Autologous monocyte-derived DC vaccination combined with cisplatin in stage III and IV melanoma patients: a prospective, randomized phase 2 trial.

BACKGROUND: Autologous dendritic cell (DC) vaccines can induce tumor-specific T cells, but their effect can be counteracted by immunosuppressive mechanisms. Cisplatin has shown immunomodulatory effects in vivo which may enhance efficacy of DC vaccination. METHODS: This is a prospective, randomized, open-label phase 2 study (NCT02285413) including stage III and IV melanoma patients receiving 3 biweekly vaccinations of gp100 and tyrosinase mRNA-loaded monocyte-derived DCs with or without cisplatin. Primary objectives were to study immunogenicity and feasibility, and secondary objectives were to assess toxicity and survival. RESULTS: Twenty-two stage III and 32 stage IV melanoma patients were analyzed. Antigen-specific CD8+ T cells were found in 44% versus 67% and functional T cell responses in 28% versus 19% of skin-test infiltrating lymphocytes in patients receiving DC vaccination with and without cisplatin, respectively. Four patients stopped cisplatin because of toxicity and continued DC monotherapy. No therapy-related grade 3 or 4 adverse events occurred due to DC monotherapy. During combination therapy, one therapy-related grade 3 adverse event, decompensated heart failure due to fluid overload, occurred. The clinical outcome parameters did not clearly suggest significant differences. CONCLUSIONS: Combination of DC vaccination and cisplatin in melanoma patients is feasible and safe, but does not seem to result in more tumor-specific T cell responses or improved clinical outcome, when compared to DC vaccination monotherapy.

Preclinical exploration of combining plasmacytoid and myeloid dendritic cell vaccination with BRAF inhibition.

BACKGROUND: Melanoma is the most lethal type of skin cancer and its incidence is progressively increasing. The introductions of immunotherapy and targeted therapies have tremendously improved the treatment of melanoma. Selective inhibition of BRAF by vemurafenib results in objective clinical responses in around 50 % of patients suffering from BRAFV600 mutated melanoma. However, drug resistance often results in hampering long-term tumor control. Alternatively, immunotherapy by vaccination with natural dendritic cells (nDCs) demonstrated long-term tumor control in a proportion of patients. We postulate that the rapid tumor debulking by vemurafenib can synergize the long-term tumor control of nDC vaccination to result in an effective treatment modality in a large proportion of patients. Here, we investigated the feasibility of this combination by analyzing the effect of vemurafenib on the functionality of nDCs. METHODS: Plasmacytoid DCs (pDCs) and myeloid DCs (mDCs) were isolated from PBMCs obtained from buffy coats from healthy volunteers or vemurafenib-treated melanoma patients. Maturation of pDCs, mDCs and immature monocyte-derived DCs was induced by R848 in the presence or absence of vemurafenib and analyzed by FACS. Cytokine production and T cell proliferation induced by mature DCs were analyzed. RESULTS: Vemurafenib inhibited maturation and cytokine production of highly purified nDCs of healthy volunteers resulting in diminished allogeneic T cell proliferation. This deleterious effect of vemurafenib on nDC functionality was absent when total PBMCs were exposed to vemurafenib. In patients receiving vemurafenib, nDC functionality and T cell allostimulatory capacity were unaffected. CONCLUSION: Although vemurafenib inhibited the functionality of purified nDC of healthy volunteers, this effect was not observed when nDCs were matured in the complete PBMC fraction. This might have been caused by increased vemurafenib uptake in absence of other cell types. In accordance, nDCs isolated from patients on active vemurafenib treatment showed no negative effects. In conclusion, our results pave the way for a combinatorial treatment strategy and, we propose that combining vemurafenib with nDC vaccination represent a powerful opportunity that deserves more investigation in the clinic.

Cisplatin inhibits frequency and suppressive activity of monocytic myeloid-derived suppressor cells in cancer patients.

