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Highly specific blood-brain barrier transmigrating single-domain antibodies selected by an in vivo phage display screening

2021Journal article Open access
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dc.contributor.authorAguiar, Sandra I
dc.contributor.authorDias, Joana N. R.
dc.contributor.authorAndré, Ana
dc.contributor.authorSilva, Marta
dc.contributor.authorMartins, Diana
dc.contributor.authorCarrapiço, Belmira
dc.contributor.authorCastanho, Miguel A. R. B.
dc.contributor.authorCarrico, Joao Andre
dc.contributor.authorCavaco, Marco
dc.contributor.authorGaspar, Maria Manuela
dc.contributor.authorNobre, Rui Jorge
dc.contributor.authorPereira de Almeida, Luís
dc.contributor.authorOliveira, Soraia
dc.contributor.authorGano, Lurdes
dc.contributor.authorCorreia, João D. G.
dc.contributor.authorCarlos F. Barbas, III
dc.contributor.authorGonçalves, João Rafael
dc.contributor.authorNeves, Vera
dc.contributor.authorAires da Silva, Frederico
dc.date.accessioned2023-05-15T13:30:04Z
dc.date.available2023-05-15T13:30:04Z
dc.date.issued2021
dc.description© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).pt_PT
dc.description.abstractA 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.pt_PT
dc.description.sponsorshipThis research was funded by: the Portuguese Funding Agency, Fundacão para a Ciência e a Tecnologia, FCT IP: 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. CIISA has provided support through Project UIDB/00276/2020, funded by FCT. FAS and JG dedicate this study to the memory of Professor Carlos F. Barbas 3rd, an outstanding mentor and friend.pt_PT
dc.description.versioninfo:eu-repo/semantics/publishedVersionpt_PT
dc.identifier.citationPharmaceutics 2021, 13, 1598pt_PT
dc.identifier.doi10.3390/pharmaceutics13101598pt_PT
dc.identifier.eissn1999-4923
dc.identifier.urihttp://hdl.handle.net/10451/57424
dc.language.isoengpt_PT
dc.peerreviewedyespt_PT
dc.publisherMDPIpt_PT
dc.relationPTDC/BBB-BIO/0508/2014pt_PT
dc.relationDL57/2016/CP1438/CT0002pt_PT
dc.relationDevelopment of immunoliposomal therapy for treatment of Pneumococcal meningitis
dc.relationTrans-BBB peptides for targeting brain metastasis
dc.relationNot Available
dc.relationResearch Institute for Medicines
dc.relationResearch Institute for Medicines
dc.relationTargeting the transporters of cationic amino acids for cancer radiotheranostics: experimental and computational chemistry approach
dc.relationCNC. IBILI
dc.relationCenter for Innovative Biomedicine and Biotechnology
dc.relationCentre for Interdisciplinary Research in Animal Health
dc.relation.publisherversionhttps://www.mdpi.com/journal/pharmaceuticspt_PT
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/pt_PT
dc.subjectBrain targeting antibodiespt_PT
dc.subjectBlood-brain barrierpt_PT
dc.subjectin vivo phage displaypt_PT
dc.subjectSingle-domain antibodiespt_PT
dc.subjectDrug deliverypt_PT
dc.titleHighly specific blood-brain barrier transmigrating single-domain antibodies selected by an in vivo phage display screeningpt_PT
dc.typejournal article
dspace.entity.typePublication
oaire.awardTitleDevelopment of immunoliposomal therapy for treatment of Pneumococcal meningitis
oaire.awardTitleTrans-BBB peptides for targeting brain metastasis
oaire.awardTitleNot Available
oaire.awardTitleResearch Institute for Medicines
oaire.awardTitleResearch Institute for Medicines
oaire.awardTitleTargeting the transporters of cationic amino acids for cancer radiotheranostics: experimental and computational chemistry approach
oaire.awardTitleCNC. IBILI
oaire.awardTitleCenter for Innovative Biomedicine and Biotechnology
oaire.awardTitleCentre for Interdisciplinary Research in Animal Health
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oaire.awardURIinfo:eu-repo/grantAgreement/FCT//SFRH%2FBPD%2F100522%2F2014/PT
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oaire.awardURIinfo:eu-repo/grantAgreement/FCT/3599-PPCDT/PTDC%2FQUI-NUC%2F30147%2F2017/PT
oaire.awardURIinfo:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/UID%2FNEU%2F04539%2F2019/PT
oaire.awardURIinfo:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/UIDB%2F04539%2F2020/PT
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oaire.citation.issue10pt_PT
oaire.citation.startPage1598pt_PT
oaire.citation.titlePharmaceuticspt_PT
oaire.citation.volume13pt_PT
oaire.fundingStreamInvestigador FCT
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person.familyNamede Jesus Guilherme Gaspar
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rcaap.rightsopenAccesspt_PT
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