Deletion of SNX9 alleviates CD8 T cell exhaustion for effective cellular cancer immunotherapy.

Tumor-specific T cells are frequently exhausted by chronic antigenic stimulation. We here report on a human antigen-specific ex vivo model to explore new therapeutic options for T cell immunotherapies. T cells generated with this model resemble tumor-infiltrating exhausted T cells on a phenotypic and transcriptional level. Using a targeted pooled CRISPR-Cas9 screen and individual gene knockout validation experiments, we uncover sorting nexin-9 (SNX9) as a mediator of T cell exhaustion. Upon TCR/CD28 stimulation, deletion of SNX9 in CD8 T cells decreases PLCγ1, Ca2+, and NFATc2-mediated T cell signaling and reduces expression of NR4A1/3 and TOX. SNX9 knockout enhances memory differentiation and IFNγ secretion of adoptively transferred T cells and results in improved anti-tumor efficacy of human chimeric antigen receptor T cells in vivo. Our findings highlight that targeting SNX9 is a strategy to prevent T cell exhaustion and enhance anti-tumor immunity.

Deciphering molecular and cellular ex vivo responses to bispecific antibodies PD1-TIM3 and PD1-LAG3 in human tumors.

BACKGROUND: Next-generation cancer immunotherapies are designed to broaden the therapeutic repertoire by targeting new immune checkpoints including lymphocyte-activation gene 3 (LAG-3) and T cell immunoglobulin and mucin-domain containing-3 (TIM-3). Yet, the molecular and cellular mechanisms by which either receptor functions to mediate its inhibitory effects are still poorly understood. Similarly, little is known on the differential effects of dual, compared with single, checkpoint inhibition. METHODS: We here performed in-depth characterization, including multicolor flow cytometry, single cell RNA sequencing and multiplex supernatant analysis, using tumor single cell suspensions from patients with cancer treated ex vivo with novel bispecific antibodies targeting programmed cell death protein 1 (PD-1) and TIM-3 (PD1-TIM3), PD-1 and LAG-3 (PD1-LAG3), or with anti-PD-1. RESULTS: We identified patient samples which were responsive to PD1-TIM3, PD1-LAG3 or anti-PD-1 using an in vitro approach, validated by the analysis of 659 soluble proteins and enrichment for an anti-PD-1 responder signature. We found increased abundance of an activated (HLA-DR+CD25+GranzymeB+) CD8+ T cell subset and of proliferating CD8+ T cells, in response to bispecific antibody or anti-PD-1 treatment. Bispecific antibodies, but not anti-PD-1, significantly increased the abundance of a proliferating natural killer cell subset, which exhibited enrichment for a tissue-residency signature. Key phenotypic and transcriptional changes occurred in a PD-1+CXCL13+CD4+ T cell subset, in response to all treatments, including increased interleukin-17 secretion and signaling toward plasma cells. Interestingly, LAG-3 protein upregulation was detected as a unique pharmacodynamic effect mediated by PD1-LAG3, but not by PD1-TIM3 or anti-PD-1. CONCLUSIONS: Our in vitro system reliably assessed responses to bispecific antibodies co-targeting PD-1 together with LAG-3 or TIM-3 using patients' tumor infiltrating immune cells and revealed transcriptional and phenotypic imprinting by bispecific antibody formats currently tested in early clinical trials.

NK cells with tissue-resident traits shape response to immunotherapy by inducing adaptive antitumor immunity.

T cell-directed cancer immunotherapy often fails to generate lasting tumor control. Harnessing additional effectors of the immune response against tumors may strengthen the clinical benefit of immunotherapies. Here, we demonstrate that therapeutic targeting of the interferon-γ (IFN-γ)-interleukin-12 (IL-12) pathway relies on the ability of a population of natural killer (NK) cells with tissue-resident traits to orchestrate an antitumor microenvironment. In particular, we used an engineered adenoviral platform as a tool for intratumoral IL-12 immunotherapy (AdV5-IL-12) to generate adaptive antitumor immunity. Mechanistically, we demonstrate that AdV5-IL-12 is capable of inducing the expression of CC-chemokine ligand 5 (CCL5) in CD49a+ NK cells both in tumor mouse models and tumor specimens from patients with cancer. AdV5-IL-12 imposed CCL5-induced type I conventional dendritic cell (cDC1) infiltration and thus increased DC-CD8 T cell interactions. A similar observation was made for other IFN-γ-inducing therapies such as Programmed cell death 1 (PD-1) blockade. Conversely, failure to respond to IL-12 and PD-1 blockade in tumor models with low CD49a+ CXCR6+ NK cell infiltration could be overcome by intratumoral delivery of CCL5. Thus, therapeutic efficacy depends on the abundance of NK cells with tissue-resident traits and, specifically, their capacity to produce the DC chemoattractant CCL5. Our findings reveal a barrier for T cell-focused therapies and offer mechanistic insights into how T cell-NK cell-DC cross-talk can be enhanced to promote antitumor immunity and overcome resistance.

