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A double punch against SARS-CoV-2 Monoclonal antibodies are an important weapon in the battle against COVID-19. However, these large proteins are difficult to produce in the needed quantities and at low cost. Attention has turned to nanobodies, which are aptly named, single-domain antibodies that are easier to produce and have the potential to be administered by inhalation. Koenig et al. describe four nanobodies that bind to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and prevent infection of cells (see the Perspective by Saelens and Schepens). Structures show that the nanobodies target two distinct epitopes on the SARS-CoV-2 spike protein. Multivalent nanobodies neutralize virus much more potently than single nanobodies, and multivalent nanobodies that bind two epitopes prevent the emergence of viral escape mutants. Science, this issue p. eabe6230; see also p. 681, SARS-CoV-2–neutralizing nanobodies were combined to design potent multivalent nanobodies., INTRODUCTION The global scale and rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pose unprecedented challenges to society, health care systems, and science. In addition to effective and safe vaccines, passive immunization by antibody-related molecules offers an opportunity to harness the vertebrate immune system to fight viral infections in high-risk patients. Variable domains of heavy-chain–only antibodies (VHHs), also known as nanobodies, are suitable lead molecules in such efforts, as they are small, extremely stable, easy to engineer, and economic to produce in simple expression systems. RATIONALE We engineered improved multivalent nanobodies neutralizing SARS-CoV-2 on the basis of two principles: (i) detailed structural information of their epitopes and binding modes to the viral spike protein and (ii) mechanistic insights into viral fusion with cellular membranes catalyzed by the spike. RESULTS Nanobodies specific for the receptor binding domain (RBD) of SARS-CoV-2 spike were identified by phage display using nanobody libraries from an alpaca and a llama immunized with the RBD and inactivated virus. Four of the resulting nanobodies—VHHs E, U, V, and W—potently neutralize SARS-CoV-2 and SARS-CoV-2–pseudotyped vesicular stomatitis virus. X-ray crystallography revealed that the nanobodies bind to two distinct epitopes on the RBD, interfaces “E” and “UVW,” which can be synergistically targeted by combinations of nanobodies to inhibit infection. Cryo–electron microscopy (cryo-EM) of trimeric spike in complex with VHH E and VHH V revealed that VHH E stabilizes a conformation of the spike with all three RBDs in the “up” conformation (3-up), a state that is typically associated with activation by receptor binding. In line with this observation, we found that VHH E triggers the fusion activity of spike in the absence of the cognate receptor ACE2. VHH V, by contrast, stabilizes spike in a 2-up conformation and does not induce fusion. On the basis of the structural information, we designed bi- and trivalent nanobodies with improved neutralizing properties. VHH EEE most potently inhibited infection, did not activate fusion, and likely inactivated virions by outcompeting interaction of the virus with its receptor. Yet evolution experiments revealed emergence of escape mutants in the spike with single–amino acid changes that were completely insensitive to inhibition by VHH EEE. VHH VE also neutralized more efficiently than VHH E or VHH V alone; stabilized the 3-up conformation of spike, as determined by cryo-EM; and more strongly induced the spike fusogenic activity. We conclude that the premature activation of the fusion machinery on virions was an unexpected mechanism of neutralization, as enhanced neutralization could not be attributed simply to better blocking of virus-receptor interactions. Activation of spike in the absence of target membranes likely induces irreversible conformational changes to assume the energetically favorable postfusion conformation without catalyzing fusion per se. Simultaneous targeting of two independent epitopes by VHH VE largely prevented the emergence of resistant escape mutants in evolution experiments. CONCLUSION Our results demonstrate the strength of the modular combination of nanobodies for neutralization. Premature activation of spike by nanobodies reveals an unusual mode of neutralization and yields insights into the mechanism of fusion. Bivalent nanobodies neutralize by inducing postfusion conformation of the SARS-CoV-2 spike. On virions, SARS-CoV-2 spike trimers are mostly in an inactive configuration with all RBDs in the down conformation (left). Binding of bivalent nanobody VE stabilizes the spike in an active conformation with all RBDs up (middle), triggering premature induction of the postfusion conformation, which irreversibly inactivates the spike protein (right)., The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread, with devastating consequences. For passive immunization efforts, nanobodies have size and cost advantages over conventional antibodies. In this study, we generated four neutralizing nanobodies that target the receptor binding domain of the SARS-CoV-2 spike protein. We used x-ray crystallography and cryo–electron microscopy to define two distinct binding epitopes. On the basis of these structures, we engineered multivalent nanobodies with more than 100 times the neutralizing activity of monovalent nanobodies. Biparatopic nanobody fusions suppressed the emergence of escape mutants. Several nanobody constructs neutralized through receptor binding competition, whereas other monovalent and biparatopic nanobodies triggered aberrant activation of the spike fusion machinery. These premature conformational changes in the spike protein forestalled productive fusion and rendered the virions noninfectious.