An Affibody Molecule Is Actively Transported into the Cerebrospinal Fluid via Binding to the Transferrin Receptor.

Sebastian W Meister,Hanna Lindberg,Hanna Tegel,Stefan Ståhl,John Löfblom,Melanie Dannemeyer,Linnea C Hjelm

doi:10.3390/ijms21082999

Sebastian W Meister, Hanna Lindberg + Show 5 more

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https://doi.org/10.3390/ijms21082999

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Abstract

The use of biotherapeutics for the treatment of diseases of the central nervous system (CNS) is typically impeded by insufficient transport across the blood–brain barrier. Here, we investigate a strategy to potentially increase the uptake into the CNS of an affibody molecule (ZSYM73) via binding to the transferrin receptor (TfR). ZSYM73 binds monomeric amyloid beta, a peptide involved in Alzheimer’s disease pathogenesis, with subnanomolar affinity. We generated a tri-specific fusion protein by genetically linking a single-chain variable fragment of the TfR-binding antibody 8D3 and an albumin-binding domain to the affibody molecule ZSYM73. Simultaneous tri-specific target engagement was confirmed in a biosensor experiment and the affinity for murine TfR was determined to 5 nM. Blockable binding to TfR on endothelial cells was demonstrated using flow cytometry and in a preclinical study we observed increased uptake of the tri-specific fusion protein into the cerebrospinal fluid 24 h after injection.

Highlights

The blood–brain barrier (BBB) is defined as the structural, physiological, and molecular mechanisms regulating the exchange of molecules between the systemic circulation and the brain [1]
The albumin-binding domain used in this study (ABD035 [21]; denoted only ABD hereinafter) has been engineered to femtomolar affinity for human serum albumin by directed evolution [21]
The fusion of scFv8D3 to ZSYM73-ABD led to an 9-fold increase in cerebrospinal fluid (CSF) bioavailability after 24 h indicating an active transport mechanism into CSF. In this present study we explored a strategy that could potentially increase the brain uptake of an affibody molecule via transferrin receptor-mediated transcytosis in the future

Summary

Introduction

The blood–brain barrier (BBB) is defined as the structural, physiological, and molecular mechanisms regulating the exchange of molecules between the systemic circulation and the brain [1]. Potential receptor-mediated transcytosis (RMT) mechanisms of endogenous ligands have been investigated with the aim to increase the brain uptake of biologics in a non-invasive manner. To this end, therapeutic macromolecules have been conjugated to antibodies against receptors or transporters expressed on the BBB. In order to achieve optimal brain exposure of the therapeutic cargo, the antibody needs to dissociate from the receptor during or after transcytosis. This can be achieved by utilizing an antibody with moderate to low affinity for TfR [14]. Monovalent binding to the heterodimeric TfR has been shown to induce a more favorable route of intracellular sorting compared to bivalent TfR binding [12,15]

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