Lilly Biotechnology Center, Lilly Research Laboratories, Eli Lilly and Company, 10290 Campus Point Drive, San Diego, California, 92121, USA.
Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana, 46285, USA.
AAPS J. 2020 Apr 16;22(3):68. doi: 10.1208/s12248-020-00444-1.
Treatment-emergent antidrug antibodies (TE-ADA) pose a major challenge to the development of biotherapeutics. The antidrug antibody responses are highly orchestrated and involve many types of immune cells and biological processes. Biological drug internalization and processing by antigen-presenting cells (APCs) are two initial and critical steps in the cascade of events leading to T cell-dependent ADA production. The assays thus far described in literature to evaluate immunogenicity potential/risk as a function of APC activity mainly focus on internalization of labeled drug candidates in vitro. Herein, we describe a high-throughput Förster Resonance Energy Transfer (FRET)-based assay for assessing both internalization and processing using CD14+ monocyte-derived dendritic cells (DCs) as APCs. Antigen-binding fragment F(ab')2 against IgG fragment crystallizable gamma (Fcγ) was labeled with the activatable FRET pair TAMRA-QSY7 as a universal probe for antibodies and proteins with a fragment crystallizable (Fc) domain. The assay was qualified using six mAbs of known clinical immunogenicity and one IgG1 isotype antibody using Design of Experiment (DoE). Correlation analysis of internalization and clinical immunogenicity data showed that this FRET-based internalization assay was able to detect clinically immunogenic antibodies. This method provides a tool for analyzing/screening the immunogenicity risk of biological candidates by assessing one of the critical components of the ADA formation process within the broader context of an immunogenicity risk assessment strategy.
治疗中出现的抗药物抗体（TE-ADA）对生物治疗药物的开发构成了重大挑战。抗药物抗体反应是高度协调的，涉及许多类型的免疫细胞和生物过程。生物药物被抗原呈递细胞（APCs）内化和处理是导致 T 细胞依赖性 ADA 产生的级联反应中的两个初始和关键步骤。迄今为止，文献中描述的评估免疫原性潜力/风险的测定法主要集中在体外评估标记候选药物的内化。在此，我们描述了一种高通量的基于Förster 共振能量转移（FRET）的测定法，用于评估使用 CD14+单核细胞衍生的树突状细胞（DCs）作为 APCs 的内化和处理。针对 IgG 片段结晶γ（Fcγ）的抗原结合片段 F(ab')2 用可活化的 FRET 对 TAMRA-QSY7 进行标记，作为具有片段结晶（Fc）结构域的抗体和蛋白质的通用探针。使用 Design of Experiment (DoE)，使用六种具有已知临床免疫原性的 mAb 和一种 IgG1 同型抗体对该测定法进行了资格鉴定。内化和临床免疫原性数据的相关分析表明，该基于 FRET 的内化测定法能够检测出临床上免疫原性的抗体。该方法提供了一种工具，可通过评估 ADA 形成过程中的一个关键组成部分，在免疫原性风险评估策略的更广泛背景下，分析/筛选生物候选物的免疫原性风险。
