Total Stimulus Molecular Enhanced Bifunctional Antibody Mediated Killer T Cells

Background and Objective T lymphocyte is the major effector cell of antitumor immunity and its activation determine the outcomes of antitumor treatments. To prime T cell needs two primary signals. One is the specific signal mediated by MHC-Ag complex, and the second one is the costimulatory signals provided by molecules such as B7and4-1BBL. If costimulatory signal was not served to activated T lymphocyte, T cell may lead to activation induced cell death (AICD) or annergy which finally impair antitumor immunity. Tumor cells often expressed low level of costimulatory molecules to provide insufficient signals to antigen presenting cells or lymphocytes, hence to escape immunosurveilence. owever, such mechanism also serve us a new stragergy to treat tumor. We can promote the activation of lymphocytes through B7or4-1BBL costimulatory molecules, enhance the antitumor response, obtain better outcomes.Methods and Result In the first part, the cytotoxicity of PBL regulated by ex4-1BBL、ex4-1BBL\anti-CD20fusion protein and anti-CD3/anti-Pgp diabody or anti-CD3/anti-CD20diabody was observed with the Calcein-Am method or Nonradioactived Cytotox96assay kit. Both demonstrate the increased cytotoxicity on their targtet cells (K562/A02or Raji) at different E:T ratio. Ex4-1BBL\anti-CD20fusion protein is more effective than ex4-1BBL. In addition, in vivo the same study was performed to observe the antitumor immunity of PBL against multidrug resistant K562/A02and Raji xenografts in nude mice. Tumor growth was drmatically inhibited by the combined therapy of ex4-1BBL and anti-CD3/anti-Pgp, and no tumor relaps was observed again, even100days after treatment initiation.In the second part, the expression of CD80or CD86on target cell which were stimulated by Ara-C in different concentration for72h were evaluated by FACS. Furthermore, the inhibition of growth of target cells by Ara-C in different concentration was analyzed by MTT. Determing the concentration of Ara-C which can stimulate the CD80or CD86increasing while having no effect on growth. Then combing Ara-C at the concentration and Diabody to do non-radioactive cytotoxicity assay samely with Raji cells. Ara-C in final concentration of0.25μmol/L which was incapable of inhibiting the growth of target cells increased the expression of CD80(B7.1) on cells but no effect on CD86(B7.2). The cytotoxity of T cells mediated by Diabody associated with Ara-C raised apparently than the Diabody and alone (p<0.05).It was improved on the Raji xenografts in nude mice.Conclusion Our results demonstrated that ex4-1BBL, as a immunoadjuvant, can modulate the PBL activation and enhance the outcomes of PBL-based antitumor biotherapy. Ex4-1BBL may become a new agent for bio-immunotherapy. Ara-C in final concentration of0.25μmol/L which was incapable of inhibiting the growth of target cells increased the expression of CD80(B7.1) on target cells. The cytotoxity of T cells mediated by Diabody associated with Ara-C was increased20%than by Diabody alone.