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|Title:||Highly Specific Blood-Brain Barrier Transmigrating Single-Domain Antibodies Selected by an In Vivo Phage Display Screening||Authors:||Aguiar, Sandra Isabel
Dias, Joana N. R.
André, Ana Santos
Silva, Marta Lisete
Castanho, Miguel A. R. B.
Gaspar, Maria Manuela
Nobre, Rui J.
Almeida, Luís Pereira de
Correia, João D. G.
|Keywords:||brain targeting antibodies; blood-brain barrier; in vivo phage display; single-domain antibodies; drug delivery||Issue Date:||2-Oct-2021||Publisher:||MDPI||Project:||IF/01010/2013 and PTDC/BBB-BIO/0508/2014 to FAS, SFRH/BPD/100522/2014 and DL57/2016/CP1438/CT0002 to SIA, PD/BD/128281/2017, PTDC/BIA-BQM/5027/2020 and Dl 57/2016/CP1451/CT0023 to MC and VN, UIDB/04138/2020 and UIDP/04138/2020 to MMG, UID/Multi/04349/2020 and PTDC/QUI-NUC/30147/2017 to LG and JDGC, UID/NEU/04539/2019, UIDB/04539/2020 and ViraVector (CENTRO-01-0145-FEDER-022095) to RJN and LPA
Gilead Génese 2019 has provided support through project PGG/050/2019 to JNRD
|Serial title, monograph or event:||Pharmaceutics||Volume:||13||Issue:||10||Abstract:||A 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.||URI:||https://hdl.handle.net/10316/105338||ISSN:||1999-4923||DOI:||10.3390/pharmaceutics13101598||Rights:||openAccess|
|Appears in Collections:||IIIUC - Artigos em Revistas Internacionais|
I&D CNC - Artigos em Revistas Internacionais
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