Using microdialysis to analyse the passage of monovalent nanobodies through the blood-brain barrier.
OBJECTIVE
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.


METHODS
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 (99m) Tc-labelled nanobodies using, respectively, a sensitive VSG-based nanobody-detection elisa, radioactivity measurement in collected microdialysates and SPECT image analysis.


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
Nanobodies can enter the brain parenchyma from the systemic circulation, especially in pathological conditions where the blood-brain barrier integrity is compromised.
Department of Animal Health, Institute of Tropical Medicine Antwerp, Antwerp, Belgium.
G
G
Caljon
V
V
Caveliers
T
T
Lahoutte
B
B
Stijlemans
G H
GH
Ghassabeh
J
J
Van Den Abbeele
I
I
Smolders
P
P
De Baetselier
Y
Y
Michotte
S
S
Muyldermans
S
S
Magez
R
R
Clinckers
D000818:Animals; D000913:Antibodies, Protozoan / Q000008:administration & dosage* / Q000378:metabolism*; D001812:Blood-Brain Barrier / Q000000981:diagnostic imaging / Q000276:immunology* / Q000469:parasitology; D008297:Male; D017551:Microdialysis / Q000379:methods; D049329:Nanostructures; D051381:Rats; D017208:Rats, Wistar; D017256:Technetium Tc 99m Sestamibi / Q000493:pharmacokinetics; D015899:Tomography, Emission-Computed, Single-Photon; D014346:Trypanosoma brucei brucei / Q000276:immunology; D014353:Trypanosomiasis, African / Q000000981:diagnostic imaging / Q000276:immunology / Q000469:parasitology; D055114:X-Ray Microtomography
D000913:Antibodies, Protozoan; D017256:Technetium Tc 99m Sestamibi