Evidence indicated that the x gene of human HBV can cause cancer in transgenic mice, moreover, HBxAg was so far the most frequent and strong antigen among those HBV markers expressed in hepatocellular carcinoma (HCC) tissues. Aiming to enhance killing of HCC by effector cells, we established an anti-HBx/anti-CD3 hybrid-hybridomas by fusion of anti-HBx hybridoma cells with FITC-labeled anti-CD3 HAT sensitive cells, and followed by FACStar sterile cell sorting, HAT selection and eventually verified by ELISA and double bridging assay. Using two color cytometric analysis, we found that bispecific monoclonal antibody (BsAb) remarkably enhanced in vitro effector-target cell conjugates (48.3% vs. 8.5%). In in vivo study, BsAb retargeting effector cells were significantly more effective than that of effector cells alone in shrinkage of LTNM4 HCC xenografts (HBxAg positive) in nude mice, not only in fresh inoculated tumors but also in established tumors (p<0.01, p<0.01, respectively). Besides, pronounced apoptotic cell death and infiltration of lymphocytes in the peripheral of tumor nodules can also be witnessed in the tissues treated by BsAb plus effector cells, but not in the controls. The results demonstrated that antiHBx/anti-CD3 BsAb was able to redirect effector cells for lysis of HBxAg positive HCC cells both in vitro and in vivo and it also indicated that shrinkage of tumors in nude mice with therapy of BsAb retargeting effector cells was partially due to initiation of apoptotic cell death.

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