Using microdialysis to analyse the passage of monovalent nanobodies through the blood–brain barrier

Abstract

BACKGROUND AND PURPOSE Nanobodies are promising antigen‐binding moieties for molecular imaging and therapeutic purposes because of their favourable pharmacological and pharmacokinetic properties. However, the capability of monovalent nanobodies to reach targets in the CNS remains to be demonstrated. EXPERIMENTAL APPROACH We have assessed the blood–brain barrier permeability of Nb_An33, a nanobody against the Trypanosoma brucei brucei variant‐specific surface glycoprotein (VSG). This analysis was performed in healthy rats and in rats that were in the encephalitic stage of African trypanosomiasis using intracerebral microdialysis, single photon emission computed tomography (SPECT) or a combination of both methodologies. This enabled the quantification of unlabelled and 99mTc‐labelled nanobodies using, respectively, a sensitive VSG‐based nanobody‐detection elisa, radioactivity measurement in collected microdialysates and SPECT image analysis. KEY RESULTS The combined read‐out methodologies showed that Nb_An33 was detected in the brain of healthy rats following i.v. injection, inflammation‐induced damage to the blood–brain barrier, as in the late encephalitic stage of trypanosomiasis, significantly increased the efficiency of passage of the nanobody through this barrier. Complementing SPECT analyses with intracerebral microdialysis improved analysis of brain disposition. There is clear value in assessing penetration of the blood–brain barrier by monovalent nanobodies in models of CNS inflammation. Our data also suggest that rapid clearance from blood might hamper efficient targeting of specific nanobodies to the CNS. CONCLUSIONS AND IMPLICATIONS Nanobodies can enter the brain parenchyma from the systemic circulation, especially in pathological conditions where the blood–brain barrier integrity is compromised.

A novel platform for engineering blood‐brain barrier‐crossing bispecific biologics

“…Based on these considerations, the measured CSF levels of Bi‐FC5‐hFc are likely an underestimation of its ISF levels. Since direct measurement of ISF unbound levels of therapeutic antibodies by micro‐dialysis is not feasible due to their size (56), we used in situ detection by immunofluorescence to demonstrate the presence of Bi‐FC5‐hFc in the neuropil, colocalizing with vascular and neuronal markers.…”

Chaperone Nanobodies Protect Gelsolin Against MT1-MMP Degradation and Alleviate Amyloid Burden in the Gelsolin Amyloidosis Mouse Model

“…and reduce Aβ40 levels in the brain of mice. 40 Nanobodies can be produced at very low cost and have also been demonstrated to penetrate the blood-brain barrier [41][42][43] without requiring specific targeting to a receptor. Therefore, side effects elicited by receptor function disruption can be avoided.…”

The maturation of antibody technology for the HIV epidemic

“…They are very stable, with a long shelf life and remain active at 37 °C for weeks. Nbs can cross the blood–brain barrier 29 . Modified Nbs with a single stabilising disulphide bond are resistant to pepsin and chymotrypsin, raising the possibility that they could be delivered orally as therapeutics 28 .…”

“…As detailed above, studies using in vitro infection experiments and animal models indicate that the Fc portion of the many bNAbs is necessary for their full protective capacity when used as infusions or vaginal microbicides. The Fc region can also extend the serum half‐life of antibodies, from hours to days, however this can also be achieved by a number of alternative methods including PEGylation, glycosylation or fusion to albumin (or albumin‐binding proteins) 29 . One alternative to making IgG antibodies is to make recombinant antibodies that contain an Fc receptor.…”