Development of an antibody-dependent cellular cytotoxicity reporter assay for measuring anti-Middle East Respiratory Syndrome antibody bioactivity

Abstract

Middle East Respiratory Syndrome coronavirus (MERS-CoV) is a highly virulent pathogen that causes Middle East Respiratory Syndrome (MERS). Anti-MERS-CoV antibodies play an integral role in the prevention and treatment against MERS-CoV infections. Bioactivity is a key quality attribute of therapeutic antibodies, and high accuracy and precision are required. The major methods for evaluating the antiviral effect of antiviral antibodies include neutralization assays using live viruses or pseudoviruses are highly variable. Recent studies have demonstrated that the antibody-dependent cellular cytotoxicity (ADCC) activity of antiviral antibodies is more consistent with the virus clearance effect in vivo than neutralization activity. However, no reports evaluating the ADCC activity of anti-MERS antibodies have been published to date. Here, we describe the development of a robust and reliable cell-based reporter gene assay for the determination of ADCC activity of anti-MERS antibodies using 293T/MERS cells stably expressing the spike protein of MERS-CoV (MERS-S) as target cells and the engineered Jurkat/NFAT-luc/FcγRIIIa stably expressing FcγRIIIA and NFAT reporter gene as effector cells. According to the ICH-Q2 analytical method guidelines, we carefully optimized the experimental conditions and assessed the performance of our assay. In addition, we found that the ADCC activity of afucosylated anti-MERS antibodies is higher than their fucosylated counterparts. The establishment of this ADCC determination system provides a novel method for evaluating the bioactivity of anti-MERS antibodies and improving ADCC activity through modification of N-glycosylation of the Fc segment.

Profile of Immunoglobulin G N-Glycome in COVID-19 Patients: A Case-Control Study

“…Despite SARS-CoV-2, two coronavirus-induced epidemics have appeared over the past two decades, including SARS-CoV in 2003 and MERS-CoV in 2012. Researchers have observed an increase in ADCC activity induced by afucosylated anti-MERS antibodies than their fucosylated counterparts, which provides a novel perspective for evaluating the role of the modification of N -glycosylation of the Fc segment in improving ADCC activity ( 37 ). Moreover, mechanistic experiments demonstrated that during dengue infection, afucosylated IgG promotes FcγRIIIa signaling, and then enhances replication of the virus in monocytes ( 38 ).…”

Cell-based reporter assays for measurements of antibody-mediated cellular cytotoxicity and phagocytosis against SARS-CoV-2 spike protein

“…The nuclear factor of activated T-cells (NFAT) signaling pathway is essential for Fcγ receptors (FcγRs) activation, which plays a critical role in ADCC and ADCP ( Aramburu et al, 1995 , Oh-hora and Rao, 2009 ); the NFAT-reporter systems are commonly used as surrogate ADCC or ADCP effectors. Human FcγRIIa (CD16a) or FcγRIIIa (CD32a)-expressing NFAT reporter cells are widely-used effector cells for ADCC or ADCP measurements for therapeutic monoclonal antibodies (mAbs) ( Cao et al, 2020 , Cheng et al, 2014 , Liu et al, 2021 , Parekh et al, 2012 ). In this study, we generated cells with stable expression of full-length SARS-CoV-2 spikes as target cells and developed NFAT-reporter cell-based assays for measurements of ADCC and ADCP activities of antibodies.…”

Development of a reporter gene assay for antibody dependent cellular cytotoxicity activity determination of anti‐rabies virus glycoprotein antibodies

“…The ADCC effector cells Jurkat/NFAT‐luc/FcγRⅢa were developed as described previously 14 . Briefly, Jurkat T cells were first transfected by electroporation with plasmid pcDNA3.1, which expresses the human FcγRⅢa, and G418‐resistant cell clones were screened by fluorescence‐activated cell sorting for FcγRⅢa expression.…”

“…The traditional ADCC assay that uses peripheral blood mononuclear cells or NK cells isolated from human blood as effector cells is not suitable for routine quantitative measurement of ADCC activity due to poor reproducibility and low sensitivity. Our laboratory has established several reporter gene‐based bioactivity determination methods, 10–13 including the ADCC assay for anti‐MERS antibodies, 14 and these methods have obvious advantages compared with traditional methods. For RABV, to date, only a few studies have demonstrated that anti‐RABV antibodies and lymphocytes from human peripheral blood could mediate the lysis of infected cells by ADCC 15,16 …”