基于抗体Fab片段特异性识别多肽配基的体内Infliximab精准富集策略研究

Infliximab (IFX), an immunoglobulin G (IgG) based tumor necrosis factor alpha (TNF-α) blocking antibody, has been developed for the treatment of several autoimmune diseases. However, many patients lose response to IFX therapy because of the inadequate drug level in blood or the formation of anti-drug antibodies. Therapeutic drug monitoring of IFX in patients' serum has shown great importance to these therapies. LC-MS/MS considered as the most promising monitoring technology still have to overcome some major challenges such as the low analyte concentrations and the interference of large amount of endogenous proteins. Previous studies found that antigen-binding fragment (Fab)-specific peptide ligands can specifically bind target mAb. Based on the complex of IFX-Fab and TNF-α, a biomimetic design strategy for Fab-specific peptide ligands of IFX was developed in this study. Peptide immobilized affinity monoliths were then designed here for the high-performance purification and quantification of IFX in complex sample matrices. After a systematically optimization and evaluation, an accurate and rapid enrichment method will finally be developed for the purification of IFX in patients' serum. Furthermore, different sample preparation methods for IFX were systematically compared in terms of their potential value and performance. Affinity enrichment method based on Fab-specific peptide ligands has many advantages over conventional precipitation and immunoaffinity chromatography, such as low cost, better ligand stability and longer life time etc. This research not only provides a tool for the PK/PD studies of IFX, but also shows great significance to the development of purification method for other mAbs.

英夫利昔（Infliximab）已成为治疗炎症性疾病的重要生物药物，系统的PK/PD研究对其临床应用和质量控制至关重要。但体内Infliximab的血药浓度低、样品组成复杂且含有高同源性的IgG，如何精准富集体内样本中的Infliximab是临床血药浓度监测方面所面临的难题。本项目拟针对Infliximab的抗原结合片段通过计算机辅助筛选等技术设计特异性多肽配基；将多肽配基的特异性和聚合物整体材料的优异理化性质相结合开发特异性多肽修饰的高亲和整体富集柱；选取最佳的整体富集柱用于溃疡性结肠炎患者血清样品中Infliximab药物的精准富集与定量分析；通过与多种传统富集方法的系统比对研究，建立高效、高选择性的体内Infliximab精准富集纯化新策略。本项目的开展不仅为Infliximab的临床分析提供了新方法新技术，而且有望推动其它单抗精准富集策略研究，对生物药的生产研发和临床应用意义重大。

英夫利昔单抗（Infliximab）已成为治疗炎症性疾病的重要生物药物，系统的PK/PD研究对其临床应用和质量控制至关重要。如何精准富集复杂样本中的Infliximab是实现上述目标所面临的难题。本项目针对Infliximab的不同特定位点（包含互补性决定区和一致性结合位点等）通过计算机辅助筛选等技术设计特异性多肽配基，进而开发出一系列特异性多肽修饰的高亲和整体富集柱，实现复杂生物样本中Infliximab的高效富集纯化。所取得的成果如下：.1. 采用分子对接和分子动力学模拟等方法，成功设计一系列能与Infliximab不同特定位点特异性结合的新型多肽配基，并通过微量热泳动技术对“配基-单抗”之间的亲和力进行系统研究，探讨了多肽配基的实际应用潜力，从而初步建立了集成计算机辅助筛选和亲和实验验证的多肽配基筛选新方法。.2. 采用多步柱后衍生化法、两步法和一步法等方法结合整体聚合物制备技术，成功制备出不同特异性多肽配基修饰的高亲和富集材料，如HT-25、CW-13或VIM功能化亲和整体材料，完善了其制备技术体系。.3. 成功将上述亲和富集材料应用于复杂体系中Infliximab的高效富集纯化，初步建立了目标单抗精准富集新方法；并在此基础上，将特异性多肽识别理念拓展应用于非霍奇金淋巴瘤患者体内Rituximab的多肽生物传感器的构建与临床应用。.综上所述，本研究成功开发了特异性识别复杂体系中Infliximab的高亲和肽功能化富集整体柱，实现了目标单抗药的高效富集纯化；并将其设计理念拓展到其他单抗药的富集与分析，有望为抗体药物研发和临床分析提供重要技术支撑。