Development, characterisation, and testing of CNS deliverable TRAIL-receptor agonists for the treatment of Glioblastoma (GBM)

Glioblastoma (GBM) is a grade IV glioma, which is the most malignant and aggressive form of glioma. It accounts for 80% of the primary malignant brain tumours with a median survival time of just ~14 months. Therefore, GBM is presented as a highly challenging tumour and continuous efforts are required to find innovative and more effective treatment options. Tumour Necrosis Factor Related Apoptosis Inducing Ligand (TRAIL)-based therapeutics potently induce apoptosis in cancer cells, including GBM cells, by binding and activating TRAIL receptors (TRAIL-R1 and R2). However, the blood-brain barrier (BBB) is a major obstacle for these biologics to enter the central nervous system (CNS). The BBB is mainly made up of tightly connected endothelial cells and therefore the penetration of large biologics into the CNS is generally controlled and prevented by the presence of the BBB, with approximately 0.1% of injected antibody doses reaching the brain parenchyma. Receptor-mediated transcytosis is a mode of transport capable of carrying large proteins and lipoproteins across the BBB. Therefore, in this research work, studies were performed to investigate if antibody-based fusion proteins that combine the apoptosis-inducing TRAIL with transcytosis-inducing angiopep-2 could be developed. It was observed that the addition of the ANG2 moiety does not interfere with the potent apoptosis induction of TRAIL and these hexavalent TRAIL-receptor agonists demonstrated robust cytotoxicity against GBM cells. TRAIL receptor quantification demonstrated that the BBB cells do indeed express TRAIL receptors although in significantly reduced amounts compared to cancer cells. In cytotoxicity studies, BBB cells remained highly resistant to this fusion protein in response to clinically relevant doses of TRAIL-receptor agonists. Binding studies indicated that ANG2 is active in these constructs, however, control peptides and TRAIL-blocking experiments demonstrated that TRAIL-ANG2 fusion construct binding to BBB cells is mainly TRAIL-mediated. ...