Monoclonal Antibody 9C4 Recognizes Epithelial Cellular Adhesion Molecule, a Cell Surface Antigen Expressed in Early Steps of Erythropoiesis

Objective: 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.