Auranofin Synergizes with the PARP Inhibitor Olaparib to Induce ROS-Mediated Cell Death in Mutant p53 Cancers.

Auranofin (AF) is a potent, off-patent thioredoxin reductase (TrxR) inhibitor that efficiently targets cancer via reactive oxygen species (ROS)- and DNA damage-mediated cell death. The goal of this study is to enhance the efficacy of AF as a cancer treatment by combining it with the poly(ADP-ribose) polymerase-1 (PARP) inhibitor olaparib (referred to as 'aurola'). Firstly, we investigated whether mutant p53 can sensitize non-small cell lung cancer (NSCLC) and pancreatic ductal adenocarcinoma (PDAC) cancer cells to AF and olaparib treatment in p53 knock-in and knock-out models with varying p53 protein expression levels. Secondly, we determined the therapeutic range for synergistic cytotoxicity between AF and olaparib and elucidated the underlying molecular cell death mechanisms. Lastly, we evaluated the effectiveness of the combination strategy in a murine 344SQ 3D spheroid and syngeneic in vivo lung cancer model. We demonstrated that high concentrations of AF and olaparib synergistically induced cytotoxicity in NSCLC and PDAC cell lines with low levels of mutant p53 protein that were initially more resistant to AF. The aurola combination also led to the highest accumulation of ROS, which resulted in ROS-dependent cytotoxicity of mutant p53 NSCLC cells through distinct types of cell death, including caspase-3/7-dependent apoptosis, inhibited by Z-VAD-FMK, and lipid peroxidation-dependent ferroptosis, inhibited by ferrostatin-1 and alpha-tocopherol. High concentrations of both compounds were also needed to obtain a synergistic cytotoxic effect in 3D spheroids of the murine lung adenocarcinoma cell line 344SQ, which was interestingly absent in 2D. This cell line was used in a syngeneic mouse model in which the oral administration of aurola significantly delayed the growth of mutant p53 344SQ tumors in 129S2/SvPasCrl mice, while either agent alone had no effect. In addition, RNA sequencing results revealed that AF- and aurola-treated 344SQ tumors were negatively enriched for immune-related gene sets, which is in accordance with AF's anti-inflammatory function as an anti-rheumatic drug. Only 344SQ tumors treated with aurola showed the downregulation of genes related to the cell cycle, potentially explaining the growth inhibitory effect of aurola since no apoptosis-related gene sets were enriched. Overall, this novel combination strategy of oxidative stress induction (AF) with PARP inhibition (olaparib) could be a promising treatment for mutant p53 cancers, although high concentrations of both compounds need to be reached to obtain a substantial cytotoxic effect.

Humoral and Cellular Immune Responses against SARS-CoV-2 after Third Dose BNT162b2 following Double-Dose Vaccination with BNT162b2 versus ChAdOx1 in Patients with Cancer.

PURPOSE: Patients with cancer display reduced humoral responses after double-dose COVID-19 vaccination, whereas their cellular response is more comparable with that in healthy individuals. Recent studies demonstrated that a third vaccination dose boosts these immune responses, both in healthy people and patients with cancer. Because of the availability of many different COVID-19 vaccines, many people have been boosted with a different vaccine from the one used for double-dose vaccination. Data on such alternative vaccination schedules are scarce. This prospective study compares a third dose of BNT162b2 after double-dose BNT162b2 (homologous) versus ChAdOx1 (heterologous) vaccination in patients with cancer. EXPERIMENTAL DESIGN: A total of 442 subjects (315 patients and 127 healthy) received a third dose of BNT162b2 (230 homologous vs. 212 heterologous). Vaccine-induced adverse events (AE) were captured up to 7 days after vaccination. Humoral immunity was assessed by SARS-CoV-2 anti-S1 IgG antibody levels and SARS-CoV-2 50% neutralization titers (NT50) against Wuhan and BA.1 Omicron strains. Cellular immunity was examined by analyzing CD4+ and CD8+ T-cell responses against SARS-CoV-2-specific S1 and S2 peptides. RESULTS: Local AEs were more common after heterologous boosting. SARS-CoV-2 anti-S1 IgG antibody levels did not differ significantly between homologous and heterologous boosted subjects [GMT 1,755.90 BAU/mL (95% CI, 1,276.95-2,414.48) vs. 1,495.82 BAU/mL (95% CI, 1,131.48-1,977.46)]. However, homologous-boosted subjects show significantly higher NT50 values against BA.1 Omicron. Subjects receiving heterologous boosting demonstrated increased spike-specific CD8+ T cells, including higher IFNγ and TNFα levels. CONCLUSIONS: In patients with cancer who received double-dose ChAdOx1, a third heterologous dose of BNT162b2 was able to close the gap in antibody response.

