Anti-tnf-alpha Small Molecule Inhibitors And Anti Cd20fab-of Tnf¦Á Fusion Protein Expression Vector Construction, Expression And Activity Of Preliminary Determination,

Tumor necrosis factor-α(TNF-α) is a multifunctional cytokine secreted by macrophages, NK and T cells, and TNF signaling may either induce cellular activation, apoptosis, or necrosis. TNF-a was thought to be a potent anticancer agent due to its cytotoxicity against a number of tumor cell lines in vitro and tumor in vivo. TNF-αwas also known as a strong endogenous mediator of inflammation, and under pathological circumstance, overexpression of TNF-αsustains inflammatory phenomena characteristics of some disorders, such as tissue damage, grave septic shock and autoimmunity disease. As a key cytokine in regulation of physiologic equilibrium, TNF-αplays a "double-edged sward" role on cancer and other diseases in clinic. According to the bi-effect of TNF-α, this study would be divided into two parts. First, the design, scan of small molecular inhibitors of TNF-α. Second, the expression and bioactivity research of antiCD20Fab-TNFa fusion protein.During the past 30 years, elucidation of the pathogenesis of rheumatoid arthritis, Crohn's disease, psoriasis and ankylosing spondylitis at the cellular and molecular levels has revealed that these diseases share common mechanisms and are more closely related than was previously recognized. And TNF-αis at the key position. So how to block TNF-α's function would be the main way to treat these diseases. Till now, three kinds of anti-TNF-αantagonists which are all biologic agents are using in clinic. Although these macromolecules have many advantages such as good specificity and affinity, they also have many defects in stabilities. Therefore, to find new oral small-molecular inhibitors targeting TNF-αhas become a tough problem to be solved in clinic.In this topic, we use the method of computer-aided drug design (CADD) to screening 300-500 small-molecular inhibitors in a compound library. After that we choose 80 compounds which are representative and available to do the further research including the preliminary screening on inhibitory effects on TNF-αmediated cytotoxic of L929; the inhibitory effects in QD labeled TNF-αbinding to the receptor; the inhibitory effects of the compounds on TNF-αmediated cytotoxic effect in different concentrations; the inhibitory effects of the compounds on TNF-αmediated PCD of L929. After the experiment, we find one small-molecular which has better activities. The further research shows that this compound could block the binding between QD labeled TNF-αand its receptor which also has a strong inhibitory effect on TNF-α(1ng/mL) mediated cytotoxic of L929 (IC50 value=10umol/L). The research shows that the small-molecular we chose could block TNF-αmediated PCD of L929 in a dose dependent way.Although TNF-αplays a very important role in autoimmune disease, people realize it as a cytokine which can cause the tumor cell's necrosis at the beginning. The experiments in vitro show that TNF-αhas a strong effect in cell cytotoxity and proliferation resistance. Therefore, TNF-αwas used by human to treat malignancy in clinic during 80s last century. But the later research shows that TNF-αsystemic administration may lead to many adverse reactions which limit its application. Till now, it has a very slow progress in TNF-αtreatment of malignancy directly. The only use of TNF-αmay be the method of isolate limb perfusion (ILP) in melanoma and sarcoma treatment.Nowadays, to transport drugs to the direct tissue using monoclonal antibody has become a major method in targeted therapy. The research indicates that transporting TNF-αto blood vessel of tumor tissue using McAbs could destroy the neovessels which can decrease the tumor. CD20 is a cell-surface marker expressed on mature B cells and most malignant B cells. It is a mainly functional molecular in regulating the proliferation and differentiation of B cells. The characteristic expression manner, bio-function and existing form make it become the main target in B lymphoma treatment. The research indicates that anti-CD20 McAb could directly block the proliferation of B lymphoma cells as well as the apoptosis induction of the tumor cells which is related with the Ca²⁺. It also shows that the dimerization or multimerization of CD20 molecular could strengthen this effect.In the study, we construct the antiCD20Fab-TNFαfusion protein vector successfully which is based on the previous study in our lab. Then we express the fusion protein in E. coli expression system and investigate its bio-activities. The study indicates that the CD20Fab fragment of fusion protein has combining capacity with Raji (CD20⁺B lymphoma cells) while the TNF-αalso has the cytotoxic effect on L929 cell line. In this study, we expect that utilizing the self- multimerization of TNF-αin soluble could lead the multimerization of CD20 coupled with it.