Ian F. Hermans

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.

CD8+ tissue-resident memory T cells provide rapid frontline protection at pathogen invasion sites, making them attractive targets for vaccine-mediated immunity. We previously developed an NKT cell-adjuvanted mRNA lipoplex vaccine capable of inducing liver Trm cells and sterile protection against malaria in mice. Here, we show that type I interferon (IFN-I) signalling through dendritic cells — not T cells — is a key brake on liver Trm induction by this vaccine. Optimising mRNA manufacturing to reduce immunostimulatory contaminants substantially dampened IFN-I production, boosted antigen expression in the lymphoid tissues, and drove significantly greater Trm accumulation. These enhanced responses translated into superior protection against parasite challenge. Our findings identify DC-intrinsic IFN-I signalling as a tractable target, and mRNA manufacturing quality as a critical and underappreciated lever, for maximising Trm-based vaccine efficacy.

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.

Malaria is caused by Plasmodium species transmitted by Anopheles mosquitoes. Following a mosquito bite, Plasmodium sporozoites migrate from skin to liver, where extensive replication occurs, emerging later as merozoites that can infect red blood cells and cause symptoms of disease. As liver tissue-resident memory T cells (Trm cells) have recently been shown to control liver-stage infections, we embarked on a messenger RNA (mRNA)-based vaccine strategy to induce liver Trm cells to prevent malaria. Although a standard mRNA vaccine was unable to generate liver Trm or protect against challenge with Plasmodium berghei sporozoites in mice, addition of an agonist that recruits T cell help from type I natural killer T cells under mRNA-vaccination conditions resulted in significant generation of liver Trm cells and effective protection. Moreover, whereas previous exposure of mice to blood-stage infection impaired traditional vaccines based on attenuated sporozoites, mRNA vaccination was unaffected, underlining the potential for such a rational mRNA-based strategy in malaria-endemic regions.

Aim: We have previously reported that polyfunctional T cell responses can be induced to the cancer testis antigen NY-ESO-1 in melanoma patients injected with mature autologous monocyte-derived dendritic cells (DCs) loaded with long NY-ESO-1-derived peptides together withα-galactosylceramide (α-GalCer), an agonist for type 1 Natural Killer T (NKT) cells.Objective: To assess whether inclusion ofα-GalCer in autologous NY-ESO-1 long peptide-pulsed DC vaccines (DCV + α-GalCer) improves T cell responses when compared to peptide-pulsed DC vaccines withoutα-GalCer (DCV).Design, setting and participants: Single-centre blinded randomised controlled trial in patients ≥ 18 years old with histologically confirmed, fully resected stage II–IV malignant cutaneous melanoma, conducted between July 2015 and June 2018 at the Wellington Blood and Cancer Centre of the Capital and Coast District Health Board.Interventions: Stage I. Patients were randomised to two cycles of DCV or DCV + α-GalCer (intravenous dose of 10 × 10⁶cells, interval of 28 days).Stage II.Patients assigned to DCV + α-GalCer were randomised to two further cycles of DCV + α-GalCer or observation, while patients initially assigned to DCV crossed over to two cycles of DCV + α-GalCer.Outcome measures: Primary:Area under the curve (AUC) of mean NY-ESO-1-specific T cell count detected by ex vivo IFN-γ ELISpot in pre- and post-treatment blood samples, compared between treatment arms at Stage I.Secondary:Proportion of responders in each arm at Stage I; NKT cell count in each arm at Stage I; serum cytokine levels at Stage I; adverse events Stage I; T cell count for DCV + α-GalCer versus observation at Stage II, T cell count before versus after cross-over.Results: Thirty-eight patients gave written informed consent; 5 were excluded before randomisation due to progressive disease or incomplete leukapheresis, 17 were assigned to DCV, and 16 to DCV + α-GalCer. The vaccines were well tolerated and associated with increases in mean total T cell count, predominantly CD4⁺T cells, but the difference between the treatment arms was not statistically significant (difference − 6.85, 95% confidence interval, − 21.65 to 7.92;P = 0.36). No significant improvements in T cell response were associated with DCV + α-GalCer with increased dosing, or in the cross-over. However, the NKT cell response toα-GalCer-loaded vaccines was limited compared to previous studies, with mean circulating NKT cell levels not significantly increased in the DCV + α-GalCer arm and no significant differences in cytokine response between the treatment arms.Conclusions: A high population coverage of NY-ESO-1-specific T cell responses was achieved with a good safety profile, but we failed to demonstrate that loading withα-GalCer provided an additional advantage to the T cell response with this cellular vaccine design. Clinical trial registration: ACTRN12612001101875. Funded by the Health Research Council of New Zealand.

