Humanized TFR1 knock-in mice: A powerful platform for evaluating TFR1-targeting brain shuttles

Background: The development of therapeutic antibodies and antibody-based diagnostics for the neurological disorders and gliomas is largely hampered by the presence of blood-brain barrier (BBB). While the BBB restricts most large molecules, it permits the transport of essential nutrients through selective transport mechanisms, creating opportunities to delivery drugs into the central nervous system using brain shuttle technologies. Transferrin receptor 1 (TFR1), which is expressed on brain endothelial cells (BECs), is one of the most extensively studied targets for receptor-mediated transcytosis (RMT) and has been validated as a gateway for brain drug delivery. To support the development of TFR1-targeting antibody or peptide-based brain shuttles, we generated a humanized TFR1 knock-in mouse model (hTFR1), serving as a robust preclinical platform for evaluating the efficacy, safety, and PK/PD profiles of TFR1-targeted therapeutics. Methods: The hTFR1 knock-in mouse model was generated by inserting the full-length human TFR1 coding sequence plus WPRE/polyA stop cassette into mouse Cd71 gene locus in C57BL/6 background via ES cell-based gene targeting, in which full-length human TFR1 proteins are expressed on the surface of targeted cells. To characterize the human TFR1 expression in the hTFR1 knockin mice, we assessed its localization on the brain endothelial cells by immunofluorescence (IF) staining and performed flow cytometry analysis to characterize its expression on activated T cells. Finally, we administered Trontinemab and the anti-human TFR1 antibodies generated via our SmocMab platform to hTFR1 mice through i.v. injection to evaluate th PK characteristics and brain uptake capability across the BBB. Results: Human TFR1 expression in hTFR1 mice was confirmed in brain microvessels and was upregulated on activated T lymphocytes. For PK analysis, brain tissues and serum samples were collected 24 hours after a single intravenous dose (10 mg/kg) of the human-specific TFR1 antibodies or a control antibody. Antibody concentrations were quantified by ELISA. The human-specific TFR1-binding antibody showed accelerated serum clearance and increased brain uptake compared with the control antibody, demonstrating its enhanced ability to penetrate the BBB. Conclusions: Our humanized TFR1 knockin mice provides a powerful tool for preclinical evaluation of TFR1-targeting therapeutic antibodies.