Monoclonal antibody 9C4 recognizes epithelial cellular adhesion molecule, a cell surface antigen expressed in early steps of erythropoiesis

Objectives Monoclonal antibody (mAb) 9C4 detects a surface antigen expressed on immature erythroid progenitor cells and epithelial tumor cell lines. The aim of this study was to identify the recognized surface antigen and to analyze a potential role of this molecule in early steps of erythropoiesis. Materials and Methods. A pituitary-derived retroviral cDNA library was used to generate viruses and infect NIH-3T3 fibroblasts. The transfected cells were stained with mAb 9C4; positive cells were sorted by FACS; and a clonal cell line binding mAb 9C4 was established. cDNA encoding the 9C4-binding protein was amplified by polymerase chain reaction and cloned. Reactivity of mAb 9C4 with human bone marrow (BM) cells was analyzed by flow cytometry. Results. Sequence analysis of the isolated cDNA uncovered a 100% identity with the epithelial cellular adhesion molecule (Ep-CAM). Two-color flow cytometric analysis revealed that almost 100% of Ep-CAM+ BM cells coexpressed CD105, E-cadherin, and high levels of CD71. Fractions of Ep-CAM+ BM cells also were CD34+ but lacked glycophorin A expression, suggesting that Ep-CAM+ cells represent immature erythroid cells. Reverse transcriptase polymerase chain reaction analysis of BM mononuclear cells revealed that the 9C4+ erythroblast population but not the 9C4− fraction expressed Ep-CAM mRNA. Peripheral blood CD34+ cells induced in vitro to differentiate into the erythroid lineage showed strong Ep-CAM expression on days 3 to 7 of culture. The addition of Ep-CAM–specific mAbs 9C4 or KS1/4 to the culture resulted in two- to three-fold up-regulation of Ep-CAM protein expression. Conclusion. mAb 9C4 recognizes Ep-CAM, a molecule expressed in the early steps of erythropoiesis.