Optimization of the Solvent and In Vivo Administration Route of Auranofin in a Syngeneic Non-Small Cell Lung Cancer and Glioblastoma Mouse Model.

The antineoplastic activity of the thioredoxin reductase 1 (TrxR) inhibitor, auranofin (AF), has already been investigated in various cancer mouse models as a single drug, or in combination with other molecules. However, there are inconsistencies in the literature on the solvent, dose and administration route of AF treatment in vivo. Therefore, we investigated the solvent and administration route of AF in a syngeneic SB28 glioblastoma (GBM) C57BL/6J and a 344SQ non-small cell lung cancer 129S2/SvPasCrl (129) mouse model. Compared to daily intraperitoneal injections and subcutaneous delivery of AF via osmotic minipumps, oral gavage for 14 days was the most suitable administration route for high doses of AF (10-15 mg/kg) in both mouse models, showing no measurable weight loss or signs of toxicity. A solvent comprising 50% DMSO, 40% PEG300 and 10% ethanol improved the solubility of AF for oral administration in mice. In addition, we confirmed that AF was a potent TrxR inhibitor in SB28 GBM tumors at high doses. Taken together, our results and results in the literature indicate the therapeutic value of AF in several in vivo cancer models, and provide relevant information about AF's optimal administration route and solvent in two syngeneic cancer mouse models.

Expression of SARS-CoV-2-Related Surface Proteins in Non-Small-Cell Lung Cancer Patients and the Influence of Standard of Care Therapy.

In this study, we aimed to study the expression of SARS-CoV-2-related surface proteins in non-small-cell lung cancer (NSCLC) cells and identify clinicopathological characteristics that are related to increased membranous (m)ACE2 protein expression and soluble (s)ACE2 levels, with a particular focus on standard of care (SOC) therapies. ACE2 (n = 107), TMPRSS2, and FURIN (n = 38) protein expression was determined by immunohistochemical (IHC) analysis in NSCLC patients. sACE2 levels (n = 64) were determined in the serum of lung cancer patients collected before, during, or after treatment with SOC therapies. Finally, the TCGA lung adenocarcinoma (LUAD) database was consulted to study the expression of ACE2 in EGFR- and KRAS-mutant samples and ACE2 expression was correlated with EGFR/HER, RAS, BRAF, ROS1, ALK, and MET mRNA expression. Membranous (m)ACE2 was found to be co-expressed with mFURIN and/or mTMPRSS2 in 16% of the NSCLC samples and limited to the adenocarcinoma subtype. TMPRSS2 showed predominantly atypical cytoplasmic expression. mACE2 and sACE2 were more frequently expressed in mutant EGFR patients, but not mutant-KRAS patients. A significant difference was observed in sACE2 for patients treated with targeted therapies, but not for chemo- and immunotherapy. In the TCGA LUAD cohort, ACE2 expression was significantly higher in EGFR-mutant patients and significantly lower in KRAS-mutant patients. Finally, ACE2 expression was positively correlated with ERBB2-4 and ROS1 expression and inversely correlated with KRAS, NRAS, HRAS, and MET mRNA expression. We identified a role for EGFR pathway activation in the expression of mACE2 in NSCLC cells, associated with increased sACE2 levels in patients. Therefore, it is of great interest to study SARS-CoV-2-infected EGFR-mutated NSCLC patients in greater depth in order to obtain a better understanding of how mACE2, sACE2, and SOC TKIs can affect the course of COVID-19.