Intratumoural administration of unmethylated cytosine-phosphate-guanine motifs (CpG) to stimulate toll-like receptor (TLR)-9 has been shown to induce tumour regression in preclinical studies and some efficacy in the clinic. Because activated natural killer T (NKT) cells can cooperate with pattern-recognition via TLRs to improve adaptive immune responses, we assessed the impact of combining a repeated dosing regimen of intratumoural CpG with a single intratumoural dose of the NKT cell agonist α-galactosylceramide (α-GalCer). The combination was superior to CpG alone at inducing regression of established tumours in several murine tumour models, primarily mediated by CD8⁺ T cells. An antitumour effect on distant untreated tumours (abscopal effect) was reliant on sustained activity of NKT cells and was associated with infiltration of KLRG1⁺ NKT cells in tumours and draining lymph nodes at both injected and untreated distant sites. Cytometric analysis pointed to increased exposure to type I interferon (IFN) affecting many immune cell types in the tumour and lymphoid organs. Accordingly, antitumour activity was lost in animals in which dendritic cells (DCs) were incapable of signaling through the type I IFN receptor. Studies in conditional ablation models showed that conventional type 1 DCs and plasmacytoid DCs were required for the response. In tumour models where the combined treatment was less effective, the addition of tumour-antigen derived peptide, preferably conjugated to α-GalCer, significantly enhanced the antitumour response. The combination of TLR ligation, NKT cell agonism, and peptide delivery could therefore be adapted to induce responses to both known and unknown antigens.

Objectives: Metastasis is the principal cause of breast cancer mortality. Vaccines targeting breast cancer antigens have yet to demonstrate clinical efficacy, and there remains an unmet need for safe and effective treatment to reduce the risk of metastasis, particularly for people with triple‐negative breast cancer (TNBC). Certain glycolipids can act as vaccine adjuvants by specifically stimulating natural killer T (NKT) cells to provide a universal form of T‐cell help.Methods: We designed and made a series of conjugate vaccines comprising a prodrug of the NKT cell‐activating glycolipid α‐galactosylceramide covalently linked to tumor‐expressed peptides, and assessed these using E0771‐ and 4T1‐based breast cancer models in vivo. We employed peptides from the model antigen ovalbumin and from clinically relevant breast cancer antigens HER2 and NY‐ESO‐1.Results: Glycolipid‐peptide conjugate vaccines that activate NKT cells led to antigen‐presenting cell activation, induced inflammatory cytokines, and, compared with peptide alone or admixed peptide and α‐galactosylceramide, specifically enhanced CD8⁺ T‐cell responses against tumor‐associated peptides. Primary tumor growth was delayed by vaccination in all tumor models. Using 4T1‐based cell lines expressing HER2 or NY‐ESO‐1, a single administration of the relevant conjugate vaccine prevented tumor colonisation of the lung following intravenous inoculation of tumor cells or spontaneous metastasis from breast, respectively.Conclusion: Glycolipid‐peptide conjugate vaccines that activate NKT cells prevent lung metastasis in breast cancer models and warrant investigation as adjuvant therapies for high‐risk breast cancer.

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.