Search

Cancer vaccination and immunotherapy revolutionised the treatment of cancer, a result of decades of research into the immune system in health and disease. However, despite recent breakthroughs in treating otherwise terminal cancer, only a minority of patients respond to cancer immunotherapy and some cancers are largely refractive to immunotherapy treatment. This is due to numerous issues intrinsic to the tumour, its microenvironment, or the immune system. CD4+ and CD8+ αβ T-cells emerged as the primary effector cells of the anti-tumour immune response but their function in cancer patients is often compromised. This review details the mechanisms by which T-cell responses are hindered in the setting of cancer and refractive to immunotherapy, and details many of the approaches under investigation to direct T-cell function and improve the efficacy of cancer vaccination and immunotherapy.

Dacarbazine chemotherapy has been the mainstay of melanoma treatment for >30 years. In the early 2000s, carboplatin (with or without other agents, such as paclitaxel) was the most commonly used second-line therapy in the UK. The aim of the present study was to report a significant response rate to second-line carboplatin in patients from three UK institutions who had been previously treated and failed to respond to dacarbazine, and investigate whether sequential therapy may be more effective compared with combination therapy. A total of 104 patients were identified, the majority of whom were treated with carboplatin (area under the curve 5-6) every 3 weeks for a maximum of 6 cycles. A total of 102 patients were evaluable for response, among whom 11 patients had an objective response (1 complete response and 10 partial responses) and 15 had stable disease, giving an overall response rate of 11% and disease control rate of 26%. The median progression-free survival was 1.8 months (range, 0.2-36+ months) and the median overall survival was 4.6 months (range, 0.2-36+ months). Surprisingly, the majority of the patients who benefited from second-line carboplatin therapy were those with visceral metastases, the survival of whom would not be expected to exceed 6 months after first-line treatment.

Background Vγ9Vδ2 T-cells are a subset of cells with a crucial role in immunosurveillance which can be activated and expanded by multiple means to stimulate effector responses, often exploited in cancer immunotherapy. Little is known about the expression of checkpoint molecules on this cell population and whether the ligation of these molecules can regulate their activity. The aim of this study was to assess the expression of activatory and inhibitory markers on Vγ9Vδ2 T-cells to assess potential avenues of regulation to target with immunotherapy. Methods PBMCs were isolated from healthy donors and the expression of activatory and inhibitory receptors was assessed on Vγ9Vδ2 T-cells by flow cytometry at baseline, following 24 hours activation and 14 days expansion using zoledronic acid (ZA) and Bacillus Calmette-Guerin (BCG), both with IL-2. Activation and expansion of Vδ2 cells was assessed by expression of CD69 and by frequency of Vδ2 cells, respectively. Production of effector molecules was also assessed following coculture with various tumour cell targets. The effect of immune checkpoint blockade on Vγ9Vδ2 T-cells was also assessed. Results Vγ9Vδ2 T-cells constitutively expressed high levels of NK-associated activatory markers NKG2D and DNAM1 which remained high following stimulation with ZA and BCG. Vγ9Vδ2 T-cells expressed variable levels of checkpoint inhibitor molecules at baseline with high levels of BTLA, KLRG1 and NKG2A and intermediate levels of PD1, TIGIT and VISTA. Expression of checkpoint receptors were modulated following activation and expansion with ZA and BCG with decreased expression of BTLA and upregulation of numerous markers including PD1, TIGIT, TIM3, LAG3 and VISTA. Expression of these markers is further modulated upon coculture with tumour cell lines with changes reflecting activation of these cells with Vγ9Vδ2 T-cells expressing inhibitory receptors PD1 and NKG2A producing the highest level of TNF. Conclusions Our data reveals unique characteristics of Vδ2 in terms of their expression of immune checkpoints, which provide a mechanism which may be utilised by tumour cells to subvert Vγ9Vδ2 T-cell cytotoxicity. Our work suggests different profiles of immune checkpoints dependent on the method of stimulation. This highlights importance of expansion method in the function of Vγ9Vδ2 T-cells. Furthermore, this work suggests important candidates for blockade by immune checkpoint therapy in order to increase the successful use of Vγ9Vδ2 T-cells in cancer immunotherapy.

Determining the expression of genes in response to different classes of chemotherapeutic drugs may allow for a better understanding as to which may be used effectively in combination. In the present study, the human colorectal cancer cell line HCT116 was cultured with equi-active concentrations of a series of anti-cancer agents. Gene expression profiles were then measured by whole-genome microarray. Although each drug induced a unique signature of gene expression in tumour cells, there were marked similarities between certain drugs, even in those from different classes. For example, the antimalarial agent artesunate and the platinum-containing alkylating agent, oxaliplatin, produced a very similar mRNA expression pattern in HCT116 cells with ~14,000 genes being affected by the two drugs in the same way. Furthermore, the overall correlation of gene responses between two agents could predict whether their use in combination would lead to a greater or lesser effect on cell number, determined experimentally, than predicted by single agent experiments. The results indicated that even when working through different mechanisms, combining drugs that initiate a similar transcriptional response may constitute the best option for determining drug-combination strategies for the treatment of cancer.

This study presents the background, rationale and method of action of Biovacc-19, a candidate vaccine for corona virus disease 2019 (Covid-19), now in advanced preclinical development, which has already passed the first acute toxicity testing. Unlike conventionally developed vaccines, Biovacc-19’s method of operation is upon nonhuman-like (NHL) epitopes in 21.6% of the composition of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)’s spike protein, which displays distinct distributed charge including the presence of a charged furin-like cleavage site. The logic of the design of the vaccine is explained, which starts with empirical analysis of the aetiology of SARS-CoV-2. Mistaken assumptions about SARS-CoV-2’s aetiology risk creating ineffective or actively harmful vaccines, including the risk of antibody-dependent enhancement. Such problems in vaccine design are illustrated from past experience in the human immunodeficiency viruses domain. We propose that the dual effect general method of action of this chimeric virus’s spike, including receptor binding domain, includes membrane components other than the angiotensin-converting enzyme 2 receptor, which explains clinical evidence of its infectivity and pathogenicity. We show the nonreceptor dependent phagocytic general method of action to be specifically related to cumulative charge from insertions placed on the SARS-CoV-2 spike surface in positions to bind efficiently by salt bridge formations; and from blasting the spike we display the NHL epitopes from which Biovacc-19 has been down-selected.

