Cancer patients with low or absent pre-existing anti-tumour immunity (“cold” tumours) respond poorly to treatment with immune checkpoint inhibitors (ICPI). In order to render these patients susceptible to ICPI, initiation of de novo tumour-targeted immune responses is required. This involves triggering of inflammatory signalling, innate immune activation including recruitment and stimulation of dendritic cells (DCs), and ultimately priming of tumour-specific T cells. The ability of tumour localised therapies to trigger these pathways and act as in situ tumour vaccines is being increasingly explored, with the aspiration of developing combination strategies with ICPI that could generate long-lasting responses. In this effort, it is crucial to consider how therapy-induced changes in the tumour microenvironment (TME) act both as immune stimulants but also, in some cases, exacerbate immune resistance mechanisms. Increasingly refined immune monitoring in pre-clinical studies and analysis of on-treatment biopsies from clinical trials have provided insight into therapy-induced biomarkers of response, as well as actionable targets for optimal synergy between localised therapies and ICB. Here, we review studies on the immunomodulatory effects of novel and experimental localised therapies, as well as the re-evaluation of established therapies, such as radiotherapy, as immune adjuvants with a focus on ICPI combinations.
BackgroundCombination herpes simplex virus (HSV) oncolytic virotherapy and BRAF inhibitors (BRAFi) represent promising immunogenic treatments for BRAF mutant melanoma, but an improved understanding of the immunobiology of combinations is needed to improve on the benefit of immune checkpoint inhibitors (ICI).MethodsUsing a BRAFV600E-driven murine melanoma model, we tested the immunogenicity of HSV/BRAFi in immunocompetent C57BL mice. In addition to standard FACS analysis, we used the ‘Timer of Cell Kinetics and Activity’ system, which can analyze the temporal dynamics of different T cell subsets. This immune data was used to inform the selection of ICI for triple combination therapy, the effects of which were then further characterized using transcriptomics.ResultsAdding BRAFi treatment to HSV improved anti-tumor effects in vivo but not in vitro. Immune characterization showed HSV or dual therapy led to fewer intratumoral Treg, although with a more activated phenotype, together with more effector CD8 +T cells. Tocky analysis further showed that HSV/BRAFi dual treatment reduced the Tocky signal (reflecting engagement with cognate antigen), in both Treg and conventional subsets of CD4+, but not in CD8 +cells. However, a higher percentage of Treg than of conventional CD4 +maintained frequent engagement with antigens on treatment, reflecting a predominance of suppressive over effector function within the CD4 +compartment. The only T cell subset which correlated with a reduction in tumor growth was within Tocky signal positive conventional CD4+, supporting their therapeutic role. Targeting CD25 high, antigen-engaged Treg with a depleting anti-CD25 ICI, achieved complete cures in 100% of mice with triple therapy. Transcriptomic analysis confirmed reduction in Foxp3 on addition of anti-CD25 to HSV/BRAFi, as well as increases in expression of genes reflecting interferon signaling and cytotoxic activity.ConclusionsCombination HSV/BRAFi is an immunogenic therapy for BRAF mutant melanoma, but cannot fully control tumors. Dual therapy results in changes in T cell dynamics within tumors, with relatively maintained antigen signaling in Treg compared with conv CD4+. Antigen-engaged CD4 +effectors correlate with tumor growth control, and depletion of Treg by addition of an anti-CD25 ICI, releasing suppression of conventional CD4 +effectors by Treg, enhances survival and activates immune signaling within tumors.
The blockade of the immune checkpoints PD-1 and CTLA-4 enhances T cell response. However, it is largely unknown how antigen-reactive T cells regulate their checkpoint expression in vivo and whether and how the checkpoint blockade can change activation dynamics of tumour-reactive T cells. To address this, here we used Nr4a3-Timer-of-cell-kinetics-and-activity (Tocky), which allows analysis of temporal changes of activated T cells following TCR signalling in vivo. By analysing melanoma-bearing Nr4a3 Tocky mice, we elucidate hidden dynamics of tumour-reactive T cells in the steady-state. Checkpoint blockade depleted highly activated effector Treg, while promoting unique effector T cell populations, and thus differentially modulating activation of tumour-reactive T cell populations. Furthermore, multidimensional analysis and seamless analysis of Tocky and scRNA-seq revealed a full spectrum of T cell dynamics in response to tumour burden and treatment with checkpoint blockade. Lastly, we propose a rational design of combinatorial therapy to further enhance T cell activities.
