新型抗CD133xCD3双特异性抗体治疗晚期结直肠癌的基础研究

发布时间：2018-04-16 19:16

本文选题：CD133 + CD3　；参考：《华中科技大学》2016年博士论文

【摘要】：目的：构建并表达纯度较高的抗CD133xCD3双特异性抗体,通过体内及体外实验检测其对结直肠癌细胞的靶向杀伤效应,并探讨其可能的作用机制,期待为晚期结直肠癌患者找到一种更安全有效的治疗方法。方法：1.利用基因重组技术构建人鼠嵌合型抗人CD133xCD3双特异性抗体(MS133)及抗人CD133单克隆抗体(AC133)。2.利用瞬时转染表达MS133及AC133,通过Protein A亲和层析和SP阳离子交换层析纯化抗体,Western blot检测蛋白表达,SDS-PAGE和SEC-HPLC检测抗体纯度。3.通过流式的方法检测MS133分别与细胞表面抗原位点CD133和CD3的结合能力,并利用CFSE、PI染料进行细胞染色,检测MS133包被激活的T细胞(activated T cell, ATC)对结直肠癌细胞(HCT116、HT29.SW480)的杀伤情况和对正常组织细胞MCF-10A的毒性效应。4.选取CD133高表达细胞株HCT116为研究对象,使用CCK8试剂盒检测MS133对其增殖的影响,并通过定量ELISA的方法检测细胞杀伤过程中相关细胞因子(IFN-y、TNF-a、GM-CSF、IL-4)的分泌情况。5.为了验证MS133的体内药效,我们建立了两种重症免疫缺陷小鼠(NOD/SCID)动物模型,首先是MS133包被ATC细胞与HCT116细胞混合共注射模型；更进一步验证是将HCT116细胞与ATC细胞混合接种于小鼠皮下,尾静脉抗体(MS133、hOKT3、hIgG)给药模型。结果：1.制备的双特异性抗体MS133分别用SDS-PAGE和SEC-HPLC检测,纯度为84%和95%,抗体产量约为10mg/L。2. MS133与CD3阳性细胞(Jurkat、ATC)和CD133阳性细胞(HCT116、HT29)均能很好的结合。MS133包被ATC细胞可高效杀伤CD133高表达结直肠癌细胞株HCT116和HT29,对CD133低表达结直肠癌细胞株SW480及正常组织细胞株MCF-10A杀伤作用弱。3.在效应细胞与靶细胞比例较高时,MS133表现出显著的HCT116增殖抑制效应。另外,MS133靶向杀伤HCT116过程中,产生较高水平IFN-y、GM-CSF细胞因子的分泌。4.小鼠体内实验中,MS133包被ATC细胞可完全抑制小鼠移植瘤的生长；此外,低剂量MS133注射可显著抑制小鼠移植瘤的生长,瘤体积及瘤重均小于单抗治疗组,并且小鼠体重未出现明显变化。结论：1.MS133体外包被激活的T细胞可有效杀伤CD133高表达结直肠癌细胞,杀伤作用的强弱与细胞表面CD133表达水平相一致。2.MS133包被ATC细胞可特异性杀伤CD133高表达结直肠癌细胞,并且对CD133低表达的结直肠癌细胞和正常组织细胞毒性作用较小。3.MS133介导ATC细胞靶向杀伤CD133高表达结直肠癌细胞HCT116的同时,IFN-r和GM-CSF分泌量也明显增加,可能是MS133高效杀伤肿瘤细胞的一个机制。4.小鼠体内实验证实,MS133介导ATC细胞的靶向杀伤能够有效抑制小鼠HCT116移植瘤的生长。因此,MS133作为一种新的治疗性抗体,对于靶向治疗晚期结直肠癌具有良好的应用潜能。
[Abstract]:Objective: to construct and express a high purity anti CD133xCD3 bispecific antibody, and to investigate its target killing effect on colorectal cancer cells in vivo and in vitro, and to explore its possible mechanism.Hope to find a more safe and effective treatment for patients with advanced colorectal cancer.Method 1: 1.Human mouse chimeric anti-human CD133xCD3 bispecific antibody (MS133) and anti-human CD133 monoclonal antibody (AC133N. 2) were constructed by gene recombination technique.MS133 and AC133were expressed by transient transfection. The antibody was purified by Protein A affinity chromatography and SP cation exchange chromatography. Western blot was used to detect the protein expression. SDS-PAGE and SEC-HPLC were used to detect the purity of antibody.The binding ability of MS133 to cell surface antigen sites (CD133 and CD3) was detected by flow cytometry.To detect the cytotoxicity of activated T cell (ATC) with MS133 on human colorectal cancer cell line HCT116 (HT29.SW480) and the toxic effect of ATCon on MCF-10A in normal tissue.The high expression CD133 cell line HCT116 was selected as the research object. The effect of MS133 on proliferation was detected by CCK8 kit, and the secretion of GM-CSFIL-4, a related cytokine, was detected by quantitative ELISA.In order to verify the efficacy of MS133 in vivo, we established two kinds of animal models of MS133 in severe immunodeficiency mice. Firstly, MS133 coated ATC cells and HCT116 cells were co-injected into each other.It was further verified that HCT116 cells and ATC cells were inoculated subcutaneously in mice and injected with MS133hOKT3hIgG.The result is 1: 1.The prepared bispecific antibody MS133 was detected by SDS-PAGE and SEC-HPLC, the purity was 84% and 95%, and the antibody production was about 10 mg / L. 2.Both MS133 and CD3 positive cells (Jurkatron) and CD133 positive cells (HCT116 (HT29)) could effectively kill HCT116 and HT29, HCT116 and HT29 cells with high expression of CD133, and had a weak killing effect on SW480 cell line with low CD133 expression and MCF-10A cell line with normal tissue cell line.When the ratio of effector cells to target cells was high, MS133 showed significant inhibitory effect on HCT116 proliferation.In addition, a high level of IFN-ytGM-CSF cytokines was produced during the targeted killing of HCT116 by MS133.In vivo experiment, ATC cells coated with MS-133 could completely inhibit the growth of transplanted tumor in mice, in addition, low dose MS133 injection could significantly inhibit the growth of transplanted tumor in mice, and the tumor volume and tumor weight were lower than those in the monoclonal antibody group.And the weight of mice did not change significantly.Conclusion: 1. The activated T cells outsourced by MS133 can effectively kill CD133 overexpression colorectal cancer cells. The killing effect is consistent with the level of CD133 expression on the surface of the cells. 2.MS133 coated ATC cells can specifically kill CD133 overexpression colorectal cancer cells.Moreover, the cytotoxicity of low expression of CD133 in colorectal cancer cells and normal tissues was less. 3.MS133 mediated ATC cells targeted killing CD133 high expression colorectal cancer cell HCT116, and the secretion of IFN-r and GM-CSF was also significantly increased.It may be a mechanism of MS133 killing tumor cells. 4.In vivo experiments in mice demonstrated that the targeted killing of ATC cells mediated by M S 133 could effectively inhibit the growth of mouse HCT116 xenografts.Therefore, as a new therapeutic antibody, MS133 has a good potential for targeted treatment of advanced colorectal cancer.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.34

【相似文献】