich domains within the novel DD-O232v3 coding regions. We are investigating the functional significance of these regions, especially with respect to their potential role in cell-to-cell or cell-matrix adhesion. DD-O232v3 is expressed as a 48 kDa glycoprotein in transiently transfected 293T cells. Cell surface biotinylation and immunofluorescence experiments with live cells confirm that the exogenously-expressed DD-O232v3 predominantly localizes to the plasma membrane. Furthermore, cells overexpressing Ep-CAM or DD-O232v3 are efficiently killed by incubation first with a specific monoclonal antibody, followed by a toxin-conjugated secondary antibody. These data suggest that both proteins are capable of internalizing bound antibodies. Literature reports have shown that overexpression of Ep-CAM enhances cell proliferation and promotes colony formation in transfected cells. Experiments to better understand the role of DD-O232v3 in cell growth and transformation are underway. Taken together, the cancer-specific overexpression, cell surface localization and the potential to internalize, make DD-O232v3 a promising target for therapeutic antibodies. We are currently generating and testing specific monoclonal antibodies against the novel regions of DD-O232v3 to validate their potential as anti-tumor agents.