Browsing by Author "Oliveira, Soraia"
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- Highly specific blood-brain barrier transmigrating single-domain antibodies selected by an in vivo phage display screeningPublication . Aguiar, Sandra I; Dias, Joana N. R.; André, Ana; Silva, Marta; Martins, Diana; Carrapiço, Belmira; Castanho, Miguel A. R. B.; Carrico, Joao Andre; Cavaco, Marco; Gaspar, Maria Manuela; Nobre, Rui Jorge; Pereira de Almeida, Luís; Oliveira, Soraia; Gano, Lurdes; Correia, João D. G.; Carlos F. Barbas, III; Gonçalves, João Rafael; Neves, Vera; Aires da Silva, FredericoA 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.
- Rabbit derived VL single-domains as promising scaffolds to generate antibody–drug conjugatesPublication . André, Ana; Dias, Joana N. R.; Aguiar, Sandra I; Nogueira, Sara; Bule, Pedro; Carvalho, Joana; António, João P. M.; Cavaco, Marco; Neves, Vera; Oliveira, Soraia; Vicente, Gonçalo; Carrapiço, Belmira; Braz, Berta São; Rütgen, Barbara; Gano, Lurdes; Correia, João D. G.; Castanho, Miguel A. R. B.; Gonçalves, João Rafael; Gois, Pedro M. P.; Gil, Solange; Tavares, Luis; Silva, Frederico Aires daAntibody-drug conjugates (ADCs) are among the fastest-growing classes of therapeutics in oncology. Although ADCs are in the spotlight, they still present significant engineering challenges. Therefore, there is an urgent need to develop more stable and effective ADCs. Most rabbit light chains have an extra disulfide bridge, that links the variable and constant domains, between Cys80 and Cys171, which is not found in the human or mouse. Thus, to develop a new generation of ADCs, we explored the potential of rabbit-derived VL-single-domain antibody scaffolds (sdAbs) to selectively conjugate a payload to Cys80. Hence, a rabbit sdAb library directed towards canine non-Hodgkin lymphoma (cNHL) was subjected to in vitro and in vivo phage display. This allowed the identification of several highly specific VL-sdAbs, including C5, which specifically target cNHL cells in vitro and present promising in vivo tumor uptake. C5 was selected for SN-38 site-selective payload conjugation through its exposed free Cys80 to generate a stable and homogenous C5-DAB-SN-38. C5-DAB-SN-38 exhibited potent cytotoxicity activity against cNHL cells while inhibiting DNA-TopoI activity. Overall, our strategy validates a platform to develop a novel class of ADCs that combines the benefits of rabbit VL-sdAb scaffolds and the canine lymphoma model as a powerful framework for clinically translation of novel therapeutics for cancer.