Blood-brain barrier transport using a high-affinity, brain-selective VNAR (Variable Domain of New Antigen Receptor) antibody targeting transferrin receptor 1

摘要: 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.MethodsA 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. Phage formatted VNARs cross-reactive to recombinant human and mouse TfR1-ECD were fused to Fc domain of human IgG1 (hFc) and tested for TfR1-ECD binding by ELISA and surface plasmon resonance. The pharmacokinetics and biodistribution of VNAR-hFcs were studied in mice by ELISA and immunolabeling following intravenous (IV) injection and cardiac perfusion. Functional activity was measured by body temperature reduction following the IV injection of neurotensin fused to a TXB2-hFc (TXB2-hFc-NT).ResultsTXB2 was identified as a high-affinity, species cross-reactive VNAR antibody against TfR1-ECD, that does not to compete with transferrin or ferritin for receptor binding. IV dosing of 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-NT at 25 nmol/kg resulted in a rapid and reversible pharmacological response as measured by …