Vδ2+ T cells are a subpopulation of γδ T cells in humans that are cytotoxic towards cells which accumulate isopentenyl pyrophosphate. The nitrogen-containing bisphosphonate, zoledronic acid (ZA), can induce tumour cell lines to accumulate isopentenyl pyrophosphate, thus rendering them more susceptible to Vδ2+ T cell cytotoxicity. However, little is known about whether ZA renders other, non-malignant cell types susceptible. In this study we focussed on macrophages (Mϕs), as these cells have been shown to take up ZA. We differentiated peripheral blood monocytes from healthy donors into Mϕs and then treated them with IFN-γ or IL-4 to generate M1 and M2 Mϕs, respectively. We characterised these Mϕs based on their phenotype and cytokine production and then tested whether ZA rendered them susceptible to Vδ2+ T cell cytotoxicity. Consistent with the literature, IFN-γ-treated Mϕs expressed higher levels of the M1 markers CD64 and IL-12p70, whereas IL-4-treated Mϕs expressed higher levels of the M2 markers CD206 and chemokine (C–C motif) ligand 18. When treated with ZA, both M1 and M2 Mϕs became susceptible to Vδ2+ T cell cytotoxicity. Vδ2+ T cells expressed perforin and degranulated in response to ZA-treated Mϕs as shown by mobilisation of CD107a and CD107b to the cell surface. Furthermore, cytotoxicity towards ZA-treated Mϕs was sensitive—at least in part—to the perforin inhibitor concanamycin A. These findings suggest that ZA can render M1 and M2 Mϕs susceptible to Vδ2+ T cell cytotoxicity in a perforin-dependent manner, which has important implications regarding the use of ZA in cancer immunotherapy. Electronic supplementary material The online version of this article (doi:10.1007/s00262-017-2011-1) contains supplementary material, which is available to authorized users.

BackgroundTo compare the clinical efficacy of New York Esophageal squamous cell carcinoma-1 (NY-ESO-1) vaccine with ISCOMATRIX adjuvant versus ISCOMATRIX alone in a randomized, double-blind phase II study in participants with fully resected melanoma at high risk of recurrence.MethodsParticipants with resected stage IIc, IIIb, IIIc and IV melanoma expressing NY-ESO-1 were randomized to treatment with three doses of NY-ESO-1/ISCOMATRIX or ISCOMATRIX adjuvant administered intramuscularly at 4-week intervals, followed by a further dose at 6 months. Primary endpoint was the proportion free of relapse at 18 months in the intention-to-treat (ITT) population and two per-protocol populations. Secondary endpoints included relapse-free survival (RFS) and overall survival (OS), safety and NY-ESO-1 immunity.ResultsThe ITT population comprised 110 participants, with 56 randomized to NY-ESO-1/ISCOMATRIX and 54 to ISCOMATRIX alone. No significant toxicities were observed. There were no differences between the study arms in relapses at 18 months or for median time to relapse; 139 vs 176 days (p=0.296), or relapse rate, 27 (48.2%) vs 26 (48.1%) (HR 0.913; 95% CI 0.402 to 2.231), respectively. RFS and OS were similar between the study arms. Vaccine recipients developed strong positive antibody responses to NY-ESO-1 (p≤0.0001) and NY-ESO-1-specific CD4+and CD8+responses. Biopsies following relapse did not demonstrate differences in NY-ESO-1 expression between the study populations although an exploratory study demonstrated reduced (NY-ESO-1)+/Human Leukocyte Antigen (HLA) class I+double-positive cells in biopsies from vaccine recipients performed on relapse in 19 participants.ConclusionsThe vaccine was well tolerated, however, despite inducing antigen-specific immunity, it did not affect survival endpoints. Immune escape through the downregulation of NY-ESO-1 and/or HLA class I molecules on tumor may have contributed to relapse.

A 64 year old male heating engineer was investigated for a persistent cough and found to have epithelioid mesothelioma with pleural effusion, lung nodules and increased thoracic lymph nodes. He declined standard of care treatment following his own research and he was enrolled in a named patient programme of IMM-101. He was advised to correct his low vitamin D3 level and to start using anti-inflammatories such as aspirin, bromelain and low dose Naltrexone. At review one year later a CT scan showed no change and he continued on the regimen. Four years after the diagnosis a CT scan showed that there was a modest but definite progression of the left malignant pleural thickening, and a new right-sided effusion, enlargement of several intrathoracic nodes which had been noted on the early scans. The chest wall lump eventually broke down and required local radiotherapy. He then developed abdominal pain and found to have peritoneal disease. Last year he obtained the cannabinoids CBD and THC which slowed down the disease and a CT scan after he had been on this for six months, showed that his disease was fairly stable with marginal progression. Keywords: Mesothelioma, Anti-inflammatory, Immunotherapy, IMM-101-015

Background/aim Cannabinoids are widely used in the management of pain, nausea and cachexia in cancer patients. However, there has been no objective clinical evidence of any anticancer activity yet. The aim of this study was to assess the effects of pharmaceutical-grade synthetic cannabidiol on a range of cancer patients. Patients and methods We analysed the data routinely collected, as part of our treatment program, in 119 cancer patients over a four-year period. Results Clinical responses were seen in 92% of the 119 cases with solid tumours including a reduction in circulating tumour cells in many cases and in other cases, a reduction in tumour size, as shown by repeat scans. No side-effects of any kind were observed when using pharmaceutical grade synthetic cannabidiol. Conclusion Pharmaceutical-grade synthetic cannabidiol is a candidate for treating breast cancer and glioma patients.

