Research Project
10235342
Abstract SARS-CoV-2 has spread rapidly across the globe. While vaccines are in development and existing drugs have shown some efficacy in reducing disease severity, there is an urgent need for molecules that can serve in both a prophylactic and therapeutic function. Proteins are well suited for this task because they can be engineered to have high-specificity and high-affinity for targets, and genetic protein fusion bestows multiple functions on a single protein molecule. A nanobody (Nb) has been identified with high affinity for the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, which mediates viral entry into cells via the angiotensin converting enzyme 2 (ACE2) receptor. Nbs are small heavy chain, single domain camelid antibodies that are stable over wide conditions, express well in multiple systems, can be humanized to reduce immunogenicity, and can be fused to other proteins. The goal of this proposal is to develop a protein-based therapeutic platform that can be taken either prophylactically or post-infection to reduce the severity of viral infection. The overall hypothesis is that a self-assembling hexameric coiled-coli (Hex) can be fused to 12-24 anti-RBD Nbs to create a complex with high avidity that will neutralize virus and prevent entry and/or aggregate virus into traps that can be cleared. Further, anti-albumin Nb will be incorporated in the fusion design to investigate any benefit of increased circulation time. Three aims have been set to meet the goal and test the hypothesis. (1) Design, fabricate and characterize Hex-Nb assemblies made from fusion proteins with different numbers of anti-RBD and anti-albumin Nbs. (2) Characterize binding, neutralization, and trapping of SARS-CoV-2 pseudovirus by Hex-Nb fusion assemblies in vitro. (3) Determine pharmacokinetics, biodistribution and humoral immunogenicity of Hex-nanobody fusion protein in mice following intravenous injection. From this work, an innovative protein-based therapeutic will be constructed from functional nanobodies that inhibits SARS-CoV-2 pseudovirus infection and extends in vivo lifetime, providing proof of concept for Hex assemblies to serve as prophylactics or therapeutics to prevent or treat viral infections. These results will enable future preclinical work, including viral challenge studies and direct airway administration of the Hex-Nb assemblies. Overall, this work will provide a strategy to respond to both the current and future pandemics with highly multivalent engineered protein complexes.
