Nanobodies for Chronic Hepatitis B Virus Infection - SUMMARY Hepatitis B virus (HBV) infection remains a major global health challenge, affecting over 250 million people worldwide and causing nearly 1 million deaths each year. Chronic HBV infection is a well-established risk factor for various forms of liver cancer, including hepatocellular carcinoma. Although a protective vaccine is available, a substantial subset of individuals fails to develop protective immunity, allowing chronic infection to persist. A key feature of chronic HBV infection is the persistent existence of hepatitis B surface antigen (HBsAg), which contributes to immune exhaustion and sustained viral persistence. Removal of circulating HBsAg is increasingly recognized as a critical prerequisite for immune restoration and durable viral control. We propose an innovative therapeutic strategy involving HBsAg-specific nanobodies (Nbs), single-domain antibody fragments derived from camelids, to reduce circulating HBsAg. This strategy has the potential to restore immune function and move the field closer to a functional cure for chronic HBV. To initiate the development of therapeutic Nbs against HBV surface antigens, we utilized our well-established phage display platform from a library obtained from an animal immunized with HBsAg to successfully generate multiple Nbs that specifically bind HBsAg. These candidates have undergone rigorous characterization, including ELISA and biolayer interferometry (BLI), with several demonstrating high binding affinity. More importantly, we have tested subsets of these Nbs for their ability to neutralize HBV and found that they displayed remarkable antiviral activity, highlighting their strong therapeutic potential for targeting circulating HBsAg. The overarching goal of this application is to further characterize these lead Nb candidates, engineer multivalent constructs, and enhance their antiviral potency and biological performance. In Aim 1, we will comprehensively evaluate the neutralizing activities of our Nbs using complementary approaches, including a high-throughput-compatible HBV strain expressing Nanoluciferase, as well as microscopy-based neutralization assays. In parallel, we will define the binding epitopes of the most promising Nbs using competitive BLI, hydrogen-deuterium exchange mass spectrometry (HDX-MS), and AlphaFold-based structural modeling to generate detailed maps of Nb-antigen interactions. In Aim 2, we will engineer our Nbs to enhance their therapeutic properties using three complementary strategies: multivalent assembly to boost avidity and efficacy, Fc fusion to extend half-life and enable effector functions, and fusion to a human serum albumin-specific Nb to further improve pharmacokinetics. Additionally, we will evaluate our top- performing Nbs for their ability to neutralize circulating HBsAg in vivo. Together, these antiviral functional assays, combined with biochemical and AI-driven epitope mapping, will lay the groundwork for future comprehensive preclinical testing of this novel Nb-based therapeutic approach, with the ultimate goal of achieving a functional cure for chronic HBV infection through combination treatment strategies.
