A single centre phase II trial to assess the immunological activity of TroVax® plus pemetrexed/cisplatin in patients with malignant pleural mesothelioma - the SKOPOS trial.

Vaccines in combination with chemotherapy have been shown to be safe in different tumor types. We investigated the immunological activity of the TroVax® vaccine in combination with pemetrexed-cisplatin chemotherapy in malignant pleural mesothelioma (MPM). In this first line, open-label, single-arm, phase 2 study, patients with locally advanced or metastatic MPM were enrolled. Eligible patients received up to 9 intramuscular injections of TroVax®, starting two weeks before chemotherapy and continuing at regular intervals during and after chemotherapy to 24 weeks. The primary endpoint was the induction of cellular or humoral anti-5T4 immune response (defined as a doubling of either response at any of six follow-up time points), with a target response rate of 64%. Of 27 patients, enrolled between Feb 2013-Dec 2014, 23 (85%) received at least three doses of TroVax® and one cycle of chemotherapy and were included in the per-protocol analysis (PPA). 22/23 patients (95.6%) developed humoral or cellular immune response to 5T4. Thus, the study reached its primary endpoint. Disease control was observed in 87% of patients (partial response: 17.4%, stable disease: 69.6%). The median progression-free survival was 6.8 months and median overall survival 10.9 months. Treatment-related adverse events were comparable to those observed in patients with chemotherapy alone. Translational immunology studies revealed a circulating baseline immune signature that was significantly associated with long-term (>20 months in n = 8/23, 34.8%) survival. In this phase 2 trial, TroVax® with pemetrexed-cisplatin chemotherapy showed robust immune activity, acceptable safety and tolerability to warrant further investigation in a phase 3 setting.

Dominant immunosuppression of dendritic cell function by prostate-cancer-derived exosomes.

Exosomes are a distinct population of extracellular vesicles of endocytic origin with a protein repertoire similar to the parent cell. Although tumour-derived exosomes harbour immunosuppressive characteristics, they also carry tumour antigens and thus potentially contribute to immune activation. The aim of this study was to examine the impact of prostate cancer exosomes on tumour antigen cross-presentation. DU145 cells, transduced with shRNA to knockdown Rab27a (DU145KD) that inhibits exosome secretion, triggered significantly stronger tumour-antigen-specific T cell responses when loaded onto dendritic cells (DC) than control DU145 cells. Enhanced T cell response was prevented by adding purified exogenous DU145 exosomes to DU145KD cells, demonstrating that the dominant effect of tumour exosomes is immunosuppression and not antigen delivery. CD8+ T cell responses were impaired via exosomal regulation of DC function; exosomes triggered the expression of CD73, an ecto-5-nucleotidase responsible for AMP to adenosine hydrolysis, on DC. CD73 induction on DC that constitutively express CD39 resulted in an ATP-dependent inhibition of TNFα- and IL-12-production. We identified exosomal prostaglandin E2 (PGE2) as a potential driver of CD73 induction, as inhibition of PGE2 receptors significantly reduced exosome-dependent CD73 induction. The results reveal a hitherto unknown suppression of DC function via exosomal PGE2, adding a new element to tumour exosome-immune cell cross-talk. Abbreviations: AMP: adenosine monophosphate; ATP: adenosine triphosphate; BLCL: B lymphoblastoid cell line; CME: exosomes enriched from cell line conditioned media; DC: dendritic cell; DMSO: dimethyl-sulfoxide; DU145C: DU145 cells with irrelevant knockdown control; DU145KD: DU145 cells with Rab27a knockdown; ELISA: enzyme-linked immunosorbent assay; FBS: fetal bovine serum; GM-CSF: granulocyte-monocyte colony stimulating factor; HLA: human lymphocyte antigen; IL: interleukin; LPS: lipopolysaccharide; mfi: mean fluorescence intensity; PBMC: peripheral blood mononuclear cells; PBS: phosphate buffer solution; PGE2: prostaglandin E2; TRF: time-resolved fluorescence.

