Esparza Thomas J, Su Shiran, Francescutti Caroline M, Rodionova Elvira, Kim Joong Hee, Brody David L
National Institute of Neurological Disorders and Stroke, Bethesda, MD, United States of America.
Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc, Bethesda, MD, United States of America.
bioRxiv. 2023 Apr 27:2023.04.26.538462. doi: 10.1101/2023.04.26.538462.
The blood brain barrier limits entry of macromolecular diagnostic and therapeutic cargos. Blood brain barrier transcytosis via receptor mediated transport systems, such as the transferrin receptor, can be used to carry macromolecular cargos with variable efficiency. Transcytosis involves trafficking through acidified intracellular vesicles, but it is not known whether pH-dependent unbinding of transport shuttles can be used to improve blood brain barrier transport efficiency.
A mouse transferrin receptor binding nanobody, NIH-mTfR-M1, was engineered to confer greater unbinding at pH 5.5 vs 7.4 by introducing multiple histidine mutations. The histidine mutant nanobodies were coupled to neurotensin for functional blood brain barrier transcytosis testing via central neurotensin-mediated hypothermia in wild-type mice. Multi-nanobody constructs including the mutant M1 and two copies of the P2X7 receptor-binding 13A7 nanobody were produced to test proof-of-concept macromolecular cargo transport using quantitatively verified capillary depleted brain lysates and histology.
The most effective histidine mutant, M1 -neurotensin, caused >8°C hypothermia after 25 nmol/kg intravenous injection. Levels of the heterotrimeric construct M1 -13A7-13A7 in capillary depleted brain lysates peaked at 1 hour and were 60% retained at 8 hours. A control construct with no brain targets was only 15% retained at 8 hours. Addition of the albumin-binding Nb80 nanobody to make M1 -13A7-13A7-Nb80 extended blood half-life from 21 minutes to 2.6 hours. At 30-60 minutes, biotinylated M1 -13A7-13A7-Nb80 was visualized in capillaries using histochemistry, whereas at 2-16 hours it was detected in diffuse hippocampal and cortical cellular structures. Levels of M1 -13A7-13A7-Nb80 reached more than 3.5 percent injected dose/gram of brain tissue after 30 nmol/kg intravenous injection. However, higher injected concentrations did not result in higher brain levels, compatible with saturation and an apparent substrate inhibitory effect.
The pH-sensitive mouse transferrin receptor binding nanobody M1 may be a useful tool for rapid and efficient modular transport of diagnostic and therapeutic macromolecular cargos across the blood brain barrier in mouse models. Additional development will be required to determine whether this nanobody-based shuttle system will be useful for imaging and fast-acting therapeutic applications.
血脑屏障限制了大分子诊断和治疗性药物的进入。通过受体介导的转运系统（如转铁蛋白受体）进行血脑屏障转胞吞作用，可用于携带效率各异的大分子药物。转胞吞作用涉及通过酸化的细胞内囊泡进行运输，但尚不清楚运输穿梭体的pH依赖性解离是否可用于提高血脑屏障的运输效率。
通过引入多个组氨酸突变，对小鼠转铁蛋白受体结合纳米抗体NIH-mTfR-M1进行改造，使其在pH 5.5时比pH 7.4时具有更大的解离度。将组氨酸突变纳米抗体与神经降压素偶联，通过野生型小鼠中枢神经降压素介导的体温过低来进行功能性血脑屏障转胞吞作用测试。构建包括突变体M1和两个拷贝的P2X7受体结合纳米抗体13A7的多纳米抗体构建体，使用定量验证的无毛细血管脑裂解物和组织学来测试概念验证性大分子药物运输。
最有效的组氨酸突变体M1-神经降压素在静脉注射25 nmol/kg后引起超过8°C的体温过低。无毛细血管脑裂解物中三聚体构建体M1-13A7-13A7的水平在1小时达到峰值，8小时时保留60%。无脑靶向的对照构建体在8小时时仅保留15%。添加白蛋白结合纳米抗体Nb80制成M1-13A7-13A7-Nb80，使血液半衰期从21分钟延长至2.6小时。在30 - 60分钟时，使用组织化学在毛细血管中观察到生物素化的M1-13A7-13A7-Nb80，而在2 - 16小时时在弥漫性海马和皮质细胞结构中检测到。静脉注射30 nmol/kg后，M1-13A7-13A7-Nb80的水平达到超过3.5%注射剂量/克脑组织。然而，更高的注射浓度并未导致更高的脑内水平，这与饱和及明显的底物抑制作用相符。
pH敏感的小鼠转铁蛋白受体结合纳米抗体M1可能是在小鼠模型中快速高效地跨血脑屏障进行诊断和治疗性大分子药物模块化运输的有用工具。需要进一步研发以确定这种基于纳米抗体的穿梭系统是否对成像和快速起效的治疗应用有用。
