新型抗EGFR“前抗体偶联药物”的制备及功能研究

发布时间：2018-04-19 21:19

本文选题：抗体偶联药物 + 前抗体　；参考：《第二军医大学》2016年博士论文

【摘要】：近年来,抗体偶联药物(ADC)已在临床肿瘤治疗中展现出良好的治疗效果。但是,针对EGFR靶点的抗体偶联药物的研发却面临着巨大挑战,其原因正是由于EGFR在人体内分布较为广泛,从肿瘤到正常组织如上皮组织的许多组织均有表达。前期,我们利用Pro-Antibody技术开发出针对EGFR靶点的前抗体,该抗体具有在肿瘤微环境中被特异性激活并靶向EGFR的特性,命名为PanP。在本研究中我们基于前期的成果,首次构建并表达了针对EGFR靶点的前抗体偶联药物(pro-antibody-drug conjugate,PDC)PanP-DM1,简称PDC。期望通过前抗体技术和抗体偶联药物这两种技术的联合运用,使该PDC既能获得肿瘤组织EGFR位点的特异性结合活性,又能获得比常规抗体更强的肿瘤细胞杀伤效应,降低抗EGFR抗体的皮肤毒性反应。本课题在PDC药物制备完成后首先使用SEC-UPLC、LC-MS等技术对其进行了表征,并结合UV/VIS方法完成了DAR值的测定。SEC-UPLC结果显示,我们制备的PDC纯度在95%以上,与PanP相比无明显差异;LC-MS对其脱糖后完整蛋白的分析结果也显示,该PDC精确分子量符合理论序列分子量,去卷积质谱谱图符合抗体偶联药物特征性多簇峰型结构,呈正态分布,以携带2~4个DM1为主;通过质谱峰强度积分并且结合UV/VIS测定法对DAR值进行测定,结果表明我们制备的PDC其DAR值在4.2左右,符合常规ADC药物的最佳药物-抗体偶联比例(Drug-antibody Ratio,DAR)。另外,我们的实验数据显示,偶联DM1之后的PDC药物依然保持了能被肿瘤组织特异性酶(uPA)酶切激活的特性。在体外、体内功能研究中,我们制备的新型PDC药物也表现出比PanP更强的抗肿瘤活性,尤其是在小鼠移植瘤模型中,使用PDC抗体治疗的实验组小鼠在治疗结束后,肿瘤组织完全消除。为进一步研究该PDC药物的抗肿瘤作用机制,我们通过流式细胞术,研究了细胞周期阻滞效应,结果表明PDC被肿瘤细胞内吞后能在溶酶体的作用下释放大量的微管抑制剂药物——DM1,使细胞周期停滞在G2/M期,从而诱导了细胞的凋亡。最后我们还通过观测小鼠体重减低情况对该药物的非特异毒性(安全性)进行了初步评估。通过本课题,我们构建、表达并制备了一种新型的抗EGFR前抗体偶联药物——PanP-DM1,其不但具有肿瘤靶向特异性,而且具有增强的高肿瘤杀伤活性以及更好的安全性,从而具有良好的成药性及临床应用前景。
[Abstract]:In recent years, antibody-coupled drug ADChas shown a good therapeutic effect in clinical tumor therapy. However, the development of antibody-coupled drugs targeting EGFR targets is facing great challenges. The reason is that EGFR is widely distributed in human body and is expressed in many tissues from tumor to normal tissue such as epithelial tissue. In the early stage, we developed a pre-antibody against EGFR target by using Pro-Antibody technique. The antibody was specifically activated in tumor microenvironment and targeted to EGFR, and was named PanP. In this study, we first constructed and expressed pro-antibody-drug conjugate drug PDC-PanP-DM1 (PDC-PanP-DM1) against EGFR targets. It is expected that the combined use of preantibody and antibody-coupled drugs will enable the PDC to obtain both the specific binding activity of EGFR sites in tumor tissues and the killing effect of tumor cells stronger than conventional antibodies. Reduce the skin toxicity of anti-EGFR antibody. SEC-UPLC- LC-MS and other techniques were used to characterize PDC drug preparation. The results of DAR determination of SEC-UPLC showed that the purity of PDC was over 95%. There was no significant difference between LC-MS and PanP in the analysis of the complete protein. The exact molecular weight of the PDC was in accordance with the theoretical molecular weight, and the deconvolution mass spectrogram was consistent with the characteristic multi-cluster structure of the antibody-coupled drugs, and showed a normal distribution. The results showed that the DAR value of our prepared PDC was about 4.2, which was in accordance with the best drug-antibody coupling ratio of conventional ADC drugs. In addition, our experimental data showed that the PDC drug could be activated by tumor tissue specific enzyme (UPA) digestion after coupling with DM1. In vitro and in vivo functional studies, our new PDC drug also showed stronger anti-tumor activity than PanP, especially in mice transplanted tumor model, the experimental group treated with PDC antibody after treatment. Tumor tissue was completely eliminated. In order to further study the anti-tumor mechanism of PDC, we studied cell cycle arrest by flow cytometry. The results showed that PDC could release a large amount of microtubule inhibitor DM1 after endocytosis by lysosome, resulting in cell cycle arrest in G _ 2 / M phase, thus inducing cell apoptosis. Finally, we evaluated the non-specific toxicity (safety) of the drug by observing the weight loss in mice. Through this study, we constructed, expressed and prepared a novel anti-preantibody coupling drug, PanP-DM1, which not only has tumor targeting specificity, but also has enhanced tumor killing activity and better safety. So it has good drug properties and clinical application prospect.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R943

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