Background\ud The use of checkpoint inhibitors (ipilimumab, pembrolizumab, nivolumab) has revolutionised the treatment of metastatic melanoma. However still more than the half the patients do not respond to single-agent immunotherapy. This has led to the development of combining these agents in an attempt to enhance the anti-cancer activity. More than 300 different studies with 15 different drug doses are currently ongoing. Combining different checkpoint inhibitors (CPIs) does indeed lead to an increase in response rate, but this is associated with significant toxicity. IMM-101 is a heat killed Mycobacterium preparation which induces marked immune modulation and little systemic toxicity. It has been reported as having activity in melanoma as single agent and in pancreatic cancer in combination with gemcitabine, the latter in a randomised study.\ud \ud Methods\ud Here we report the effect of adding CPIs to 3 patients who had previously been on IMM-101, either as a trial or a named patient programme and a patient who received the IMM-101 together with nivolumab.\ud \ud Results\ud All 4 patients had rapid and very good responses, three of them maintained over 18 months with no significant additional toxicity.\ud \ud Conclusions\ud The rapid and complete clinical responses seen in these patients may suggest that IMM-101 is activating a complementary pathway which is synergistic with CPI treatment.

The antigenic makeup of tumour cells can have a profound effect on the progression of cancer and success of immunotherapies. Therefore, one strategy to improve the efficacy of cancer treatments is to augment the antigens displayed by tumours. The present study explores how the recognition of tumour cells may be altered by non-cytotoxic concentrations of gemcitabine (GEM). Testing a panel of chemotherapeutics in human cancer cell lines in vitro, it was found that GEM increased surface expression of HLA-A,B,C and that underlying this were specific increases in β-2-microglobulin and immunoproteasome subunit proteins. Furthermore, the peptide antigen repertoire displayed on HLA class I was altered, revealing a number of novel antigens, many of which that were derived from proteins involved in the DNA-damage response. Changes in the nature of the peptide antigens eluted from HLA-A,B,C after GEM treatment consisted of amino acid anchor-residue modifications and changes in peptide length which rendered peptides likely to favour alternative HLA-alleles and increased their predicted immunogenicity. Signalling through the MAPK/ERK and NFκB/RelB pathways was associated with these changes. These data may explain observations made in previous in vivo studies, advise as to which antigens should be used in future vaccination protocols and reinforce the idea that chemotherapy and immunotherapy could be used in combination.

Pancreatic cancer is one of the most aggressive and lethal types of cancer, and more effective therapeutic agents are urgently needed. Overexpressed cell surface antigens are ideal targets for therapy with monoclonal antibody (mAb)-based drugs, but none have been approved for the treatment of pancreatic cancer. Here, we report development of two novel mouse mAbs, KU42.33C and KU43.13A, against the human pancreatic cancer cell line BxPC-3. Using ELISA, flow cytometry, competitive assay and immunoprecipitation followed by mass spectrometry, we discovered that these two mAbs target two distinct epitopes on the external domain of CD109 that are overexpressed by varying amounts in human pancreatic cancer cell lines. Treatment with these two naked antibodies alone did not affect tumour cell growth or migration in vitro. Of the two mAbs, only KU42.33C was useful in determining the expression of CD109 in tumour cells by Western blot and immunohistochemistry. Interestingly, immunohistochemistry of human pancreatic carcinoma tissue arrays with mAb KU42.33C showed that 94% of the 65 human pancreatic adenocarcinoma cases were CD109 positive, with no expression in normal pancreatic tissues. Our results suggest that these two novel mAbs are excellent tools for determining the expression level of CD109 in the tumour specimens and sera of patients with a wide range of cancers, in particular pancreatic cancer, and for investigating its diagnostic, prognostic and predictive value. Further research is warranted and should aim to unravel the therapeutic potential of the humanised forms or conjugated versions of such antibodies in patients whose tumours overexpress CD109 antigen.

Phytocannabinoids possess anticancer activity when used alone, and a number have also been shown to combine favourably with each other in vitro in leukaemia cells to generate improved activity. We have investigated the effect of pairing cannabinoids and assessed their anticancer activity in cell line models. Those most effective were then used with the common anti-leukaemia drugs cytarabine and vincristine, and the effects of this combination therapy on cell death studied in vitro. Results show a number of cannabinoids could be paired together to generate an effect superior to that achieved if the components were used individually. For example, in HL60 cells, the IC50 values at 48 h for cannabidiol (CBD) and tetrahydrocannabinol (THC) when used alone were 8 and 13 µM, respectively; however, if used together, it was 4 µM. Median-effect analysis confirmed the benefit of using cannabinoids in pairs, with calculated combination indices being

The CD4 (or T4) surface antigen of human T lymphocytes is an important part of the receptor for the human immunodeficiency virus (HIV). After binding to the receptor, the HIV may enter the T cell and induce the formation of syncytia. In an attempt to identify the receptor site more closely, monoclonal antibodies (Mab's) to CD4 were tested for their ability to block HIV infection in a syncytium formation assay, and the CD4 epitopes so identified were mapped by antibody cross-blocking. The antibodies that showed strong inhibition of HIV fell into two main families while a third group of Mab's blocked syncytia formation weakly or not at all. Several different isolates of HIV as well as the laboratory strain CBL1 grown in CEM cells were used to induce the syncytia. The data indicate that only some epitopes of CD4 are important for virus binding and imply that the virus-binding site for CD4 is conserved in different isolates of HIV with substantially divergent env gene sequences. Preliminary studies of patients suggest that polymorphism of these epitopes does not play a role in determining susceptibility to infection.