Targeting hedgehog signaling in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) remains a leading cause of cancer related death. The urgent need for effective therapies is highlighted by the lack of adequate targeting. In PDAC, hedgehog (Hh) signaling is known to be aberrantly activated, which prompted the pathway as a possible target for effective treatment for PDAC patients. Unfortunately, specific targeting of upstream molecules within the Hh signaling pathway failed to bring clinical benefit. This led to the ongoing debate on Hh targeting as a therapeutic treatment for PDAC patients. Additionally, concurrent non-canonical activation routes also result in translocation of Gli transcription factors into the nucleus. Therefore, different downstream targets of the Hh signaling pathway were identified and evaluated in preclinical and clinical research. In this review we summarize the variety of Hh signaling antagonists in different preclinical models of PDAC. Furthermore, we discuss published and ongoing clinical trials that evaluated Hh antagonists and point out the current hurdles and future perspectives in the light of redesigning Hh-targeting therapies for the treatment of PDAC patients.

A systematic review on poly(I:C) and poly-ICLC in glioblastoma: adjuvants coordinating the unlocking of immunotherapy.

Immunotherapy is currently under intensive investigation as a potential breakthrough treatment option for glioblastoma. Given the anatomical and immunological complexities surrounding glioblastoma, lymphocytes that infiltrate the brain to develop durable immunity with memory will be key. Polyinosinic:polycytidylic acid, or poly(I:C), and its derivative poly-ICLC could serve as a priming or boosting therapy to unleash lymphocytes and other factors in the (immuno)therapeutic armory against glioblastoma. Here, we present a systematic review on the effects and efficacy of poly(I:C)/poly-ICLC for glioblastoma treatment, ranging from preclinical work on cellular and murine glioblastoma models to reported and ongoing clinical studies. MEDLINE was searched until 15 May 2021 to identify preclinical (glioblastoma cells, murine models) and clinical studies that investigated poly(I:C) or poly-ICLC in glioblastoma. A systematic review approach was conducted according to PRISMA guidelines. ClinicalTrials.gov was queried for ongoing clinical studies. Direct pro-tumorigenic effects of poly(I:C) on glioblastoma cells have not been described. On the contrary, poly(I:C) changes the immunological profile of glioblastoma cells and can also kill them directly. In murine glioblastoma models, poly(I:C) has shown therapeutic relevance as an adjuvant therapy to several treatment modalities, including vaccination and immune checkpoint blockade. Clinically, mostly as an adjuvant to dendritic cell or peptide vaccines, poly-ICLC has been demonstrated to be safe and capable of eliciting immunological activity to boost therapeutic responses. Poly-ICLC could be a valuable tool to enhance immunotherapeutic approaches for glioblastoma. We conclude by proposing several promising combination strategies that might advance glioblastoma immunotherapy and discuss key pre-clinical aspects to improve clinical translation.

Auranofin reveals therapeutic anticancer potential by triggering distinct molecular cell death mechanisms and innate immunity in mutant p53 non-small cell lung cancer.

Auranofin (AF) is an FDA-approved antirheumatic drug with anticancer properties that acts as a thioredoxin reductase 1 (TrxR) inhibitor. The exact mechanisms through which AF targets cancer cells remain elusive. To shed light on the mode of action, this study provides an in-depth analysis on the molecular mechanisms and immunogenicity of AF-mediated cytotoxicity in the non-small cell lung cancer (NSCLC) cell line NCI-H1299 (p53 Null) and its two isogenic derivates with mutant p53 R175H or R273H accumulation. TrxR is highly expressed in a panel of 72 NSCLC patients, making it a valid druggable target in NSCLC for AF. The presence of mutant p53 overexpression was identified as an important sensitizer for AF in (isogenic) NSCLC cells as it was correlated with reduced thioredoxin (Trx) levels in vitro. Transcriptome analysis revealed dysregulation of genes involved in oxidative stress response, DNA damage, granzyme A (GZMA) signaling and ferroptosis. Although functionally AF appeared a potent inhibitor of GPX4 in all NCI-H1299 cell lines, the induction of lipid peroxidation and consequently ferroptosis was limited to the p53 R273H expressing cells. In the p53 R175H cells, AF mainly induced large-scale DNA damage and replication stress, leading to the induction of apoptotic cell death rather than ferroptosis. Importantly, all cell death types were immunogenic since the release of danger signals (ecto-calreticulin, ATP and HMGB1) and dendritic cell maturation occurred irrespective of (mutant) p53 expression. Finally, we show that AF sensitized cancer cells to caspase-independent natural killer cell-mediated killing by downregulation of several key targets of GZMA. Our data provides novel insights on AF as a potent, clinically available, off-patent cancer drug by targeting mutant p53 cancer cells through distinct cell death mechanisms (apoptosis and ferroptosis). In addition, AF improves the innate immune response at both cytostatic (natural killer cell-mediated killing) and cytotoxic concentrations (dendritic cell maturation).

