Peredo Aguilar, Maria Azucena
(2023)
Part A - PD-1/PD-L1 blockade in cancer immunotherapy/ Part B - Evaluation of PD-1/PD-L1 immune checkpoint inhibitors in a luciferase-reporter cell-based assay.
MRes thesis, University of Nottingham.
Abstract
Part A:
Molecules that downregulate the immune response are key during cancer treatment because they pose a mechanism to avoid immunotherapy, also called immune checkpoints. In that regard, the PD-1/PD-L1 pathway has been thoroughly studied to deepen our understanding of the function of these immune checkpoint molecules. This signalling pathway involves the interaction of programmed cell death (PD-1), which is a surface protein found mainly in T and B cells, and programmed cell death ligand 1 (PD-L1) found in antigen-presenting cells. However, there is clear evidence demonstrating the overexpression of PD-L1 in some cancer cells, and when this protein interacts with PD-1, it leads to T-cell inactivation and thus helps cancer cells escape clearance by the immune system. In this literature review, the knowledge around the components of the PD-1/PD-L1 pathway and how these mediate immune evasion in the tumour microenvironment will be summarized. Given the importance of this signalling pathway, the potential for developing cancer therapeutics targeting this biological process started to grow. The predominant PD-1/PD-L1 inhibitors in the market are monoclonal antibodies, but there are other peptides and non-peptide molecules in research that seem promising in terms of their effectiveness and produce few immune adverse effects. To test the activity of such inhibitors, an array of assays can be used to characterize the molecules in different contexts, from a physicochemical to a cellular and organ level. Despite the favourable results, not all patients respond to PD-1/PD-L1 inhibitors and the reason for that, although not fully understood, is still an ongoing challenge. Overall, the complexity of the immune regulation mediated by PD-1 and PD-L1 is not completely unveiled but is an active area of cancer immunotherapy research.
Part B:
Interaction between programmed cell death 1 (PD-1) and programmed cell death-ligand 1 (PD-L1) represents an important immune checkpoint that prevents overactive immune responses to self-antigens. However, this mechanism has been hijacked by cancer cells to avoid the immune response. Since the discovery of this mechanism, promising immunotherapies targeting either PD-1 or PD-L1 have been developed, eventually leading to the approval of a series of monoclonal antibodies to treat distinct types of cancer. Nonetheless, many small molecule inhibitors (SMI) are in development because of their advantages over antibodies. Here, a commercially available anti-immune checkpoint cell-based assay from InvivoGen (PD-1/PD-L1 Bio-IC™) was used to test the activity of antibodies and SMI targeting the PD-1/PD-L1 complex. This system takes advantage of a luciferase reporter gene controlled under NFAT response elements, which enables monitoring of T cell activation when the PD-1/PD-L1 axis is blocked. Although most of the inhibitors showed a dose-dependent change in luminescence, antibodies produced a larger assay window compared to SMI. Antibodies showed no impact on cell viability measured with an ATP- based assay, although most SMI severely impacted cell viability at concentrations above 10 µM. Despite efforts to improve the small assay window obtained when screening SMI through use of different target to effector cell ratios or changing the content of serum proteins in the media, the related experiments failed to identify improved assay conditions. Factors such as different mechanisms of action between antibodies and small molecules, and the strong inhibition carried by the overexpression of PD-1 and PD-L1 might account for the small assay window. Despite this, we have validated this cell assay system for screening small molecule and antibody inhibitors of PD-1/PD-L1.
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