Drug-resistance is a major contributing factor for the poor prognosis in patients with pancreatic cancer. We have shown previously that the irreversible ErbB family blocker afatinib, is more effective than the reversible EGFR tyrosine kinase inhibitor erlotinib in inhibiting the growth of human pancreatic cancer cells. The aim of this study was to develop human pancreatic cancer cell (BxPc3) variants with acquired resistance to treatment with gemcitabine, afatinib, or erlotinib, and to investigate the molecular changes that accompany the acquisition of a drug-resistant phenotype. We also investigated the therapeutic potential of various agents in the treatment of such drug-resistant variants. Three variant forms of BxPc3 cells with acquired resistance to gemcitabine (BxPc3GEM), afatinib (BxPc3AFR) or erlotinib (BxPc3OSIR) were developed following treatment with increasing doses of such drugs. The expression level, mutational and phosphorylation status of various growth factor receptors and downstream cell signaling molecules were determined by FACS, human phopsho-RTK array, and western blot analysis while the sulforhodamine B assay was used for determining the effect of various agents on the growth of such tumours. We found that all three BxPc3 variants with acquired resistance to gemcitabine (BxPc3GEM), afatinib (BxPc3AFR) or erlotinib (BxPc3OSIR) also become less sensitive to treatment with the two other agents. Acquisition of resistance to these agents was accompanied by upregulation of p-c-MET, p-STAT3, CD44, increased autocrine production of EGFR ligand amphiregulin and differential activation status of EGFR tyrosine residues as well as downregulation of total and p-SRC. Of all therapeutic interventions examined, including the addition of an anti-EGFR antibody ICR62, an anti-CD44 monoclonal antibody, and of STAT3 or c-MET inhibitors, only treatment with the STAT3 inhibitor Stattic produced a higher growth inhibitory effect in all three drug-resistant variants. In addition, treatment with a combination of afatinib with either c-MET inhibitor Crizotinib or Stattic resulted in an additive or synergistic growth inhibition in all three variants. Our results suggest that activation of STAT3 may play an important role in the acquisition of resistance to gemcitabine and HER inhibitors in pancreatic cancer and warrant further studies on the therapeutic potential of STAT3 inhibitors in such a setting.

Attenuated and heat-killed mycobacteria display demonstrable activity against cancer in the clinic; however, the induced immune response is poorly characterised and potential biomarkers of response ill-defined. We investigated whether three mycobacterial preparations currently used in the clinic (BCG and heat-killed Mycobacterium vaccae and Mycobacterium obuense) can stimulate anti-tumour effector responses in human γδ T-cells. γδ T-cell responses were characterised by measuring cytokine production, expression of granzyme B and cytotoxicity against tumour target cells. Results show that γδ T-cells are activated by these mycobacterial preparations, as indicated by upregulation of activation marker expression and proliferation. Activated γδ T-cells display enhanced effector responses, as shown by upregulated granzyme B expression, production of the TH1 cytokines IFN-γ and TNF-α, and enhanced degranulation in response to susceptible and zoledronic acid-treated resistant tumour cells. Moreover, γδ T-cell activation is induced by IL-12, IL-1β and TNF-α from circulating type 1 myeloid dendritic cells (DCs), but not from type 2 myeloid DCs or plasmacytoid DCs. Taken together, we show that BCG, M. vaccae and M. obuense induce γδ T-cell anti-tumour effector responses indirectly via a specific subset of circulating DCs and suggest a mechanism for the potential immunotherapeutic effects of BCG, M. vaccae and M. obuense in cancer. Electronic supplementary material The online version of this article (doi:10.1007/s00262-011-1121-4) contains supplementary material, which is available to authorized users.

Background: Some cancer patients are immuno-compromised, and it has been long felt that immune-intervention is not compatible with standard chemotherapies. However, increasing evidence suggests that standard chemotherapy drugs may stimulate beneficial changes in both the immune system and tumour. Methods: We have assessed the expression of human leucocyte antigen class 1 (HLA1) on tumour cells before and after chemotherapy agents (cyclophosphamide, oxaliplatin or gemcitabine). In addition, we show that chemotherapy-stressed tumour cells may release cytokines that enhance the interactions between dendritic cells (DCs) and T cells into growth media. Results: Here we report that some chemotherapy agents can increase HLA1 expression in tumour cells, even when expression is low. Increases were associated with killing by cytotoxic T cells, which were negated by HLA1-blockade. Furthermore, T-cell function, as indicated by increased proliferation, was enhanced as supernatants derived from tumours treated with chemotherapy augmented DC-maturation and function. Conclusion: There is evidence that a facet of immune surveillance can be restored by appropriate chemotherapy agents. Also, tumours exposed to some chemotherapy may secrete cytokines that can mature DCs, which ultimately enhances T-cell responses.

This review compares the optimal use of vaccines vs. other forms of immunotherapy, which includes cytokines, such as IL-2, monoclonal antibodies, such as the 'checkpoint inhibitors', against CTLA-4 and PD-1. The review includes both prophylactic and therapeutic vaccines using a variety of technologies. It is already established that vaccines can be enhanced by other immunotherapies, such as cytokines (IL-2) and there is scope for combining both of these with the 'checkpoint' antibodies. Moreover, both can be enhanced with other modalities, such as radiotherapy, ablative therapy and both high and low dose chemotherapies.

Summary Background Advanced melanoma is an aggressive disease with a poor prognosis. Approved therapy is limited in the U.K. and, until recently, no treatment had improved survival over best supportive care. A deeper understanding of current clinical practice will help new agents find a place in future treatment pathways. Objectives To document U.K. clinical practice for the treatment of patients with unresectable stage III/IV (advanced) melanoma. Methods MELODY (melanoma treatment patterns and outcomes among patients with unresectable stage III/IV disease: a retrospective longitudinal survey) compiled registries of consecutive patients with malignant melanoma (any stage) between 1 July 2005 and 30 June 2006 from France, Italy and the U.K. Patients with advanced melanoma and ≥ 2 months of follow-up were eligible for analysis. Results There were 220 eligible patients identified in the U.K., of whom 117 (53·2%) received systemic therapy outside of clinical trials. Over half of these patients received dacarbazine as first- or second-line therapy. Healthcare-resource utilization was extensive and patients had short survival times: 1- and 2-year survival rates after first-line systemic treatment were 45·5% [95% confidence interval (CI) 37·1–53·6] and 24·7% (95% CI 17·7–32·3), respectively. Conclusions Systemic and palliative treatments used to manage advanced melanoma in the U.K. are associated with considerable healthcare resource utilization and poor short-term survival.

