Koenig et al. identified and engineered variable domains of heavy-chain-only antibodies (nanobodies) associated with the spike protein receptor-binding domain (RBD) of SARS-CoV-2. Engineered nanobodies solve issues with conventional antibodies, such as high production costs, low thermostability, and difficult modifiability. Researchers identified the nanobodies by screening alpaca and llama nanobody libraries using phage display. Their work produced 23 potential nanobodies, which were assessed for their neutralizing activity. Four nanobodies – E, U, H, and V – neutralized SARS-CoV-2-pseudotyped virus infection in a dose-dependent manner. A competitive binding assay found that the nanobodies bind to two distinct interfaces of the RBD; U, H, V competed for the same epitope and were able to bind concurrently with the E nanobody. X-ray crystallography and cryo-electron microscopy revealed that E stabilized the spike protein complex in an “up” conformation. As a result, the spike fusion process was triggered, which caused a premature change in the spike protein, irreversibly preventing infection. Flow cytometry data shows that U, H, and V bound outcompeted ACE2 for the RBD, keeping the spike protein in the “down” conformation. Significantly, the “down” conformation is thought to be less accessible to neutralizing antibodies for binding. Koenig et al. manipulated the nanobodies to create multivalent nanobodies with over 100 fold neutralizing monovalent nanobody activity. Since E nanobodies do not compete directly with U, H, and V, combinations of such nanobodies yield strong synergistic neutralizing ability.