A potent synthetic nanobody targets RBD and protects mice from SARS-CoV-2 infection

SARS-CoV-2, the causative agent of COVID-19¹, recognizes host cells by attaching its receptor-binding domain (RBD) to the host receptor ACE2^2–7. Neutralizing antibodies that block RBD-ACE2 interaction have been a major focus for therapeutic development^8–18. Llama-derived single-domain antibodies (nanobodies, ∼15 kDa) offer advantages including ease of production and possibility for direct delivery to the lungs by nebulization¹⁹, which are attractive features for bio-drugs against the global respiratory disease. Here, we generated 99 synthetic nanobodies (sybodies) by in vitro selection using three libraries. The best sybody, MR3 bound to RBD with high affinity (K_D = 1.0 nM) and showed high neutralization activity against SARS-CoV-2 pseudoviruses (IC₅₀ = 0.40 μg mL⁻¹). Structural, biochemical, and biological characterization of sybodies suggest a common neutralizing mechanism, in which the RBD-ACE2 interaction is competitively inhibited by sybodies. Various forms of sybodies with improved potency were generated by structure-based design, biparatopic construction, and divalent engineering. Among these, a divalent MR3 conjugated with the albumin-binding domain for prolonged half-life displayed highest potency (IC₅₀ = 12 ng mL⁻¹) and protected mice from live SARS-CoV-2 challenge. Our results pave the way to the development of therapeutic nanobodies against COVID-19 and present a strategy for rapid responses for future outbreaks.