The overall aim of this Grand Challenges project is to identify nanobodies (NBs) that have the ability to cross the blood-brain barrier (BBB) or the blood-cerebrospinal fluid barrier (BCSFB) by receptor-mediated transcytosis (RMT), a process in which binding of a molecule (e.g. a nanobody) to its receptor enables transportation over the BBB. The proposed discovery pipeline has resulted in the identification of 50 potential candidate RMT receptors, of which 36 of have been used to generate nanobodies that bind these receptors. 25 of these are enriched in vitro and will be further explored as novel targets. Additional methods are being developed to deliver impact:

• An unbiased approach against any known target was developed by generating nanobodies against human brain capillaries, extracellular vesicles and choroid plexus (CP) tissues, in camelids. To date, 3 different nanobody families have been identified that are able to cross the BBB via RMT. Enrichment of the current immune libraries for binding to the receptor targets and ultimately crossing the BBB is performed via phage display and sampling of the brain interstitial fluid in non-human primates. So far, two novel receptors have been identified that could be targeted for crossing. Nanobodies against these targets have been generated and are currently being validated for their crossing potential.
• Several technologies are being evaluated to detect and quantify nanobodies able to cross the BBB:
  • A new reported system in mice is being evaluated in which nanobodies can be detected in vivo.
  • An ongoing collaboration with Baggerman lab (VITO) is evaluating whether nanobodies can be detected and quantified in vivo in mixtures using state-of-the-art mass spectroscopy. 
  • In an ongoing collaboration, SINTEF (Norway) is developing a method to detect nanobodies labelled with 13C carbon in in vivo samples.
  • A unique probing system (modified nanoprobe) was developed which allows continuous sampling in the brain. Validation of the probe is currently ongoing.