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

Publication . 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, Frederico

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.

2021Journal article

Open access

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

Publication . Aguiar, Sandra Isabel; Dias, Joana N. R.; S. André, Ana; Silva, Lisete M.; Martins, Diana; Carrapiço, Belmira; Castanho, Miguel; Carriço, João; Cavaco, Marco; Aires da Silva, Frederico; [et al.]

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.

2021-10-02Journal article

Open access

Conjugation of a blood brain barrier peptide shuttle to an Fc domain for brain delivery of therapeutic biomolecules

Publication . Cavaco, Marco; Frutos, Silvia; Oliete, Paula; Valle, Javier; Andreu, David; Castanho, Miguel A. R. B.; Vila-Perelló, Miquel; Neves, Vera

The frequency of brain disease has increased significantly in the past years. After diagnosis, therapeutic options are usually limited, which demands the development of innovative therapeutic strategies. The use of antibody−drug conjugates (ADCs) is promising but highly limited by the existence of the blood−brain barrier (BBB). To overcome the impermeability of this barrier, antibody fragments can be engineered and conjugated to BBB peptide shuttles (BBBpS), which are capable of brain penetration. Herein, we linked the highly efficient BBBpS, PepH3, to the IgG fragment crystallizable (Fc) domain using the streamlined expressed protein ligation (SEPL) method. With this strategy, we obtained an Fc-PepH3 scaffold that can carry different payloads. Fc-PepH3 was shown to be nontoxic, capable of crossing an in vitro cellular BBB model, and able to bind to the neonatal Fc receptor (FcRn), which is responsible for antibody long half-life (t1/2). Overall, we demonstrated the potential of Fc-PepH3 as a versatile platform readily adaptable to diverse drugs of therapeutic value to treat different brain conditions.

2021Journal article

Open access

Exosomes and brain metastases: a review on their role and potential applications

Publication . Oliveira, Filipa; Castanho, Miguel A. R. B.; Neves, Vera

Brain metastases (BM) are a frequent complication in patients with advanced stages of cancer, associated with impairment of the neurological function, quality of life, prognosis, and survival. BM treatment consists of a combination of the available cancer therapies, such as surgery, radiotherapy, chemotherapy, immunotherapy and targeted therapies. Even so, cancer patients with BM are still linked to poor prognosis, with overall survival being reported as 12 months or less. Intercellular communication has a pivotal role in the development of metastases, therefore, it has been extensively studied not only to better understand the metastization process, but also to further develop new therapeutic strategies. Exosomes have emerged as key players in intercellular communication being potential therapeutic targets, drug delivery systems (DDS) or biomarkers. In this Review, we focus on the role of these extracellular vesicles (EVs) in BM formation and their promising application in the development of new BM therapeutic strategies.

2021Journal article

Open access

Development of breast cancer spheroids to evaluate cytotoxic response to an anticancer peptide

Publication . Cavaco, Marco; Fraga, Patrícia; Valle, Javier; Andreu, David; Castanho, Miguel A. R. B.; Neves, Vera

Breast cancer (BC) is the most commonly diagnosed cancer in women and one of the most common causes of cancer-related deaths. Despite intense research efforts, BC treatment still remains challenging. Improved drug development strategies are needed for impactful benefit to patients. Current preclinical studies rely mostly on cell-based screenings, using two-dimensional (2D) cell monolayers that do not mimic in vivo tumors properly. Herein, we explored the development and characterization of three-dimensional (3D) models, named spheroids, of the most aggressive BC subtypes (triple-negative breast cancer-TNBC; and human-epidermal growth receptor-2-HER2+), using the liquid overlay technique with several selected cell lines. In these cell line-derived spheroids, we studied cell density, proliferation, ultrastructure, apoptosis, reactive oxygen species (ROS) production, and cell permeabilization (live/dead). The results showed a formation of compact and homogeneous spheroids on day 7 after seeding 2000 cells/well for MDA-MB-231 and 5000 cells/well for BT-20 and BT-474. Next, we compared the efficacy of a model anticancer peptide (ACP) in cell monolayers and spheroids. Overall, the results demonstrated spheroids to be less sensitive to treatment than cell monolayers, revealing the need for more robust models in drug development.

2021Journal article

Open access