Browsing by Author "Krishna Moorthy, Nivetha"

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Open Access
Development, characterisation, and testing of CNS deliverable TRAIL-receptor agonists for the treatment of Glioblastoma (GBM)
(2022) Krishna Moorthy, Nivetha; Morrison, Markus (Prof. Dr.)
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. TRAIL-agonists bind cells effectively at sub-nanomolar concentrations, whereas angiopep-2 binds its target Lrp1 with an affinity of 313 nM. As a result of the binding studies and the difference in affinity, it was hypothesized that low TRAIL receptor expression on BBB endothelial cells may interfere with efficient transport of TRAIL-ANG2 fusion proteins, which was indeed observed by transwell transport studies. However, ANG2-mediated transport can be restored by blocking the TRAIL moieties in the fusion proteins. Overall, this study showed that TRAIL-ANG2 fusion proteins are highly potent in inducing apoptosis in GBM cells, but it requires TRAIL-R masking or other innovative strategies to achieve efficient CNS-transport and utilize them for the treatment of GBM.
Open Access
Low-level endothelial TRAIL-receptor expression obstructs the CNS-delivery of angiopep-2 functionalised TRAIL-receptor agonists for the treatment of glioblastoma
(2021) Krishna Moorthy, Nivetha; Seifert, Oliver; Eisler, Stephan; Weirich, Sara; Kontermann, Roland E.; Rehm, Markus; Fullstone, Gavin
Glioblastoma (GBM) is the most malignant and aggressive form of glioma and is associated with a poor survival rate. Latest generation Tumour Necrosis Factor Related Apoptosis-Inducing Ligand (TRAIL)-based therapeutics potently induce apoptosis in cancer cells, including GBM cells, by binding to death receptors. However, the blood–brain barrier (BBB) is a major obstacle for these biologics to enter the central nervous system (CNS). We therefore investigated if antibody-based fusion proteins that combine hexavalent TRAIL and angiopep-2 (ANG2) moieties can be developed, with ANG2 promoting receptor-mediated transcytosis (RMT) across the BBB. We demonstrate that these fusion proteins retain the potent apoptosis induction of hexavalent TRAIL-receptor agonists. Importantly, blood–brain barrier cells instead remained highly resistant to this fusion protein. Binding studies indicated that ANG2 is active in these constructs but that TRAIL-ANG2 fusion proteins bind preferentially to BBB endothelial cells via the TRAIL moiety. Consequently, transport studies indicated that TRAIL-ANG2 fusion proteins can, in principle, be shuttled across BBB endothelial cells, but that low TRAIL receptor expression on BBB endothelial cells interferes with efficient transport. Our work therefore demonstrates that TRAIL-ANG2 fusion proteins remain highly potent in inducing apoptosis, but that therapeutic avenues will require combinatorial strategies, such as TRAIL-R masking, to achieve effective CNS transport.