Targeting the PD-1 Axis with Pembrolizumab for Recurrent or Metastatic Cancer of the Uterine Cervix: A Brief Update.

Even though cervical cancer is partly preventable, it still poses a great public health problem throughout the world. Current therapies have vastly improved the clinical outcomes of cervical cancer patients, but progress in new systemic treatment modalities has been slow in the last years. Especially for patients with advanced disease this is discouraging, as their prognosis remains very poor. The pathogen-induced nature, the considerable mutational load, the involvement of genes regulating the immune response, and the high grade of immune infiltration, suggest that immunotherapy might be a promising strategy to treat cervical cancer. In this literature review, we focus on the use of PD-1 blocking therapy in cervical cancer, pembrolizumab in particular, as it is the only approved immunotherapy for this disease. We discuss why it has great clinical potential, how it opens doors for personalized treatment in cervical cancer, and which trials are aiming to expand its clinical use.

The Search for an Interesting Partner to Combine with PD-L1 Blockade in Mesothelioma: Focus on TIM-3 and LAG-3.

Malignant pleural mesothelioma (MPM) is an aggressive cancer that is causally associated with previous asbestos exposure in most afflicted patients. The prognosis of patients remains dismal, with a median overall survival of only 9-12 months, due to the limited effectiveness of any conventional anti-cancer treatment. New therapeutic strategies are needed to complement the limited armamentarium against MPM. We decided to focus on the combination of different immune checkpoint (IC) blocking antibodies (Abs). Programmed death-1 (PD-1), programmed death ligand-1 (PD-L1), T-cell immunoglobulin mucin-3 (TIM-3), and lymphocyte activation gene-3 (LAG-3) blocking Abs were tested as monotherapies, and as part of a combination strategy with a second IC inhibitor. We investigated their effect in vitro by examining the changes in the immune-related cytokine secretion profile of supernatant collected from treated allogeneic MPM-peripheral blood mononuclear cell (PBMC) co-cultures. Based on our in vitro results of cytokine secretion, and flow cytometry data that showed a significant upregulation of PD-L1 on PBMC after co-culture, we chose to further investigate the combinations of anti PD-L1 + anti TIM-3 versus anti PD-L1 + anti LAG-3 therapies in vivo in the AB1-HA BALB/cJ mesothelioma mouse model. PD-L1 monotherapy, as well as its combination with LAG-3 blockade, resulted in in-vivo delayed tumor growth and significant survival benefit.

Ribonucleic Acid Engineering of Dendritic Cells for Therapeutic Vaccination: Ready 'N Able to Improve Clinical Outcome?

Targeting and exploiting the immune system has become a valid alternative to conventional options for treating cancer and infectious disease. Dendritic cells (DCs) take a central place given their role as key orchestrators of immunity. Therapeutic vaccination with autologous DCs aims to stimulate the patient's own immune system to specifically target his/her disease and has proven to be an effective form of immunotherapy with very little toxicity. A great amount of research in this field has concentrated on engineering these DCs through ribonucleic acid (RNA) to improve vaccine efficacy and thereby the historically low response rates. We reviewed in depth the 52 clinical trials that have been published on RNA-engineered DC vaccination, spanning from 2001 to date and reporting on 696 different vaccinated patients. While ambiguity prevents reliable quantification of effects, these trials do provide evidence that RNA-modified DC vaccination can induce objective clinical responses and survival benefit in cancer patients through stimulation of anti-cancer immunity, without significant toxicity. Succinct background knowledge of RNA engineering strategies and concise conclusions from available clinical and recent preclinical evidence will help guide future research in the larger domain of DC immunotherapy.