Prostaglandin E2-mediated adenosinergic effects on CD14+ cells: Self-amplifying immunosuppression in cancer.

CD39 and CD73 are surface-expressed ectonucleotidases that hydrolyze ATP in a highly regulated, serial manner into ADP, AMP and adenosine. The end product, adenosine, has both tumor-promoting and immunosuppressive effects. The aim of this study was to determine CD73 expression on immune cells in pleural effusion (PE) in order to have a better understanding of the immune environment in mesothelioma. PE- or blood-derived CD14+ cells of mesothelioma patients and healthy donors were analyzed by flow cytometry for the expression of CD39 and CD73. CD73-induction was studied by exposure of CD14+ cells to the soluble fraction of PE (sPE), while the signaling mechanism, responsible for CD73 induction, by phosphoflow cytometry and receptor-inhibition studies. We observed CD73 expression on CD14+ cells in PE but not peripheral blood of mesothelioma patients or healthy donors. CD73 expression was inducible on CD14+ cells with sPE, cyclic-AMP (cAMP)-inducers (forskolin and prostaglandin-E2 (PGE2)) and adenosine. Inhibition of PGE2 receptors or adenosine A2 receptors blocked CD73-induction by sPE. sPE treatment triggered protein kinase A and p38 activation. However, signal-transducer and activator of transcription 3 (STAT3)-blocking led to enhanced CD73 expression, demonstrating a hitherto unknown negative control of purinergic signaling by STAT3 in CD14+ cells. TNFα production by CD73+ CD14+ cells was significantly impaired in the presence of AMP, confirming immunosuppressive function. Taken together, CD73 expression can be induced by PGE2, cAMP or adenosine on human CD14+ cells. We suggest that targeting this autocrine loop is a valid therapeutic approach in mesothelioma that may also enhance immunotherapy.

Cross-Presentation of the Oncofetal Tumor Antigen 5T4 from Irradiated Prostate Cancer Cells--A Key Role for Heat-Shock Protein 70 and Receptor CD91.

Immune responses contribute to the success of radiotherapy of solid tumors; however, the mechanism of triggering CD8(+) T-cell responses is poorly understood. Antigen cross-presentation from tumor cells by dendritic cells (DC) is a likely dominant mechanism to achieve CD8(+) T-cell stimulation. We established a cross-presentation model in which DCs present a naturally expressed oncofetal tumor antigen (5T4) from irradiated DU145 prostate cancer cells to 5T4-specific T cells. The aim was to establish which immunogenic signals are important in radiation-induced cross-presentation. Radiation (12 Gy) caused G2-M cell-cycle arrest and cell death, increased cellular 5T4 levels, high-mobility protein group-B1 (HMGB1) release, and surface calreticulin and heat-shock protein-70 (Hsp70) expression in DU145 cells. DCs phagocytosed irradiated tumor cells efficiently, followed by upregulation of CD86 on phagocytic DCs. CD8(+) 5T4-specific T cells, stimulated with these DCs, proliferated and produced IFNγ. Inhibition of HMGB1 or the TRIF/MyD88 pathway only had a partial effect on T-cell stimulation. Unlike previous investigators, we found no evidence that DCs carrying Asp299Gly Toll-like receptor-4 (TLR4) single-nucleotide polymorphism had impaired ability to cross-present tumor antigen. However, pretreatment of tumor cells with Hsp70 inhibitors resulted in a highly statistically significant and robust prevention of antigen cross-presentation and CD86 upregulation on DCs cocultured with irradiated tumor cells. Blocking the Hsp70 receptor CD91 also abolished cross-presentation. Together, the results from our study demonstrate that irradiation induces immunologically relevant changes in tumor cells, which can trigger CD8(+) T-cell responses via a predominantly Hsp70-dependent antigen cross-presentation process.

