Browsing by Author "Neves, Vera"
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- Antibodies for the treatment of brain metastases, a dream or a reality?Publication . Cavaco, Marco; Gaspar, Diana; Castanho, Miguel A. R. B.; Neves, VeraThe incidence of brain metastases (BM) in cancer patients is increasing. After diagnosis, overall survival (OS) is poor, elicited by the lack of an effective treatment. Monoclonal antibody (mAb)-based therapy has achieved remarkable success in treating both hematologic and non-central-nervous system (CNS) tumors due to their inherent targeting specificity. However, the use of mAbs in the treatment of CNS tumors is restricted by the blood-brain barrier (BBB) that hinders the delivery of either small-molecules drugs (sMDs) or therapeutic proteins (TPs). To overcome this limitation, active research is focused on the development of strategies to deliver TPs and increase their concentration in the brain. Yet, their molecular weight and hydrophilic nature turn this task into a challenge. The use of BBB peptide shuttles is an elegant strategy. They explore either receptor-mediated transcytosis (RMT) or adsorptive-mediated transcytosis (AMT) to cross the BBB. The latter is preferable since it avoids enzymatic degradation, receptor saturation, and competition with natural receptor substrates, which reduces adverse events. Therefore, the combination of mAbs properties (e.g., selectivity and long half-life) with BBB peptide shuttles (e.g., BBB translocation and delivery into the brain) turns the therapeutic conjugate in a valid approach to safely overcome the BBB and efficiently eliminate metastatic brain cells.
- Antibody approaches to treat brain diseasesPublication . Neves, Vera; Silva, Frederico Aires da; Real, Sofia Corte; Castanho, Miguel A. R. B.Recombinant antibodies are an emerging class of therapeutics with substantial promise to treat central nervous system (CNS) diseases such as Alzheimer's, Parkinson's, stroke, and brain cancers. However, poor blood–brain barrier (BBB) permeability hinders the progress of antibody therapies for conditions in which the target is located in the brain. Nonetheless, antibodies can nowa-days be engineered to enhance their therapeutic applications and translocation of the BBB using the natural portals of the brain. This paper reviews advances in the development and engineering of therapeutic BBB-crossing antibodies and their high potential for treatment of CNS disorders.
- Antimigratory and antiproliferative effects of the brain‐targeted peptide conjugate PepH3‐vCPP2319 on triple negative breast cancer cell culturesPublication . Gonçalves, Catarina; Castanho, Miguel A. R. B.; Cavaco, Marco; Neves, VeraTriple-negative breast cancer (TNBC) is an aggressive breast cancer subtype affecting mostly younger women with a poor 5-year overall survival. It is characterized by a high metastization rate, particularly to the brain, where the blood-brain barrier (BBB) hinders the pharmaceuticals delivery. New anticancer drugs able to inhibit cell migration are required to effectively prevent the development of metastasis. PepH3-vCPP2319 (AGILKRW(Ahx)WRRRYRRWRRRRRQRRRPRR-amide), consisting of the conjugation of the BBB peptide shuttle (BBBpS) PepH3 (AGILKRW-amide) to the anticancer peptide (ACP) vCPP2319 (WRRRYRRWRRRRRQRRRPRR-amide), was reported to have high anticancer activity (IC50 = 5.0 μM) toward highly aggressive TNBC cells (MDA-MB-231) paired with 2-fold increased accumulation in the brain when compared to unconjugated vCPP2319. Herein, we demonstrate that PepH3-vCPP2319 inhibits cell migration and proliferation in wound healing assays, outperforming the gold standard small chemical inhibitor, iCRT-3. The concentration required to inhibit cell migration is 10-fold lower for PepH3-vCPP2319 (0.5 μM) when compared with iCRT-3 (50 μM). Likewise, PepH3-vCPP2319 at 2.5 μM was more efficient in preventing cell proliferation when compared with 50 μM iCRT-3, with 45% reduction in spheroid diameter. This study sheds light on the antimetastatic potential of PepH3-vCPP2319 through abrogation of cell migration to distant sites, including the brain.
- Conjugation of a blood brain barrier peptide shuttle to an Fc domain for brain delivery of therapeutic biomoleculesPublication . Cavaco, Marco; Frutos, Silvia; Oliete, Paula; Valle, Javier; Andreu, David; Castanho, Miguel A. R. B.; Vila-Perelló, Miquel; Neves, VeraThe 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.
