| Epithelial cell adhesion molecule (EpCAM) is a typeâ… transmembrane glycoprotein with molecular weight of 40kDa. It was designated as CD326 at the 8th International Conference on HLDA (Human Leucocyte Differentiation Antigens) in 2004. EpCAM includes two main functional regions: intracellular domain (Ep-ICD) and extracellular region (EpEx). EpEx as the ectodomain of EpCAM serves important roles in cell adhesion, migration and proliferation, whereas Ep-ICD, actting as the mitogenic signal transducer of EpCAM, can induce gene transcription and lead to increased cell proliferation. The expression of EpCAM is restricted to simple epithelia in normal tissues. However, in pathological situations, EpCAM can be expressed in a variety of tumor cells. EpCAM has been displayed as a surface marker of some kind of cancer stem cells as well as normal stem cells. Futhermore, EpCAM as the morphoregulatory protein may play roles in carcinogenesis from cancer stem cells. The expression pattern and level of EpCAM change notably during the course of carcinogenesis, inflammatory diseases and even in embryogenesis. What is more, the changes of expression pattern and level of this molecule in carcinoma correlate with the changes in survival rate of patients with some tumors. Thus, the expression of EpCAM may have a diagnostic value for some certain carcinoma types. Moreover, EpCAM-directed therapeutics is one of hotspots for cancer treatment by therapeutic antibodies. With the development of molecular biological and immunological techniques, many strategies for EpCAM-target treatments of cancer have been developed, from the murine monoclonal antibody (mAb) to chimeric antibody, fully human antibody and single-chain antibody fragments. Among these, Catumaxomab was approved in the European Union (EU) for the treatment of malignant ascites in patients with EpCAM-positive carcinomas in 2009. Till now, EpCAM-target cancer therapy has not been reported in China.In this paper, we focuse on the development and application of the single-chain antibody(single-chain fragment variable, scFv) against EpCAM. From the group of the mAbs against EpCAM, which had been developed by our lab and were designated as CD326 at HLDA 8, five hybridoma clones (FMU-EpCAM-2A9,4E4,4A11,2D7 and 4F6)were selected, which display high binding activity to the EpCAM on the cell surface. Then from 4 hybridoma cell lines (FMU-EpCAM-2A9,4E4,2D7 and 4F6), variable regions of immunoglobulin gene were amplified by reverse transcription polymerase chain reaction (RT-PCR). Variable regions of FMU-EpCAM-2A9 were chosen as template to build scFv (GST-scFv2A9). Prokaryotic expression vector GST-scFv2A9 was constructed and recombinant protein was expressed and purified. Flow cytometry and ELISA were used to identify antigen-binding activity of GST-scFv2A9. Recombinant protein GST-scFv2A9 was proved to have antigen-binding activity with recombinant EpCAM antigen, as well as the natural EpCAM molecule on the cell surface. Next, an EpCAM-specific recombinant immunotoxin was developed and the antitumor activity of this immunotoxin was investigated in vitro. Prokaryotic expression vector of the immunotoxin (GST-scFv2A9-PE or GST-APE) was constructed by genetically fusing a truncated form (PE38KDEL) of Pseudomonas aeruginosa exotoxin with the scFv (GST-scFv2A9). After expressing and purifying the recombinant immunotoxin, the GST-tag was cut off by thrombin. Flow cytometry and ELISA were performed to verify antigen-binding activity of immunotoxin with recombinant EpCAM and the natural EpCAM on the cell surface. The results showed that the immunotoxin had similar antigen-binding activity with scFv (GST-scFv2A9). Cytotoxicity of immunotoxin APE was measured in vitro, and the results showed that immunotoxin APE potently and specifically reduced the viability of EpCAM-positive carcinoma cells (HHCC). Altogether, we describe the development of an EpCAM-targeting single-chain antibody with potent activity against tumor cells, which may lay the foundation for future development of therapeutic antibody for the treatment of EpCAM positive tumors with independent intellectual property rights.In addition, we analyzed the expression of EpEx and Ep-ICD in pancreatic cancer tissue of different histological grade. For the first time, we found the expression of EpEx and Ep-ICD correlated with the differentiation of pancreatic cancer. In poorly differentiated tissue with poor prognosis, reciprocal loss of membrane EpEx and Ep-ICD, while increased nuclear and cytoplasmic accumulation of Ep-ICD was observed. In well differentiated pancreatic cancer tissue, membrane EpEx expression increased, which made it possible for the well-differentiated pancreatic cancer to be selected as the therapeutic object of the EpCAM-targeting therapeutic antibodies.
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