RESUMEN
Since the delivery of biologic drugs to the brain is greatly hampered by the existence of the blood-brain barrier (BBB), brain shuttles are being developed to enhance therapeutic efficacy. As we have previously shown, efficient and selective brain delivery was achieved with TXB2, a cross-species reactive, anti-TfR1 VNAR antibody. To further explore the limits of brain penetration, we conducted restricted randomization of the CDR3 loop, followed by phage display to identify improved TXB2 variants. The variants were screened for brain penetration in mice using a 25 nmol/kg (1.875 mg/kg) dose and a single 18 h timepoint. A higher kinetic association rate to TfR1 correlated with improved brain penetration in vivo. The most potent variant, TXB4, showed a 3.6-fold improvement over TXB2, which had on average 14-fold higher brain levels when compared to an isotype control. Like TXB2, TXB4 retained brain specificity with parenchymal penetration and no accumulation in other organs. When fused with a neurotensin (NT) payload, it led to a rapid drop in body temperature upon transport across the BBB. We also showed that fusion of TXB4 to four therapeutic antibodies (anti-CD20, anti-EGFRvIII, anti-PD-L1 and anti-BACE1) improved their brain exposure between 14- to 30-fold. In summary, we enhanced the potency of parental TXB2 brain shuttle and gained a critical mechanistic understanding of brain delivery mediated by the VNAR anti-TfR1 antibody.
RESUMEN
Transfer across the blood-brain barrier (BBB) remains a significant hurdle for the development of biopharmaceuticals with therapeutic effects within the central nervous system. We established a functional selection method to identify high affinity single domain antibodies to the transferrin receptor 1 (TfR1) with efficient biotherapeutic delivery across the BBB. A synthetic phage display library based on the variable domain of new antigen receptor (VNAR) was used for in vitro selection against recombinant human TfR1 ectodomain (rh-TfR1-ECD) followed by in vivo selection in mouse for brain parenchyma penetrating antibodies. TXB2 VNAR was identified as a high affinity, species cross-reactive VNAR antibody against TfR1-ECD that does not compete with transferrin or ferritin for receptor binding. IV dosing of TXB2 when fused to human Fc domain (TXB2-hFc) at 25 nmol/kg (1.875 mg/kg) in mice resulted in rapid binding to brain capillaries with subsequent transport into the brain parenchyma and specific uptake into TfR1-positive neurons. Likewise, IV dosing of TXB2-hFc fused with neurotensin (TXB2-hFc-NT) at 25 nmol/kg resulted in a rapid and reversible pharmacological response as measured by body temperature reduction. TXB2-hFc did not elicit any acute adverse reactions, bind, or deplete circulating reticulocytes or reduce BBB-expressed endogenous TfR1 in mice. There was no evidence of target-mediated clearance or accumulation in peripheral organs except lung. In conclusion, TXB2 is a high affinity, species cross-reactive, and brain-selective VNAR antibody to TfR1 that rapidly crosses the BBB and exhibits a favorable pharmacokinetic and safety profile and can be readily adapted to carry a wide variety of biotherapeutics from blood to brain.
