IgGs, scFvs, ACE2‐huFc‐Avi, and coronavirus RBDs were expressed and purified from Expi293F cells cultured in 33% Expi293 Expression/66% FreeStyle Expression medium and grown in baffled polycarbonate shaking flasks at 37°C and 8% CO2. Cells were grown to a density of ~3 ×10^6/ml and transiently transfected using FectoPro. Briefly, 0.5 μg DNA/ml final transfection volume was added to culture medium (1/10 volume of final transfection) followed by FectoPro at a concentration of 1.3 μl/ml final transfection volume and incubated at RT for 10 min. Transfection mixtures were added to cells, which were then supplemented with d‐glucose (4 g/L final concentration) and 2‐propylpentanoic (valproic) acid (3 mM final concentration). Cells were harvested 3–5 days after transfection via centrifugation at 18,000g for 15 min. Cell culture supernatants were filtered using a 0.22 μm filter prior to purification.

Infection with SARS-CoV-2 elicits robust antibody responses in some patients, with a majority of the response directed at the receptor binding domain (RBD) of the spike surface glycoprotein. Remarkably, many patient-derived antibodies that potently inhibit viral infection harbor few to no mutations from the germline, suggesting that naïve antibody libraries are a viable means for discovery of novel SARS-CoV-2 neutralizing antibodies. Here, we used a yeast surface-display library of human naïve antibodies to isolate and characterize three novel neutralizing antibodies that target the RBD: one that blocks interaction with angiotensin-converting enzyme 2 (ACE2), the human receptor for SARS-CoV-2, and two that target other epitopes on the RBD. These three antibodies neutralized SARS-CoV-2 spike-pseudotyped lentivirus with IC₅₀ values as low as 60 ng/ml in vitro. Using a biolayer interferometry-based binding competition assay, we determined that these antibodies have distinct but overlapping epitopes with antibodies elicited during natural COVID-19 infection. Taken together, these analyses highlight how in vitro selection of naïve antibodies can mimic the humoral response in vivo, yielding neutralizing antibodies and various epitopes that can be effectively targeted on the SARS-CoV-2 RBD.