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

SARS-CoV-2, the causative agent of COVID-19 ¹ , 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 nebulization ¹⁹ , 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 (IC ₅₀ = 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 (IC ₅₀ = 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.