Abstract
BackgroundPreclinical models to determine blood to brain transport ability of therapeutics are often ambiguous. In this study a method is developed that relies on CNS target-engagement and is able to rank brain-penetrating capacities. This method led to the discovery of an anti-transferrin receptor nanobody that is able to deliver a biologically active peptide to the brain via receptor-mediated transcytosis.MethodsVarious nanobodies against the mouse transferrin receptor were fused to neurotensin and injected peripherally in mice. Neurotensin is a neuropeptide that causes hypothermia when present in the brain but is unable to reach the brain from the periphery. Continuous body temperature measurements were used as a readout for brain penetration of nanobody-neurotensin fusions after its peripheral administration. Full temperature curves were analyzed using two-way ANOVA with Dunnett multiple comparisons tests.ResultsOne anti-transferrin receptor nanobody coupled to neurotensin elicited a drop in body temperature following intravenous injection. Epitope binning indicated that this nanobody bound a distinct transferrin receptor epitope compared to the non-crossing nanobodies. This brain-penetrating nanobody was used to characterize the in vivo hypothermia model. The hypothermic effect caused by neurotensin is dose-dependent and could be used to directly compare peripheral administration routes and various nanobodies in terms of brain exposure.ConclusionThis method led to the discovery of an anti-transferrin receptor nanobody that can reach the brain via receptor-mediated transcytosis after peripheral administration. This method could be used to assess novel proteins for brain-penetrating capabilities using a target-engaging readout.
Highlights
Preclinical models to determine blood to brain transport ability of therapeutics are often ambiguous
Delivery of biologics to the central nervous system (CNS) has been a major challenge. This is partly due to the fact that the CNS is physically separated from the periphery by several barriers, including the blood–brain barrier (BBB), a monolayer of endothelial cells supported by astrocytes and pericytes [1]
We explore whether it is possible to use a nanobody to reach the brain using new nanobodies raised against the mouse Transferrin receptor transferrin receptor (TfR), a known receptor-mediated transcytosis target
Summary
Preclinical models to determine blood to brain transport ability of therapeutics are often ambiguous. In this study a method is developed that relies on CNS target-engagement and is able to rank brain-penetrating capacities. This method led to the discovery of an anti-transferrin receptor nanobody that is able to deliver a biologically active peptide to the brain via receptor-mediated transcytosis. Delivery of biologics to the central nervous system (CNS) has been a major challenge This is partly due to the fact that the CNS is physically separated from the periphery by several barriers, including the blood–brain barrier (BBB), a monolayer of endothelial cells supported by astrocytes and pericytes [1]. Nutrients are able to cross the BBB via specific receptors expressed on the luminal side of brain endothelial cells via receptor-mediated transcytosis (RMT). Wouters et al Fluids Barriers CNS (2020) 17:62 currently there are no therapeutic proteins approved for clinical use that cross the BBB to exert their effect [8]
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