This approach confirmed the results that we obtained by ELISA and showed that 1C and G4 likely belong to Group 1, and 3A belongs to Group 2 in terms of the binding competition (Supplementary Fig. Introduction SARS-CoV-2 is a coronavirus that causes the human disease COVID-19, which is contagious and can rapidly spread to cause mild to severe infection, including death [CDC (https://www.cdc.gov/coronavirus/types.html)1]. The spread of this newly emergent virus has reached a pandemic level with a significant public impact on the world, leading to more than 25 million infections and more than a 0.85 million deaths worldwide [World Health Organization (WHO) (https://www.who.int/emergencies/diseases/novel-coronavirus-2019)]. In addition to threatening human health, COVID-19 has also caused a significant socio-economic impact around the world [United Nations (https://www.undp.org/content/undp/en/home/coronavirus/socio-economic-impact-of-covid-19.html)]. Although there are relatively successful diagnostic methods to detect the SARS-CoV-2 infection in humans, there are currently no successful therapies that can interfere with virus replication. The small antiviral molecule Remdesivir (Gilead) which inhibits 5-hydroxymethyl tolterodine (PNU 200577) the RNA-dependent RNA polymerase of SARS-CoV-2 decreases the recovery time in patients with COVID-192, but it most likely cannot completely stop or prevent SARS-CoV-2 infections in humans. Another small antiviral molecule, GRL-0617, shows promise in interfering with the SARS-CoV-2 replication by inhibiting the papain-like protease, however, it is yet to be tested in clinical trials3. Moreover, there are no FDA-approved vaccines to prevent SARS-CoV-2 infections in humans, although several groups are currently in the pursuit such vaccines [WHO (https://www.who.int/publications/m/item/draft-landscape-of-covid-19-candidate-vaccines)]. Therefore, rapid development of therapeutics and preventative strategies has become an essential and urgent need 5-hydroxymethyl tolterodine (PNU 200577) to fight the COVID-19 pandemic. The trimeric spike (S) proteins that protrude through the envelope of the SARS-CoV-2 virion mediate virus entry into the host cells by interacting with the human ACE2 receptor4C9. Therefore, a major target for anti-SARS-CoV-2 neutralizing antibodies in development are to block the interaction of SARS-CoV-2 S1 protein with ACE2. In particular, two popular strategies 5-hydroxymethyl tolterodine (PNU 200577) have been employed to discover and develop monoclonal IgG antibodies that can recognize SARS-CoV-2 S1 protein mainly by binding to its receptor binding domain (RBD)10C15. The first commonly used method is to clone the antibody V genes from the B cells of surviving COVID-19 patients who have mounted a natural immune response against SARS-CoV-210,11,13. This strategy has yielded a number of neutralizing monoclonal antibodies; however, it is important to note that the patients antibody repertoire condition and the timing of blood sample collection play a critical role in its success. The other well-recognized and classic approach for antibody generation is by immunizing 5-hydroxymethyl tolterodine (PNU 200577) humanized mice15. Additionally, new SARS-CoV-2 antibodies were developed by screening cross-neutralizing antibodies for the SARS-CoV-2 S1 protein binders from the antibodies that 5-hydroxymethyl tolterodine (PNU 200577) were initially tested or developed to treat SARS by blocking SARS-CoV S/ACE2 or MERS by blocking MERS-CoV S/CD26 interactions12,14. One of the cross-binders is a single domain antibody/nanobody (VHH) generated from SARS-CoV S-immunized llama14. Moreover, VHHs against SARS-CoV-2 have also been generated from the llama VHH libraries16. The approach of using camelid antibody VHHs is advantageous because the VHH regions are easy to produce, are stable, and are smaller sized, which increases the possibility to target unique epitopes that are not accessible to conventional VH/VL antibodies17,18. In our recently published and follow-up studies, we identified more than 80 VHH binders against SARS-CoV-2 S1 protein from na?ve and synthetic humanized llama VHH libraries19, out of which 19 had S/ACE2 blocking ability. Then, we analyzed the synergistic effects of combining pairs of S/ACE2 blocking candidates, which led to the construction of bi-specific VHH-Fc antibodies which are significantly more potent than the individual monoclonal VHH-Fcs in SARS-CoV-2 Sema3e S1 RBD binding and S/ACE2 blocking19. Based on our findings with the bi-specific VHH-Fc and computer-aided epitope modeling predictions, we reasoned that.
