br Acknowledgement br Introduction There has been great prog
Introduction There has been great progress in the treatment of autoimmune disease during the past several decades. However, the majority of autoimmune diseases have no known cure. B lymphocytes and autoantibodies play important roles in many autoimmune diseases, including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), multiple sclerosis (MS), and myasthenia gravis (MG). Recent data suggested that B lymphocytes play more varied roles than previously thought in the immune response. Along with their traditional roles as precursors of antibody-producing plasma cells, B cells have also been found to produce cytokines and chemokines, present gnrh antagonist to T cells, assist the activation of T cells, and result in a breakdown of self-tolerance (Dalakas, 2006, Dorner and Burmester, 2003). Therefore, selectively eliminating B cells may be a promising therapeutic goal for many autoimmune conditions (Edwards and Cambridge, 2006, Eisenberg and Albert, 2006). Selective B lymphocyte elimination has been achieved with the use of Rituximab (Sabahi and Anolik, 2006). Rituximab is a chimeric murine/human monoclonal antibody directed against the CD20 surface antigen of B cells. It has been used for the treatment of MG, especially those severe, refractory MG patients and those who resistant to conventional immunotherapy. Several authors reported encouraging results that treatment with Rituximab marked improved the patient's clinical symptoms, decreased the levels of AChR-Ab and peripheral B lymphocytes, and discontinued or reduced of prednisolone and other drugs. (Baek et al., 2007, Evoli et al., 2008, Hain et al., 2006, Illa et al., 2008, Kerkeni et al., 2008, Thakre et al., 2007, Wylam et al., 2003, Zaja et al., 2000). However, this chimeric antibody shows clinical efficacy for the MG but still carries risk of infection and immunodeficiency because it targets all B cells besides autoreactive B cells that produce autoantibodies (Bournia et al., 2009, Garcia-Carrasco et al., 2009). Therefore, the ideal B-cell-targeted therapy must be one that removes disease-associated B cells selectively. An urgent problem that needs to be solved is how to specifically deplete the disease-associated B cells without impairing the entire immune system. Our aim was to design a protein with the potential to eliminate autoreactive B cells thought to be the root cause of pathogenic autoantibodies. Herein we report the construction and expression of an AChR-Fc fusion protein that consists of the extracellular domain of AChR α1 subunit (Hα1-210) and the CH2 and CH3 domains of the human IgG1 heavy chain. This fusion protein is expected to selectively eliminate most, if not all, AChR-reactive B cells by cross-linking the B cell receptor (BCR) that has an identical specificity with that of the secreted antibody and inhibitory receptor FcγRIIB on the surface of B cells. We detected the binding ability and cytotoxicity of AChR-Fc fusion protein in an AChR-specific hybridoma cell line in vitro. Additionally, the fusion protein was tested in vitro for cytotoxicity against B cells from AChR-immunized rat spleen cells.
Materials and methods
Discussion In the present study, we tested the hypothesis that an AChR-Fc fusion protein could be used to selectively target and eliminate AChR-reactive B cells in two different systems. AChR-Fc could inhibit proliferation and induce apoptosis of hybridoma cells producing anti-AChR antibody in vitro, and could also eliminate AChR-reactive B cells from AChR-immunized Lewis rats ex vivo. Our very preliminary data may shed new light on a possible treatment for MG. Dendritic cells, T cells, and complement also play important roles in the pathogenesis of MG. However, autoreactive B cells are the downstream check-point in the pathogenesis of MG. AChR-reactive B cells are the ultimate functional mechanism that act by secreting anti-AChR autoantibodies to destroy AChR and cause MG. If AChR-reactive B cells are eliminated, there should be long-term remission.