- Development of breast cancer spheroids to evaluate cytotoxic response to an anticancer peptidePublication . Cavaco, Marco; Fraga, Patrícia; Valle, Javier; Andreu, David; Castanho, Miguel A. R. B.; Neves, VeraBreast 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.
- DPepH3, an improved peptide shuttle for receptor-independent transport across the blood-brain barrierPublication . Cavaco, Marco; Valle, Javier; Silva, Rúben; Correia, João D. G.; Castanho, Miguel A. R. B.; Andreu, David; Neves, VeraBackground: The use of peptides as drug carriers across the blood-brain barrier (BBB) has increased significantly during the last decades. PepH3, a seven residue sequence (AGILKRW) derived from the α-helical domain of the dengue virus type-2 capsid protein, translocates across the BBB with very low toxicity. Somehow predictably from its size and sequence, PepH3 is degraded in serum relatively fast. Among strategies to increase peptide half-life (t1/2), the use of the enantiomer (wholly made of D-amino acid residues) can be quite successful if the peptide interacts with a target in non-stereospecific fashion. Methods: The goal of this work was the development of a more proteolytic-resistant peptide, while keeping the translocation properties. The serum stability, cytotoxicity, in vitro BBB translocation, and internalization mechanism of DPepH3 was assessed and compared to the native peptide. Results: DPepH3 demonstrates a much longer t1/2 compared to PepH3. We also confirm that BBB translocation is receptor-independent, which fully validates the enantiomer strategy chosen. In fact, we demonstrate that internalization occurs trough macropinocytosis. In addition, the enantiomer demonstrates to be non-cytotoxic towards endothelial cells as PepH3. Conclusion: DPepH3 shows excellent translocation and internalization properties, safety, and improved stability. Taken together, our results place DPepH3 at the forefront of the second generation of BBB shuttles.
- Exosomes and brain metastases: a review on their role and potential applicationsPublication . Oliveira, Filipa; Castanho, Miguel A. R. B.; Neves, VeraBrain 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.
- 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.
- A new noncanonical anionic peptide that translocates a cellular blood-brain barrier modelPublication . Neves Coelho, Sara; Eleutério, Rute P.; Enguita, Francisco J.; Neves, Vera; Castanho, Miguel A. R. B.The capacity to transport therapeutic molecules across the blood–brain barrier (BBB) represents a breakthrough in the development of tools for the treatment of many central nervous system (CNS)-associated diseases. The BBB, while being protective against infectious agents, hinders the brain uptake of many drugs. Hence, finding safe shuttles able to overcome the BBB is of utmost importance. Herein, we identify a new BBB-translocating peptide with unique properties. For years it was thought that cationic sequences were mandatory for a cell-penetrating peptide (CPP) to achieve cellular internalization. Despite being anionic at physiological pH, PepNeg (sequence (SGTQEEY) is an efficient BBB translocator that is able to carry a large cargo (27 kDa), while maintaining BBB integrity. In addition, PepNeg is able to use two distinct methods of translocation, energy-dependent and -independent, suggesting that direct penetration might occur when low concentrations of peptide are presented to cells. The discovery of this new anionic trans-BBB peptide allows the development of new delivery systems to the CNS and contributes to the need to rethink the role of electrostatic attraction in BBB-translocation
- Novel peptides derived from dengue virus capsid protein translocate reversibly the blood−brain barrier through a receptor-free mechanismPublication . Neves, Vera; Silva, Frederico Aires da; Morais, Maurício; Gano, Lurdes; Ribeiro, Elisabete; Pinto, Antónia; Aguiar, Sandra; Gaspar, Diana; Fernandes, Célia; Correia, João D. G.; Castanho, Miguel A. R. B.The delivery of therapeutic molecules to the central nervous system is hampered by poor delivery across the blood-brain barrier (BBB). Several strategies have been proposed to enhance transport into the brain, including invasive techniques and receptor-mediated transport (RMT). Both approaches have several drawbacks, such as BBB disruption, receptor saturation, and off-target effects, raising safety issues. Herein, we show that specific domains of Dengue virus type 2 capsid protein (DEN2C) can be used as trans-BBB peptide vectors. Their mechanism of translocation is receptor-independent and consistent with adsorptive-mediated transport (AMT). One peptide in particular, named PepH3, reaches equilibrium distribution concentrations across the BBB in less than 24 h in a cellular in vitro assay. Importantly, in vivo biodistribution data with radiolabeled peptide derivatives show high brain penetration. In addition, there is fast clearance from the brain and high levels of excretion, showing that PepH3 is a very good candidate to be used as a peptide shuttle taking cargo in and out of the brain.
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