Construction Of Anti TNF-α×antiED-B Bispecific Antibody And Activity Analysis

Rheumatoid arthritis (RA) is a joint disease systemic autoimmune diseases, mainly for symmetry, chronic polyarthritis, chronic inflammation of synovial hyperplasia, the formation of pannus, eventually leading to joint deformity and loss of function. TNF-α involved in RA of synovial tissue hyperplasia, inflammatory and autoimmune responses and other pathophysiological processes, occupies a central position of pro-inflammatory factor in the pathogenesis of RA. Antagonists of TNF-α including neutralizing antibodies have been proven to be effective therapeutic agent for rheumatoid arthritis. U.S. FDA approved Etanercept, Infliximab and Adalimumab, three kinds of biological therapeutic agents against TNF-α, which the latter two are anti-TNF-alpha monoclonal antibody. The three biological therapeutic agents in inflammatory joint local play the role of treatment of RA, but also inhibit other organizations and organs of the body’s normal immune response, increase the chance of microbial infection and tumorigenesis. Select the appropriate drug design methods to make the drug selective distribution of inflammatory joints in RA, to reduce or eliminate its immunosuppressive effects on normal tissues and joints, can effectively improve the treatment of RA biotherapeutic agent. Precursor RNA of Human fibronectin (FN) selectivly splice exon, can produce different subtypes of FN, respectively is the extra domain A (ED-A), extra domain B (ED-B), and type Ⅲ repeat unit connected fragment (ⅢCS), wherein, the retention of ED-B domain FN called ED-B FN. Recently the accumulated data indicate that ED-B FN in patients with RA synovium showing specific expression, research on targeting effect of anti-ED-B monoclonal antibody in experimental models of RA mice found that can specificily localized in the inflammation of joints in mice, confirmed the feasibility of ED-B as a target antigen. Base on the tissue specificity of ED-B FN expression and the information on the tumor vascular targeting therapy, make use of antibody targeting ED-B FN to make drug selectively distribute in the RA inflammatory joint should have a good prospect of application of the treatment of RA.In this study, using genetic engineering methods to construct the anti-TNF-α×anti-ED-B bivalent bispecific antibody prokaryotic expression vector, transformed into E. coli BL21(DE3) Star, using0.5mM IPTG induced target gene expressed, by SDS-PAGE and Western-blotting analysis of target protein expression, the bispecific antibody to insoluble inclusion bodies form in E. coli was highly expressed; using ultrasound to break bacterium to prepare inclusion body proteins, dissolved by8M urea and purified by affinity chromatography through Ni-NTA column, inclusion body protein refolding by slowly dialysis to remove denaturant. obtained the purity of more than95%of the soluble form bispecific antibody aEBTA; analyzed the biological activity of bispecific antibody aEBTA in vitro, the direct ELISA assay bispecific antibody aEBTA is capable of specific binding of TNF-α; MTT assay bispecific antibody aEBTA neutralize the cytotoxicity of TNF-a on mice into fiber cells (L929), concentration of aEBTA was about5μg/ml of50%inhibition (TNF-α killing concentration of lng/ml); immunohistochemical staining detect the aEBTA can effectively combine with the ED-B in synovial.This study has successfully constructed a bivalent bispecific antibody against TNF-α at the same time resistance to the ED-B FN, activity in vitro experiments show that can be better to combine with the two antigens, laid the foundation for activity in vivo experiments in animal models to study the effect of drug treatment, proposed the use of bispecific antibodies in RA treatment is an exploratory study, put forward new ideas for drug design of RA.