基于药物—受体原位结合动力学的单克隆抗体药物耐药性机理研究

Drug discovery industry has witnessed the rapid raise of monoclonal antibody (mAb) drugs in the treatment of cancers and self-immune diseases in the past decades, which have now occupied more than half of the biological drug market. Although the action mechanisms of mAb drugs are complicated and greatly vary case by case, the strong and specific interactions between mAb drugs and their targets, which are often membrane receptors, are one of the key factors. Despite of their success in the market, clinical performances of many mAb drugs were greatly compromised by unexpected drug resistance issues. Significant percentage of patients, who used to recover quite well, stopped responding to the mAb drugs after a certain term of drug registration. It has been long time hypothesized that the drug-receptor interactions were interrupted in the resistant cancer cells, causing the failure of mAb therapy. However, traditional biomolecule interaction assays, such as surface plasmon resonance and quartz crystal microbalance, measure the binding kinetics by extracting, purifying and immobilizing the membrane receptors on a solid surface. Such in vitro approaches eliminate the influences of biochemical environments on the drug-receptor interactions and thus were not able to examine the real interactions occurring in the cell membranes. In this project, we utilize a novel surface plasmon resonance microscopy technique, which was recently developed in our group, to study the in situ drug-receptor binding kinetics, as well as to elucidate the mechanisms of durg resistance associated with three FDA-approved mAb drugs, trastuzumab, panitumumab and cetixumab. Compared to the traditional in vitro methods, the present technique quantifies the binding kinetics in the native cellular membrane environment of the receptors, which reduces false-positive and negative incidence in drug screening, and provides valuable information regarding the drug resistance mechanisms.

单克隆抗体药物（单抗药物）以其特异性好、副作用小等优点广泛用于癌症的临床治疗，是当前最具发展潜力的生物药之一。其功效的发挥依赖于单抗药物与疾病相关受体分子的特异性结合。然而临床实践表明，许多病人在接受单抗药物治疗后产生明显的耐药性，严重降低了单抗疗法的成效。测定耐药性细胞中单抗药物—受体原位结合动力学常数能够直接判定药物分子是否仍能与受体有效结合，对于阐明耐药性机理具有重要意义。传统方法主要研究基于纯化蛋白的离体相互作用，没有考虑细胞表面特定化学环境的影响，因而不能区分药敏性和耐药性细胞表面原位结合过程的异同。基于此，本项目拟以申请人最近发展的表面等离子共振显微镜技术测定单抗药物—受体原位结合动力学常数，探索细胞化学环境对其影响规律，比较药敏性和耐药性细胞中药物—受体结合过程的异同，以曲妥珠单抗、帕尼单抗和西妥昔单抗为模型药物研究单抗药物的耐药性机理，为耐药性问题的解决指明方向。

单克隆抗体药物（以下简称单抗药物）是近二十年来获得快速发展和广泛关注的一种新型生物药。单抗药物具有靶向性好、副作用较小的独特优点，使其在癌症和自免疫疾病的临床治疗中获得了日益广泛的应用。本项目提出在单细胞水平上对单抗药物—受体结合动力学进行原位测定，定量获取单细胞表面受体的表达水平，以此探索单抗药物耐药性的形成机理。本项目主要开展了以下几方面的研究工作。在方法研究方面，优化了现有光学检测系统性能，建立了细胞样品制备和图像数据处理方法，提高了检测灵敏度和准确度。在应用研究方面，以两种重要的疾病相关膜蛋白(HER2)和表皮生长因子受体(EGFR)为模型体系，在单细胞水平上测定了抗体—膜受体的原位结合动力学常数，研究了不同蛋白质调控方法对受体表达水平和空间分布的影响规律，探索了本方法在耐药性产生机理和病理样本临床分析中的应用潜力。.本项目所取得的主要研究成果及其科学意义如下。第一，完善了表面等离子共振显微成像技术的理论基础，建立了单细胞中疾病相关受体蛋白（以HER2和EGFR为模型）表达水平的免标记定量分析方法，对细胞信号转导、药物研发和蛋白质工程等领域研究具有重要意义。第二，以抗肿瘤药物赫赛汀为模型药物，揭示了乳腺肿瘤细胞耐药性形成的分子机制，为发展赫赛汀组合疗法、抑制赫赛汀耐药性指明了方向。第三，测定了经荧光标记和纳米粒子标记的单抗药物分子与肿瘤细胞的结合动力学常数，证实了标记过程对蛋白质相互作用的显著影响，进一步显示了本项目所发展的免标记分析技术的重要意义。.就本项目所取得的成果在Angew. Chem. Int. Ed.和Anal. Chem.等期刊上发表学术论文7篇（其中6篇影响因子大于5.0）。提交中国发明专利申请1项，正在审理中。

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