靶向表皮生长因子受体的新型前抗体制备及抗肿瘤功能研究

发布时间：2018-11-20 07:33

【摘要】：目的：帕妥木单抗(Panitumumab)是一种已上市的特异性靶向表皮生长因子受体(EGFR)的抗体。然而由于其是一种IgG2亚类抗体,抗体依赖的细胞介导的细胞毒作用(ADCC)较弱。因此我们将帕妥木单抗的可变区移植到IgG1亚类的骨架区后得到新型抗体Pan,以提高其ADCC活性。接下来,利用ProbodyTM技术对Pan进行改造，，构建了可被尿激酶型纤溶酶原激活因子(uPA)激活的新型前抗体Pan-P,以期能减轻抗EGFR抗体引起的毒副反应。综上所述,本课题旨在构建具有强抑瘤效能且可被选择性激活的前抗体,以达到在增强疗效的同时降低其皮肤毒性的目的。方法：首先,利用基因工程技术将帕妥木单抗的可变区基因连接到IgG1亚类抗体Fc段,转入CHO细胞中表达、纯化得到新型抗体Pan。利用以下手段对其进行表征分析：SDS-PAGE等分析其分子量和纯度；Biacore和ELISA测定抗体的亲和力和抗原结合力；CCK-8试验测定抗体对A431细胞的生长抑制效应；LC-MS测定Pan的糖型比例；ADCC报告基因试验评估其体外ADCC活性。最后,在荷瘤小鼠体内评估其抑瘤活性。其次,利用ProbodyTM技术对Pan抗体进行改造。改造方法为在其轻链N端添加了封闭肽、连接肽及酶切底物肽等序列而得到了前抗体Pan-P。利用以下方法对其进行表征分析：SDS-PAGE、LC-MS分析其可被uPA特异性酶切的特性；Biacore、ELISA及FACS表征其可被uPA酶切且激活的特性；CCK-8试验测定其激活后对A431及DiFi细胞生长抑制的活性；冰冻组织原位染色技术评估Pan-P在结直肠癌患者肿瘤组织的酶切激活特性；同时在荷瘤小鼠体内评估Pan-P的酶切激活后靶向性。最后,在荷瘤小鼠体内评估其抑瘤活性。结果：我们获得了Pan抗体,经分析其纯度大于99%。经测定,Pan的亲和力约为7.4×10-10M,与帕妥木单抗相似。同时,CCK-8试验也证实其保持了帕妥木单抗对A431细胞的生长抑制效能。更重要的是,我们发现在Pan的糖型比例中,非岩藻糖修饰的糖型比率为33%左右,这可能有助于增强其ADCC活性。在ADCC报告基因试验中,Pan介导ADCC效应的效能显著高于帕妥木单抗。尤其值得注意的是,Pan显示的体内抑瘤效能较之帕妥木单抗更强。在此基础上改造得到的前抗体Pan-P可被酶切激活而恢复完全功能活性。具体结果如下：SDS-PAGE、LC-MS等试验证实uPA可对Pan-P进行特异性酶切。Biacore、ELISA及CCK-8等试验证实Pan-P可被酶切激活而恢复与Pan相似的功能活性。更重要的是，Pan-P在结直肠癌患者肿瘤组织中亦可被酶切激活。最后,在荷瘤小鼠体内证实了Pan-P的靶向性及抑瘤活性。结论：综上所述,我们最终得到了具有酶激活特性及增强的抗肿瘤效能的前抗体Pan-P。未来用于结直肠癌等的治疗时,将有望增强治疗效果并缓解抗EGFR拮抗剂对正常组织的毒性,提升治疗指数。
[Abstract]:Objective: Patumumab (Panitumumab) is a specific antibody targeting epidermal growth factor receptor (EGFR). However, because it is a IgG2 subclass antibody, antibody dependent cell mediated cytotoxicity of (ADCC) is weak. So we transplanted the variable region of Patumumab into the skeleton region of IgG1 subclass and obtained a new antibody Pan, to improve its ADCC activity. After that, Pan was modified by ProbodyTM technique to construct a novel prokaryotic antibody (Pan-P,) activated by urokinase type plasminogen activator (uPA) in order to reduce the toxicity caused by anti-EGFR antibody. In conclusion, the aim of this study is to construct proantibodies which have strong tumor inhibition and can be selectively activated in order to enhance the therapeutic effect and reduce the skin toxicity. Methods: firstly, the variable region gene of the patumab antibody was linked to the Fc segment of IgG1 subclass antibody by genetic engineering technique, and expressed in CHO cells. The novel antibody Pan. was purified and purified. The molecular weight and purity of A431 cells were analyzed by SDS-PAGE, Biacore and ELISA were used to determine the affinity and antigen binding ability of A431 cells, and the growth inhibition of A431 cells was determined by CCK-8 assay. LC-MS and ADCC reporter gene assay were used to determine the proportion of Pan and ADCC activity in vitro. Finally, tumor inhibitory activity was evaluated in tumor-bearing mice. Secondly, Pan antibody was modified by ProbodyTM technique. The pre-antibody Pan-P. was obtained by adding the sequence of blocking peptide, ligating peptide and digesting substrate peptide to the N-terminal of its light chain. The characterization analysis was carried out by the following methods: SDS-PAGE,LC-MS, Biacore,ELISA and FACS were used to characterize the activity of the enzyme digested by uPA, and the characteristics of uPA specific enzyme digestion were analyzed by SDS-PAGE,LC-MS, Biacore,ELISA and FACS, respectively, and the results showed that it could be digested and activated by uPA. The growth inhibition activity of A431 and DiFi cells was determined by CCK-8 assay, and the activation characteristics of Pan-P in tumor tissue of colorectal cancer were evaluated by in situ staining technique. At the same time, the target activity of Pan-P was evaluated in tumor-bearing mice. Finally, tumor inhibitory activity was evaluated in tumor-bearing mice. Results: Pan antibody was obtained and its purity was higher than 99%. The affinity of Pan was about 7.4 脳 10 ~ (-10) M, which was similar to that of Patumumab. At the same time, CCK-8 test also confirmed that it maintained the growth inhibitory effect of Patumumab on A 431 cells. More importantly, we found that the ratio of non-fucose-modified sugars to Pan is about 33%, which may help to enhance its ADCC activity. In the ADCC reporter gene test, the efficiency of Pan mediated ADCC effect was significantly higher than that of Patumumab. In particular, Pan showed stronger tumor inhibition than Patumumab. On this basis, the pre-antibody Pan-P can be activated by enzyme digestion to restore its full functional activity. The results were as follows: SDS-PAGE,LC-MS et al confirmed that uPA could specifically endonuclease Pan-P, and Biacore,ELISA and CCK-8 showed that Pan-P could be activated by enzyme digestion to restore the functional activity similar to Pan. More importantly, Pan-P can also be activated by enzyme digestion in tumor tissues of colorectal cancer patients. Finally, Pan-P targeting and tumor-inhibiting activity were confirmed in tumor-bearing mice. Conclusion: to sum up, we finally got pre-antibody Pan-P. with enzyme activation and enhanced anti-tumor effect. In future treatment of colorectal cancer, it is expected to enhance the therapeutic effect, alleviate the toxicity of EGFR antagonist to normal tissue, and improve the therapeutic index.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R730.5

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