Moores Cancer Center, University of California San Diego, La Jolla, CA, 92093-0815, USA.
Cancer Chemother Pharmacol. 2010 Oct;66(5):919-25. doi: 10.1007/s00280-009-1240-1. Epub 2010 Jan 20.
Monoclonal antibodies (mAb) are an important and growing class of cancer therapeutics, but pharmacokinetic analyses have in many cases been constrained by the lack of standard and robust pharmacologic assays. The goal of this project was to develop a general method for the production of immunoassays that can measure the levels of therapeutic monoclonal antibodies in biologic samples at relevant concentrations.
Alemtuzumab and rituximab are monoclonal approved for the treatment of B-cell malignancies and were used as a model system. Phage-displayed peptide libraries were screened for peptide sequences recognized by alemtuzumab (anti-CD52) or rituximab (anti-CD20). Synthetic biotinylated peptides were used in enzyme-linked immunosorbent assays (ELISA). Peptides directly synthesized on polymer resin beads were used in an immunofluorescent-based assay.
Peptide mimetope sequences were recovered for both mAb and confirmed by competitive staining and kinetic measurements. A peptide-based ELISA method was developed for each. The assay for rituximab had a limit of detection of 4 microg/ml, and the assay for alemtuzumab had a limit of detection of 1 microg/ml. Antibody-specific staining of peptide conjugated beads could be seen in a dose-dependent manner.
Phage-displayed peptide libraries can be a source of highly specific mimetopes for therapeutic mAb. The biotinylated forms of those peptides are compatible with conventional ELISA methods with sensitivities comparable to other assay methods and sufficient for pharmacological studies of those mAb given at high dose. The process outlined here can be applied to any mAb to enable improved pharmacokinetic analysis during the development and clinical use of this class of therapies.
单克隆抗体（mAb）是一类重要且不断发展的癌症治疗药物，但在许多情况下，由于缺乏标准和稳健的药理学检测方法，其药代动力学分析受到限制。本项目的目的是开发一种通用方法，用于生产免疫分析，可以在相关浓度下测量生物样品中治疗性单克隆抗体的水平。
阿仑单抗和利妥昔单抗是批准用于治疗 B 细胞恶性肿瘤的单克隆抗体，被用作模型系统。用噬菌体展示肽文库筛选与阿仑单抗（抗-CD52）或利妥昔单抗（抗-CD20）识别的肽序列。合成的生物素化肽用于酶联免疫吸附测定（ELISA）。直接在聚合物树脂珠上合成的肽用于基于免疫荧光的测定。
回收了两种 mAb 的肽模拟序列，并通过竞争染色和动力学测量进行了验证。为每个 mAb 开发了基于肽的 ELISA 方法。利妥昔单抗的检测限为 4μg/ml，阿仑单抗的检测限为 1μg/ml。可以以剂量依赖性方式观察到肽偶联珠的抗体特异性染色。
噬菌体展示肽文库可以作为治疗性 mAb 的高度特异性模拟物的来源。这些肽的生物素化形式与常规 ELISA 方法兼容，灵敏度与其他检测方法相当，足以用于高剂量给药时这些 mAb 的药代动力学研究。这里概述的过程可以应用于任何 mAb，以在这类疗法的开发和临床应用中改善药代动力学分析。