The potential and controversy of targeting STAT family members in cancer.

The Signal Transducer and Activator of Transcription (STAT) family of proteins consists of transcription factors that play a complex and essential role in the regulation of physiologic cell processes, such as proliferation, differentiation, apoptosis and angiogenesis, and serves to organize the epigenetic landscape of immune cells. To date, seven STAT genes have been identified in the human genome; STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b and STAT6. They all account for diverse effects in response to extracellular signaling proteins, mainly by altering gene transcription in the effector cells. Members of the STAT family have been implicated in human cancer development, progression, metastasis, survival and resistance to treatment. Particularly STAT3 and STAT5 are of interest in cancer biology. They are currently considered as oncogenes, but their signaling is embedded into a complex and delicate balance between different (counteracting) transcription factors, and thus, in some contexts they can have a tumor suppressive role. Assessing STAT signaling mutations as well as screening for aberrant STAT pathway activation may have a role to predict sensitivity to immunotherapy and targeted STAT inhibition. In the present comprehensive review of the literature, we discuss in-depth the role of each STAT family member in cancer, assemble cutting-edge information on the use of these molecules as potential biomarkers and targets for treatment, and address why their clinical implementation is controversy.

Efficient and Non-genotoxic RNA-Based Engineering of Human T Cells Using Tumor-Specific T Cell Receptors With Minimal TCR Mispairing.

Genetic engineering of T cells with tumor specific T-cell receptors (TCR) is a promising strategy to redirect their specificity against cancer cells in adoptive T cell therapy protocols. Most studies are exploiting integrating retro- or lentiviral vectors to permanently introduce the therapeutic TCR, which can pose serious safety issues when treatment-related toxicities would occur. Therefore, we developed a versatile, non-genotoxic transfection method for human unstimulated CD8+ T cells. We describe an optimized double sequential electroporation platform whereby Dicer-substrate small interfering RNAs (DsiRNA) are first introduced to suppress endogenous TCR α and ß expression, followed by electroporation with DsiRNA-resistant tumor-specific TCR mRNA. We demonstrate that double sequential electroporation of human primary unstimulated T cells with DsiRNA and TCR mRNA leads to unprecedented levels of transgene TCR expression due to a strongly reduced degree of TCR mispairing. Importantly, superior transgenic TCR expression boosts epitope-specific CD8+ T cell activation and killing activity. Altogether, DsiRNA and TCR mRNA double sequential electroporation is a rapid, non-integrating and highly efficient approach with an enhanced biosafety profile to engineer T cells with antigen-specific TCRs for use in early phase clinical trials.

Natural killer cells and their therapeutic role in pancreatic cancer: A systematic review.

Pancreatic cancer is among the three deadliest cancers worldwide with the lowest 5-year survival of all cancers. Despite all efforts, therapeutic improvements have barely been made over the last decade. Even recent highly promising targeted and immunotherapeutic approaches did not live up to their expectations. Therefore, other horizons have to be explored. Natural Killer (NK) cells are gaining more and more interest as a highly attractive target for cancer immunotherapies, both as pharmaceutical target and for cell therapies. In this systematic review we summarise the pathophysiological adaptions of NK cells in pancreatic cancer and highlight possible (future) therapeutic NK cell-related targets. Furthermore, an extensive overview of recent therapeutic approaches with an effect on NK cells is given, including cytokine-based, viro- and bacteriotherapy and cell therapy. We also discuss ongoing clinical trials that might influence NK cells. In conclusion, although several issues regarding NK cells in pancreatic cancer remain unsolved and need further investigation, extensive evidence is already provided that support NK cell oriented approaches in pancreatic cancer.

Dendritic Cells and Programmed Death-1 Blockade: A Joint Venture to Combat Cancer.