Dual G9A/EZH2 Inhibition Stimulates Antitumor Immune Response in Ovarian High-Grade Serous Carcinoma.

Ovarian high-grade serous carcinoma (HGSC) prognosis correlates directly with presence of intratumoral lymphocytes. However, cancer immunotherapy has yet to achieve meaningful survival benefit in patients with HGSC. Epigenetic silencing of immunostimulatory genes is implicated in immune evasion in HGSC and re-expression of these genes could promote tumor immune clearance. We discovered that simultaneous inhibition of the histone methyltransferases G9A and EZH2 activates the CXCL10-CXCR3 axis and increases homing of intratumoral effector lymphocytes and natural killer cells while suppressing tumor-promoting FoxP3+ CD4 T cells. The dual G9A/EZH2 inhibitor HKMTI-1-005 induced chromatin changes that resulted in the transcriptional activation of immunostimulatory gene networks, including the re-expression of elements of the ERV-K endogenous retroviral family. Importantly, treatment with HKMTI-1-005 improved the survival of mice bearing Trp53-/- null ID8 ovarian tumors and resulted in tumor burden reduction. These results indicate that inhibiting G9A and EZH2 in ovarian cancer alters the immune microenvironment and reduces tumor growth and therefore positions dual inhibition of G9A/EZH2 as a strategy for clinical development.

Plinabulin, a Distinct Microtubule-Targeting Chemotherapy, Promotes M1-Like Macrophage Polarization and Anti-tumor Immunity.

Reprogramming tumor infiltrating myeloid cells to elicit pro-inflammatory responses is an exciting therapeutic maneouver to improve anti-tumor responses. We recently demonstrated that a distinct microtubule-targeting drug, plinabulin-a clinical-stage novel agent-modulates dendritic cell maturation and enhances anti-tumor immunity. Here, we investigated the effects of plinabulin on macrophage polarization in vitro and in vivo. Plinabulin monotherapy induced significant tumor growth inhibition in mice bearing subcutaneous MC38 colon cancer. Importantly, the regressing tumors were characterized by an increase in M1-like/M2-like tumor-associated macrophages (TAM) ratio. The efficacy of plinabulin remained unaltered in T cell-deficient Rag2-/- mice, suggesting an important role of macrophages in driving the drug's anti-tumor effect. Exposure of murine and healthy human macrophages to plinabulin induced polarization toward the M1 phenotype, including increased expression of co-stimulatory molecules CD80, CD86 and pro-inflammatory cytokines IL-1ß, IL-6, and IL-12. M2-associated immunosuppressive cytokines IL-10 and IL-4 were reduced. This pro-inflammatory M1-like skewing of TAMs in response to plinabulin was dependent on the JNK pathway. Functionally, plinabulin-polarized human M1 macrophages directly killed HuT 78 tumor cells in vitro. Importantly, plinabulin induced a functional M1-like polarization of tumor infiltrating macrophages in murine tumors as well as in tumor samples from ovarian cancer patients, by preferentially triggering M1 proliferation. Our study uncovers a novel immunomodulatory effect of plinabulin in directly triggering M1 polarization and proliferation as well as promoting TAM anti-tumoral effector functions.

Transcriptional analysis of multiple ovarian cancer cohorts reveals prognostic and immunomodulatory consequences of ERV expression.

BACKGROUND: Endogenous retroviruses (ERVs) play a role in a variety of biological processes, including embryogenesis and cancer. DNA methyltransferase inhibitors (DNMTi)-induced ERV expression triggers interferon responses in ovarian cancer cells via the viral sensing machinery. Baseline expression of ERVs also occurs in cancer cells, though this process is poorly understood and previously unexplored in epithelial ovarian cancer (EOC). Here, the prognostic and immunomodulatory consequences of baseline ERV expression was assessed in EOC. METHODS: ERV expression was assessed using EOC transcriptional data from The Cancer Genome Atlas (TCGA) and from an independent cohort (Hammersmith Hospital, HH), as well as from untreated or DNMTi-treated EOC cell lines. Least absolute shrinkage and selection operator (LASSO) logistic regression defined an ERV expression score to predict patient prognosis. Immunohistochemistry (IHC) was conducted on the HH cohort. Combination of DNMTi treatment with γδ T cells was tested in vitro, using EOC cell lines and patient-derived tumor cells. RESULTS: ERV expression was found to define clinically relevant subsets of EOC patients. An ERV prognostic score was successfully generated in TCGA and validated in the independent cohort. In EOC patients from this cohort, a high ERV score was associated with better survival (log-rank p=0.0009) and correlated with infiltration of CD8+PD1+T cells (r=0.46, p=0.0001). In the TCGA dataset, a higher ERV score was found in BRCA1/2 mutant tumors, compared to wild type (p=0.015), while a lower ERV score was found in CCNE1 amplified tumors, compared to wild type (p=0.019). In vitro, baseline ERV expression dictates the level of ERV induction in response to DNMTi. Manipulation of an ERV expression threshold by DNMTi resulted in improved EOC cell killing by cytotoxic immune cells. CONCLUSIONS: These findings uncover the potential for baseline ERV expression to robustly inform EOC patient prognosis, influence tumor immune infiltration and affect antitumor immunity.

