(a) Field of the Invention
The present invention relates to recombinant chimeric acetylcholine receptor polypeptides recognized by CD4 T cells of a myasthenia gravis patient and chimeric acetylcholine derivatives for the treatment of myasthenia gravis containing the same as an effective ingredient, more precisely, recombinant acetylcholine receptor polypeptides deficient in B cell epitopes, recombinant acetylcholine polypeptides in which two or more T cell epitopes are fused, a composition for the treatment of myasthenia gravis containing the above recombinant polypeptides as an effective ingredient and a treatment method for myasthenia gravis using the compositions.
(b) Description of the Related Art
Autoimmune disease is led by the immune response induced against self-components (proteins or cells) according to the loss of self-tolerance. T cells regulating immune response are matured in thymus, in which they are instructed to cause immune response by recognizing an antigen linked to their MHC (major histocompatibility complex). Basically, T cells can not respond to those cells that have their own antigens in their MHC, but from time to time, self-active T cells are generated to recognize an antigen complex produced by the combination of their MHC and the antigens therein. Autoimmune disease is not infectious but inherited. In some cases, autoimmune disease can be triggered or worsened by virus infection, aging, chromium poisoning, hormones and pregnancy.
Autoimmune disease is a chronic disease, whose cause has not been disclosed, yet. Studies on autoantigens have been underway. Most autoimmune diseases are closely related to the activities of T cells, B cells and antigen presenting cells. Approaches to treat such autoimmune diseases are largely outlined by following four methods; first, targeting a subject which is involved in the activity of an immune cell; second, regulating an antigen-specific immune response; third, reconstructing immune system by transplanting autologous or allogeneic stem cells; and fourth, transplanting an organ. Besides, to treat autoimmune diseases, conventional immunosuppressants have been widely used, which are exemplified by calcineurin inhibitors (cyclosporin, tacrolimus), antimetabolites (azathioprine, leflunomide, methotrexate, mycophenolate mofetil), antiproliferatives (sirolimus), monoclonal antibodies to T lymphocyte (basiliximab, daclizumab, muromonab-CD3) and anticytokines (anakinra, etanercept, infliximab). Recently, approximately more than 23 immunosuppressants are under clinical trial or ready for the trial (Expert Opin Emerg Drugs. 2003 May; 8(1):47-62). The gene therapy has recently been tried out to treat autoimmune diseases, for example, CD4+ T cells are infected with a vector specially designed to express cytokines (IL-4, IL-10, IL-12p40) having functions of immunoregulation, which are then amplified to be used as a cell therapy agent (Autoimmun Rev. 2002; 1(4):213-9); Curr Opin Immunol. 2001; 13(6):676-82). However, the above methods not only inhibit abnormal autoimmune responses but also suppress normal immune response to protect human body from an invading antigen, so that it might increase the chances of infectious diseases and other side effects including weakening the function of inhibiting tumor expression by immune cells.
Another attempt has been made to treat autoimmune disease. Oral administrations of autoimmune disease specific antigens have induced antigen-specific immune tolerance in various experimental autoimmune animal models. These are including following experimental autoimmune model systems; bovine/chicken collagen for the treatment of rheumatoid arthritis, myelin basic protein for the treatment of multiple sclerosis, insulin for the treatment of type-I diabetes and IRBP (interphotoreceptor retinoid-binding protein) for the treatment of autoimmune uveitis. However, the oral administration of these antigens was effective mainly in animal models but not in human clinical trials. There are still problems in the development of an oral immune regulator. The immune responses mediated by CD8 cells and B cells against orally administered proteins have not been considered.
In summary, for the development of safe and effective oral immunomodulator potential immunoactivator function of orally administered protein should be considered. For example, to develop oral immunomodulator targeting CD4 T cell immune responses, orally administered proteins should induce hypoimmune response to administered antigen-specific CD4+ T cells without stimulating immune responses mediated by antigen-specific B cells and CD8 cells.
Myasthenia gravis is one of chronic autoimmune diseases, which has the incidence of 1/5,000-10,000. Currently antigen-specific therapeutic agent for myasthenia gravis has not been developed, yet. Myasthenia gravis is developed when the normal binding acetylcholine to acetylcholine receptor (referred as “AChR” hereinafter) is interrupted in neuromuscular junction. In Myasthenia gravis anti-AChR antibody to self-AChR acts as an inhibitor of signal transduction in the neuromuscular junction. In fact, approximately 80% of myasthenia gravis patients have been reported to harbor antibodies responding to AChR. The antibody responding to AChR can be a direct cause of the disease and the generation of the antibody is essentially involved in the action of T cells. Namely generation of AChR-specific antibody is depending on AChR-reactive T cell. Therefore, any substance that can incapacitate AChR-specific T cells or B cells can be used for the treatment of myasthenia gravis. For the treatment of myasthenia gravis, non-specific treatment methods (immune inhibitor, intravenously injectable immunoglobulin, and plasmapheresis) and acetylcholine esterase inhibitor known as mestinon are in use, and the market for them is growing every year. Nevertheless, the therapeutic agents in use have side effects and show limited effects.
AChR is composed of two α, β, γ and δ (ε) chains. Most autoimmune response related T cell and B cell antigens localize in the α chain (see FIG. 1), particularly in the extracellular domain of the α chain, and B cell epitope known as MIR (main immunogenic region) and various AChR-reactive epitopes antigens are also localized in non-alpha chains.
US Patent Publication No. 2002-0081652 describes the production of a polypeptide derived from human AChR α subunit. But, the polypeptide still harbors the B cell epitopes that could be recognized by auto-AChR reactive B-cell receptors in myasthenia gravis. This could be harmful when B-cell epitope-containing human AChR α subunit is administered into a myasthenia gravis patient or animal since AChR-reactive B-cell receptors could recognizes B cells, thereof to activate AChR-reactive B cells as well as T cells. Indeed when B-cell epitope containing human AChR α subunit was administered in acute state of myathenic rats it exacerbated myasthenia by stimulating AChR-reactive T cells as well as B cells (Im et al., J Immunol., 165: 3599-3605, 2000).
The present inventors produced a polypeptide by inserting P3A sequence which is composed of 25 amino acids expressed only in some of acetylcholine receptors into the region between the 58th and the 59th amino acids among 1st-205th amino acids of human AChR α subunit, and then confirmed that the produced polypeptide characteristically has the structure reducing antigenicity of B cell epitope against myasthenia gravis. The present inventors further induced immune tolerance against an autoantigen by the oral administration of the produced polypeptide to an animal model with myasthenia gravis (Im et al., J Clin. Invest., 165:3599-3605, 2000). And also, the present inventors made an attempt to treat myasthenia gravis by the intranasal administration of the produced polypeptide to an animal model with myasthenia gravis (Im et al., J Neuroimmunol., 111(1-2) :161-168, 2000).