Project 2: Nanobodies as Novel Entry Inhibitors of Pandemic Viruses

Project 2 - Nanobodies as Novel Entry Inhibitors of Pandemic Viruses Summary Nanobodies (Nbs) are single domain antibodies derived from camelid heavy chain-only antibodies. Due to their small size, Nbs have many advantages over conventional antibodies as human therapeutics, such as their access to cryptic sites on targets, ease of production, superior pharmacokinetics, and strong physical and chemical stabilities. Nbs also have many advantages over small molecule drugs as human therapeutics, such as their high on-target specificity and low side effects. Several Nb drugs have been clinically approved to treat diseases in humans, confirming the safety and efficacy of Nbs as human therapeutics. A novel coronavirus (CoV) SARS-CoV-2 has caused the global COVID-19 pandemic. The fast emergence of many SARS-CoV-2 variants calls for urgent need of potent and broad-spectrum anti-COVID-19 therapeutics. Compared to small- molecule antiviral drugs, Nbs are particularly powerful in battling SARS-CoV-2 variants because they can be quickly adapted to new viral variants through phage display. Moreover, other pathogenic viruses also demonstrate pandemic potential, such as Ebola filovirus (EBOV), Lassa arenavirus (LASV)/Machupo arenavirus (MACV), and Zika flavivirus (ZIKV). These RNA viruses all contain a surface glycoprotein that mediates virus entry into host cells, thus the viral glycoprotein serves as a key therapeutic target. The current AViDD RFA program specifically includes Nb antiviral drugs as one of its missions. Therefore, Project 2 proposes to develop highly effective Nbs that target conserved epitopes of viral glycoproteins as novel inhibitors to block viral entry of these pandemic viruses. In our prior studies, we have developed several potent anti-CoV Nbs, including a series of anti-SARS-CoV-2 Nb candidate drugs named Nanosota-1. We have established camelid Nb phage display library platforms for Nb screening. We hypothesize that Nbs with high potency, good stability, low production costs, minimal side effects, superior pharmacokinetics, and broad antiviral spectrum can be developed as novel antiviral therapeutics. This project has three specific aims. In Aim 1, we will screen for antiviral Nbs using naïve or immunized Nb phage display libraries. We will also use in vitro affinity maturation to optimize the target-binding affinity of discovered Nbs. In Aim 2, based on structural information, we will engineer Nbs to further improve their target-binding affinity and antiviral potency. We will also improve Nb's pharmacokinetics and minimize their side effects. In Aim 3, we will test and validate Nb candidate drugs in animal models against viral infections. This project is built upon a strong research team with complementary expertise in coronaviruses, filoviruses, arenaviruses, and flaviviruses, solid preliminary data, well-established platforms, and full support from the administration, chemistry, structural biology, and virology cores (Cores A and C-E). The overall goals of Project 2 are: (i) to discover Nb therapeutics against SARS- CoV-2 and its variants, helping ending the COVID-19 pandemic and (ii) to establish Nbs as potent, safe, and cost-effective therapeutics against other pathogenic viruses with pandemic potential.