From bee venom to blood-brain barrier shuttles. Development of minimized apamin derivatives for brain delivery of antibodies and other cargoes

• Autores: Benjamí Oller Salvia
• Directores de la Tesis: Meritxell Teixidó Turá (dir. tes.), Ernest Giralt Lledó (dir. tes.)
• Lectura: En la Universitat de Barcelona ( España ) en 2015
• Idioma: inglés
• Tribunal Calificador de la Tesis: Salvador Borrós Gómez (presid.), Ernesto Nicolás (secret.), Paula Alexandra de Carvalho Gomes (voc.)
• Materias:
  • Química
    • Bioquímica
      • Péptidos
• Enlaces
  • Tesis en acceso abierto en:  TDX  Dipòsit Digital de la UB 
• Resumen

  • Brain delivery is a major unmet challenge because most drugs cannot cross the blood-brain barrier (BBB). Despite the restrictive nature of this barrier, brain vasculature reaches essentially every neural cell to supply it with nutrients. BBB-shuttles are molecules that take advantage of endogenous transport mechanisms to deliver cargoes into the brain parenchyma. However, the efficiency and selectivity of current shuttles needs to be improved. It has recently been shown that one way to enhance their transport capacity is to make them protease-resistant. The main goals of this thesis were to find new protease-resistant BBB-shuttle peptides and also to increase the transport monoclonal antibodies across the barrier. A source of peptides with high resistance to proteases is venoms. Moreover, some components, such as the potassium channel blocker apamin, have an effect on the CNS without disrupting the BBB, which makes them good BBB-shuttle candidates. Firstly, we proved that apamin and the most similar analogue described not to be toxic had a similar permeability in a bovine cell-based BBB model, which indicated that the residues implied in toxicity were not required for the transport across the BBB. Then we generated a simplified analogue, MiniAp-1, which crossed the cell monolayer mainly through an active mechanism with higher permeability than apamin. This result encouraged us to prepare additional analogues and one of them, MiniAp-4, had a permeability 3-fold that of apamin. This increase in transport could be partly explained by its high resistance to serum proteases and its different conformational preferences with respect to the other analogues. We then demonstrated that MiniAp-4 significantly improved the transport of GFP, quantum dots and gold nanoparticles in a human cellbased model of the BBB. Finally, we showed that this peptide was capable of enhancing 7.6-fold the delivery of cyanine-5.5 in mice brain. Furthermore, we detected the targeted probe in the brain parenchyma. In parallel, we aimed to increase the transport of monoclonal antibodies against glioblastoma across the BBB. We set up five reactions to conjugate peptide shuttles to different parts of two antibodies. Modification of the transaminated N-termini through oxime ligation provided a peptide/antibody ratio (PAR) lower than 1. Conjugation of the fucose present in the glycan chains yielded a PAR of 2 with one antibody but was not applied to the other because of its complex glycosylation pattern. Conversely, we achieved a PAR of 4 on partially reduced interchain cystines of both antibodies. Hence, we used this method to generate a small library of BBB-shuttle-antibody conjugates. The thioether bond linking the shuttles to the antibodies proved stable at least for 24 h in human serum. We also linked peptides containing cysteines to the lysines using cuppercatalyzed azide-alkyne cycloaddition (CuAAC). No conjugation significantly reduced antibody affinity except for N-terminal ligation. When the library of conjugates was assayed in the human cell-based BBB model we observed a significant increase of the transport with most peptides. MiniAp-1 and RVG29 were the shuttles providing the highest enhancement. Regarding the modification site, conjugation to lysines using CuAAC was the most suitable, probably because shuttles are more accessible to their receptors. In conclusion, in this thesis we have shown that cyclic mini-apamins, with reduced toxicity and immunogenicity, are highly resistant to serum proteases and are capable of crossing a tight brain endothelial cell monolayer. MiniAp-4 efficiently delivers diverse cargoes across the BBB in a human cell-based model and in mice. We have also proved that some BBB-shuttles, particularly MiniAp-1 and RVG29, can enhance the transport of monoclonal antibodies in a cell-based model and that peptides linked to the lysines using CuAAC provide the highest increase.