Research Areas: Pharmacology & PharmacyA major bottleneck in the successful development of central nervous system (CNS) drugs
is the discovery and design of molecules that can cross the blood-brain barrier (BBB). Nano-delivery
strategies are a promising approach that take advantage of natural portals of entry into the brain
such as monoclonal antibodies (mAbs) targeting endogenous BBB receptors. However, the main
selected mAbs rely on targeting broadly expressed receptors, such as the transferrin and insulin
receptors, and in selection processes that do not fully mimic the native receptor conformation, leading
to mistargeting and a low fraction of the administered dose effectively reaching the brain. Thus, there
is an urgent need to identify new BBB receptors and explore novel antibody selection approaches that
can allow a more selective delivery into the brain. Considering that in vitro models fail to completely
mimic brain structure complexity, we explored an in vivo cell immunization approach to construct a
rabbit derived single-domain antibody (sdAb) library towards BBB endothelial cell receptors. The
sdAb antibody library was used in an in vivo phage display screening as a functional selection of
novel BBB targeting antibodies. Following three rounds of selections, next generation sequencing
analysis, in vitro brain endothelial barrier (BEB) model screenings and in vivo biodistribution studies,
five potential sdAbs were identified, three of which reaching >0.6% ID/g in the brain. To validate the
brain drug delivery proof-of-concept, the most promising sdAb, namely RG3, was conjugated at the
surface of liposomes encapsulated with a model drug, the pan-histone deacetylase inhibitor panobinostat (PAN). The translocation efficiency and activity of the conjugate liposome was determined in
a dual functional in vitro BEB-glioblastoma model. The RG3 conjugated PAN liposomes enabled an
efficient BEB translocation and presented a potent antitumoral activity against LN229 glioblastoma cells without influencing BEB integrity. In conclusion, our in vivo screening approach allowed the selection of highly specific nano-antibody scaffolds with promising properties for brain targeting and drug delivery.info:eu-repo/semantics/publishedVersio