Mesothelioma is an aggressive asbestos induced cancer with extremely poor prognosis and limited treatment options. Immune checkpoint blockade (ICPB) has demonstrated effective therapy in melanoma and is now being applied to other cancers, including mesothelioma. However, the efficacy of ICPB and which immune checkpoint combinations constitute the best therapeutic option for mesothelioma have yet to be fully elucidated. Here, we used our well characterised mesothelioma tumour model to investigate the efficacy of different ICBP treatments to generate effective therapy for mesothelioma. We show that tumour resident regulatory T cell co-express high levels of CTLA-4, OX40 and GITR relative to T effector subsets and that these receptors are co-expressed on a large proportion of cells. Targeting any of CTLA-4, OX40 or GITR individually generated effective responses against mesothelioma. Furthermore, the combination of αCTLA-4 and αOX40 was synergistic, with an increase in complete tumour regressions from 20% to 80%. Other combinations did not synergise to enhance treatment outcomes. Finally, an early pattern in T cell response was predictive of response, with activation status and ICP receptor expression profile of T effector cells harvested from tumour and dLN correlating with response to immunotherapy. Taken together, these data demonstrate that combination ICPB can work synergistically to induce strong, durable immunity against mesothelioma in an animal model.

While chemotherapy remains the first-line treatment for many cancers, it is still unclear what distinguishes responders from non-responders. Here, we characterize the chemotherapy-responsive tumor microenvironment in mice, using RNA sequencing on tumors before and after cyclophosphamide, and compare the gene expression profiles of responders with progressors. Responsive tumors have an inflammatory and highly immune infiltrated pre-treatment tumor microenvironment characterized by the enrichment of pathways associated with CD4⁺ T cells, interferons (IFNs), and tumor necrosis factor alpha (TNF-α). The same gene expression profile is associated with response to cyclophosphamide-based chemotherapy in patients with breast cancer. Finally, we demonstrate that tumors can be sensitized to cyclophosphamide and 5-FU chemotherapy by pre-treatment with recombinant TNF-α, IFNγ, and poly(I:C). Thus, a CD4⁺ T cell-inflamed pre-treatment tumor microenvironment is necessary for response to chemotherapy, and this state can be therapeutically attained by targeted immunotherapy.

Background: Antibodies that target immune checkpoints such as cytotoxic T lymphocyte antigen 4 (CTLA-4), programmed cell death protein/ligand 1 (PD-1/PD-L1) are approved for treatment of multiple cancer types. Chemotherapy is often administered with immune checkpoint blockade (ICB) therapies that target CTLA-4 and/or PD-(L)1. ICB targeting other immune checkpoints such as lymphocyte activating gene-3 (LAG-3) has the potential to improve antitumor responses when combined with chemotherapy. Response to anti-PD-1 ICB is dependent on progenitor exhausted CD8⁺ T cells (T_PEX) in the tumor, but it is unclear how chemotherapy alters T_PEX proportions and phenotype. Methods: Here we investigated whether sequential chemotherapy altered T_PEX frequency and immune checkpoint expression in multiple murine tumor models. Results: Two doses of two different anti-metabolite chemotherapies increased tumor infiltrating CD4⁺, and CD8⁺ T_PEX expressing LAG-3 in multiple mouse models, which was not restricted to tumor antigen specific CD8⁺ T cells. To determine if LAG-3⁺tumor infiltrating lymphocytes (TILs) could be targeted to improve tumor control, we administered anti-LAG-3 and anti-PD-1 ICB after two doses of chemotherapy and found combination therapy generated robust antitumor responses compared with each agent alone. Both anti-LAG-3 and anti-PD-1 ICB with chemotherapy were required for the complete tumor regression observed. Conclusions: Changes in immune checkpoint expression on TILs during chemotherapy administration informs selection of ICB therapies to combine with.

