Yogi Alvaro, Hussack Greg, van Faassen Henk, Haqqani Arsalan S, Delaney Christie E, Brunette Eric, Sandhu Jagdeep K, Hewitt Melissa, Sulea Traian, Kemmerich Kristin, Stanimirovic Danica B
Human Health Therapeutics Research Centre, National Research Council Canada, Ottawa, ON K1A 0R6, Canada.
Pharmaceutics. 2022 Jul 12;14(7):1452. doi: 10.3390/pharmaceutics14071452.
The ability of drugs and therapeutic antibodies to reach central nervous system (CNS) targets is greatly diminished by the blood-brain barrier (BBB). Receptor-mediated transcytosis (RMT), which is responsible for the transport of natural protein ligands across the BBB, was identified as a way to increase drug delivery to the brain. In this study, we characterized IGF1R5, which is a single-domain antibody (sdAb) that binds to insulin-like growth factor-1 receptor (IGF1R) at the BBB, as a ligand that triggers RMT and could deliver cargo molecules that otherwise do not cross the BBB. Surface plasmon resonance binding analyses demonstrated the species cross-reactivity of IGF1R5 toward IGF1R from multiple species. To overcome the short serum half-life of sdAbs, we fused IGF1R5 to the human (hFc) or mouse Fc domain (mFc). IGF1R5 in both N- and C-terminal mFc fusion showed enhanced transmigration across a rat BBB model (SV-ARBEC) in vitro. Increased levels of hFc-IGF1R5 in the cerebrospinal fluid and vessel-depleted brain parenchyma fractions further confirmed the ability of IGF1R5 to cross the BBB in vivo. We next tested whether this carrier was able to ferry a pharmacologically active payload across the BBB by measuring the hypothermic and analgesic properties of neurotensin and galanin, respectively. The fusion of IGF1R5-hFc to neurotensin induced a dose-dependent reduction in the core temperature. The reversal of hyperalgesia by galanin that was chemically linked to IGF1R5-mFc was demonstrated using the Hargreaves model of inflammatory pain. Taken together, our results provided a proof of concept that appropriate antibodies, such as IGF1R5 against IGF1R, are suitable as RMT carriers for the delivery of therapeutic cargos for CNS applications.
血脑屏障（BBB）极大地削弱了药物和治疗性抗体到达中枢神经系统（CNS）靶点的能力。受体介导的转胞吞作用（RMT）负责天然蛋白质配体跨BBB的转运，它被确定为一种增加药物向脑内递送的方法。在本研究中，我们将IGF1R5鉴定为一种能触发RMT的配体，它是一种单域抗体（sdAb），在BBB处与胰岛素样生长因子-1受体（IGF1R）结合，并能递送原本无法穿过BBB的货物分子。表面等离子体共振结合分析证明了IGF1R5对多种物种的IGF1R具有物种交叉反应性。为了克服sdAb血清半衰期短的问题，我们将IGF1R5与人（hFc）或小鼠Fc结构域（mFc）融合。N端和C端mFc融合的IGF1R5在体外大鼠BBB模型（SV-ARBEC）中均显示出增强的跨膜迁移能力。脑脊液和血管缺失脑实质部分中hFc-IGF1R5水平的升高进一步证实了IGF1R5在体内穿过BBB的能力。接下来，我们分别通过测量神经降压素和甘丙肽的降温及镇痛特性，测试了这种载体是否能够携带具有药理活性的payload穿过BBB。IGF1R5-hFc与神经降压素的融合诱导了核心体温的剂量依赖性降低。使用炎症性疼痛的哈格里夫斯模型证明了与IGF1R5-mFc化学连接的甘丙肽对痛觉过敏的逆转作用。综上所述，我们的结果提供了一个概念验证，即合适的抗体，如针对IGF1R的IGF1R5，适合作为RMT载体用于递送中枢神经系统应用的治疗性货物。