Background: There is evidence that tumours produce substances such as cytokines and microvesicular bodies bearing bioactive molecules, which support the carcinogenic process. Furthermore, chemotherapy has also been shown to modify these exudates and in doing so, neutralise their tumourigenic influence. Methods: In the current study, we have investigated the effect of chemotherapy agents on modifying the cytokine profile and microvesicular cargo of supernatants derived from cancer cell lines. In addition, we have explored the effect of these tumour-derived supernatants on angiogenesis, and how chemotherapy can alter the supernatants rendering them less pro-angiogenic. Results: Herein, we show that supernatants contain a rich cocktail of cytokines, a number of which are potent modulators of angiogenesis. They also contain microvesicular bodies containing RNA transcripts that code for proteins involved in transcription, immune modulation and angiogenesis. These supernatants altered intracellular signalling molecules in endothelial cells and significantly enhanced their tubulogenic character; however, this was severely compromised when supernatants from tumours treated with chemotherapy was used instead. Conclusion: This study suggests tumour exudates and bioactive material from tumours can influence cellular functions, and that treatment with some chemotherapy can serve to negate these pro-tumourigenic processes.

Background: Reduced expression of class 1 human leucocyte antigens (HLA1) is often a mechanism by which tumours evade surveillance by the host immune system. This is often associated with an immune function that is unable to mount appropriate responses against disease, which can result in a state that favours carcinogenesis. Methods: In the current study, we have explored the effects of Bacillus Calmette-Guerin (BCG) on the cytokine output of leucocytes, which is a key determinant in generating antitumour action, and have also assessed the effect of these cytokine cocktails on HLA1 expression in solid tumour cell lines. Results: BCG potently activated a broad range of leucocytes, and also enhanced the production of cytokines that were Th1-predominant. Supernatants from BCG-treated leucocytes significantly increased the expression of HLA1 on the surface of cancer cell lines, which correlated with increased cytolytic T-cell activity. We also showed that the increased HLA1 expression was associated with activation of intracellular signalling pathways, which was triggered by the increases in the Th1-cytokines interferon-γ and tumour necrosis factor-α, as counteracting their effects negated the enhancement. Conclusion: These studies reaffirm the role of BCG as a putative immunotherapy through their cytokine-modifying effects on leucocytes and their capacity to enhance tumour visibility.

Background: Thalidomide and lenalidomide are FDA approved for the treatment of multiple myeloma and, along with pomalidomide, are being investigated in various other cancers. Although these agents display immunomodulatory, anti-angiogenic and anti-apoptotic effects, little is known about their primary mode of therapeutic action in patients with cancer. Methods: As part of a continuing research effort, we have investigated the effects of these agents on the metastatic capacity of murine colorectal cancer cell lines both in vivo and in vitro. Allied to these, we have studied their effects on the molecular pathways associated with metastasis. Results: Results indicate that thalidomide, lenalidomide and pomalidomide significantly inhibit the metastatic capability of colorectal carcinoma cells. Anchorage-independent growth, used as a coarse indicator of transformation, was significantly reduced, as were migratory capacity and invasive competence. In addition, an in vivo experimental metastasis model also showed that treatment with the drugs resulted in a significantly lower number of metastatic pulmonary nodules relative to control mice. Allied to these cellular and phenotypic changes were alterations in molecular markers of metastasis and in intracellular signalling competency. Conclusions: These results provide evidence that in addition to their immunomodulatory effects, thalidomide, lenalidomide and pomalidomide can impair the metastatic capacity of tumours, and that this mechanism may involve alterations to cell signalling functionality.

More than a decade ago, the pathogenesis of AIDS was reviewed in this journal, using the subtitle 'classical and alternative views', when evidence was accumulating that HIV could not cause AIDS simply through direct cytopathic mechanisms alone. Generalised immune activation after infection with HIV is now understood to be associated with and predictive of disease progression and probably represents the single most important difference between rapid progression and slow or non-progression. However, the fundamental source of this phenomenon remains undetermined. Do pathogenic events after acute infection promote an environment susceptible to increased hyperactivity or does inherent reactivity towards HIV in susceptible individuals ultimately influence these processes? New strategies aimed at eliminating HIV-induced immune activation are required, as is investigation into the clinical and immunological influence of antibodies that target HIV epitopes associated with disease and that are not necessarily neutralising. Therapeutic vaccines to prevent disease may be more practical and effective than classic prophylactic vaccination.

