Programmable Nanobody‐Targeting Chimeras Enable Intracellular Viral Protein Degradation

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.