The S2 subunit of spike encodes diverse targets for functional antibody responses to SARS-CoV-2

The SARS-CoV-2 virus responsible for the COVID-19 global pandemic has exhibited a striking capacity for viral evolution that drives continued evasion from vaccine and infection-induced immune responses. Mutations in the receptor binding domain of the S1 subunit of the spike glycoprotein have led to considerable escape from antibody responses, reducing the efficacy of vaccines and monoclonal antibody (mAb) therapies. Therefore, there is a need to interrogate more constrained regions of Spike, such as the S2 subdomain. Here, we describe a collection of S2 mAbs from two SARS-CoV-2 convalescent individuals that target multiple regions in the S2 subdomain and can be grouped into at least five epitope classes. Most did not neutralize SARS-CoV-2 with the exception of C20.119, which bound to a highly conserved epitope in the fusion peptide and showed broad binding and neutralization activity across SARS-CoV-2, SARS-CoV-1, and closely related zoonotic sarbecoviruses. Several of the S2 mAbs tested mediated antibody-dependent cellular cytotoxicity (ADCC) at levels similar to the S1 mAb S309 that was previously authorized for treatment of SARS-CoV-2 infections. Three of the mAbs with ADCC function also bound to spike trimers from HCoVs, such as MERS-CoV and HCoV-HKU1. Our findings suggest there are diverse epitopes in S2, including functional S2 mAbs with HCoV and sarbecovirus breadth that likely target functionally constrained regions of spike. These mAbs could be developed for potential future pandemics, while also providing insight into ideal epitopes for eliciting a broad HCoV response. ### Competing Interest Statement J.O. is a consultant for Aerium Therapeutics, Inc. H.Y.C reported consulting with Ellume, Merck, Abbvie, Pfizer, Medscape, Vindico, and the Bill and Melinda Gates Foundation. She has received research funding from Gates Ventures, and support and reagents from Ellume and Cepheid outside of the submitted work. J.O. and J.G are listed on a patent application (22-173-US-PSP2) and license agreement with Aerium Therapeutics, Inc for SARS-CoV-2 mAbs not described in this manuscript.