| Objective:To construct and express high purity anti-CD133xCD3 bispecific antibody, detect its targeting cytotoxicities toward colorectal cancer cells through in vitro and in vivo experiments, explore possible mechanism for this activity, and look forward to finding a more safe and effective method for advanced colorectal cancer treatment.Methods:1. The human-mouse chimeric anti-CD 133xCD3 bispecific antibody (MS133) and anti-human CD 133 monoclonal antibody (AC 133) were constructed by Recombinant DNA technology.2. MS133 and AC 133 were expressed by transient transfection and purified by ProteinA affinity chromatography and SP cation exchange chromatography, then Western blot was used to detect the expression of proteins, SDS-PAGE and SEC-HPLC were performed to analyze the purities.3. Binding ability of MS 133 to CD 133 and CD3 were measured by flow cytometry. CFSE and PI double staining were used to determine MS133-mediated cytotoxicity toward colorectal cancer cells (HCT116, HT29, SW480) and the normal cell line MCF-10A.4. Proliferation of CD133high colorectal cancer cell HCT116 was tested by CCK-8 and the cytokines (IFN-y, TNF-a, GM-CSF, IL-4) production in cytotoxicity assays were measured by quantitative ELISA.5. To determine the in vivo efficacy of MS133, we developed two kinds of NOD/SCID mice models. The first model was that MS 133 armed ATC were co-injected with HCT116 into mice. Moreover, T cells mixed with HCT116 were injected subcutaneously, and antibodies (MS 133, hOKT3, hIgG) were injected intravenously into mice for treatment.Results:1. The purities of prepared MS133 detected by SDS-PAGE and SEC-HPLC were approximately 84% and 95%, respectively. The yield of MS133 was about 10 mg/L.2. MS133 displayed high affinity to both CD133 positive cells (HCT116?HT29) and CD3 positive cells (Jurkat, ATC). Cytotoxicities mediated by MS133 toward CD133high colorectal cancer cells (HCT116, HT29) were more effective than CD133low colorectal cancer cell (SW480) and normal cell line MCF-10A.3. At high effector E:T ratio, MS133 significantly inhibited the proliferation of HCT116. Moreover, in cytotoxicity assay, MS 133 targeting HCT116 produced more cytokines such as IFN-y and GM-CSF.4. In in vivo assay, MS 133 armed ATC completely inhibited tumor growth. In addition, low-dose MS 133 injection significantly inhibited tumor growth. Tumor size and weight were significantly lower than the monoclonal antibody treatment groups, and no clear changes were observed in body weight of mice.Conclusion:1. MS133 armed ATC could effectively kill CD133high colorectal cancer cells and the ability correlated well with CD133 expression level on cell membrane.2. MS 133 armed ATC could specifically kill CD133high colorectal cancer cells, and the cytotoxicities mediated by MS133 toward CD133low colorectal cancer cells and normal cells were relatively low.3. That the amount of IFN-y and GM-CSF produced by MS133-armed ATC which targets CD133high colorectal cancer cell HCT116 were increased significantly may be a mechanism for MS133 efficient killing of tumor cells.4. The in vivo assay demonstrated that MS133 could target colorectal cancer cell HCT116 and significantly inhibit tumor growth. Therefore, as a novel therapeutic antibody, MS 133 has a good potential in the treatment of advanced colorectal cancer. |
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