| Tumor necrosis factor (TNF) is a soluble homotrimer of 17 kDa protein subunits secreted mainly by monocytes and macrophages. A membrane-bound 26 kDa precursor form of TNF also exists. TNF causes pro-inflammatory actions, inducing procoagulant activity on vascular endothelial cells, increasing the adherence of neutrophils and lymphocytes and stimulating the release of platelet activating factor from macrophages, neutrophils and vascular endothelial cells, which result in tissue injury. Recent evidence associates TNF with infections, autoimmune diseases, graft-versus host reaction, and tumors.Anti-TNF antibody is able to inhibit the binding of TNF to its receptors and to inhibit the cytotoxicity of TNF. Neutralizing mAbs to TNF have been shown in mammals other than man to abrogate adverse physiological changes and prevent death after lethal challenge in experimental endotoxemia and bacteremia.To date, experience with anti-TNF murine mAb therapy inhumans has been limited. Patients with severe septic shock were administered a murine anti-TNF mAb in a single dose from 0.4-10 mg/kg. However, about 50% of patients developed a human anti-murine antibody response due to the murine immunoglobulin. Such immunogenicity causes decreased effectiveness of continued administration and can render treatment ineffective, in patients undergoing diagnostic or therapeutic administration of murine anti-TNF antibodies.Accordingly, there is a need to provide novel TNF antibodies, which overcome the problems of murine antibody immunogenicity, and which provide reduced immunogenicity and increased neutralization activity. Beneficial effects were obtained in treatment of Rheumatoid Arthritis and Crohn's Diseases with mouse-human chimeric anti-TNF antibody.We formerly prepared a set of murine anti-human TNF monoclonal antibodies by immunizing BALB/c mice with recombinant human TNF. We selected three hybridoma cell lines named as D2, E6 and F6 for further study due to their higher titers and their ability to binding natural TNF. Ascitic fluids were prepared according to our lab's routine protocol and primarily purified by ammonium sulfate precipitation and further by anion exchange chromatography or protein A affinity chromatography. The purity of purified mAb was identified by SDS-PAGE and measured by Gel-Blot-Pro scanner. The result shown that the purities of D2, E6 and F6 mAbs were 91%, 93% and 94%, respectively.The affinity of mAbs is critical for their therapeutic use. MAb with high affinity can not only greatly reduce the amount used for treatment, but also reduce the chance of hypersensitivity in vivo. Wetested the relative affinity through determining the titers of them by using indirect ELISA method. The titers of ascitic fluid of D2, F6 and E6 mAbs were 1 X 10-6, 1 X 10-7and 1 X 10-7 and the titers of purified D2, F6 and E6 mAbs were 10, 2, and 2ng/ml respectively, which were considered relative high.It has been well accepted that the neutralizing activity is the most important parameter of therapeutic antibodies. In order to comprehensively identify the neutralizing activity of the three mAbs, we employed three separate experimental systems according to the inflammatory mechanics of TNF.The first experimental system was mAbs inhibiting TNF-induced cytotoxicity of L929. The concentration of D2, E6, and F6 mAbs to protect 50% of cells from TNF (200U/ml)-induced L929 apoptosis was 0.16, 0.40 and 0.40ug/ml, respectively.ICAM-1 up-regulation on ECV304 cells following exposure to TNF was also used as an experimental system. All three mAbs could inhibit TNF induced ICAM-1 expression. At the higher concentration (1.0 u g/ml) used, the percentages of inhibition by D2, E6, and F6 were 75.2%, 64.3% and 61.3%. At the lower concentration (0.1 u g/ml) used, the percentages of inhibition were 63.5%, 57.0% and 61.2%, respectively.At last, induction of NF- k B nuclear translocation following exposure of ECV304 cells to TNF was used as another experimental system. At the concentration of 10.0 u g/ml, the inhibition by D2, E6, and...
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