Two decades of clinical cancer research with dendritic cell (DC)-based vaccination have proved that this type of personalized medicine is safe and has the capacity to improve survival, but monotherapy is unlikely to cure the cancer. Designed to empower the patient's antitumor immunity, huge research efforts are set to improve the efficacy of next-generation DC vaccines and to find synergistic combinations with existing cancer therapies. Immune checkpoint approaches, aiming to breach immune suppression and evasion to reinforce antitumor immunity, have been a revelation in the immunotherapy field. Early success of therapeutic antibodies blocking the programmed death-1 (PD-1) pathway has sparked the development of novel inhibitors and combination therapies. Hence, merging immunoregulatory tumor-specific DC strategies with PD-1-targeted approaches is a promising path to explore. In this review, we focus on the role of PD-1-signaling in DC-mediated antitumor immunity. In the quest of exploiting the full potential of DC therapy, different strategies to leverage DC immunopotency by impeding PD-1-mediated immune regulation are discussed, including the most advanced research on targeted therapeutic antibodies, lessons learned from chemotherapy-induced immune activation, and more recent developments with soluble molecules and gene-silencing techniques. An overview of DC/PD-1 immunotherapy combinations that are currently under preclinical and clinical investigation substantiates the clinical potential of such combination strategies.

Abundant expression of TIM-3, LAG-3, PD-1 and PD-L1 as immunotherapy checkpoint targets in effusions of mesothelioma patients.

Malignant pleural mesothelioma (MPM) is an aggressive cancer with an increasing incidence, poor prognosis and limited effective treatment options. Hence, new treatment strategies are warranted which include immune checkpoint blockade approaches with encouraging preliminary data. Research on the immunological aspects of the easily accessible mesothelioma microenvironment could identify prognostic and/or predictive biomarkers and provide useful insights for developing effective immunotherapy. In this context, we investigated the immune cell composition of effusions (pleural and ascites fluids) from 11 different chemotherapy-treated MPM patients. We used multicolor flow cytometry to describe different subsets of immune cells and their expression of immune checkpoint molecules TIM-3, LAG-3, PD-1 and PD-L1. We demonstrate a patient-dependent inter- and intraspecific variation comparing pleural and ascites fluids in immune cell composition and immune checkpoint expression. We found CD4+ and CD8+ T cells, B cells, macrophages, natural killer cells, dendritic cells and tumor cells in the fluids. To the best of our knowledge, we are the first to report TIM-3 and LAG-3 expression and we confirm PD-1 and PD-L1 expression on different MPM effusion-resident immune cells. Moreover, we identified two MPM effusion-related factors with clinical value: CD4+ T cells were significantly correlated with better response to chemotherapy, while the percentage of PD-L1+ podoplanin (PDPN)+ tumor cells is a significant prognostic factor for worse outcome. Our data provide a basis for more elaborate research on MPM effusion material in the context of treatment follow-up and prognostic biomarkers and the development of immune checkpoint-targeted immunotherapy.

Interleukin-15 stimulates natural killer cell-mediated killing of both human pancreatic cancer and stellate cells.

Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer-related death in Western countries with a 5-year survival rate below 5%. One of the hallmarks of this cancer is the strong desmoplastic reaction within the tumor microenvironment (TME), orchestrated by activated pancreatic stellate cells (PSC). This results in a functional and mechanical shield which causes resistance to conventional therapies. Aiming to overcome this resistance by tackling the stromal shield, we assessed for the first time the capacity of IL-15 stimulated natural killer (NK) cells to kill PSC and pancreatic cancer cells (PCC). The potency of IL-15 to promote NK cell-mediated killing was evaluated phenotypically and functionally. In addition, NK cell and immune checkpoint ligands on PSC were charted. We demonstrate that IL-15 activated NK cells kill both PCC and PSC lines (range 9-35% and 20-50%, respectively) in a contact-dependent manner and significantly higher as compared to resting NK cells. Improved killing of these pancreatic cell lines is, at least partly, dependent on IL-15 induced upregulation of TIM-3 and NKG2D. Furthermore, we confirm significant killing of primary PSC by IL-15 activated NK cells in an ex vivo autologous system. Screening for potential targets for immunotherapeutic strategies, we demonstrate surface expression of both inhibitory (PD-L1, PD-L2) and activating (MICA/B, ULBPs and Galectin-9) ligands on primary PSC. These data underscore the therapeutic potential of IL-15 to promote NK cell-mediated cytotoxicity as a treatment of pancreatic cancer and provide promising future targets to tackle remaining PSC.

