Development and Validation of a Cell-Based Binding Neutralizing Antibody Assay for an Antibody –Drug Conjugate

AbstractThe utilization of antibody –drug conjugates (ADCs) has gained considerable attention in the field of targeted cancer therapy due to their ability to synergistically combine the specificity of monoclonal antibodies (mAbs) and the potency of small molecular drugs. However, the immunogenic nature of the antibody component with in ADCs warrants the need for robust immunogenicity testing, including a neutralizing antibody (NAb) assay. Since the mechanism of action (MOA) of the ADC is to first bind to the target cells and then release the payload intracellularly to kill the cells, the most relevant NAb assay format would b e a cell-based killing assay. However, in this paper, we present a case where a cell-based killing assay could not be developed after multiple cell lines and NAb-positive controls (PC) had been tested. Surprisingly, contrary to our expectations, all NAb PCs tested exhibited an increased killing effe ct on the target cells, instead of the expected protective response. This unexpected phenomenon most likely is due to the non-specific internalization of drug/NAb complexes via FcγRs, as an excessive amount of human IgG1 and mouse IgG2a, but not mouse IgG1, greatly inhibited drug or drug/NAb comple xes induced cell death. To overcome this obstacle, we implemented a novel cell-based binding assay utilizing the Meso Scale Discovery (MSD) platform. We also propose that anin vitro cell killing NAb assay is limited to at best monitoring the target binding and...
Source: The AAPS Journal - Category: Drugs & Pharmacology Source Type: research