Enhanced in vivo blood brain barrier transcytosis of macromolecular cargo using an engineered pH-sensitive mouse transferrin receptor binding nanobody

Fluids and Barriers of the CNS

Research

Elvira Rodionova¹  Caroline M. Francescutti¹  David L. Brody²  Shiran Su³  Joong Hee Kim⁴  Thomas J. Esparza⁴ 

[1] National Institute of Neurological Disorders and Stroke, Bethesda, MD, USA;National Institute of Neurological Disorders and Stroke, Bethesda, MD, USA;Center for Neuroscience and Regenerative Medicine, Bethesda, MD, USA;Department of Neurology, Uniformed Services University of the Health Sciences, Bethesda, MD, USA;National Institute of Neurological Disorders and Stroke, Bethesda, MD, USA;Department of Biomedical Engineering, Washington University in St. Louis, St. Louis, MO, USA;National Institute of Neurological Disorders and Stroke, Bethesda, MD, USA;Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc, Bethesda, MD, USA;

关键词: Blood brain barrier;    Transferrin receptor;    Nanobody;    Neurotensin;    Histidine;    P2X7 receptor;    Capillary depletion;    Mouse;   

DOI  :  10.1186/s12987-023-00462-z

 received in 2023-04-26, accepted in 2023-08-10,  发布年份 2023

来源: Springer

【 摘 要 】

BackgroundThe 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.MethodsA 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 in vivo functional blood brain barrier transcytosis testing via central neurotensin-mediated hypothermia in wild-type mice. Multi-nanobody constructs including the mutant M1R56H, P96H, Y102H and two copies of the P2X7 receptor-binding 13A7 nanobody were produced to test proof-of-concept macromolecular cargo transport in vivo using quantitatively verified capillary depleted brain lysates and in situ histology.ResultsThe most effective histidine mutant, M1R56H, P96H, Y102H-neurotensin, caused > 8 °C hypothermia after 25 nmol/kg intravenous injection. Levels of the heterotrimeric construct M1R56H, P96H, Y102H-13A7-13A7 in capillary depleted brain lysates peaked at 1 h and were 60% retained at 8 h. A control construct with no brain targets was only 15% retained at 8 h. Addition of the albumin-binding Nb80 nanobody to make M1R56H, P96H, Y102H-13A7-13A7-Nb80 extended blood half-life from 21 min to 2.6 h. At 30–60 min, biotinylated M1R56H, P96H, Y102H-13A7-13A7-Nb80 was visualized in capillaries using in situ histochemistry, whereas at 2–16 h it was detected in diffuse hippocampal and cortical cellular structures. Levels of M1R56H, P96H, Y102H-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.ConclusionThe pH-sensitive mouse transferrin receptor binding nanobody M1R56H, P96H, Y102H 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.

【 授权许可】

CC BY   
© BioMed Central Ltd., part of Springer Nature 2023

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