Katrina Dionne, Université de Montréal

Katrina Dionne1,2,*, Monika Chandravanshi3,4*, Guillaume Beaudoin-Bussières1,2,*, Keon-Woong Yoon7,*., Geneviève Laroche5, Catherine Bourassa2, Halima Medjahed2, Shilei Ding2, Gjordje Gbric6, Marie-Claude Carrier6, Geneviève Giroux6, William D Tolbert3,4, Martin Cloutier6, Marceline Côté5, Simon Gaudreau6, Pradeep D. Uchil7,#, Marzena Pazgier3,#, Andrés Finzi1,2,#. 1Département de Microbiologie, Infectiologie et Immunologie, Université de Montréal 2Centre de Recherche du CHUM, Montréal, Québec, Canada, H2X 0A9 3Infectious Disease Division, Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA. 4Henry M Jackson Foundation for the Advancement of Military Medicine, Bethesda, MD. 5Department of Biochemistry, Microbiology and Immunology, and Center for Infection, Immunity, and Inflammation, University of Ottawa, Ottawa, ON K1H 8M5, Canada 6Immune Biosolutions Inc, Sherbrooke, Quebec, J1E 0M8, Canada 7Department of Microbial Pathogenesis, Yale University, School of Medicine, New Haven CT 06510, USA *co-first #co-corresponding

Fighting & Responding

Context: While SARS-CoV-2 has become endemic, the threat of future outbreaks necessitates the development of therapeutic pan-coronavirus antibodies (Abs). We previously identified CV3-25, a broad anti-S2 Ab effective in a phase II clinical trial during the Omicron wave (Maranda et al., 2023, Lancet Infect Dis). Here we enhanced its breadth and potency by introducing recently-described artificial intelligence–guided mutations that enhance its breadth (Marden et al., bioRxiv, 2025). The wild-type and broad-spectrum (CV3-25BS) variant was used to generate hybrids with M55, an ACE2-like receptor with increased affinity for the spike. Methods: CV3-25, CV3-25BS, and the hybrid CV3-25-M55 were tested for recognition of Spikes from beta coronaviruses including SARS-CoV and recent SARS-CoV-2 VOCs. Neutralization was tested using pseudoviral and authentic virus assays, and Fc-effector functions were measured with a FACS-based assay. The crystal structure of CV3-25BS Fab bound to a S2 peptide was solved at 1.9 Å. In vivo efficacy against SARS-CoV-2WA1 was tested in K18-hACE2 mice under therapeutic settings. Results: CV3-25BS displayed improved breadth, binding, neutralization and Fc-effector functions. CV3-25-M55 showed a remarkable increase in neutralization potency across SARS-CoV-2 variants. Comparative structural analysis revealed CV3-25BS forms a strong network of interactions at the heavy and light chain interface that changes CDR H1 engagement with the S2 helix to drive increased affinity. CV3-25BS-M55 was the most effective in clearing established infection, preventing mortality and required Fc function for its in vivo efficacy. Importance: These engineered therapeutics Abs represent promising pan-coronavirus candidates, and a potential first line of defence against future pandemics.