Enhancement of T cell responses as a result of synergy between lower doses of radiation and T cell stimulation.

As a side effect of cancer radiotherapy, immune cells receive varying doses of radiation. Whereas high doses of radiation (>10 Gy) can lead to lymphopenia, lower radiation doses (2-4 Gy) represent a valid treatment option in some hematological cancers, triggering clinically relevant immunological changes. Based on our earlier observations, we hypothesized that lower radiation doses have a direct positive effect on T cells. In this study, we show that 0.6-2.4 Gy radiation enhances proliferation and IFN-γ production of PBMC or purified T cells induced by stimulation via the TCR. Radiation with 1.2 Gy also lowered T cell activation threshold and broadened the Th1 cytokine profile. Although radiation alone did not activate T cells, when followed by TCR stimulation, ERK1/2 and Akt phosphorylation increased above that induced by stimulation alone. These changes were followed by an early increase in glucose uptake. Naive (CD45RA(+)) or memory (CD45RA(-)) T cell responses to stimulation were boosted at similar rates by radiation. Whereas increased Ag-specific cytotoxic activity of a CD8(+) T cell line manifested in a 4-h assay (10-20% increase), highly significant (5- to 10-fold) differences in cytokine production were detected in 6-d Ag-stimulation assays of PBMC, probably as a net outcome of death of nonstimulated and enhanced response of Ag-stimulated T cells. T cells from patients receiving pelvic radiation (2.2-2.75 Gy) also displayed increased cytokine production when stimulated in vitro. We report in this study enhanced T cell function induced by synergistic radiation treatment, with potential physiological significance in a wide range of T cell responses.

Decreased HPV-specific T cell responses and accumulation of immunosuppressive influences in oropharyngeal cancer patients following radical therapy.

Oropharyngeal cancer (OPC) is a type of squamous cell head and neck cancer that is often associated with human papillomavirus (HPV) infection, suggesting the potential for immunotherapeutic targeting of HPV antigens. This study aimed to determine the effect of radical therapy on HPV-specific T cells and other immune parameters in 20 OPC patients, as a prelude to future immunotherapy studies. HPV DNA could be detected in 9/12 available tissue samples (8/9 HPV(+) samples were also p16(+)). HPV-specific T cell responses against HPV16 E6 and E7 peptides were detected by enzyme-linked immunoSPOT in 10/13 and 8/13 evaluable patients, respectively, but did not appear to correlate with HPV status. Post-treatment, both HPV E6 and E7 T cell responses were decreased (4/13 and 2/13 patients, respectively). These reductions in T cell response could not be explained by a concurrent decrease in memory T cells whose absolute numbers were relatively unaffected by radical therapy (27,975 vs. 25,661/10(5) PBMC) despite a significant decrease in overall lymphocyte counts (1.74 vs. 0.69 × 10(9)/L). Instead, there were significant increases in regulatory T cells (3.7 vs. 6.8 %) and a population of myeloid-derived suppressor cells (CD14(-)HLA-DR(-)CD15(hi), 12.38 vs. 21.92 %). This suggests that immunosuppression may contribute to the reduction in HPV-specific T cell responses post-treatment, although study of larger patient cohorts will be required to test whether this affects clinical outcome. Overall these findings suggest that HPV-targeted immunotherapy in post-therapy OPC patients will require multiple strategies to boost T cell immunity and to overcome the influence of immunosuppressive cells.

