Identification and in vivo characterization of a brain-penetrating nanobody
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Fluids and Barriers of the CNS Open Access
RESEARCH
Identification and in vivo characterization of a brain‑penetrating nanobody Y Wouters1,2, T Jaspers1,2, B De Strooper1,2,3* and M Dewilde1,2,4,5*
Abstract Background: 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. Methods: Various 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. Results: One 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. Conclusion: This 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. Keywords: Nanobody, VHH, Transferrin receptor, Neurotensin, Blood–brain barrier, Receptor-mediated transcytosis Background 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]. It is estimated that only 0.1% of circulating macromolecules is able to reach the brain *Correspondence: [email protected]; maarten. [email protected] 1 VIB Center for Brain & Disease Research, Campus Gasthuisberg O&N4, Herestraat 49, box 602, B‑3000 Leuven, Belgium Full list of author information is available at the end of the article
parenchyma, which severely limits the use of biologics to treat CNS-related diseases [2]. Transcytosis pathways involved in the delivery of essential nutrients have been explored for delivery of drugs to the brain. Nutrients are able to cross the BBB via specific receptors expressed on the lum
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