| BackgroundBispecific antibodies (BsAbs) have significant potential for cancer therapy because they can be used to retarget cytotoxic effector cells against tumor cells.The BsAbs have the binding characteristics of two parental antibodies and can bind to two different antigens. This anti-ErbB2/CD16 trivalent BsAb possesses three antigen binding sites, two antigen binding sites in the form of scFvs targeting the tumor cells overexpressing ErbB2 and a monovalent Fab fragment redirecting NK cells.The trivalent BsAb both has capacity to effectively direct the cytotoxic activities of the effector cells to tumor cells, and enhances retargeting potentials of effect cells specificly, avoids the side-effects as effect cells extensively cross-linking. In this study, the trivalent BsAb was expressed and purified. The binding characteristics and killing-cell ability of the trivalent BsAb in vitro and in vivo were analyzed.ObjectiveTo study the anti-tumor activity mediated by the anti-ErbB2/CD16 trivalent BsAb in vitro and in vivo.MethodsWe added His-tag on the anti-ErbB2/CD16 trivalent BsAb by gene engineering technology and induced expression of the BsAb with IPTG.. This BsAb was efficiently expressed in E coli BL21 strain (DE3) as inclusion bodies but was very low in the supernatant. The high purity of the protein was obtained by refolding. To compare the biological activity of the BsAb with its respective parental antibodies, we cultured CG5 cells, which stably express anti-CD16 scFv-Fc fusion protein, and HG2 cells, which stably express anti-ErbB2 scFv-Fc fusion protein, by using Spinner System , and purified antibodies by rProtein A Sepharose Fast Flow Column . Furthermore, on 2,4,6,7day, we real-time monitored the content of fusion protein in the supernatant of the transfected CHO cells in 250ml Spinner System by sandwich ELISA.Binding properties of the anti-ErbB2/CD16 trivalent BsAb were characterized by flow cytometry (FACS). Whether the binding activities to CD16 expressing cells was affected by human IgG or human serum were determined as the mean fluorescence intensities. The efficacy of the BsAb in mediating tumor cell lysis was determined using colorimetric lactate dehydrogenase (LDH) release assays.The anti-tumor activity mediated by the trivalent BsAb was derectly viewed under inverted microscope.The anti-tumor efficacy of the BsAb in vivo was studied in a tumor xenograft model and compared therapeutic efficacy of the BsAb with that of anti-ErbB2 scFv-Fc.ResultsThe high purity of the anti-ErbB2/CD16 trivalent BsAb was obtained and the concentration of the BsAb was about 1mg/ml. Two parental antibodies were also obtained and the concentration were about 2 mg/ml.The trivalent BsAb was able to bind SKBR3 cells overexpressing ErbB2 and effector cells expressing CD16 bispecificly, and this capacity was not affected by human IgG or serum. The cytotoxicity assay indicated, especially at a low E/T ratio, the BsAb was able to mediate ADCC to effectively kill tumor cells. Furthermore, the BsAb was significantly more potent to kill tumor cells than its two parental antibodies in the cytotoxicity assay. The killing mediated by the BsAb was more effective to SKBR3 cells than to MCF-7 cells and COS-7 cells, indicating that the BsAb resulted in the specific lytic activity of effector cells against ErbB2 expressing tumor cells in vitro and the killing of tumor cells was dependent on the density of ErbB2 molecules on cell surface.Three times animal experiments demonstrated that the BsAb was able to mediate effectively LAK cells to kill SKOV3 cells in high-expressive ErbB2 tumor xenograft models. The potential of inhibiting tumor of the BsAb was more potent than anti-ErbB2 scFv-Fc and bearing cancer control. The BsAb had not significant side-effects in vivo.ConclusionThe anti-ErbB2/CD16 trivalent BsAb shows potent anti-tumor activity in vitro and in vivo. The BsAb may be valuable to be further modified and optimized for the treatment of malignant cells in a minimal residual disease.
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