Anti-RANKL Monoclonal Antibody Treating CIA Rat And Exploratory Study On Regulation Of RANKL Expression

Research Backgrounds:Rheumatoid arthritis (RA) is one of the most common autoimmune diseases, characterized by chronic inflammation on bone. The main symptoms include chronic synovitis on symmetrical joints and extra-articular changes. The synovitis can induce systemic and/or local bone loss primarily and secondarily in the form of bone and cartilage erosion, destruction and systemic bone loss. There are many drugs in the RA treatment, such as non-steroidal anti-inflammatory drugs (NSAIDs), tetracycline antibiotics, adrenal corticosteroids, methotrexate (MTX), and specific agents come into sight just these years, which cover a large part of recent research. These agents include: TNF-a blocking agents, inflammatory factors specific neutralizing antibodies (IL-6,17), and other quite promising therapeutic target molecules, such as:RANK-RANKL signaling axis, Wnt signaling pathway. A lot of effort is made to explore the pathogenesis of rheumatoid and develop new agents.The metabolic balance in the bone relies on the coordinated function of osteoclasts and osteoblasts, which forms the basis for the reconstruction process in this tissue. If the balance is broke down, there would appear diverse pathological changes. In the RA the increased proportion of osteoclasts in patients with rheumatoid arthritis is considered as an important factor in bone and joint damage. Osteoblasts and osteoclasts are modulated by an important signal transduction system:RANKL-RANK-OPG signaling pathway regulation. OPG as a soluble RANKL receptor competitively inhibits its binding to RANK on the membranes of osteoclast precursor cells or mature osteoclasts. The interactions between the three molecules modulate osteoclast differentiation and activation. Most professors hold the view that osteoclast differentiation and activation modulated by the RANKL/OPG ratio and the increase of RANKL lead to pathological changes in rheumatoid arthritis in the form of bone erosion. To conclude, almost all the factors prompting bone resorption such as IL-1, IL-6, IL-11, IL-17, TNF-α. PTH, carry out their function through up-regulating RANKL expression.Based on further understanding of RANKL-RANK-OPG signaling system, the following methods for treatment of rheumatoid arthritis may be advisable theoretically:1. Overexpression of OPG or application of exogenous OPG blocks the signal transduction; 2. Block the intracellular signal transduction in osteoclast, thereby inhibiting osteoclast differentiation; 3. Decrease RANKL levels by inhibiting other factors (such as TNF-a and IL-17); 4. Use anti-RANKL antibodies which inhibit downstream signaling of RANK. At present, AMG162 developed by the Amgen Corporation has been approved by FDA in June 2nd 2010, treating osteoporosis, which is fully human monoclonal antibody in the form of the IgG2 subtype immunoglobulin, with high elective affinity. The relationship between pharmacokinetics and dose is nonlinear, which is similar to the other fully human monoclonal antibody. During clinical trial the effects is fast and lasting, while the function of inhibiting osteoclast and bone resorption is reversible. In the treatment of postmenopausal osteoporosis, compared with bisphosphonates, bone density increased significantly, reducing incidence of vertebral fractures, non-vertebral fractures and pelvic fractures, and there were no additional side effects observed compared with the placebo group. Some studies show it is more effective than OPG and Fosamax.There are still some disadvantages in the treatment choice using monoclonal antibody, such as human anti-mouse antibody response, difficulty to reach the target cells and high expense and so on. To overcome these shortcomings, the use of genetic engineering technology to produce artificial antibodies began to be accepted by most researchers. The application of single-chain antibody (Single chain antibody, scFv or single chain antigen binding protein, SCA) has many advantages:①a small molecule, low immunogenicity, difficult to induce human anti-foreign protein reaction;②easy access to micro-circulation around the solid tumor;③fast blood circulation and systemic clearance, short half-life, less renal accumulation;④no Fc segment, reaction concerning the Fc segment is abolished;⑤easy process through genetic method and mass production using gene engineering technology. In addition single-chain antibodies also could combine with toxins, pro-drug converting enzyme, radioisotope, cytokine and other effecting molecules to construct a variety of bifunctional antibody molecules; meanwhile single chain antibody is also the ideal component to construct bispecific antibodies.Based on the background knowledge, we can get a common idea that anti-RANKL antibody used in the treatment of rheumatoid arthritis may have some potential value. We try to produce monoclonal antibody against RANKL and observe its effect on rheumatoid arthritis animal models (CIA rat model), in the hope to find a new effective agent to treat rheumatoid arthritis.Contents:This study resolved on the monoclonal antibodies against RANKL. The technology involved in this study listed as follows:gene cloning, hybridoma technology, affinity purification, immunofluorescence analysis, immunohistochemistry, ELISA testing inflammatory factor in serum and synovial fluid, general observation, tissue slice and many other experimental techniques.Complete the following tasks:1. Fermentation and purification of anti-RANKL monoclonal antibodies.2. Detecting monoclonal antibody and the RANKL protein binding.3. Anti-RANKL monoclonal antibody treatment on the model of rheumatoid arthritis.4. Actively exploring intracellular RANKL expression regulation.