Cancer immunotherapies have induced long-lasting responses in cancer patients including those with melanoma and head and neck squamous cell carcinoma (HNSCC). However, the majority of treated patients does not achieve clinical benefit from immunotherapy because of systemic tumor-induced immunosuppression. Monocytic myeloid-derived suppressor cells (M-MDSCs) are implicated as key players in inhibiting anti-tumor immune responses and their frequencies are closely associated with tumor progression. Tumor-derived signals, including signaling via STAT3-COX-2, induce the transformation of monocytic precursors into suppressive M-MDSCs. In a retrospective assessment, we observed that survival of melanoma patients undergoing dendritic cell vaccination was negatively associated with blood M-MDSC levels. Previously, it was shown that platinum-based chemotherapeutics inhibit STAT signaling. Here, we show that cisplatin and oxaliplatin treatment interfere with the development of M-MDSCs, potentially synergizing with cancer immunotherapy. In vitro, subclinical doses of platinum-based drugs prevented the generation of COX-2+ M-MDSCs induced by tumor cells from melanoma patients. This was confirmed in HNSCC patients where intravenous cisplatin treatment drastically lowered M-MDSC frequency while monocyte levels remained stable. In treated patients, expression of COX-2 and arginase-1 in M-MDSCs was significantly decreased after two rounds of cisplatin, indicating inhibition of STAT3 signaling. In line, the capacity of M-MDSCs to inhibit activated T cell responses ex vivo was significantly decreased after patients received cisplatin. These results show that platinum-based chemotherapeutics inhibit the expansion and suppressive activity of M-MDSCs in vitro and in cancer patients. Therefore, platinum-based drugs have the potential to enhance response rates of immunotherapy by overcoming M-MDSC-mediated immunosuppression.

STAT Family Protein Expression and Phosphorylation State during moDC Development Is Altered by Platinum-Based Chemotherapeutics.

The STAT signaling pathway is important in dendritic cell (DC) development and function. Tumor cells can induce STAT signaling, thereby inhibiting DC maturation and immunostimulatory functions, leading to hampered efficacy of DC-based immunotherapies. Platinum-based chemotherapeutics can inhibit STAT signaling, thereby making them an interesting tool to improve DC development and function. In this study, we provide a comprehensive overview of STAT expression and phosphorylation during DC differentiation and maturation and investigate the effects of platinum drugs on STAT signaling during these processes. Monocytes were differentiated into monocyte-derived DCs (moDCs) with IL-4 and GM-CSF and matured with cytokines or TLR ligands. STAT expression and phosphorylation were analyzed by western blotting, and moDC viability and phenotype were analyzed by flow cytometry. Platinum drugs were added at day 3 of differentiation or at the start of maturation to investigate regulation of the STAT signaling pathway. All STAT proteins were expressed during moDC differentiation and STAT1, STAT5, and STAT6 were phosphorylated. No significant changes occurred in the expression and phosphorylation state of the STAT proteins during differentiation. After maturation with TLR ligands, the expression of STAT1 increased, but other STAT proteins were not affected. Phosphorylation of STAT1 and STAT3 increased during maturation, where TLR ligands induced significantly higher levels of phosphorylation than cytokines. Platinum drugs cisplatin and oxaliplatin significantly inhibited phosphorylation of STAT6 during differentiation and maturation. Treatment did not affect the phenotype or viability of the cells. As STAT6 is an important regulator of DC function, these findings suggest a role for platinum-based chemotherapeutics to enhance DC function via inhibition of STAT signaling, thereby potentially enhancing efficacy of DC-based immunotherapies.

Monitoring of dynamic changes in Keyhole Limpet Hemocyanin (KLH)-specific B cells in KLH-vaccinated cancer patients.

Keyhole limpet hemocyanin (KLH) is used as an immunogenic neo-antigen for various clinical applications and during vaccine development. For advanced monitoring of KLH-based interventions, we developed a flow cytometry-based assay for the ex vivo detection, phenotyping and isolation of KLH-specific B cells. As proof-of-principle, we analyzed 10 melanoma patients exposed to KLH during anti-cancer dendritic cell vaccination. Our assay demonstrated sensitive and specific detection of KLH-specific B cells in peripheral blood and KLH-specific B cell frequencies strongly correlated with anti-KLH serum antibody titers. Profiling of B cell subsets over the vaccination course revealed that KLH-specific B cells matured from naïve to class-switched memory B cells, confirming the prototypic B cell response to a neo-antigen. We conclude that flow-cytometric detection and in-depth phenotyping of KLH-specific B cells is specific, sensitive, and scalable. Our findings provide novel opportunities to monitor KLH-specific immune responses and serve as a blueprint for the development of new flow-cytometric protocols.

