Preparation Of Mouse Anti-human OX40 Monoclonal Antibody And Study Of Its Biological Character

Engagement of OX40/OX40L results in enhanced T cells activation, proliferation, migration and long-term survial, also generation of memory T cell and germinal center ( GC ), maturation of dendritic cells ( DCs ). Engagement of OX40/OX40L of antigen ( Ag )-specific stimulation of T cell, mediates proliferation and activation of Ag-specific effector T cell. Therefore, OX40/OX40L pathway plays an important role in autoimmune diseases, tumor and transplant rejection, which was attached much significance. Moreover, it has shown promising results that target OX40/OX40L in immune intervention.This project established one anti-OX40 monoclonal antibody and study its biological character primarily.PartⅠPreparation of one novel mouse anti-human OX40 monoclonal antibodyObjective : To preparation mouse anti-human OX40 monoclonal antibody for illuminate the expression and biological function of human OX40 molecule.Method : BALB/c mice were immunized with OX40 transfected cell OX40-L929 which highly expressed human OX40 molecule; Immunized mouse spleen B cell were fused with mouse plasmocytoma cells SP2/0. Hybridoma cells were screened with OX40-L929 cell, and L929-Mock cell as negtive control; Antibody Ig subclass analysis was performed with fast-strip kit; Indirected immunofluorescence and Western-blot were designed to identify the specificity of established mAbs; The epitopes recognition of mAbs were analyzed by competitive inhibition assay; The expression of OX40 on different human tumor cell line and T cell were detected by FCM.Results: After 4 times subcloning and selection, one monoclonal antibody, named 9H8 was obtained. The subclass of 9H8 Ig was proved to be IgG1 withкlight chain; Western-blot and immunofluorescence showed that 9H8 could bind to OX40 protein specificly and results were consistent with the commercial antibody ACT-35; The competitive inhibition test demonstrated that 9H8 recognized completely different antigen binding sites from 1F7 and ACT-35; FCM analysis revealed 9H8 could not only recognize OX40 molecule on activated T cells but also on human tumor cells SHG-44, SF-295, MCF-7, H460, HELA, SIHA, CW-2, LS-174T, Y79 U937, U266, Jurkat, etc.Conclusion: One mouse anti-human OX40 monoclonal antibody were generated successfully, which recognize different epitopes. This result will provid promising tool for the establishment of ELISA kit and research on OX40/OX40L pathway.PartⅡCo-stimulation of mouse anti-human OX40 monoclonal antibody on activated T cells in vitro.Objective: To study the role OX40 played involving in T cells proliferation, muturation and secretion of cytokine with obtained mAb.Methods: To culture human T cells which were activated by anti-CD3 mAbs in the presence of obtained mAb 9H8 in vitro, then the proliferation of T cells was analyzed by MTT assay, and gathered the culture supernate to detect the production of cytokines by ELISA method; To detect CD25 expression on human CD4+ T cells and CD8+ T cells that cultured with presence of anti-CD3 and mAb 9H8 during 0~5 days by FCM.Results: MTT test revealed that OX40 mAb 9H8 could enhance the T cell proliferation and production of cytokines without CD28 signal; Moreover, increase of CD25 expression on CD4+ T cells and CD8+ T cells were found by immunofluorescence.Conclusion: Obtained one novel mouse anti-human OX40 monoclonal antibody ,named 9H8 in this study, which has a potency to enhance proliferation and muturation of activated T cells in vitro and also up-regulate cytokines which promote the T cells differentiation. It provides one effective method to enlarge the number of effector T cells in vitro and has potential clinical value.