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

Abstract

Background: 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.

Methods: 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 in vivo functional blood brain barrier transcytosis testing via central neurotensin-mediated hypothermia in wild-type mice. Multi-nanobody constructs including the mutant M1_{R56H, 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.

Results: The most effective histidine mutant, M1_{R56H, P96H, Y102H}-neurotensin, caused > 8 °C hypothermia after 25 nmol/kg intravenous injection. Levels of the heterotrimeric construct M1_{R56H, 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 M1_{R56H, P96H, Y102H}-13A7-13A7-Nb80 extended blood half-life from 21 min to 2.6 h. At 30-60 min, biotinylated M1_{R56H, 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 M1_{R56H, 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.

Conclusion: The pH-sensitive mouse transferrin receptor binding nanobody M1_{R56H, 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.

Keywords: Blood brain barrier; Capillary depletion; Histidine; Mouse; Nanobody; Neurotensin; P2X7 receptor; Transferrin receptor.

Fig. 1

Binding and pH-dependent unbinding of M1 nanobody and histidine mutants to recombinant mTfR apical domain. Each nanobody was bound at multiple concentrations to 96 well ELISA plates coated with recombinant mTfR at pH 7.4, then washed at either pH 7.4 or pH 5.5. The original M1 has very little pH dependent unbinding, whereas the M1_P96H mutant had an average of 1.46-fold more unbinding at pH 5.5 and the M1_{R56H, P96H, Y102H} mutant had an average of 2.63-fold more unbinding at pH 5.5

Fig. 2

M1 mTfR binding nanobody–NT fusions for assessment of central nervous system target engagement. Blinded assessment of M1 variants injected iv into wild-type mice (n = 3/group). M1 and M1_P96H mutant caused modest hypothermia at 600 nmol/kg. M1_AA (non TfR binding) TfR caused no detectible hypothermia. M1_{R56H, P96H, Y102H} caused pronounced hypothermia at 25 nmol/kg

Fig. 3

BBB transcytosis of macromolecular cargos by M1_{R56H, P96H, Y102H} and brain target engagement in vivo. A Diagram of the three nanobody construct including M1_{R56H, P96H, Y102H} plus a tandem dimer of the P2X7 receptor binding nanobody 13A7 for brain target engagement. B Diagram of a control three nanobody construct including M1_{R56H, P96H, Y102H} plus a tandem dimer of the human amyloid-beta binding nanobody Nb3 which has no known targets in the mouse brain. C Time course of capillary depleted lysate levels of biotinylated M1_{R56H, P96H, Y102H}-13A7-13A7 (n = 3 per time point) after iv injection of 30 nmol/kg. D Time course of capillary depleted lysate levels of biotinylated M1_{R56H, P96H, Y102H}-Nb3-Nb3 (n = 3 per time point) after iv injection of 30 nmol/kg. E–F Time course of blood levels of the biotinylated three nanobody constructs from the same mice as C, D

Fig. 4

Blood half-life extension using an albumin-binding nanobody further enhances in vivo BBB transcytosis of macromolecular cargos by M1_{R56H, P96H, Y102H}. A Diagram of four nanobody constructs including M1_{R56H, P96H, Y102H}, a tandem dimer of the P2X7 receptor binding nanobody 13A7 for brain target engagement or the human amyloid-beta binding nanobody Nb3 with no known mouse brain targets, plus the albumin binding nanobody Nb80. B Time course of blood levels of the biotinylated four nanobody constructs demonstrating extended 2.6 h half-life. C Time course of capillary depleted lysate levels of biotinylated M1_{R56H, P96H, Y102H}-13A7-13A7-Nb80 (n = 3 per time point) after iv injection of 30 nmol/kg. D Time course of capillary depleted lysate levels of biotinylated M1_{R56H, P96H, Y102H}-Nb3-Nb3-Nb80 (n = 3 per time point) after iv injection of 30 nmol/kg. E, F Size exclusion chromatography of a biotinylated M1_P96H-13A7-13A7-Nb80 prior to injection (E) and in brain homogenates (F) from 4 different mice (gray, red, orange, and blue symbols) 2 h after iv injection into wild-type mice at 300 nmol/kg body weight demonstrating integrity of the four nanobody constructs in brain. No substantial aggregation or degradation was apparent

Fig. 5

In vivo target engagement of four nanobody constructs after iv injection. A–F In situ labeling of biotinylated M1_{R56H, P96H, Y102H}-13A7-13A7-Nb80 injected iv into wild-type mice at 600 nmol/kg body weight at 30 min showing largely labelling in structures with morphologies consistent with capillaries, and at 1, 2, 4, 8 and 16 h showing cellular labeling in hippocampus. G Positive control: ex vivo staining of naïve mouse brain slice with 50 nM biotinylated M1_{R56H, P96H, Y102H}-13A7-13A7-Nb80. H Negative control: no in situ labeling after intravenous injection of 600 nmol/kg M1_AA-13A7-13A7-Nb80 which does not bind mTfR. I Negative control: trace in situ labeling of structures with morphologies consistent with capillaries after intravenous injection of 600 nmol/kg M1_{R56H, P96H, Y102H}-Nb3-Nb3-Nb80. Nb3 has no known targets in wild-type mouse brain

Fig. 6

Capillary depleted brain lysate levels of nanobody construct 4 h after injection of a range of doses of biotinylated M1_{R56H, P96H, Y102H-}13A7-13A70-Nb80 in mice. N = 3 per dose, except for N = 2 at 1 nmol/kg and N = 2 for 1000 nmol/kg. Hill equation fit parameters were B_max = 3.94 nM, ED₅₀ = 5.6 nmol/kg, Hill coefficient = 1.6. Substrate inhibition fit parameters were B_max = 4.7 nM, ED₅₀ = 7.3 nmol/kg, K_i = 4877 nmol/kg

Fig. 7

Diagram of hypothesized mechanisms involved in BBB transcytosis of mTfR-and-P2X7-receptor binding nanobody constructs. Anti-mTfR nanobodies bind to mTfR in blood and constructs are endocytosed. Nanobody constructs unbind from mTfR in a pH-dependent fashion in intracellular vesicular compartments. Nanobody constructs are released into the brain and bind P2X7 receptor targets. (Produced using BioRender)