Tumor-associated carbohydrates and immunomodulatory lectins as targets for cancer immunotherapy.

During oncogenesis, tumor cells present specific carbohydrate chains that are new targets for cancer immunotherapy. Whereas these tumor-associated carbohydrates (TACA) can be targeted with antibodies and vaccination approaches, TACA including sialic acid-containing glycans are able to inhibit anticancer immune responses by engagement of immune receptors on leukocytes. A family of immune-modulating receptors are sialic acid-binding Siglec receptors that have been recently described to inhibit antitumor activity mediated by myeloid cells, natural killer cells and T cells. Other TACA-binding receptors including selectins have been linked to cancer progression. Recent studies have shown that glycan-lectin interactions can be targeted to improve cancer immunotherapy. For example, interactions between the immune checkpoint T cell immunoglobulin and mucin-domain containing-3 and the lectin galectin-9 are targeted in clinical trials. In addition, an antibody against the lectin Siglec-15 is being tested in an early clinical trial. In this review, we summarize the previous and current efforts to target TACA and to inhibit inhibitory immune receptors binding to TACA including the Siglec-sialoglycan axis.

Correction to: Human ovarian cancer intrinsic mechanisms regulate lymphocyte activation in response to immune checkpoint blockade.

The original version of this article unfortunately contained a mistake. Complete figure captions are missing.

Human ovarian cancer intrinsic mechanisms regulate lymphocyte activation in response to immune checkpoint blockade.

Immune checkpoint blocking antibodies are currently being tested in ovarian cancer (OC) patients and have shown some responses in early clinical trials. However, it remains unclear how human OC cancer cells regulate lymphocyte activation in response to therapy. In this study, we have established and optimised an in vitro tumour-immune co-culture system (TICS), which is specifically designed to quantify the activation of multiple primary human lymphocyte subsets and human cancer cell killing in response to PD-1/L1 blockade. Human OC cell lines and treatment naïve patient ascites show differential effects on lymphocyte activation and respond differently to PD-1 blocking antibody nivolumab in TICS. Using paired OC cell lines established prior to and after chemotherapy relapse, our data reveal that the resistant cells express low levels of HLA and respond poorly to nivolumab, relative to the treatment naïve cells. In accordance, knockdown of IFNγ receptor expression compromises response to nivolumab in the treatment naïve OC cell line, while enhanced HLA expression induced by a DNA methyltransferase inhibitor promotes lymphocyte activation in TICS. Altogether, our results suggest a 'cross resistance' model, where the acquired chemotherapy resistance in cancer cells may confer resistance to immune checkpoint blockade therapy through down-regulation of antigen presentation machinery. As such, agents that can restore HLA expression may be a suitable combination partner for immunotherapy in chemotherapy-relapsed human ovarian cancer patients.

Chemotherapy-induced apoptosis, autophagy and cell cycle arrest are key drivers of synergy in chemo-immunotherapy of epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) is the most lethal of all gynecological malignancies in the UK. Recent evidence has shown that there is potential for immunotherapies to be successful in treating this cancer. We have previously shown the effective application of combinations of traditional chemotherapy and CAR (chimeric antigen receptor) T cell immunotherapy in in vitro and in vivo models of EOC. Platinum-based chemotherapy synergizes with ErbB-targeted CAR T cells (named T4), significantly reducing tumor burden in mice. Here, we show that paclitaxel synergizes with T4 as well, and look into the mechanisms behind the effectiveness of chemo-immunotherapy in our system. Impairment of caspase activity using pan-caspase inhibitor Z-VAD reveals this chemotherapy-induced apoptotic pathway as an essential factor in driving synergy. Mannose-6-phosphate receptor-mediated autophagy and the arrest of cell cycle in G2/M are also shown to be induced by chemotherapy and significantly contributing to the synergy. Increased expression of PD-1 on T4 CAR T cells occurred when these were in culture with ovarian tumor cells; on the other hand, EOC cell lines showed increased PD-L1 expression following chemotherapy treatment. These findings provided a rationale to look into testing PD-1 blockade in combination with paclitaxel and T4 immunotherapy. Combination of these three agents in mice resulted in significant reduction of tumor burden, compared to each treatment alone. In conclusion, the mechanism driving synergy in chemo-immunotherapy of EOC is multifactorial. A deeper understanding of such process is needed to better design combination therapies and carefully stratify patients.