Tarloxotinib targets tumor hypoxia to improve therapeutic efficacy of immune checkpoint inhibition in a TLR9 dependent manner

Abstract

Immune checkpoint inhibitors can elicit deep and durable immune responses although most cancer patients fail to experience long-term remission. There is consensus that tumor hypoxia coordinates a multitude of underlying resistance mechanisms that contribute to treatment failure. Here we show that the clinical-stage hypoxia-activated prodrug tarloxotinib lowers tumor hypoxia 8-fold in the EGFR-dependent MB49 syngeneic tumor model, attracting a effector CD8+ T cell infiltrate into an oxygen enriched tumor microenvironment, leading to potentiation of checkpoint inhibitor activity. Various methodologies, including CD8+ T cell depletion and exogenous T cell priming confirmed that tarloxotinib has a positive impact on the function of activated CD8+ T cells. Whilst anti-PD-L1, anti-PD-1 and anti-CTLA-4 treatments all benefited from the remodeled tumor microenvironment, anti-PD-L1 was most responsive to tarloxotinib coadministration, providing a 100% complete response rate and >360% improvement in tumor growth delay (day 19; p<0.0001). This robust interaction was associated with MB49 tumor enrichment of CD8+ T cell infiltrate from 8% to 43% of the total CD45+ population. To further understand these promising observations, comparative RNA transcript analysis of 770 cancer/immune related genes in naive and tarloxotinib treated tumors highlighted the strong induction of gene clusters related to co-stimulatory signaling, cytokine/chemokine signaling, interferon signaling, immune cell adhesion/migration, antigen presentation and the lymphoid/myeloid compartments. Serum protein analysis confirmed upregulation of a mixture of cytokines/chemokines, including IL-6, IL-12p40, IFN{gamma}, G-CSF and MIP-1{beta}, amongst others. The source of this broad immunogenic response was identified as toll-like receptor 9 (TLR9) dependent, with the positive interaction between anti-PD-L1 and tarloxotinib blocked in MyD88 or TLR9 knockout mice. Further, the observed therapeutic interaction was still evident in MC38 and EG7.OVA syngeneic tumor models, both refractory to tarloxotinib by virtue of EGFR-signal independence. Consistent with the clinical experience of minimal systemic toxicities related to EGFR inhibition (e.g. diarrhea), mouse body weight loss was minimal across all in vivo studies and histopathology screening for evidence of lung fibrosis proved negative. Tarloxotinib therefore represents a systemically administered small molecule with an established clinical safety profile that is capable of activating TLR9 signaling within tumors whilst remodeling the microenvironment to facilitate efficacy of checkpoint blockade. What is already known on this topicSolid tumor resistance to immune checkpoint inhibitors (ICI) is orchestrated through a diverse collection of hypoxia-driven mechanisms. In preclinical models modifying the tumor microenvironment (TME) to lessen hypoxia typically improves responses to ICI. What this study addsTarloxotinib is a hypoxia-activated prodrug (HAP) of an irreversible EGFR/HER2 TKI that profoundly remodels the TME, both eliminating tumor hypoxia and elevating cytokine/chemokine production via a TLR9 dependent effect. Together, this results in a marked tumor influx of activated CD8+ T cells (and other TILs), that results in major improvements in the efficacy of ICIs, particularly anti-PD-L1. How this study might affect research, practice or policyTarloxotinib is the first example of a tumor-targeted small molecule TLR9 stimulant that restores ICI sensitivity in a range of syngeneic tumor models. Phase I/II data has demonstrated that tarloxotinib is well tolerated, with few EGFR-dependent (on-target) side-effects, reflecting minimization of normal tissue exposure with the tumor-targeted HAP approach. Importantly, it also optimizes tumor selective exposure, offering a compelling clinical rationale for evaluation of this combination in cancer patients experiencing ICI relapse or resistance.