Development of a FRET-Based Assay for Analysis of mAbs Internalization and Processing by Dendritic Cells in Preclinical Immunogenicity Risk Assessment

AbstractTreatment-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 candidatesin vitro. Herein, we describe a high-throughput F örster 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 da ta 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 component...
Source: The AAPS Journal - Category: Drugs & Pharmacology Source Type: research