Malignant melanoma is a rare cancer with an incidence of 3% in the United Kingdom. It has been previously reported to undergo spontaneous remission. The incidence of metastatic melanoma with a completely regressed or unknown primary cutaneous lesion is estimated at 3% to 15%; regression of metastases is very rare. We describe a case of a 47-year-old woman who was diagnosed with stage III malignant melanoma in March 2004. The initial lesion measured 2.5 cm in diameter, was pigmented, and was situated in the left posterior angle of the neck. The excision biopsy confirmed malignant melanoma Breslow thickness 1.75 mm, Clark level IV with vertical and lateral spread, pT2a. Following excision biopsy, the patient had a wide local excision and sentinel lymph node biopsy. The wide local excision showed no residual melanoma cells, but there were melanoma cells in 1 of the 2 sentinel lymph nodes found. She therefore proceeded to left radical neck dissection and nodal clearance. None of the 26 lymph nodes excised showed melanoma cells. The staging computed tomography (CT) scan at the time did not show any evidence of distant metastases, and the final American Joint Committee on Cancer staging was IIIA. Sixteen months later, she gave a history of nausea for several weeks and weight loss of 7 lb. Although the symptoms were settling, a CT scan was arranged, which showed metastatic disease in the lungs and liver. Initially, no treatment was instigated, and the plan was to repeat CT in 2 months. The patient, who was obviously very upset at the diagnosis, decided to change her diet by adding antioxidants in the form of Nutriedge supplement (Genesis Enterprise); green tea, 2 to 3 cups per day on the advice of her brother, who is a physician in Australia; and pineapples, 2 per week, including the minced fruit cores. Nutriedge formula contains a combination of antioxidants available over the counter, mainly αand β-carotene, vitamin C, selenium, and vitamin E. The patient maintained an active lifestyle with regular exercise as well as the nutritional supplements mentioned above. The CT scan, repeated as planned, showed almost complete resolution of lung and liver metastases (see Figures 1 and 2). Spontaneous regression of cancer is rare, and the evidence is often anecdotal. The stories of cancer resolution as a result of lifestyle change or alternative therapy are even more infrequent and should be regarded with caution. Melanoma, on the other hand, is one of the rare tumors for which spontaneous regression is not unheard of. King et al reviewed the literature and described one such case in 2001. The literature suggests that the regression is more likely to occur if there is some nonspecific event such as local infection, autoimmune illness, transfusion reaction, a biopsy, or incomplete excision. Such behavior would suggest a correlation with an immune system response, although the mechanism itself is poorly understood. It is likely to be T-lymphocyte driven following recognition of a tumor antigen presented de novo to the immune system. In the case of our patient, there is no specific event other than a lifestyle change and a healthy diet as well as an increased intake of the antioxidants, green tea, and pineapples, including the fruit cores. The evidence for use of antioxidants in cancer is extensive; however, the data remain confusing. Green tea, for example, acts via the active compounds, mainly catechins, and causes inhibition of vascular endothelial growth factor–induced angiogenesis. A large Japanese study from 1986 showed a delayed cancer onset in

SUMMARYDespite advances in our understanding of tumour immunology there is no therapy of proven survival benefit for advanced melanoma. Nevertheless, disease progression is slow in a small proportion of patients with metastatic melanoma, suggesting a contribution to outcome from host factors. Recent data have indicated the importance of the heat shock protein receptor CD91 in immune responses to, and progression of, infectious disease. Here we investigate the relationship between CD91 expression and outcome in malignancy. Rare melanoma patients were recruited with advanced disease that was progressing unusually slowly. CD91 expression on their monocytes was compared with control patients with more typical rapidly advancing metastatic disease. Th1 and Th2 cytokines, as well as innate and adaptive immune subsets, were also measured in the two groups. A significant increase in median CD91 expression levels was observed in slow progressors (P = 0·006). There were no differences in other immune subset markers or inflammatory cytokines. The ability of CD91 to internalize and cross-present tumour antigens through the major histocompatibility complex class I pathway may maintain CD8-positive cytotoxic T cell responses and contribute to slow progression of advanced melanoma.

The anti-tumour effects of thalidomide have been associated with its anti-angiogenic properties. Second generation thalidomide analogues are distinct compounds with enhanced therapeutic potential. Although these compounds are beginning to enter trials for the treatment of cancer there is very little information regarding the anti-angiogenic activity of these clinically relevant compounds. Furthermore, it is not known how the various immunomodulatory activities of these compounds relate to anti-angiogenic activity. In this study we assessed the anti-angiogenic activity of compounds from both IMiD™ and SelCID™ classes of analogues using a novel in vitro multicellular human assay system and the established rat aorta assay. Our results show that both the IMiDs and SelCIDs tested are significantly more potent than thalidomide. The anti-angiogenic potency of the analogues was not related to inhibition of endothelial cell proliferation, nor their TNF-α/PDE type 4 inhibitory properties. However, anti-migratory effects in vitro and inhibition of tumour growth in vivo was observed with the analogue IMiD-1 (clinically known as REVIMID™). Our results show that anti-angiogenic activity spans both currently defined classes of thalidomide analogue and is not related to their previously described immunomodulatory properties. Identification of the differential effects of these compounds will enable targeting of such compounds into the appropriate clinical setting. British Journal of Cancer (2002) 87, 1166–1172. doi:10.1038/sj.bjc.6600607 www.bjcancer.com © 2002 Cancer Research UK

Normal and SV40 transfected human peritoneal mesothelial cells produce IL-6 and IL-8: implication for gynaecological disease. High levels of interleukin-6 (IL-6) and interleukin-8 (IL-8) have been demonstrated in the peritoneal fluid of benign and malignant gynaecological disease. Peritoneal monocytes and macrophages, endometrial cells, endometrial and peritoneal stromal cells and tumour cells produce these cytokines in vitro. To investigate whether normal human peritoneal mesothelial cells (HPMC) produce IL-6 and IL-8, HPMC were isolated from omental biopsies. Primary HPMC (P-HPMC) were transfected with pSV3-neo encoding SV40 large T antigen (T- HPMC) to generate sufficient cells. T-HPMC preserved the characteristics of P-HPMC as assessed by phase contrast microscopy, electron microscopy, immunocytochemistry and flow cytometry (FACS) analysis. T-HPMC retained a stable phenotype up to passage 14-19, whereas P-HPMC proliferated poorly and became senescent by passage 4-6. T-HPMC and PHPMC constitutively expressed IL-6 and IL-8 at both protein and mRNA level. IL-6 and IL-8 production was stimulated by recombinant human interleukin-1beta (hIL-1beta) or human tumour necrosis factor-alpha (hTNF-alpha) alone in a dose-dependent manner. Moreover, hIL-1beta or hTNF-alpha up-regulated IL-6 and IL-8 gene expression as determined by competitive PCR. In contrast, human interferon-gamma (hIFN) or lipopolysaccharide (LPS) showed no effect. These data indicate that (1) T-HPMC lines mimic the morphological and functional features of P-HPMC, (2) P-HPMC and T-HPMC constituitively produce IL-6 and IL-8, which is enhanced by hIL-1beta and hTNF-alpha and (3) HPMC in vivo may participate in the pathogenesis of benign and malignant gynaecological disease.