CD4 T-cells require T-cell receptor (TCR) signalling for their activation and differentiation. Foxp3+ regulatory T-cells (Treg) are dependent on TCR signals for their differentiation and suppressive function. However, it is not fully known how TCR signalling controls the differentiation of polyclonal CD4 T-cells upon antigen recognition at the single-cell level in vivo. In this study, using Nr4a3-Tocky (Timer-of-cell-kinetics-and-activity), which analyses temporal changes of antigen-reactive T-cells following TCR signalling, we investigated T-cell response to Spike protein fragments (S1a, S1b, S2a, and S2b) upon immunisation. We show that S1a and S2a induced the differentiation of PD1hiCXCR5+ T follicular helper (Tfh) cells, which is related to CD4 T-cell immunogenicity. In contrast, S1b induced CD25hiGITRhiPD-1int Treg, which intermittently received TCR signalling. Using Foxp3-Tocky, which analyses Foxp3 transcriptional dynamics, the S1b-reactive Treg sustained Foxp3 transcription over time, which is a hallmark of activated Treg. Foxp3 fate-mapping showed that the S1b-reactive Treg were derived not from pre-existing thymic Treg, suggesting Foxp3 induction in non-Treg cells. Thus, the current study reveals temporally dynamic differentiation of CD4 T-cells and Treg upon immunisation in the polyclonal TCR repertoire.
Small molecule inhibitors of the MAPK pathway have demonstrated rapid responses in melanoma patients, but their use is hindered by the frequent development of acquired resistance. Oncolytic viruses are a novel class of immunotherapeutics which selectively replicate inside cancer cells, causing lysis and subsequent activation of anti-tumor immunity. BRAF and MEK inhibitors have previously been shown to synergise with oncolytic viruses to kill melanoma cells, but the consequences of these interactions on immune responses have not been assessed. Here, we show that the combination of PLX4720 (BRAFV600E inhibitor) and intratumoral oncolytic herpes virus HSV1716 led to a significant tumor reduction and improved survival over single-agent treatment in a murine BRAFV600E melanoma model. The combination led to a significant increase of PD-L1+ neutrophils and monocytes at an early time point following therapy administration, suggesting an inflammatory switch in the tumor microenvironment. Importantly, the combination led to a significant increase in cytotoxic CD8+ cells at a later time point, which was accompanied by an increase in CD25+ Foxp3+ T-regulatory (Treg) cells. Addition of an anti-CD25 antibody to the PLX4720/HSV1716 doublet led to complete eradication of melanoma tumors in vivo. Current efforts are focused on deciphering T-cell dynamics during this therapy using a novel technology, Timer of cell kinetics and activity (Tocky). Tocky uses a fluorescent Timer protein which spontaneously changes its emission spectrum from blue to red, reporting real-time activation of the T-cell receptor and subsequent activation of immune cells in vivo. Using Tocky, we are identifying specific immune subsets that are stimulated following therapy and the real-time dynamics of antigen recognition and subsequent cell activation. These findings form the basis of future testing of drug-virus combinations in an effort to identify potent cytotoxic, as well as immuno-modulatory, strategies and their detailed mechanism of action. Citation Format: Galabina Bozhanova, Victoria Jennings, Malin Pedersen, Joan Kyula, Emmanuel Patin, Jehanne Hassan, Masahiro Ono, Kevin Harrington, Alan Melcher. Mutant BRAF small molecule inhibition enhances oncolytic herpes virus immunotherapy through increased immune cell recruitment and activation in melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3415.
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