Pasteurized akkermansia muciniphila improves irritable bowel syndrome-like symptoms and related behavioral disorders in mice.

Gut - brain communications disorders in irritable bowel syndrome (IBS) are associated with intestinal microbiota composition, increased gut permeability, and psychosocial disturbances. Symptoms of IBS are difficult to medicate, and hence much research is being made into alternative approaches. This study assesses the potential of a treatment with pasteurized Akkermansia muciniphila for alleviating IBS-like symptoms in two mouse models of IBS with different etiologies. Two clinically relevant animal models were used to mimic IBS-like symptoms in C57BL6/J mice: the neonatal maternal separation (NMS) paradigm and the Citrobacter rodentium infection model. In both models, gut permeability, colonic sensitivity, fecal microbiota composition and colonic IL-22 expression were evaluated. The cognitive performance and emotional state of the animals were also assessed by several tests in the C. rodentium infection model. The neuromodulation ability of pasteurized A. muciniphila was assessed on primary neuronal cells from mice dorsal root ganglia using a ratiometric calcium imaging approach. The administration of pasteurized A. muciniphila significantly reduced colonic hypersensitivity in both IBS mouse models, accompanied by a reinforcement of the intestinal barrier function. Beneficial effects of pasteurized A. muciniphila treatment have also been observed on anxiety-like behavior and memory defects in the C. rodentium infection model. Finally, a neuroinhibitory effect exerted by pasteurized A. muciniphila was observed on neuronal cells stimulated with two algogenic substances such as capsaicin and inflammatory soup. Our findings demonstrate novel anti-hyperalgesic and neuroinhibitory properties of pasteurized A. muciniphila, which therefore may have beneficial effects in relieving pain and anxiety in subjects with IBS.

Ilixadencel, a Cell-based Immune Primer, plus Sunitinib Versus Sunitinib Alone in Metastatic Renal Cell Carcinoma: A Randomized Phase 2 Study.

Background: The prognosis of patients with synchronous metastatic renal cell carcinoma (mRCC) is poor. Whereas single-agent tyrosine kinase inhibition (TKI) is clearly insufficient, the effects can be enhanced by combinations with immune checkpoint inhibitors. Innovative treatment options combining TKI and other immune-stimulating agents could prove beneficial. Objective: To evaluate the clinical effects on metastatic disease when two doses of allogeneic monocyte-derived dendritic cells (ilixadencel) are administrated intratumorally followed by nephrectomy and treatment with sunitinib compared with nephrectomy and sunitinib monotherapy, in patients with synchronous mRCC. Design setting and participants: A randomized (2:1) phase 2 multicenter trial enrolled 88 patients with newly diagnosed mRCC to treatment with the combination ilixadencel/sunitinib (ILIXA/SUN; 58 patients) or sunitinib alone (SUN; 30 patients). Outcome measurements and statistical analysis: The primary endpoints were 18-mo survival rate and overall survival (OS). A secondary endpoint was objective response rate (ORR) assessed up to 18 mo after enrollment. Statistic evaluations included Kaplan-Meier estimates, log-rank tests, Cox regression, and stratified Cochran-Mantel-Haenszel tests. Results and limitations: The median OS was 35.6 mo in the ILIXA/SUN arm versus 25.3 mo in the SUN arm (hazard ratio 0.73, 95% confidence interval 0.42-1.27; p = 0.25), while the 18-mo OS rates were 63% and 66% in the ILIXA/SUN and SUN arms, respectively. The confirmed ORR in the ILIXA/SUN arm were 42.2% (19/45), including three patients with complete response, versus 24.0% (six/25) in the SUN arm (p = 0.13) without complete responses. The study was not adequately powered to detect modest differences in survival. Conclusions: The study failed to meet its primary endpoints. However, ilixadencel in combination with sunitinib was associated with a numerically higher, nonsignificant, confirmed response rate, including complete responses, compared with sunitinib monotherapy. Patient summary: We studied the effects of intratumoral vaccination with ilixadencel followed by sunitinib versus sunitinib only in a randomized phase 2 study. The combination treatment showed numerically higher numbers of confirmed responses, suggesting an immunologic effect.

Phase I trial evaluating safety and efficacy of intratumorally administered inflammatory allogeneic dendritic cells (ilixadencel) in advanced gastrointestinal stromal tumors.

