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Development of Bioluminescent Cell-based Assay Platforms for Quantitative Measurement of ADCC/ADCP Activities for SARS-CoV-2 Antibodies

Jun Wang, Pete Stecha, Jim Hartnett, Brad Swanson, Frank Fan, Mei Cong and Zhijie Jey Cheng
Promega Corporation, 2800 Woods Hollow Rd, Madison, WI 53711

SARS-CoV-2 is a newly emerged coronavirus responsible for the current COVID-19 pandemic which results in 80 million cases of infection worldwide with almost 1.8 million deaths as if December 2020. SARS-CoV-2 relies on its surface spike protein to bind to human host cell receptor angiotensin-converting enzyme 2 (ACE2) which is a step critical for viral entry, and thus spike protein has been the main target of antiviral mAb therapy and vaccine development. However, the mechanisms of anti-spike neutralizing antibodies are still not fully understood. Besides neutralization by blocking its interaction with ACE2, anti-spike antibodies may have additional anti-viral activities mediated by Fc domain, including antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Therefore, it is very important to develop quantitative methods to help understand how anti-spike antibodies play protective and potential pathogenic roles to guide drug design and clinical development.

Previously we developed two cell-based assay platforms for quantitative measurement of antibody Fc functions: FcgR reporter bioassays using engineered reporter effector cell lines to measure ADCC and ADCP activities via activating FcγRs (FcgRI, FcgRIIa, FcgRIIIa), and improved PBMC ADCC cytotoxicity assay using primary PBMC and engineered HiBiT-expressing target cells. To specifically measure the Fc functions for SARS-CoV-2 spike antibodies, we further developed two engineered S protein-expressing stable cell lines. A panel of commercial recombinant human or chimeric anti-spike antibodies were selected in the study, with some of their sequences originally derived from patients recovered from COVID-19 infection. Four anti-spike antibodies including three anti-S1 Ab and an anti-S2 Ab, showed positive ADCC and ADCP reporter activities when a CHO-K1 cell line stably expressing SARS-CoV-2 spike protein was used as target cells in reporter-based ADCC and ADCP assays. Similar results and Fc functions were confirmed for both anti-S1 and anti-S2 Abs in PBMC ADCC assay using a CHO-K1 cells stably expressing SARS-CoV-2 spike protein and a HaloTag-HiBiT protein with the latter serving as an indication of target cell lysis when being released and detected in the medium after ADCC occurs. Our results demonstrate that these two cell-based assay platforms combined with engineered spike-expressing target cells can potentially serve as valuable tools to help understand Fc-mediated effector functions for anti-spike antibodies in therapeutic drug development and also from the patient’s samples after vaccine administration.