Overexpression and potential targeting of the oncofoetal antigen 5T4 in malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is resistant to conventional treatments. Novel, targeted treatments are hampered by the relative lack of MPM-associated tumour antigens. The aim of this study was to evaluate the level of expression and the relevance of 5T4 as a tumour-associated antigen in MPM. 5T4 expression was assessed by Western blotting, flow cytometry, immuno-cytochemistry and -histochemistry in 11 mesothelioma cell lines, 21 tumour biopsies, and ex vivo tumour cells obtained from the pleural fluid (PF) of 10 patients. 5T4 antibody levels were also determined in the plasma of patients and healthy donors. The susceptibility of MPM cells to 5T4-specific T-cell-mediated killing was determined using an HLA-A2(+), CD8(+) T-cell line, developed against the 5T4(17-25) peptide. We report here that cell surface 5T4 expression was detected in all mesothelioma cell lines and PF cell samples. Mesothelin and CD200, a suggested mesothelioma marker, were co-expressed with 5T4 on tumour cells in PF. Immunohistochemistry confirmed overexpression of 5T4, similar to mesothelin, on tumour cells but not on reactive stroma in all tissue sections tested. Median 5T4 antibody levels were 46% higher in patient than in healthy donor plasma, indicating immune recognition. Importantly, 5T4-specific CD8(+) T-cells were able to kill four out of six HLA-A2(+) MPM cell lines but not an HLA-A2(-) cell line, demonstrating immune recognition of MPM-associated 5T4 antigen at the effector T-cell level. We conclude that 5T4 is a potential new antigen for targeted therapies such as immunotherapy in MPM, as it is overexpressed on mesothelioma cells and recognised by 5T4-specific cytotoxic T-cells. Our findings have been translated into a Phase II clinical trial applying 5T4-targeted therapies in MPM patients.

Cancer exosomes express CD39 and CD73, which suppress T cells through adenosine production.

Extracellular adenosine is elevated in cancer tissue, and it negatively regulates local immune responses. Adenosine production from extracellular ATP has attracted attention as a mechanism of regulatory T cell-mediated immune regulation. In this study, we examined whether small vesicles secreted by cancer cells, called exosomes, contribute to extracellular adenosine production and hence modulate immune effector cells indirectly. We found exosomes from diverse cancer cell types exhibit potent ATP- and 5'AMP-phosphohydrolytic activity, partly attributed to exosomally expressed CD39 and CD73, respectively. Comparable levels of activity were seen with exosomes from pleural effusions of mesothelioma patients. In such fluids, exosomes accounted for 20% of the total ATP-hydrolytic activity. Exosomes can perform both hydrolytic steps sequentially to form adenosine from ATP. This exosome-generated adenosine can trigger a cAMP response in adenosine A(2A) receptor-positive but not A(2A) receptor-negative cells. Similarly, significantly elevated cAMP was also triggered in Jurkat cells by adding exosomes with ATP but not by adding exosomes or ATP alone. A proportion of healthy donor T cells constitutively express CD39 and/or CD73. Activation of T cells by CD3/CD28 cross-linking could be inhibited by exogenously added 5'AMP in a CD73-dependent manner. However, 5'AMP converted to adenosine by exosomes inhibits T cell activation independently of T cell CD73 expression. This T cell inhibition was mediated through the adenosine A(2A) receptor. In summary, the data highlight exosome enzymic activity in the production of extracellular adenosine, and this may play a contributory role in negative modulation of T cells in the tumor environment.

Temperature-, concentration- and cholesterol-dependent translocation of L- and D-octa-arginine across the plasma and nuclear membrane of CD34+ leukaemia cells.

