Site-Specific Molecular Engineering of Nanobody–Glucoside\nConjugates for Enhanced Brain Tumor Targeting

Nanobodies\nplay an increasingly prominent role in cancer imaging\nand therapy. However, their <i>in vivo</i> efficacy is often\nconstrained by inadequate tumor penetration and rapid clearance from\nthe bloodstream, particularly in brain tumors due to the intractable\nblood–brain barrier (BBB). Glycosylation is a favorable strategy\nfor modulating the biological functions of nanobodies, including permeability\nand pharmacokinetics, but it also leads to heterogeneous glycan structures,\nwhich affect the targeting ability, stability, and quality of nanobodies.\nHere, we describe a post-translational modification strategy to produce\nprecisely engineered and homogeneous nanobody–glucoside conjugates\nfor effective BBB penetration and brain tumor targeting. Specifically,\nwe employ an enzymatic method and click chemistry to functionalize\nnanobodies with glucoside and poly(ethylene glycol) (PEG), facilitating\nefficient transcytosis into the brain via glucose transporter-1 (GLUT1).\nFurthermore, we rationally select a near-infrared (NIR) fluorophore\nfor labeling to maintain the metabolic pathway and biodistribution\nof nanobodies and assess their potency in two tumor models. The resulting\nnanobody–glucoside conjugates demonstrate a remarkable increase\nin BBB penetration and brain tumor accumulation, which are ∼2.9-fold\nhigher in the transgenic mouse model and ∼5.7-fold higher in\nthe orthotopic glioma model compared to unmodified nanobodies. This\nstudy provides a promising approach for the production of nanobody\ntherapeutic agents for central nervous system (CNS) delivery.