Transfer across the blood-brain barrier (BBB) remains a significant hurdle for the development of biopharmaceuticals with therapeutic effects within the central nervous system. We established a functional selection method to identify high affinity single domain antibodies to the transferrin receptor 1 (TfR1) with efficient biotherapeutic delivery across the BBB. A synthetic phage display library based on the variable domain of new antigen receptor (VNAR) was used for in vitro selection against recombinant human TfR1 ectodomain (rh-TfR1-ECD) followed by in vivo selection in mouse for brain parenchyma penetrating antibodies. TXB2 VNAR was identified as a high affinity, species cross-reactive VNAR antibody against TfR1-ECD that does not compete with transferrin or ferritin for receptor binding. IV dosing of TXB2 when fused to human Fc domain (TXB2-hFc) at 25 nmol/kg (1.875 mg/kg) in mice resulted in rapid binding to brain capillaries with subsequent transport into the brain parenchyma and specific uptake into TfR1-positive neurons. Likewise, IV dosing of TXB2-hFc fused with neurotensin (TXB2-hFc-NT) at 25 nmol/kg resulted in a rapid and reversible pharmacological response as measured by body temperature reduction. TXB2-hFc did not elicit any acute adverse reactions, bind, or deplete circulating reticulocytes or reduce BBB-expressed endogenous TfR1 in mice. There was no evidence of target-mediated clearance or accumulation in peripheral organs except lung. In conclusion, TXB2 is a high affinity, species cross-reactive, and brain-selective VNAR antibody to TfR1 that rapidly crosses the BBB and exhibits a favorable pharmacokinetic and safety profile and can be readily adapted to carry a wide variety of biotherapeutics from blood to brain.

中文翻译：

---

使用靶向转铁蛋白受体 1 的高亲和力脑选择性 VNAR 抗体进行血脑屏障转运

跨血脑屏障 (BBB) 的转移仍然是开发在中枢神经系统内具有治疗作用的生物药物的重大障碍。我们建立了一种功能选择方法来识别针对转铁蛋白受体 1 (TfR1) 的高亲和力单域抗体，并通过 BBB 进行有效的生物治疗。基于新抗原受体 (VNAR) 可变域的合成噬菌体展示文库用于体外选择重组人 TfR1 胞外域 (rh-TfR1-ECD)，然后在小鼠体内选择脑实质穿透抗体。TXB2 VNAR 被鉴定为针对 TfR1-ECD 的高亲和力、物种交叉反应性 VNAR 抗体，不与转铁蛋白或铁蛋白竞争受体结合。在小鼠中以 25 nmol/kg (1.875 mg/kg) 与人 Fc 结构域 (TXB2-hFc) 融合时静脉注射 TXB2 导致快速结合脑毛细血管，随后转运到脑实质并特异性摄取到 TfR1 阳性神经元. 同样，与神经降压素 (TXB2-hFc-NT) 融合的 TXB2-hFc 以 25 nmol/kg 的 IV 给药导致快速和可逆的药理学反应，如通过体温降低所测量的。TXB2-hFc 不会在小鼠中引起任何急性不良反应、结合或消耗循环网织红细胞或减少 BBB 表达的内源性 TfR1。除肺外，没有证据表明外周器官中靶介导的清除或积聚。总之，TXB2 是一种高亲和力、物种交叉反应性、