Blood‐brain barrier transport using a high affinity, brain‐selective VNAR antibody targeting transferrin receptor 1

Abstract

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. 2 | STOCKI eT al.

A single domain shark antibody targeting the transferrin receptor 1 delivers a TrkB agonist antibody across the blood brain barrier to provide full neuroprotection in a mouse model of Parkinson’s Disease

“…The TfR1 binding TXB2 VNAR can carry cargos, including a human IgG Fc domain (TXB2-hFc) as well as biologically active neurotensin, across the BBB. Importantly the TXB2-hFc does not compete for transferrin binding, clear the TfR1 from capillaries, or lyse reticulocytes 33 . TXB4 is a version of TXB2 with some modifications in the CDR3 that improved brain penetration (JLR and PS, manuscript in preparation).…”

“…There is considerable interest in the possibility of utilizing receptor mediated transcytosis pathways that exist on brain endothelial cells that form the BBB to carry biotherapeutics from the blood to the brain parenchyma with the transferrin receptor 1 (TfR1) being the most widely studied 32 . TXB4 is a single domain shark variable new antigen receptor (VNAR) antibody specific to TfR1 and was developed from previously reported parental TXB2 VNAR 33 by restricted randomisation of CDR3 domain that led to improved brain penetration (JLR & PS, manuscript in preparation). TXB2 when fused to human IgG1 Fc (TXB2-hFc) readily crossed the intact BBB, reaching low nanomolar concentrations with a half-life of several days in the brain where it can be found to localised to TfR1 expressing neurons.…”

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“…Biological therapy with infusing TXB2-hFc to neurotensin in mice does not induce any adverse effect or specific clearance from organs. It is also shown that TXB2 is a selective variable domain of new antigen receptor (VNAR) to transferrin receptor 1 (TfR1) crossing BBB with safe pharmacological and pharmacokinetic profile [ 194 ].…”

Brain Barriers and brain fluids research in 2020 and the fluids and barriers of the CNS thematic series on advances in in vitro modeling of the blood–brain barrier and neurovascular unit

“…The Ullmann et al [140] study demonstrated delivery of iduronate 2-sulfatase to the brain in a mouse model of mucopolysaccharidosis type II, another lysosomal storage disorder, and improvement of brainrelated pathology. Stocki et al [141] also identified single domain antibodies with high affinity for the transferrin receptor, one of which crosses the blood-brain interface and is taken up by neurons. Intravenous administration of the antibody fused with neurotensin caused a reduction in body temperature (i.e., the construct induced a physiological response).…”