Antibodies targeting functional domains of the SARS-CoV-2 Spike protein

John S. Kenney, Kurt DeShayes, Catherine Vo, Emmeline Truong, Rick Chang, Opel Arenas, Shyairra Dodd, Huda Abushanab, Ron Gamatero, Edgar Rodriguez, Glen Lin, Leonel Santibanez-Vargas, Emily Keshner, and Joshua K. Lowitz. 

Antibody Solutions, 3033 Scott Blvd., Santa Clara, CA 95054. 

Infectivity of SARS-CoV-2 is regulated by three regions on the spike protein: a receptor binding motif (RBM), a subunit (S)1/S2 cleavage site acted upon by the host protease furin, and an S2’ cleavage acted upon by host protease TMPRSS2. The relative contribution of each site to SARS-CoV-2 infectivity and progression to COVID-19 progression has been the subject of debate. All three sites on the spike protein are potential sites of therapeutic intervention. We sought to generate antibodies with fine specificity to each of these sites that could be used to analyze virus infection and serve as potential therapeutic antibody leads.

In order to focus antibody discovery on the three sites, we designed peptides spanning each site that could be used to raise antibodies with fine specificity.  Molecular modeling was used to identify peptides that would best match the secondary structure contained within the native spike protein.  Mice were immunized with peptide carrier protein conjugates and promising monoclonal antibodies were isolated using our Hybridoma Library(TM) platform. Screening was performed with a range of recombinant soluble spike proteins or trimer transfected cells as well as functional assays for inhibition of spike protein binding to ACE2 or cleavage by furin.

We describe results for 3 antibodies, each raised to one of the 3 sites.  The RBM antibody binds monomeric and trimeric forms of soluble spike protein containing the RBM and spike trimer expressed on cells. It has nM affinity for spike protein and can block the binding of spike protein to soluble ACE2 receptor. The S1/S2 site antibody binds only to spike protein which has an intact, un-mutated S1/S2 site.  It shows sub-nM affinity for spike protein and inhibits cleavage of spike protein by furin. The S2’ site antibody binds only to spike proteins with an intact S2’ site.  It has sub-nM affinity for spike protein.

In summary, we’ve identified 3 unique domain-specific, high-affinity antibodies to the CoV-2 spike protein. They are sensitive to mutations and have functional activity. They should be useful as tools to dissect the mechanisms of SARS-CoV-2 infectivity and serve as potential leads for COVID-19 therapeutics.