Development of a novel monoclonal antibody to B7-H4: characterization and biological activity

OBJECTIVE B7-H4, a member of the B7 family of immunoregulatory receptors, may participate in the negative regulation of cell-mediated immunity. Aberrant B7-H4 expression is detected in some tumors and it plays a role in the occurrence and development of tumors. The aim of this study was to elucidate the functional and structural properties of B7-H4. METHODS We developed a monoclonal antibody (mAb) against the extracellular domain of B7-H4 through immunization of Balb/c mice with 3T3-mB7-H4 cells which expressed extrinsic B7-H4. A stable hybridoma cell line was established. Then, we analysed the characterization of the mAb through Enzyme linked immunosorbent assay (ELISA), Immunoprecipitation (IP), western blotting, Immunohistochemical (IHC), and tested the biological activity of the mAb. RESULTS ELISA, IP, and western blotting analyses indicated that the mAb specifically recognized B7-H4. In addition, flow cytometry demonstrated that the mAb exhibits excellent reactivity when applied to leukemic cells. IHC staining revealed that the mAb stained in a predominantly diffuse plasmalemmal or cytoplasmic pattern when applied to certain tumor tissues. The preliminary results of the mAb's biological activity showed that the mAb could effectively inhibit the function of B7-H4 in the inhibition of T cell, while promoting the growth of T cells and the secretion of Interleukin-2 (IL-2), Interleukin-4 (IL-4), Interleukin-10 (IL-10) and Interferon-? (IFN-γ). CONCLUSION This mAb will be a valuable tool for the further investigation of B7-H4 function.