Brain-penetrating nanobodies illustrate routes for delivering biologics to the brain

Monoclonal antibodies are commonly used as a treatment for a variety of diseases, but very few are used to treat neurological conditions. One reason is that their delivery into the brain is hampered by the blood-brain barrier. Small antibody fragment from camelids, called Nanobody®, are good alternative to these conventional antibodies. In the present work, we study how brain-penetrating nanobodies reach the brain after peripheral administration in mice by developping different approaches. We used nanobodies specific to a metabotropic glutamate (mGlu) receptor, the mGlu2 subtype, as a model. Firstly, we show that different nanobodies can enter the brain independently of their electric charge and the presence of the mGlu2 receptor. This was shown by a drop in temperature measured by telemetry, after the administration of the nanobodies coupled to neurotensin. Secondly, to measure the concentration of nanobodies in the brain and describe their pharmacokinetic profile, we developed an innovative time-resolved FRET-based method. This method relies on a pair of classical antibodies which recognize nanobodies specifically, coupled to donor and acceptor fluorophores. Finally, autoradiography shows that the nanobodies accumulate in brain regions near the ventricles. Using a two-photon microscopy, we clearly observe their leakage through the fenestrated capillaries of the median eminence. Altogether, our data indicate that nanobodies enter the brain passively through the circumventricular organs. This finding may be useful for therapeutic interventions involving antibodies in the brain.