Delineating the mechanisms by which cell-penetrating peptides, such as HIV-Tat peptide, oligoarginines and penetratin, gain access to cells has recently received intense scrutiny. Heightened interest in these entities stems from their ability to enhance cellular delivery of associated macromolecules, such as genes and proteins, suggesting that they may have widespread applications as drug-delivery vectors. Proposed uptake mechanisms include energy-independent plasma membrane translocation and energy-dependent vesicular uptake and internalization through endocytic pathways. In the present study, we investigated the effects of temperature, peptide concentration and plasma membrane cholesterol levels on the uptake of a model cell-penetrating peptide, L-octa-arginine (L-R8) and its D-enantiomer (D-R8) in CD34+ leukaemia cells. We found that, at 4-12 degrees C, L-R8 uniformly labels the cytoplasm and nucleus, but in cells incubated with D-R8 there is additional labelling of the nucleolus which is still prominent at 30 degrees C incubations. At temperatures between 12 and 30 degrees C, the peptides are also localized to endocytic vesicles which consequently appear as the only labelled structures in cells incubated at 37 degrees C. Small increases in the extracellular peptide concentration in 37 degrees C incubations result in a dramatic increase in the fraction of the peptide that is localized to the cytosol and promoted the binding of D-R8 to the nucleolus. Enhanced labelling of the cytosol, nucleus and nucleolus was also achieved by extraction of plasma membrane cholesterol with methyl-beta-cyclodextrin. The data argue for two, temperature-dependent, uptake mechanism for these peptides and for the existence of a threshold concentration for endocytic uptake that when exceeded promotes direct translocation across the plasma membrane.

Effects of Na+/H+ exchanger inhibitors on subcellular localisation of endocytic organelles and intracellular dynamics of protein transduction domains HIV-TAT peptide and octaarginine.

Protein transduction domains such as those derived from the HIV protein TAT have great potential as vectors for delivery of therapeutic entities such as genes and proteins into cells. Extensive studies have shown that a major fraction of the most studied variants enters cells via an endocytic mechanism. However, controversy surrounds the exact uptake mechanism and whether a specific pathway is utilised. Studies showing inhibition of uptake of protein transduction domains in the presence of ion-transport inhibitors such as amiloride and its more potent analogue 5-(N-ethyl-N-isopropyl) amiloride (EIPA) suggest a link between peptide internalisation and macropinocytosis. In this study, using immunolabelling of early and late components of the endocytic pathway, we show that treatment of cells with EIPA and to a lesser extent amiloride affects the morphology and subcellular location of early, late endosomes and lysosomes. Enlarged early and late endocytic structures were observed in EIPA-treated cells, and these organelles accumulated in a perinuclear region. Results from experiments investigating the effects of EIPA on distribution of fluorescent octaarginine were in agreement with the immunolocalisation studies. Treatment of the CD34(+) leukaemia cell line KG1a with EIPA in the presence of fluorescent conjugates of HIV-TAT peptide and octaarginine showed distinct vesicular staining in agreement with untreated cells but EIPA-treated cells were additionally characterized by increased localization of the peptides in the cytosol. At levels previously shown to inhibit uptake of HIV-TAT peptide and octaarginine in other cell lines, EIPA was without major effect on uptake of both peptides in KG1a cells.

Intracellular traffic and fate of protein transduction domains HIV-1 TAT peptide and octaarginine. Implications for their utilization as drug delivery vectors.

Transduction domains such as those derived from the HIV-TAT protein are candidate vectors for intracellular delivery of therapeutic macromolecules such as DNA and proteins. The mechanism by which they enter cells is controversial, and very little spatial information regarding the downstream fate of these peptides from the plasma membrane is available. We studied endocytic traffic of fluorescent conjugates of HIV-TAT peptide and octaarginine in human hematopoietic cell lines K562 (CD34-) and KG1a (CD34+) and substantiated our findings in epithelia cells. Both peptides were efficiently internalized to endocytic pathways of both hematopoietic cell lines; however, comparative analysis of the intracellular location of the peptides with endocytic probes revealed major differences in spatial organization of their endocytic organelles and their interaction with the peptides at low temperatures. Double labeling confocal microscopy demonstrates that prelabeled lysosomes of all the tested cells are accessible to internalized peptides within 60 min of endocytic uptake. Incubation of cells with nocodazole and cytochalasin D inhibited peptide traffic from early to late endosomal structures, demonstrating a cytoskeletal requirement for lysosomal delivery. Disruption of Golgi and endoplasmic reticulum dynamics was without effect on peptide localization, suggesting that endosomes and lysosomes rather than these organelles are the major acceptor compartments for these molecules.