The knock-in mice. including severe acute respiratory syndrome (SARS),1 Middle East respiratory syndrome (MERS)2, and coronavirus disease 2019 (COVID-19).3,4 One of the challenges to control coronaviruses is that they evolve constantly, even though slower than HIV and influenza.5 Analyses of over 28,000 gene sequences of SARS-CoV-2 spike protein (S-protein) in May 2020 exposed a D614G amino acid substitution (SARS-CoV-2-D614G) that was rare before March 2020, but increased greatly in frequency as the pandemic spread worldwide, reaching over 74% of all published sequences by June 20206 and 81% by May 2021 (GISAID). Development of coronaviruses renders them ability to evade virus-specific medications.7,8 Recently, the emergence of multiple mutant variants of SARS-CoV-2, including B.1.1.7 (UK), B.1.351 (South Africa), P.1 (Brazil)9, and B.1.61710 (India) manifests such challenge. In fact, a monoclonal antibody against SARS-CoV-2, bamlanivimab, has been revoked Emergency Use Authorization for expected poor overall performance against variants currently popular in the US (FDA news). In theory, broad-spectrum coronavirus therapeutics can withstand viral mutations and be potentially utilized in long term campaigns against different coronavirus outbreaks. The key to developing broad-spectrum coronavirus therapeutics is definitely to identify broad-spectrum anti-viral focuses on. Although RNA polymerase is definitely a broad anti-RNA virus target, it suffers from low specificity and effectiveness.11,12 By employing a multi-dimensional approach, Gordon et al. proposed a set of potential pan viral target for coronaviruses, but the druggability of these targets are yet to be evaluated.13 ACE2 fusion proteins can act as decoy receptors to capture SARS-CoV-2,14,15 but the affinity and developability of these proteins are generally less than antibodies. Recently, Rappazzo et al. generated a set of monoclonal antibodies that bound to a large panel of coronaviruses, but their neutralizing capabilities have not been tested yet.16 The infection of SARS-CoV-2 is triggered by binding of their envelope spike glycoproteins (S-protein) to angiotensin-converting enzyme 2 (ACE2) molecules indicated on sponsor cells.17,18 The S-protein consists of two subunits: (1) S1-subunit (also called S1-protein) at N-terminal, containing the receptor-binding domain (RBD) responsible for ACE2 binding; (2) S2-subunit at C-terminal responsible for membrane fusion.18 The RBD of SARS-CoV-2 has been heavily targeted by antibodies as well as small molecule Cladribine approaches,19C23 but the RBD-targeting approaches are prone to drug resistance caused by viral evolution and are not broad-spectrum. ACE2 is definitely a Rabbit polyclonal to ADI1 type-I transmembrane glycoprotein that takes on important tasks in maintaining blood Cladribine pressure homeostasis in the renin-angiotensin system.24,25 Cladribine It is a shared receptor for multiple coronaviruses, such as SARS-CoV-2, SARS-CoV, HCoV-NL63,17,26,27 bat coronavirus RaTG13,28 pangolin coronavirus GX/P2V/2017 and GD/1/2019.29 SARS-CoV, a detailed sibling of SARS-CoV-2 in the coronavirus family, was the culprit that caused SARS outbreak in 2003,3 while HCoV-NL63 infects human much more frequently but causes only chilly symptoms with moderate clinical effects.30 Binding of coronavirus to ACE2 not only facilitates viral entry into the sponsor cells, but also down-regulates ACE2 expression.31,32 Previous results revealed the RBD-binding site of ACE2 does not overlap with its catalytic site,33C35 it is therefore hypothesized that targeting the RBD-binding site on ACE2 with antibodies can block the access of all ACE2-dependent coronaviruses, while sparing ACE2s physiological activities. Such antibodies can be theoretically utilized in controlling both current and long term coronavirus outbreaks and tolerate viral mutations. By focusing on ACE2, additionally, the antibody could be evaluated in HCoV-NL63 individuals even when COVID-19 individuals are no longer available for medical tests. To test the hypothesis, we generated a monoclonal antibody, namely 3E8, to target the RBD-binding site on ACE2. The restorative potentials and security profiles of 3E8 were investigated and the key binding sites of 3E8 on human being ACE2 molecule were exposed by cryo-EM and mutation studies to aid long term drug discovery effort. Results Antibody generation by hybridoma technology BALB/c mice were immunized with Fc-tagged human being ACE2 protein and the sera were screened for binding to ACE2 (supplementary Fig. 1a) and obstructing of SARS-CoV-2 S1-subunit and ACE2 connection (supplementary Fig. 1b). Hybridoma cells were constructed and the supernatants were screened.