Programmable Nanobody‐Targeting Chimeras Enable Intracellular Viral Protein Degradation

ABSTRACT Chronic hepatitis B virus (HBV) infection remains a major global health challenge, largely because current antiviral therapies seldom achieve sustained clearance of the viral surface antigen (HBsAg), a central driver of immune tolerance and viral persistence. Here, we present a programmable, nanobody‐based targeted protein degradation platform, termed Nanobody‐Targeting Chimera (Nab‐TAC), designed for the intracellular clearance of HBV antigens. To systematically identify functional degradation modules, we developed a split‐luciferase complementation‐based quantitative screening platform that benchmarks degron motifs and proximity‐inducing effectors, enabling comprehensive ranking of proteasome‐ and lysosome‐targeting elements across diverse cellular contexts and subcellular compartments. By coupling phage display‐derived nanobodies and single‐chain variable fragments with these optimized degradation modules, we engineered a panel of Nab‐TAC constructs that efficiently degrade viral HBsAg in hepatocytes. Notably, the lead construct, E3‐FCGR3B, achieved robust intrahepatic antigen clearance and markedly reduced circulating HBsAg levels in an HBV hydrodynamic injection mouse model. Together, these findings establish Nab‐TAC as a modular and versatile platform for targeted antiviral protein degradation and highlight its potential as a therapeutic strategy toward a functional cure for chronic HBV infection.