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.
Highlights
Over the past two decades, three coronaviruses have appeared in the world and their outbreaks have caused considerable global health panic
The pseudovirus neutralization activity mediated by the Fab segment and the binding affinities with FcγRIIIa mediated by the Fc segment of several anti-Middle East Respiratory Syndrome (MERS)-CoV antibodies with the same antigen binding site and different Fc segments were determined. 2E6 is a neutralizing antibody against MERS-CoV targeting the receptor-binding domain of the spike protein, and were generated by mice immunized with recombinant MERS-S and subsequently selection of hybridoma cell lines. 2E6 can block virus entry in vitro with high efficacy[20]
Afucosylated humanized 2E6 (h2E6) antibody (AF-h2E6) were produced by a Chinese hamster ovary (CHO) host cell line lacking the fucose synthesis ability while the h2E6 were produced by a wildtype CHO cell line
Summary
Over the past two decades, three coronaviruses have appeared in the world and their outbreaks have caused considerable global health panic. Several vaccines and drugs for the prevention or treatment of MERS-CoV infection are currently under investigation, but none have been approved for clinical use[5,6,7,8,9]. Among these vaccines and drugs, passive immunotherapy in vivo using neutralizing monoclonal antibodies (mAbs) has been reported to be effective in the prophylaxis and treatment of MERS-CoV infections[9]. The major method for evaluating the anti-virus effect of the antibody drugs includes neutralization assays using live viruses[19,27] or pseudoviruses[17,24], and animal infection protection e xperiments[22,24]. To date, no reports evaluating the ADCC activity of anti-MERS antibody have been published
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