Monocyte-Derived Dendritic Cells with Silenced PD-1 Ligands and Transpresenting Interleukin-15 Stimulate Strong Tumor-Reactive T-cell Expansion.

Although allogeneic stem cell transplantation (allo-SCT) can elicit graft-versus-tumor (GVT) immunity, patients often relapse due to residual tumor cells. As essential orchestrators of the immune system, vaccination with dendritic cells (DC) is an appealing strategy to boost the GVT response. Nevertheless, durable clinical responses after DC vaccination are still limited, stressing the need to improve current DC vaccines. Aiming to empower DC potency, we engineered monocyte-derived DCs to deprive them of ligands for the immune checkpoint regulated by programmed death 1 (PD-1). We also equipped them with interleukin (IL)-15 "transpresentation" skills. Transfection with short interfering (si)RNA targeting the PD-1 ligands PD-L1 and PD-L2, in combination with IL15 and IL15Rα mRNA, preserved their mature DC profile and rendered the DCs superior in inducing T-cell proliferation and IFNγ and TNFα production. Translated into an ex vivo hematological disease setting, DCs deprived of PD-1 ligands (PD-L), equipped with IL15/IL15Rα expression, or most effectively, both, induced superior expansion of minor histocompatibility antigen-specific CD8+ T cells from transplanted cancer patients. These data support the combinatorial approach of in situ suppression of the PD-L inhibitory checkpoints with DC-mediated IL15 transpresentation to promote antigen-specific T-cell responses and, ultimately, contribute to GVT immunity. Cancer Immunol Res; 5(8); 710-5. ©2017 AACR.

Prognostic and predictive aspects of the tumor immune microenvironment and immune checkpoints in malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is an aggressive cancer with a poor prognosis and an increasing incidence, for which novel therapeutic strategies are urgently required. Since the immune system has been described to play a presumed role in the protection against MPM, characterization of its tumor immune microenvironment (TME) and immune checkpoints can identify new immunotherapeutic targets and their predictive and/or prognostic value. To characterize the TME and the immune checkpoint expression profile, we performed immunohistochemistry (IHC) on formalin-fixed paraffin embedded (FFPE) tissue sections from 54 MPM patients (40 at time of diagnosis; 14 treated with chemotherapy). We stained for PD-1, PD-L1, TIM-3, LAG-3, CD4, CD8, CD45RO, granzyme B, FoxP3 and CD68. Furthermore, we analyzed the relationship between the immunological parameters and survival, as well as response to chemotherapy. We found that TIM-3, PD-1 and PD-L1 were expressed on both immune and tumor cells. Strikingly, PD-1 and PD-L1 expression on tumor cells was only seen in unpretreated samples. No LAG-3 expression was observed. CD45RO expression in the stroma was an independent negative predictive factor for response on chemotherapy, while CD4 and TIM-3 expression in lymphoid aggregates were independent prognostic factors for better outcome. Our data propose TIM-3 as a promising new target in mesothelioma. Chemotherapy influences the expression of immune checkpoints and therefore further research on the best combination treatment schedule is required.

The tumor-associated antigen RHAMM (HMMR/CD168) is expressed by monocyte-derived dendritic cells and presented to T cells.

We formerly demonstrated that vaccination with Wilms' tumor 1 (WT1)-loaded autologous monocyte-derived dendritic cells (mo-DCs) can be a well-tolerated effective treatment in acute myeloid leukemia (AML) patients. Here, we investigated whether we could introduce the receptor for hyaluronic acid-mediated motility (RHAMM/HMMR/CD168), another clinically relevant tumor-associated antigen, into these mo-DCs through mRNA electroporation and elicit RHAMM-specific immune responses. While RHAMM mRNA electroporation significantly increased RHAMM protein expression by mo-DCs, our data indicate that classical mo-DCs already express and present RHAMM at sufficient levels to activate RHAMM-specific T cells, regardless of electroporation. Moreover, we found that RHAMM-specific T cells are present at vaccination sites in AML patients. Our findings implicate that we and others who are using classical mo-DCs for cancer immunotherapy are already vaccinating against RHAMM.