Zhe Fu

Background Immune checkpoint inhibitor therapy can elicit deep and durable immune responses in patients with cancer, although most do not experience long-term remission. Tumor hypoxia contributes to this treatment failure. We tested whether the clinical-stage hypoxia-activated prodrug tarloxotinib enhances immune checkpoint inhibitor therapy outcomes.Method Syngeneic mouse tumor models were used to assess the effect of tarloxotinib and immune checkpoint inhibitors on the tumor microenvironment, immune cell tumor infiltration, cytokine and chemokine expression, and tumor growth.Results Tarloxotinib lowered tumor hypoxia by eightfold in the epidermal growth factor receptor (EGFR)-dependent MB49 syngeneic mouse tumor model of bladder cancer, attracting CD8 + T cells to the tumor, and potentiating checkpoint inhibitor activity. Various methodologies, including CD8+ T cell depletion and exogenous T cell priming, confirmed that tarloxotinib improves the function of activated CD8+ T cells. While anti-programmed death-ligand 1 (anti-PD-L1), anti-programmed death protein-1, and anti-cytotoxic T-lymphocyte antigen-4 treatments were all effective in the remodeled tumor microenvironment, anti-PD-L1 was most responsive to tarloxotinib coadministration, providing a 100% complete response rate with more than 360% improvement in tumor growth delay (p<0.0001) and an increasing CD8+ T cell infiltrate from 8% to 43% of the total CD45+ population in MB49 tumors. RNA transcript analysis of 770 cancer and immune-related genes in naïve and tarloxotinib-treated tumors highlighted the induction of gene clusters related to co-stimulatory signaling, cytokine/chemokine signaling, interferon signaling, immune cell adhesion/migration, antigen presentation, and lymphoid/myeloid compartments. Serum protein analysis confirmed upregulation of several cytokines/chemokines, including interleukin (IL)-6, IL-12p40, interferon-γ, and macrophage inflammatory protein-1β. This broad immunogenic response was dependent on toll-like receptor 9 (TLR9), with the positive interaction between anti-PD-L1 and tarloxotinib being blocked in myeloid differentiation primary response 88- or TLR9-knockout mice. Surprisingly, this observed therapeutic interaction was also evident in EGFR-negative MC38 (and EG7.OVA) syngeneic tumor models, which was associated with suppression of tumor hypoxia rather than TLR9 signaling, indicating two independent mechanisms of action. Consistent with the clinical experience of minimal systemic toxicities related to normal tissue EGFR inhibition (eg, diarrhea, pruritus), mouse body-weight loss was minimal across all in vivo studies.Conclusions Tarloxotinib represents a systemically administered small molecule with an established clinical safety profile that is capable of stimulating TLR9 signaling within tumors while remodeling the microenvironment to facilitate efficacy of checkpoint inhibitors.

Background: Tumor-targeted therapy causes impressive tumor regression, but the emergence of resistance limits long-term survival benefits in patients. Little information is available on the role of the myeloid cell network, especially dendritic cells (DC) during tumor-targeted therapy. Methods: Here, we investigated therapy-mediated immunological alterations in the tumor microenvironment (TME) and tumor-draining lymph nodes (LN) in the D4M.3A preclinical melanoma mouse model (harboring the V-Raf murine sarcoma viral oncogene homolog B (BRAF)^V600E mutation) by using high-dimensional multicolor flow cytometry in combination with multiplex immunohistochemistry. This was complemented with RNA sequencing and cytokine quantification to characterize the immune status of the tumors. The importance of T cells during tumor-targeted therapy was investigated by depleting CD4⁺ or CD8⁺ T cells in tumor-bearing mice. Tumor antigen-specific T-cell responses were characterized by performing in vivo T-cell proliferation assays and the contribution of conventional type 1 DC (cDC1) to T-cell immunity during tumor-targeted therapy was assessed using Batf3^−/− mice lacking cDC1. Results: Our findings reveal that BRAF-inhibitor therapy increased tumor immunogenicity, reflected by an upregulation of genes associated with immune activation. The T cell-inflamed TME contained higher numbers of activated cDC1 and cDC2 but also inflammatory CCR2-expressing monocytes. At the same time, tumor-targeted therapy enhanced the frequency of migratory, activated DC subsets in tumor-draining LN. Even more, we identified a cDC2 population expressing the Fc gamma receptor I (FcγRI)/CD64 in tumors and LN that displayed high levels of CD40 and CCR7 indicating involvement in T cell-mediated tumor immunity. The importance of cDC2 is underlined by just a partial loss of therapy response in a cDC1-deficient mouse model. Both CD4⁺ and CD8⁺ T cells were essential for therapy response as their respective depletion impaired therapy success. On resistance development, the tumors reverted to an immunologically inert state with a loss of DC and inflammatory monocytes together with the accumulation of regulatory T cells. Moreover, tumor antigen-specific CD8⁺ T cells were compromised in proliferation and interferon-γ-production. Conclusion: Our results give novel insights into the remodeling of the myeloid landscape by tumor-targeted therapy. We demonstrate that the transient immunogenic tumor milieu contains more activated DC. This knowledge has important implications for the development of future combinatorial therapies.

Hypoxia-activated prodrugs (HAP) are a promising class of antineoplastic agents that can selectively eliminate hypoxic tumor cells. This study evaluates the hypoxia-selectivity and antitumor activity of CP-506, a DNA alkylating HAP with favorable pharmacologic properties. Stoichiometry of reduction, one-electron affinity, and back-oxidation rate of CP-506 were characterized by fast-reaction radiolytic methods with observed parameters fulfilling requirements for oxygen-sensitive bioactivation. Net reduction, metabolism, and cytotoxicity of CP-506 were maximally inhibited at oxygen concentrations above 1 μmol/L (0.1% O2). CP-506 demonstrated cytotoxicity selectively in hypoxic 2D and 3D cell cultures with normoxic/anoxic IC50 ratios up to 203. Complete resistance to aerobic (two-electron) metabolism by aldo-keto reductase 1C3 was confirmed through gain-of-function studies while retention of hypoxic (one-electron) bioactivation by various diflavin oxidoreductases was also demonstrated. In vivo, the antitumor effects of CP-506 were selective for hypoxic tumor cells and causally related to tumor oxygenation. CP-506 effectively decreased the hypoxic fraction and inhibited growth of a wide range of hypoxic xenografts. A multivariate regression analysis revealed baseline tumor hypoxia and in vitro sensitivity to CP-506 were significantly correlated with treatment response. Our results demonstrate that CP-506 selectively targets hypoxic tumor cells and has broad antitumor activity. Our data indicate that tumor hypoxia and cellular sensitivity to CP-506 are strong determinants of the antitumor effects of CP-506.

The magnitude of the host immune response can be regulated by either stimulatory or inhibitory immune checkpoint molecules. Receptor-ligand binding between inhibitory molecules is often exploited by tumours to suppress anti-tumour immune responses. Immune checkpoint inhibitors that block these inhibitory interactions can relieve T-cells from negative regulation, and have yielded remarkable activity in the clinic. Despite this success, clinical data reveal that durable responses are limited to a minority of patients and malignancies, indicating the presence of underlying resistance mechanisms. Accumulating evidence suggests that tumour hypoxia, a pervasive feature of many solid cancers, is a critical phenomenon involved in suppressing the anti-tumour immune response generated by checkpoint inhibitors. In this review, we discuss the mechanisms associated with hypoxia-mediate immunosuppression and focus on modulating tumour hypoxia as an approach to improve immunotherapy responsiveness.