Modulating Subcellular Localization to Preserve the Stability and Functionality of Intracellular Nanobodies

<b>Background:</b> Antibodies have revolutionized therapeutics and diagnostics, but their applications are largely restricted to extracellular targets due to challenges in intracellular delivery and stability. Nanobodies, with their small size and lack of disulfide bonds, hold great promise for intracellular use but face challenges such as aggregation and rapid degradation in the cytosol. <b>Methods:</b> To overcome this, we engineered nanobodies by fusing them with subcellular localization motifs to redirect their localization within cells, including the mitochondrial surface, endoplasmic reticulum surface, endomembrane system, and cytoskeleton. <b>Results:</b> Our results demonstrate that nanobodies located in the cytoskeleton or endomembrane exhibit significantly reduced degradation rates and enhanced stability, while maintaining their target-binding capacity. Mechanistically, these modifications lowered ubiquitination levels and prolonged functional activity. <b>Conclusions:</b> This work provides a novel strategy to enhance the intracellular stability and efficacy of nanobodies, expanding their potential applications in functional proteomics, disease research, and therapeutic development.