本文選題：CD133 + CD3�。� 參考：《華中科技大學(xué)》2016年博士論文

【摘要】：目的：構(gòu)建并表達(dá)純度較高的抗CD133xCD3雙特異性抗體,通過體內(nèi)及體外實(shí)驗(yàn)檢測(cè)其對(duì)結(jié)直腸癌細(xì)胞的靶向殺傷效應(yīng),并探討其可能的作用機(jī)制,期待為晚期結(jié)直腸癌患者找到一種更安全有效的治療方法。方法：1.利用基因重組技術(shù)構(gòu)建人鼠嵌合型抗人CD133xCD3雙特異性抗體(MS133)及抗人CD133單克隆抗體(AC133)。2.利用瞬時(shí)轉(zhuǎn)染表達(dá)MS133及AC133,通過Protein A親和層析和SP陽(yáng)離子交換層析純化抗體,Western blot檢測(cè)蛋白表達(dá),SDS-PAGE和SEC-HPLC檢測(cè)抗體純度。3.通過流式的方法檢測(cè)MS133分別與細(xì)胞表面抗原位點(diǎn)CD133和CD3的結(jié)合能力,并利用CFSE、PI染料進(jìn)行細(xì)胞染色,檢測(cè)MS133包被激活的T細(xì)胞(activated T cell, ATC)對(duì)結(jié)直腸癌細(xì)胞(HCT116、HT29.SW480)的殺傷情況和對(duì)正常組織細(xì)胞MCF-10A的毒性效應(yīng)。4.選取CD133高表達(dá)細(xì)胞株HCT116為研究對(duì)象,使用CCK8試劑盒檢測(cè)MS133對(duì)其增殖的影響,并通過定量ELISA的方法檢測(cè)細(xì)胞殺傷過程中相關(guān)細(xì)胞因子(IFN-y、TNF-a、GM-CSF、IL-4)的分泌情況。5.為了驗(yàn)證MS133的體內(nèi)藥效,我們建立了兩種重癥免疫缺陷小鼠(NOD/SCID)動(dòng)物模型,首先是MS133包被ATC細(xì)胞與HCT116細(xì)胞混合共注射模型；更進(jìn)一步驗(yàn)證是將HCT116細(xì)胞與ATC細(xì)胞混合接種于小鼠皮下,尾靜脈抗體(MS133、hOKT3、hIgG)給藥模型。結(jié)果：1.制備的雙特異性抗體MS133分別用SDS-PAGE和SEC-HPLC檢測(cè),純度為84%和95%,抗體產(chǎn)量約為10mg/L。2. MS133與CD3陽(yáng)性細(xì)胞(Jurkat、ATC)和CD133陽(yáng)性細(xì)胞(HCT116、HT29)均能很好的結(jié)合。MS133包被ATC細(xì)胞可高效殺傷CD133高表達(dá)結(jié)直腸癌細(xì)胞株HCT116和HT29,對(duì)CD133低表達(dá)結(jié)直腸癌細(xì)胞株SW480及正常組織細(xì)胞株MCF-10A殺傷作用弱。3.在效應(yīng)細(xì)胞與靶細(xì)胞比例較高時(shí),MS133表現(xiàn)出顯著的HCT116增殖抑制效應(yīng)。另外,MS133靶向殺傷HCT116過程中,產(chǎn)生較高水平IFN-y、GM-CSF細(xì)胞因子的分泌。4.小鼠體內(nèi)實(shí)驗(yàn)中,MS133包被ATC細(xì)胞可完全抑制小鼠移植瘤的生長(zhǎng)；此外,低劑量MS133注射可顯著抑制小鼠移植瘤的生長(zhǎng),瘤體積及瘤重均小于單抗治療組,并且小鼠體重未出現(xiàn)明顯變化。結(jié)論：1.MS133體外包被激活的T細(xì)胞可有效殺傷CD133高表達(dá)結(jié)直腸癌細(xì)胞,殺傷作用的強(qiáng)弱與細(xì)胞表面CD133表達(dá)水平相一致。2.MS133包被ATC細(xì)胞可特異性殺傷CD133高表達(dá)結(jié)直腸癌細(xì)胞,并且對(duì)CD133低表達(dá)的結(jié)直腸癌細(xì)胞和正常組織細(xì)胞毒性作用較小。3.MS133介導(dǎo)ATC細(xì)胞靶向殺傷CD133高表達(dá)結(jié)直腸癌細(xì)胞HCT116的同時(shí),IFN-r和GM-CSF分泌量也明顯增加,可能是MS133高效殺傷腫瘤細(xì)胞的一個(gè)機(jī)制。4.小鼠體內(nèi)實(shí)驗(yàn)證實(shí),MS133介導(dǎo)ATC細(xì)胞的靶向殺傷能夠有效抑制小鼠HCT116移植瘤的生長(zhǎng)。因此,MS133作為一種新的治療性抗體,對(duì)于靶向治療晚期結(jié)直腸癌具有良好的應(yīng)用潛能。
[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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R735.34

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