Novel nanobodies to prevent and treat SARS-CoV-2 and other pathogenic human coronaviruses

SummaryA novel coronavirus (CoV), SARS-CoV-2, is causing global pandemics with growing numbers of daily casesand deaths. SARS-CoV-2 is closely related to SARS-CoV, which caused the 2002-2003 SARS epidemic, andless closely related to MERS-CoV, which causes a high mortality rate in infected patients. No therapeuticagents or vaccines have been approved to control the infections of these CoVs in humans, calling forimmediate efforts to develop effective countermeasures. The CoV spike (S) proteins are important targets fortherapeutics. They guide virus entry into host cells by binding to a host receptor through their S1 subunits andfusing the viral and host cell membranes through their S2 subunits. The S1 subunits contain a receptor-bindingdomain (RBD). Both RBD and S2 region of S protein can elicit neutralizing antibodies. The RBD is the majortarget to induce potent neutralizing antibodies, but it diverges among different CoVs, whereas the S2 region ofS protein is more conserved among different CoVs. Nanobodies (Nbs) are single-domain antibodies derivedfrom camelid antibodies. They are emerging as novel therapeutic agents, numbers of which have beenapproved or tested in clinical trials to prevent and treat other human diseases. Nbs possess many uniqueadvantages as therapeutic agents: they have high physical and chemical stabilities, excellent tissuepenetration capability (superior pharmacokinetics), easy expression with great production yields, androbustness for storage and transportation. Moreover, Nbs can potentially recognize epitopes (e.g. hidden orpartially hidden epitopes) that are not accessible to conventional antibodies. In this proposal, we will designand develop Nbs as therapeutic agents against SARS-CoV-2 and other pathogenic human CoVs using phagedisplay and structural biology as guiding tools. We propose to develop highly efficacious Nbs against SARS-CoV-2 to stop the current COVID-19 pandemic and also broad-spectrum neutralizing Nbs targeting future CoVinfections. We have established Nb libraries and identified several neutralizing Nbs targeting the RBDs ofSARS-CoV and MERS-CoV, providing a solid foundation for the proposed studies. In our previous work, weextensively characterized the structures and functions of MERS-CoV and SARS-CoV S proteins, and we haverecently characterized the RBD of SARS-CoV-2 and solved its structure in complex with viral receptor, pavingthe way for rapid screening and identification of SARS-CoV-2 S protein-based Nbs. The specific aims of thisproposal are to: 1) develop highly efficacious Nbs targeting SARS-CoV-2 S (RBD/S2) protein; develop broad-spectrum Nbs against CoV infections, 2) characterize these CoV S-targeting Nbs, and 3) evaluate in vivoefficacy of these Nbs against SARS-CoV-2 and other CoV infections. Overall, this proposal will develophighly efficacious Nbs targeting SARS-CoV-2 S protein, aiming to stop the current COVID-19 pandemic. It willalso develop broad-spectrum Nbs targeting future CoV infections. This proposal has important implications forcombating pathogenic coronaviruses and neutralizing their threat to human health.