Preparation Of A Novel Anti-human Monoclonal Antibody And Its Application

CD40, a 48KD cell membrane molecule, is a type I glycoprotein, and belongs to the tumor necrosis factor receptor (TNFR) superfamily. CD40 is expressed in many cell types including B cells, monocytes, dendritic cells, endothelial cells, epithelial ceils and also many carcinoma cells. CD40 is a pivotal costimulatory molecule in immune response, and with its ligand (CD40L) transduces the second signal the activation of T and B cell needs. To date, an increasing number of studies indicate that signals mediated by CD40 will lead to B cell proliferation, Ig secretion and its class switch; dendritic cell activation and maturation; adhesion molecules expression on endothelial cells, inflammatory factors secretion, and the accumulation of white cells to the inflammatory sites.Recent reports show the association of CD40 signaling with many human diseases, including autoimmune diseases, infectious diseases, allograft rejection, cancers and atherosclerosis. The blockade of CD40/CD40L interaction by CD40L antibodies, has been investigated to prolong the survival of experimentally transplanted organs. However, this agent was recently halted because of unexpected complication. Thus blocking CD40 mAb may provide an alternative way to inhibit the CD40 costimulatory signal. The soluble form of CD40 (sCD40), which co-exists with the membrane-anchored form (mCD40), is a natural antagonist of mCD40/CD40L interaction. sCD40 production is an active process regulated by the engagement of mCD40 and its proteolytic cleavage by TACE or a related MP disintegrin. The levels of sCD40 increasing in some liver diseases have been used as a serum index of apoptosis in liver. Contin and colleagues by analysis sCD40 in uraemic patients suggested that the sCD40 compromised the humoral immune response in vivo. Although the crucial mechanism triggering the sCD40 production and its role in theprocesses of immune response still needs further investigation, the detection of sCD40 levels has been shown to be a very important tool in the diagnosis of many diseases.In this study we prepared another anti-human CD40 mAb 3G3 by using CD40 highly expressing MM cell line XG2 as immunogen. 3G3 was shown to bind a variety of CD40 positive cells but not CD40 negative cells. Competition experiments showed that 3G3 recognized a different epitope of CD40 molecule from that of 5C11, 3B2 or mAb89. Functional studies showed that 3G3 interfered with the homotypic aggregation induced by the agonistic CD40 mAb 5C11 in lymphoma Daudi cells line. What might be more important, 3G3 inhibited the proliferation of lymphocytes in mixed lymphocyte reaction assay. Thus 3G3 may be of great potential in the design of more effective and safer therapy through manipulation of CD40/CD40L interaction.In our current study, we used 5C11 mAb as capture antibody and 3G3 mAb as detection and set up an ELISA kit. What we found was that significantly higher levels of sCD40 were detected in sera of patients with hyperthyroidism, chronic nephritis, rheumatoid arthritis and lung cancer as compared to that of healthy individuals..In conclusion, we generated a novel blocking monoclonal antibody recognizing both membrane and soluble CD40 molecule. This anti-CD40 mAb has immunosuppressive characteristics via blocking CD40/CD40L interaction, which might be valuable in the design of alternative agent to prevent organ or tissue transplant rejection through the blockage of CD40/CD40L costimulatory signals; the sCD40 kit, composed of two different epitope-recognizing antibodies, provides a sensitive tool for the detection of soluble CD40 in patient serum, which may serve as an index in the clinical diagnosis for a variety of autoimmune diseases and cancers.