抗体药物及免疫复合物质谱定量分析方法与药代动力学研究

In comparison with small-molecule agents, therapeutic antibody exhibits high targeting specificity, low off-target toxicity, low clinical risk, and prolonged efficacy. Therefore, antibody engineering has been emerging as one of the most promising fields in drug development over the next decade. Despite of the significant advances, the exact mechanisms of the absorption, distribution, and elimination of antibody drugs have yet to be fully elucidated. Traditional immunoassays may suffer from endogenous interferences, lengthen development, and low successful rate. Liquid chromatography/mass spectrometry (LC/MS) based methods have emerged as a promising alternative for protein quantification in biological matrixes, because these methods provide high specificity, high sensitivity, and multiplexing capability and are often not matrix-selective. Nevertheless, there are several challenges associated with the development of LC/MS-based methods. To address these fundamental challenges, we propose a generally applicable strategy that enables high-throughput, streamlined method development for sensitive, selective, and reliable quantification of therapeutic antibody and immune complexes in biological samples. As a proof of concept, this strategy will be applied to the characterization of anti-methotrexate IgG1 antibody(AMI)/bis-methotrexate (Bis-MTX) complexes in preclinical models with regard to binding stoichiometry and in vivo kinetics of immune complex formation/disposition/elimination. This work represents the first extensive pharmacokinetic investigation of antibody drug and immune complexes. The proposed procedure has the potential to develop quantitative methods for a large number of antibody drugs in a short time period. This proposal is adaptable to high-throughput development of sensitive and accurate methods for the quantification of therapeutic proteins in various pharmaceutical and clinical matrixes.

抗体药物凭借其明确的药理活性、药效特异性以及优异的药动学性质迅速成为目前制药工业和药学研究领域最大的研究热点。缺乏准确高效的体内分析方法是目前制约抗体药物研发的主要技术瓶颈。传统的免疫方法存在易受干扰、开发周期长、成本高等缺陷，难以满足现代药物开发需求。质谱技术的进展为抗体药物分析提供了有效的研究平台。但目前采用质谱方法对抗体药物进行定量处于起步阶段，分析流程存在许多亟需完善之处。本项目在前期工作基础上，拟选择AMI为模型药物，构建AMI/Bis-MTX免疫复合物体内模型，针对抗体药物分析中的关键问题，探索和优化以质谱技术为核心的蛋白绝对定量方法，完善并规范基于选择反应监测技术的定向蛋白质组学质谱方法开发策略，建立准确、灵敏、高效的质谱分析平台对抗体药物及免疫复合物体内动力学过程进行系统研究。

单克隆抗体药物近来取得突破性进展，成为目前制药工业和药学研究领域最大的研究热点。缺乏准确高效的体内分析方法是制约抗体药物研发的主要技术瓶颈。质谱技术的进展为抗体药物分析提供了有效的研究平台。但目前采用质谱方法对抗体药物进行定量处于起步阶段，分析流程存在许多亟需完善之处。本项目在前期工作基础上，选择多种抗肿瘤免疫抗体（包括抗肿瘤细胞表面抗原抗体8c2、Ras蛋白HIV-Tat融合抗体GAP161、抗甲氨喋呤单克隆抗体ACA-06等）为模型药物，立足技术体系和平台建设、分析策略优化以及药物动力学评估等不同层面进行创新研究，完成了既定实验目标，取得了以下进展：1）以质谱技术为核心结合新型富集、纯化、分离（包括亲和纯化、非变性胶分离等）体系，成功构建针对抗体药物的准确、灵敏、高效的质谱定量分析平台；2）针对抗体药物分析中的关键问题，探索和优化以质谱技术为核心的蛋白绝对定量方法，建立了基于选择反应监测技术的定向蛋白质组学质谱定量即时优化策略(On-the-fly orthogonal array optimization procedure)；3）系统详细考察影响抗体药物质谱分析定量结果的关键校准因素，规范和完善了针对蛋白质谱绝对定量方法的评价标准；4）完成包括抗甲氨喋呤单克隆抗体ACA-06、抗肿瘤细胞表面抗原抗体（8c2）、抗体药物融合蛋白（GAP161）、抗体免疫复合物等多种抗体药物的临床前药物动力学研究。研究结果对于深入认识抗体药物及免疫复合物的体内动力学特征有指导意义，为抗体药物临床治疗提供理论基础和数据支持。