NewroBus for the brain: humanized TfR1-targeting nanobodies with high BBB permeability and cargo transport capacity

Effective delivery of therapeutics to the brain is restricted by the blood-brain barrier (BBB). A strategy to overcome this limitation involves taking advantage of receptor-mediated transcytosis pathways, such as those mediated by transferrin receptor 1 (TfR1), which is highly expressed on brain endothelial cells and naturally transports iron-bound transferrin across the BBB. To exploit this mechanism, we immunized camelids with human TfR1 and cloned 470 VHH nanobody sequences from their B cells. From this repertoire, 24 nanobodies (TfR1b-Nbs) were identified that bind human TfR1 on the cell membrane. These nanobodies were screened for binding to human TfR1, lack of interference with transferrin binding and TfR1-mediated iron uptake, and the ability to cross the BBB via human TfR1-mediated transcytosis in newly generated humanized Tfr1h knock-in rats. To improve developability and reduce potential immunogenicity, selected TfR1b-Nbs were humanized and optimized with computational and artificial intelligence (AI) algorithms, enhancing humanness, solubility, and VHH-nativeness. Eight optimized TfR1b-Nbs retained BBB permeability and were fused to humanized anti-TNF nanobody inhibitors (TNFI- or TNFI-{beta}), generating 16 heterodimers. Fusion to these TNFIs served as a functional readout, confirming that TfR1b-Nbs can shuttle biologically active, BBB-impermeable payloads into the central nervous system (CNS). All heterodimers demonstrated CNS delivery after intravenous administration, and selected constructs also reached the brain via subcutaneous injection, maintaining high serum and cerebrospinal fluid (CSF) levels for up to 72 hours. A pilot study with one heterodimer showed that chronic administration in rats humanized for both transferrin and TfR1 caused no hematological toxicity or signs of anemia - a key safety concern when targeting TfR1. These results establish humanized TfR1b-Nbs - designated NewroBus - as promising BBB shuttles for the safe and effective therapeutic delivery of biologics to the brain.