IMM-101 is an immunotherapeutic agent in clinical development as an adjunctive treatment in cancer. It comprises heat-killed whole cell Mycobacterium obuense and is being developed by Immodulon Therapeutics Ltd, a biopharmaceutical company dedicated to the discovery and development of immuno-oncology therapies. Cancer immunotherapy is based on the evidence that the host immune system, when appropriately modulated, is able to generate protective responses against malignant cells. Such responses, including both innate and adaptive immune components, may affect the establishment, growth and spread of tumours. Hence, therapies which boost general immunosurveillance (e.g. bacterial preparations) through sustained activation of the immune system provide therapeutic potential. IMM-101 has undergone a Phase I safety and tolerability trial in melanoma (NCT01559819), with a number of patients deciding to continue treatment with IMM-101 (NCT01559818). Two Phase II trials are ongoing in advanced, inoperable pancreatic cancer (NCT01303172) and advanced pre-treated colorectal cancer (NCT01539824). Here we describe "A Randomised, Open-Label, Proof-of-Concept, Phase II Trial Comparing Gemcitabine with and without IMM-101 in Advanced Pancreatic Cancer". This study, investigating the efficacy of IMM-101 as an adjunctive immunotherapy, allows for 12 cycles of treatment over approximately 12 months. The primary efficacy endpoint is overall survival but safety and tolerability, clinical signs and symptoms of disease, progression-free survival and overall response rate are also recorded. Sera, collected monthly, are available for measurements of tumour burden markers, immunological status and relevant mediators, in an effort to identify potential prognostic factors for future assessment. Recruitment to the trial with 22 sites in 5 countries finished in July 2013 when 142 patients had been screened and 110 randomised, 75 to the investigational group and 35 to the control arm in line with the planned 2:1 active:control ratio. The treatment phase of the study ended in July 2014, with 12 patients from the active group and 1 from the control group having completed 12 cycles of treatment. The remaining patients withdrew from the study before this time point. Patients completing the study were invited to enter a follow-on study, in which they continued to receive IMM-101 and 12, including 1 control patient, were able to do this. Endpoints will be analysed using the safety and intent-to-treat analysis sets. Final results, which will reveal whether patients receiving the IMM-101 immunotherapeutic product in combination with Gemcitabine have significantly improved overall survival compared to those receiving Gemcitabine alone, are expected to be available early in 2015.

Immune deficiency has recently been identified as one of the hallmarks of cancer, and as such, strategies that rectify this are therapeutically attractive. A potential immunotherapy is IMM-101, which is a suspension of heat-killed Mycobacterium obuense that has recently undergone a Phase I trial of safety and tolerability in melanoma patients (NCT01559819). It is also currently undergoing Phase II trials in pancreatic cancer in combination with Gemcitabine ({"type":"clinical-trial","attrs":{"text":"NCT01303172","term_id":"NCT01303172"}}NCT01303172) and colorectal cancer in combination with radiation therapy ({"type":"clinical-trial","attrs":{"text":"NCT01539824","term_id":"NCT01539824"}}NCT01539824).

Meeting abstracts Chemotherapy and Immunotherapy were always considered incompatible in the treatment of cancer. However, analysis of sequential use has revealed that there may be significant additive potential if not synergistic, in enhancing the immune response to cancer. There are many reasons

SUMMARYWe investigated the in vitro effect of the water-soluble, highly stable thalidomide analogue CC-3052 on HIV-1 expression and TNF-α production in latently infected promonocytic U1 cells, acutely infected T cells and monocyte-derived human macrophages (MDM), and in mitogen-stimulated ex vivo cultures from patients with primary acute HIV-1 infection. HIV-1 expression was assessed by Northern blot analysis of RNAs, and ELISA for p24 antigen release and reverse transcriptase (RT) activity. TNF-α expression was evaluated by RT-polymerase chain reaction (PCR)-ELISA for mRNA and ELISA for protein secretion. We demonstrated that CC-3052 is able to inhibit HIV-1 expression, as evaluated by mRNA, p24 release and RT activity, in phorbol myristate acetate (PMA)- and cytokine-stimulated U1 cells. Furthermore, CC-3052 inhibited HIV-1 expression, as evaluated by p24 and RT activity, in acutely infected MDM and T cells. As far as TNF-α is concerned, CC-3052 significantly reduced TNF-α mRNA and protein secretion in PMA-stimulated U937 and U1 cells, and in PMA-stimulated uninfected and acutely infected MDM. Consistently, the addition of CC-3052 reduced TNF-α production in phytohaemagglutinin (PHA) and lipopolysaccharide (LPS)-stimulated whole blood cultures from patients during the primary acute phase of HIV-1 infection. Since TNF-α is among the most potent enhancers of HIV-1 expression, the effect of CC-3052 on TNF-α may account for its inhibitory activity on HIV-1 expression. Given the well documented immunopathological role of TNF-α and its correlation with viral load, advanced disease and poor prognosis, CC-3052 could be an interesting drug for the design of therapeutic strategies in association with anti-retroviral agents.

