Cancer Monoclonal Antibody Developed By The Sgb-8 And Nfat5 Hybridoma Antibodies Generated Impact

The thesis consists of two parts of investigations: (1) Downregulation of NFAT5 by RNA interference reduces monoclonal antibody productivity of hybridoma cells; (2) Preparation and characterization of a monoclonal antibody (mAb) that specifically combines with the esophageal cancer cells.(1) AIM: To prepare a monoclonal antibody that specifically combines with the esophageal cancer cells for future diagnosis and treatment of this disease. METHODS: BALB/c mice were immunized with Eca 109 (a human esophageal cancer cell line) cells, and a mAb was prepared with hybridoma technique; Biological characteristic of the mAb was analyzed with SDS-PAGE, indirect ELISA and immunohistochemistrical methods; RT-PCR and DNA sequencing techniques were applied to clone gene sequence of the variable region of the mAb. Results: A hybridoma cell line that secretes a monoclonal antibody, SGB-8, specific for antigens expressed on the surface of Eca 109 cells was obtained. The mAb belonged to IgG 2a and its purity is more than 90%. It specifically recognized esophageal cancer cells while had no cross reaction with normal adult and fetal tissues. In addition, the gene sequence of the variable region of the mAb was cloned and prepared for further study of antibody humanization. Conclusion: The mAb, SGB-8, has been prepared successfully and it will provide a useful reagent for clinical therapy and further research.(2) Hybridoma cells display an increase in antibody productivity following exposure to hypertonic conditions. However, the mechanism is not clearly understood. In the present study, we hypothesize that nuclear factor of activated T cells 5 (NFAT5)/ tonicity enhancer binding protein (TONEBP) functions to increase the antibody productivity of hybridoma cells. NFAT5 is an osmosensitive mammalian transcription factor. However its ubiquitous expression in various organs that are not bathed in hypertonic milieu suggests that NFAT5 may optimize cell growth and function under isotonic conditions. In this study, the expression of NFAT5 in hybridoma cells was determined by western blot analysis, and we found that it increased significantly in hypertonic medium. To further define the function of NFAT5 in hybridoma cells, RNA interference technique was used to downregulate the expression of NFAT5 in SGB-8 cells (a hybridoma cell line). In isotonic medium, downregulation of NFAT5 resulted in reduced antibody productivity of hybridoma cells while cell proliferation was not influenced. The results presented here demonstrate that NFAT5 not only plays an important role in osmotic stress response pathway in hybridoma cells, but also is essential for optimal antibody productivity.