Fig. 4
Blood half-life extension using an albumin-binding nanobody further enhances in vivo BBB transcytosis of macromolecular cargos by M1R56H, P96H, Y102H. A Diagram of four nanobody constructs including M1R56H, 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 M1R56H, 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 M1R56H, 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 M1P96H-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