Tarloxotinib amplifies the anti-tumour efficacy of immune checkpoint inhibitors through multiple mechanisms

Abstract

The magnitude of a T-cell response is regulated by various stimulatory and inhibitory signals via receptors expressed on the cell surface. The interaction between inhibitory receptors and their ligands can be exploited by tumours to suppress anti-tumour immune responses. Immune checkpoint inhibitors (ICIs) are monoclonal antibodies that function by blocking the inhibitory interactions between these immunosuppressive molecules, thereby relieving T-cells from negative regulation. This therapeutic approach is capable of eliciting robust and durable antitumour immune responses in advance-staged cancer patients. However, clinical studies have shown that durable responses are limited to a minority of patients and malignancies, indicating underlying resistance mechanisms to ICIs. Accumulating evidence suggests that oxygen deficiency (tumour hypoxia) within tumours is an important phenomenon that can suppress the anti-tumour immune response via multiple mechanisms. It was therefore hypothesised that the administration of tarloxotinib, a hypoxia-activated prodrug (HAP) of the potent irreversible EGFR/HER2 inhibitor tarloxotinib-E, may improve the efficacy of ICIs in EGFR-dependent murine syngeneic tumour models through lowering of tumour hypoxic fraction. Through in vitro evaluations, the MB49 syngeneic murine bladder cancer cell line was identified to be the most sensitive cell line to tarloxotinib under anoxia. The pharmacokinetic and pharmacodynamic properties of tarloxotinib were subsequently characterised in MB49 monolayer and tumour xenograft models. Tarloxotinib treatment inhibited both in vitro and in vivo EGFR signalling in MB49 cells, significantly delayed the growth of established subcutaneous MB49 tumours in C57BL/6 mice and significantly reduced the hypoxic tumour fraction. The MB49 tumour model was therefore identified as the most suitable cell line for the in vivo evaluations of the combination of tarloxotinib and ICIs. In vivo studies showed that the combination of tarloxotinib with ICIs including αPD-1, αPDL1 and αCTLA led to much improved anti-tumour activities compared to the respective single agent treatments. Flow cytometric analysis focusing on the combination of tarloxotinib and αPD-L1 showed that the combination treatment increased the tumour infiltration of effector Tcells and enhanced their production of IFN-γ. T-cells were demonstrably spared from exposure to the hypoxic tumour microenvironment and the expression of PD-L1 on MB49 cells was reduced. Experiments revealed this combinatorial activity required both the functional release of tarloxotinib-E and inhibition of the PD1/PD-L1 pathway, and was CD8+ T-cell dependent. Further evaluations highlighted enhanced production of serum cytokines including IL-2, IL-6, G-CSF, IFN-γ, MCP-1, MIP-1α and MIP-1β, and induction of multiple pathway genes predominantly in immune-related pathways (e.g. antigen presentation, interferon signalling, co-stimulatory signalling, cytotoxicity and immune cell adhesion). These activities could be explained, at least in part, by the discovery that tarloxotinib-E exhibited TLR9 agonist activity in both murine and human HEK293-TLR9 reporter cell lines. This unexpected finding raised important questions as to the respective roles for EGFR signal inhibition, hypoxia suppression and innate immune activation in the synergistic activity of tarloxotinib when combined with ICIs. Overall, results presented in this thesis using the syngeneic MB49 tumour model demonstrate the therapeutic potential of combining the HAP tarloxotinib with ICIs, particularly αPD-L1. Future investigation in other EGFR-driven cancer models is required to further delineate its mechanism of action and to determine the optimal combination strategy and dosing schedule. These promising data also encourage the design of clinical trials incorporating the HAP and immunotherapy treatment modalities for clinical translation to improve patient prognosis.