The Mechanism Involved In TmTNF-α Antibody Competitively Inhibiting Conversion Of TmTNF-α To STNF-α

Tumor necrosis factor-alpha(TNF-α) is an important inflammatory cytokine mainly secreted by activated macrophages, but also expressed by many tumor cells. TNF-α exists two forms, namely transmembrane tumor necrosis factor alpha(tmTNF-α) and secreted tumor necrosis factor alpha(sTNF-α), and they play completely different biological effects in inflammation and tumor. sTNF-α is a pro-inflammatory cytokine, and can induce tumor cell growth(physiological level or low level) or necrosis(high level), where as tmTNF-α has anti-inflammatory effect, and can induce tumor cell apoptosis. The substances which mediated the process of tmTNF-α transferring to sTNF-α mainly include tumor necrosis factor-α converting enzyme(TACE, also known as ADAM17) and MMP7, serine protease was also reported to have the similar effect. Based on the previous work in our lab, we have developed a monoclonal antibody specific to tmTNF-?(tmTNF-α antibody) which can bind the tm TNF-α on the surface of cell, and enhance the effect of anti-tumor, killing tumor cells and inhibiting the growth of tumor, but it has no cross-reactivity to sTNF-α. Thus, the antibody is expected to be developed clinically a biological drugs targeted to TNF-α expressing-tumors(such as breast cancer, leukemia, etc.).Currently, it remains unclear that the mechanism involved in biological action of the tmTNF-α antibody. It was showed that the tmTNF-α antibody recognized an epitope which close to the action site of TACE. So we supposed that the antibody may competitively block the binding of TACE to tmTNF-α by steric hindrance, thereby inhibiting the secretion of sTNF-α and promoting the expression of tmTNF-α. In order to clarify this hypothesis, first it was maked sure that the antibody could promote tmNF-α expression and inhibiting sTNF-α secretion, and then the human His-tagged MMP7 full-length protein and TACE mutant protein were obtainted by molecular cloning and protein purification techniques, and lastly the results of protein binding experiment showed that the anti- tmTNF-αcould competitively inhibit MMP7 or TACE binding to tmTNF-α. Therefore, this study provides theoretical and experimental evidence for the development of clinical applications of the tmTNF-α antibody.