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

FASEB J. 2021 Feb;35(2):e21172. doi: 10.1096/fj.202001787R. Epub 2020 Nov 25.

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.

Keywords: blood-brain barrier (BBB); brain delivery; transcytosis; transferrin receptor 1 (TfR1); variable domain of new antigen receptor (VNAR).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibody Affinity*
  • Antigens, CD / genetics
  • Antigens, CD / immunology*
  • Antigens, CD / metabolism
  • Bacteriophages / immunology
  • Biological Transport / genetics
  • Biological Transport / immunology*
  • Blood-Brain Barrier / immunology*
  • Blood-Brain Barrier / metabolism*
  • Cross Reactions
  • Female
  • HEK293 Cells
  • Humans
  • Mice
  • Mice, Inbred BALB C
  • Receptors, Antigen / immunology
  • Receptors, Antigen / metabolism
  • Receptors, Transferrin / genetics
  • Receptors, Transferrin / immunology*
  • Receptors, Transferrin / metabolism
  • Recombinant Proteins / immunology
  • Recombinant Proteins / metabolism
  • Single-Chain Antibodies / immunology*
  • Single-Chain Antibodies / pharmacokinetics
  • Transfection

Substances

  • Antigens, CD
  • CD71 antigen
  • Receptors, Antigen
  • Receptors, Transferrin
  • Recombinant Proteins
  • Single-Chain Antibodies
  • Tfrc protein, mouse