Direct inhibition of STAT signaling by platinum drugs contributes to their anti-cancer activity.

Platinum-based chemotherapeutics are amongst the most powerful anti-cancer drugs. Although their exact mechanism of action is not well understood, it is thought to be mediated through covalent DNA binding. We investigated the effect of platinum-based chemotherapeutics on signaling through signal transducer and activator of transcription (STAT) proteins, which are involved in many oncogenic signaling pathways. We performed in vitro experiments in various cancer cell lines, investigating the effects of platinum chemotherapeutics on STAT phosphorylation and nuclear translocation, the expression of STAT-modulating proteins and downstream signaling pathways. Direct binding of platinum to STAT proteins was assessed using an AlphaScreen assay. Nuclear STAT3 expression was determined by immunohistochemistry and correlated with disease-free survival in retrospective cohorts of head and neck squamous cell carcinoma (HNSCC) patients treated with cisplatin-based chemoradiotherapy (n= 65) or with radiotherapy alone (n = 32). At clinically relevant concentrations, platinum compounds inhibited STAT phosphorylation, resulting in loss of constitutively activated STAT proteins in multiple distinct cancer cell lines. Platinum drugs specifically inhibited phospho-tyrosine binding to SH2 domains, thereby blocking STAT activation, and subsequently downregulating pro-survival- and anti-apoptotic- target genes. Importantly, we found that active STAT3 in tumors directly correlated with response to cisplatin-based chemoradiotherapy in HNSCC patients (p = 0.006). These findings provide insight into a novel, non-DNA-targeted mechanism of action of platinum drugs, and could be leveraged into the use of STAT expression as predictive biomarker for cisplatin chemotherapy and to potentiate other therapeutic strategies such as immunotherapy.

Improving cancer immunotherapy by targeting the STATe of MDSCs.

Cancer immunotherapy is a promising therapeutic avenue; however, in practice its efficacy is hampered by an immunosuppressive tumor microenvironment that consists of suppressive cell types like myeloid-derived suppressor cells (MDSCs). Eradication or reprogramming of MDSCs could therefore enhance clinical responses to immunotherapy. Here, we review clinically available drugs that target MDSCs, often through inhibition of STAT signaling, which is essential for MDSC accumulation and suppressive functions. Interestingly, several drugs used for non-cancerous indications and natural compounds similarly inhibit MDSCs by STAT inhibition, but have fewer side effects than anticancer drugs. Therefore, they show great potential for combination strategies with immunotherapy.

Human CD1c(+) DCs are critical cellular mediators of immune responses induced by immunogenic cell death.

Chemotherapeutics, including the platinum compounds oxaliplatin (OXP) and cisplatin (CDDP), are standard care of treatment for cancer. Although chemotherapy has long been considered immunosuppressive, evidence now suggests that certain cytotoxic agents can efficiently stimulate antitumor responses, through the induction of a form of apoptosis, called immunogenic cell death (ICD). ICD is characterized by exposure of calreticulin and heat shock proteins (HSPs), secretion of ATP and release of high-mobility group box 1 (HMGB1). Proper activation of the immune system relies on the integration of these signals by dendritic cells (DCs). Studies on the crucial role of DCs, in the context of ICD, have been performed using mouse models or human in vitro-generated monocyte-derived DCs (moDCs), which do not fully recapitulate the in vivo situation. Here, we explore the effect of platinum-induced ICD on phenotype and function of human blood circulating DCs. Tumor cells were treated with OXP or CDDP and induction of ICD was investigated. We show that both platinum drugs triggered translocation of calreticulin and HSP70, as well as the release of ATP and HMGB1. Platinum treatment increased phagocytosis of tumor fragments by human blood DCs and enhanced phenotypic maturation of blood myeloid and plasmacytoid DCs. Moreover, upon interaction with platinum-treated tumor cells, CD1c(+) DCs efficiently stimulated allogeneic proliferation of T lymphocytes. Together, our observations indicate that platinum-treated tumor cells may exert an active stimulatory effect on human blood DCs. In particular, these data suggest that CD1c(+) DCs are critical mediators of immune responses induced by ICD.