There are many complex relationships between tu-\ud mour cells and effector cells in the immune system.\ud These interactions are controlled predominantly by cy-\ud tokines, either within the tumour environment, or sys-\ud temically where the effector cells may be stimulated as\ud a response to the presence of the tumour. Favourable\ud clinical responses in cancer patients have been shown\ud to be associated with enhanced cell-mediated immunity\ud as well as T cell infiltration in tumours. This status\ud is controlled in part by a predominantly Th1 cytokine\ud profile e.g. IFN γ , TNF α and IL-12. Conversely, pa-\ud tients with advancing cancer may have impaired cell-\ud mediated immunity as a result of an imbalance of Th1\ud and Th2 cytokines e.g. IL-4 and IL-10 [6,9,15]. Whilst\ud cytokines have long been known to orchestrate the im-\ud mune system by allowing communication between reg-\ud ulatory and effector cells, the pleiotropic nature of these\ud molecules results in a very complex environment in\ud which to study any single molecule’s properties. Several\ud in vitro protocols have been developed,which aim to closely reflect cytokine production and T cell function in vivo. However, these assays have been developed in artificial settings and as such only allow conclusions to be drawn within a defined context [11]. The aim of this report is to outline the basic proto-cols and applications for the detection of intracellular cytokines by flow cytometry, in the context of disease monitoring.

Antigen derived peptides bound on MHC class II molecules on presenting cells stimulate specific CD4 lymphocytes that are in a naive state if antigen is given for the first time, or in a memory state if antigen has been previously encountered. In order to compare clonal heterogeneity of the human CD4+ T helper repertoire in primary vs. recall responses, we have generated T cell lines in vitro by repeated stimulation of peripheral lymphocytes with primary or with recall antigens. Clonal heterogeneity was broad in the case of recall response to tetanus toxoid or PPD, with a high frequency of specific precursors (> 100 cells/10(6) lymphocytes). In contrast, T cell lines responsive to primary antigens (HIV gp120 or HIV p66) were oligoclonal as defined by TCR V beta gene usage and by spectratyping, and the precursor frequency was low (< 2 cells/10(6) lymphocytes). Primary T cell lines generated from blood samples drawn at different times from the same donor showed that clones with identical TCR CDR3 region coding sequences were expanded, suggesting that in these individuals a large progeny derived from one single precursor is present, even though a previous encounter with the antigen was not documented. Assuming an even in vivo distribution of such cells, the presence of one precursor every 10(6) CD4 lymphocytes (within the CD4 T repertoire that comprises roughly 10(11) CD4 T cells) indicates that approximately 10(5) identical T cells from the same clonal precursor account for the primary response against the model antigens we have studied.

Background Aberrant expression and activation of the IGF-IR have been reported in a variety of human cancers and have been associated with resistance to HER targeted therapy. In this study, we investigated the effect of simultaneous targeting of IGF-IR and HER (erbB) family, with NVP-AEW541 and afatinib, on proliferation of pancreatic cancer cells. Methods The sensitivity of a panel of human pancreatic cancer cell lines to treatment with NVP-AEW541 used alone or in combination with afatinib, anti-EGFR antibody ICR62, and cytotoxic agents was determined using the Sulforhodamine B colorimetric assay. Growth factor receptor expression, cell-cycle distribution and cell signalling were determined using flow cytometry and western blot analysis. Results All pancreatic cancer cell lines were found to be IGF-IR positive and NVP-AEW541 treatment inhibited the growth of the pancreatic cancer cell lines with IC50 values ranging from 342 nM (FA6) to 2.73 μM (PT45). Interestingly, of the various combinations examined, treatment with a combination of NVP-AEW541 and afatinib was superior in inducing synergistic growth inhibition of the majority of pancreatic cancer cells. Conclusion Our results indicate that co-targeting of the erbB (HER) family and IGF-IR, with a combination of afatinib and NVP-AEW541, is superior to treatment with a single agent and encourages further investigation in vivo on their therapeutic potential in IGF-IR and HER positive pancreatic cancers.

Tasquinimod is a small molecule with pleiotropic effects on the tumour microenvironment. Tasquinimod inhibits the growth and metastasis of tumour cells in vitro and in vivo. It targets the tumour microenvironment, enhancing the host immune response and inhibiting the angiogenic response. Tasquinimod influences infiltrating myeloid cells in the tumour milieu shifting the balance towards a less immunosuppressive phenotype. Myeloid-derived suppressor cells and tumour-associated macrophages are major components of the immunosuppressive microenvironment and as a result promote tumour growth and favour angiogenesis and metastasis formation. Growing evidence indicates that tasquinimod targets these myeloid cells and modulates local tumour immunity by blocking the interaction between the multifunctional protein S100A9 and its ligands receptor of advanced glycation end products and Toll-like receptor 4. Its anti-angiogenic effects are achieved at least in part through these effects on regulatory myeloid cells and also potentially through inactivating histone deacetylase-4 and reducing expression of hypoxia-inducible factor 1-controlled genes. The aim is to comprehensively review the mode of action of tasquinimod as a novel oral anti-cancer agent. Based on its unique combination of effects, tasquinimod is a novel agent with clinical therapeutic potential in various solid tumours, both alone and as part of rational combination therapy.

Vaccination usually works in infectious disease, why not in Cancer? Differences in the potency of microbial and cancer antigens, poor initiation of an immune response due to inadequate expression of tumour associated antigens, weak antigens or tolerance induction and local immune suppression were considered. There is a big difference between a therapeutic and a prophylactic vaccine.The opinion of the expert group was that an improved therapeutic efficacy can hardly be expected by further variation of types of vaccines, schedules, routes of administration and adjuvants alone. A major hurdle for developing therapeutic cancer vaccines is the need to effectively monitor the immune response and to be able to use this in an adaptive trial approach.End-points of assessment should be different from standard treatments as complete response or partial responses are usually low, unless combined with other therapies.In order to focus resources to overcome the hurdles of enhancing the therapeutic efficacy of cancer vaccines the Cancer Vaccine Clinical Trial Working Group, representing academia and the pharmaceutical and biotechnology industries has in a consensus process defined ‘A clinical development paradigm for cancer vaccines and related biologics’.