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.

中文翻译：

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脑穿透纳米抗体的鉴定和体内表征

背景 确定治疗药物的血液到大脑转运能力的临床前模型通常是不明确的。在这项研究中，开发了一种方法，该方法依赖于 CNS 目标参与并能够对大脑穿透能力进行排名。这种方法导致发现了一种抗转铁蛋白受体纳米抗体，它能够通过受体介导的胞吞作用将生物活性肽输送到大脑。方法将针对小鼠转铁蛋白受体的各种纳米抗体与神经降压素融合并注射到小鼠外周。神经降压素是一种神经肽，当存在于大脑中时会导致体温过低，但无法从外围到达大脑。在外周给药后，连续体温测量被用作纳米抗体-神经降压素融合物的脑渗透读数。使用带有 Dunnett 多重比较检验的双向 ANOVA 分析全温度曲线。结果 一种与神经降压素偶联的抗转铁蛋白受体纳米抗体在静脉注射后引起体温下降。表位合并表明，与非交叉纳米抗体相比，该纳米抗体结合了不同的转铁蛋白受体表位。这种穿透大脑的纳米抗体用于表征体内低温模型。神经降压素引起的低温效应是剂量依赖性的，可用于直接比较外周给药途径和各种纳米抗体的脑暴露情况。结论 该方法导致发现了一种抗转铁蛋白受体纳米抗体，它可以在外周给药后通过受体介导的胞吞作用到达大脑。