Abstract
Granzyme B is a serine protease that occurs abundantly in granules of cytotoxic T cells and Natural Killer (NK) cells. Upon degranulation, GRZB is released together with the pore forming protein perforin. In targeted cells, GRZB can subsequently cause apoptosis via proteolytic activation of caspases 3, 7, 8 and 10. GRZB was investigated over the last years as a biomarker for the cellular immune response, among others in oncological settings. Within the tumor micro-environment, the amount of active GRZB that is secreted by activated immune cells, is inversely correlated with the degree of immunosuppression. Therefore, GRZB is quantified in tumor biopsies of patients treated with immune checkpoint inhibitors. Moreover, significant research effort is going to the discovery of Positron Emission Tomography (PET)-probes that allow minimally-invasive imaging of GRZB. This approach is also relevant for CAR T and CAR NK cell therapy, where the amount of released GRZB correlates with therapy response. All GRZB-PET probes reported to date, however, have a peptide-derived structure and a suboptimal pharmacokinetic and/or pharmacodynamic profile. To address these limitations, we formulated following objectives: (1) synthesis of a reference molecule "VTI-1002" with nanomolar activity against the protease; (2) synthesis of inhibitors with a modified warhead moiety (e.g., a carbonitrile moiety); (3) evaluate the inhibitory potency and target selectivity of the ligands; (5) gather Structure-Activity-Relationship (SAR) data for modifications performed; (6) transform optimal Granzyme B ligands into probe molecules.
At the current stage, 30 potential inhibitors were designed, synthesized, and characterized. Recombinant human Granzyme B was expressed, and the compounds were screened against the protease. The majority of inhibitors showed potent inhibition of Granzyme B with IC50 values from 20 nM to 1 μM. The compound library was also evaluated against Caspase-3, the main off-target protease. Several Granzyme B hit compounds showed moderate inhibition of the protease, with the most potent inhibitor reaching a selectivity index of approximately 12. These findings lead to the need for further structural optimization. Future studies will also specifically address lipophilicity to understand whether the compound can cross the cell membrane.
Within the framework of the OncoProTools MSCA doctoral network, some of the inhibitors were additionally tested against Cathepsins L, S, and B, proteases that are highly expressed in the tumour microenvironment and share structural similarities with Granzyme B.
Work during the SEP-funded year, will be directed towards the development of potential PET and biotin probes, based on the most potent inhibitor sequence that is currently available. We plan to further proceed with 18F-labelling and evaluation of the probes in a pancreatic cancer model in immune-competent mice. Additionally, we plan to assess the probes in human cancer biopsies to estimate Granzyme B expression inside the tissues. Finally, we aim to publish 2 manuscripts as a first author during 2026 and to deliver a PhD thesis.
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