With cancer immunotherapy and precision medicine dynamically evolving, there is greater need for pre-clinical models that can better replicate the intact tumor and its complex tumor microenvironment (TME). Precision-cut tumor slices (PCTS) have recently emerged as an ex vivo human tumor model, offering the opportunity to study individual patient responses to targeted therapies, including immunotherapies. However, little is known about the physiologic status of PCTS and how culture conditions alter gene expression. In this study, we generated PCTS from head and neck cancers (HNC) and mesothelioma tumors (Meso) and undertook transcriptomic analyses to understand the changes that occur in the timeframe between PCTS generation and up to 72 h (hrs) in culture. Our findings showed major changes occurring during the first 24 h culture period of PCTS, involving genes related to wound healing, extracellular matrix, hypoxia, and IFNγ-dependent pathways in both tumor types, as well as tumor-specific changes. Collectively, our data provides an insight into PCTS physiology, which should be taken into consideration when designing PCTS studies, especially in the context of immunology and immunotherapy.

Background: In situ cancer vaccination is a therapeutic approach that involves stimulating the immune system in order to generate a polyclonal, anti-tumor response against an array of tumor neoantigens. Traditionally, in situ vaccination approaches have utilized adenoviral vectors to deliver immune-stimulating genes directly to the tumor microenvironment. Lipid nanoparticle (LNP)-mediated delivery methods offer several advantages over adenoviral delivery approaches, including increased safety, repeated administration potential, and enhanced tumor microenvironment activation. Methods: To explore in situ vaccination using LNPs, we evaluated LNP-mediated delivery of a reporter gene, mCherry, and an immune-stimulating gene, IFNβ, in several in vitro and in vivo models of lung cancer. Results: In vitro experiments demonstrated successful transfection of murine cancer cell lines with LNPs carrying both mCherry and IFN-β mRNA, resulting in high expression levels and IFNβ production. In vivo studies using LLC.ova flank tumors showed that intratumoral injection of IFNβ-mRNA LNPs led to significant IFNβ production within the tumor microenvironment, with minimal systemic exposure. Therapeutic efficacy was evaluated by injecting established LLC.ova flank tumors with IFNβ-mRNA LNPs bi-weekly for two weeks. Treated tumors showed significant growth inhibition compared to controls. Flow cytometric analysis of tumor-infiltrating leukocytes revealed that tumors injected with IFNβ-mRNA LNPs were associated with an increased CD8:CD4 T-cell ratio among lymphocytes, more CD69-expressing CD8 T-cells, and an increased presence of M1 macrophages. Efficacy and an abscopal effect were confirmed in a squamous cell carcinoma model, MOC1. No toxicity was observed. Conclusions: These findings show that intratumoral LNP delivery of immune-stimulating mRNA transcripts, such as IFNβ, can effectively stimulate local anti-tumor immune responses and warrants further investigation as a potential immunotherapeutic approach for cancer.

Regulatory T cells (Tregs) are essential for maintaining immune tolerance but also contribute to immune suppression within the tumor microenvironment (TME), dampening antitumor immunity in hematologic and solid tumors. As such, strategies aimed at depleting Tregs or reducing their suppressive activity are of great clinical interest. CC chemokine receptor 4 (CCR4) is highly expressed on intratumoral Tregs and mediates Treg migration into the TME. Although current therapies targeting CCR4 using monoclonal antibodies have shown some Treg depletion in clinical trials, their clinical efficacy has been limited. We therefore tested whether chimeric antigen receptor (CAR) T-cell (CART) therapy could be used to deplete Tregs. We evaluated human-specific CCR4-directed CARTs (CCR4-CARTs) previously developed for T-cell malignancies and determined whether these CARTs could deplete human Tregs ex vivo and in vivo. In patient-derived malignant pleural effusions and lung cancer tumor digests, CCR4-CARTs almost completely depleted Tregs, along with a small population of CCR4+CD4+ non-Tregs, while sparing CD8+ T cells. When tested in vivo in humanized mice, a single dose of CCR4-CARTs led to nearly complete Treg depletion. These findings support the potential of CCR4-CARTs as a selective and effective approach to Treg modulation and warrant further clinical investigation.