BACKGROUND: The majority of patients with advanced gastrointestinal stromal tumor (GIST) develop resistance to imatinib, and subsequent treatments have limited efficacy. Ilixadencel (allogeneic inflammatory dendritic cells) is a cell-based immune primer injected intratumorally that previously has been clinically investigated in metastatic renal cell carcinoma and hepatocellular carcinoma. METHODS: The trial was a single arm phase I trial assessing safety and efficacy of ilixadencel in subjects with progressing advanced/metastatic GIST despite ongoing treatment with second or later lines of tyrosine kinase inhibitors (TKI). Three patients were progressing while on sunitinib (second line), one on regorafenib (third line), and two on pazopanib (fourth line). TKI treatment was maintained throughout, while two intratumoral injections of ilixadencel (10 × 106 viable and HLA-DR expressing cells per dose) were administered. RESULTS: No severe adverse events were found to be related to ilixadencel administration. Four patients showed continued tumor progression at 3 months per RECIST 1.1 and Choi criteria. One patient (on third line regorafenib) had stable disease for 9 months and another patient (on second line sunitinib) had stable disease at end of study (12 months) as per RECIST 1.1. These two patients developed a partial response as per Choi criteria with a duration of 3 and 6 months, respectively. The median progression-free survival (PFS) was 4.0 months. CONCLUSION: Ilixadencel treatment presented an acceptable safety profile among advanced GIST patients who developed resistance to TKI. Encouraging radiological tumor responses were detected in 33% of treated patients, supporting further investigation. Clinical trial registration www.clinicaltrials.gov ; NCT: 02432846; registration date: February 22, 2016.

ACCELERATE and European Medicines Agency Paediatric Strategy Forum for medicinal product development of checkpoint inhibitors for use in combination therapy in paediatric patients.

The third multistakeholder Paediatric Strategy Forum organised by ACCELERATE and the European Medicines Agency focused on immune checkpoint inhibitors for use in combination therapy in children and adolescents. As immune checkpoint inhibitors, both as monotherapy and in combinations have shown impressive success in some adult malignancies and early phase trials in children of single agent checkpoint inhibitors have now been completed, it seemed an appropriate time to consider opportunities for paediatric studies of checkpoint inhibitors used in combination. Among paediatric patients, early clinical studies of checkpoint inhibitors used as monotherapy have demonstrated a high rate of activity, including complete responses, in Hodgkin lymphoma and hypermutant paediatric tumours. Activity has been very limited, however, in more common malignancies of childhood and adolescence. Furthermore, apart from tumour mutational burden, no other predictive biomarker for monotherapy activity in paediatric tumours has been identified. Based on these observations, there is collective agreement that there is no scientific rationale for children to be enrolled in new monotherapy trials of additional checkpoint inhibitors with the same mechanism of action of agents already studied (e.g. anti-PD1, anti-PDL1 anti-CTLA-4) unless additional scientific knowledge supporting a different approach becomes available. This shared perspective, based on scientific evidence and supported by paediatric oncology cooperative groups, should inform companies on whether a paediatric development plan is justified. This could then be proposed to regulators through the available regulatory tools. Generally, an academic-industry consensus on the scientific merits of a proposal before submission of a paediatric investigational plan would be of great benefit to determine which studies have the highest probability of generating new insights. There is already a rationale for the evaluation of combinations of checkpoint inhibitors with other agents in paediatric Hodgkin lymphoma and hypermutated tumours in view of the activity shown as single agents. In paediatric tumours where no single agent activity has been observed in multiple clinical trials of anti-PD1, anti-PDL1 and anti-CTLA-4 agents as monotherapy, combinations of checkpoint inhibitors with other treatment modalities should be explored when a scientific rationale indicates that they could be efficacious in paediatric cancers and not because these combinations are being evaluated in adults. Immunotherapy in the form of engineered proteins (e.g. monoclonal antibodies and T cell engaging agents) and cellular products (e.g. CAR T cells) has great therapeutic potential for benefit in paediatric cancer. The major challenge for developing checkpoint inhibitors for paediatric cancers is the lack of neoantigens (based on mutations) and corresponding antigen-specific T cells. Progress critically depends on understanding the immune macroenvironment and microenvironment and the ability of the adaptive immune system to recognise paediatric cancers in the absence of high neoantigen burden. Future clinical studies of checkpoint inhibitors in children need to build upon strong biological hypotheses that take into account the distinctive immunobiology of childhood cancers in comparison to that of checkpoint inhibitor responsive adult cancers.

Phase 1 Trial With the Cell-Based Immune Primer Ilixadencel, Alone, and Combined With Sorafenib, in Advanced Hepatocellular Carcinoma.

Several lines of evidence support immunotherapy in hepatocellular carcinoma (HCC). We have shown that intratumoral injections of the immune primer ilixadencel (pro-inflammatory allogeneic dendritic cells) are safe in renal-cell carcinoma. Here, we assessed ilixadencel as a single agent and combined with sorafenib in advanced HCC. Of 17 HCC patients enrolled, 12 patients received ilixadencel at the dose of 10 × 106 cells (six as monotherapy and six in combination with sorafenib), and five received ilixadencel at the dose of 20 × 106 cells as monotherapy. The primary objective was to evaluate tolerability. All patients had at least one adverse event, with 30% of such events considered as treatment-related, with one single treatment-related grade three event. The most common toxicity was grade 1 and 2 fever and chills. Eleven of 15 evaluable patients (73%) showed increased frequency of tumor-specific CD8+ T cells in peripheral blood. Overall one patient had a partial response (with ilixadencel as monotherapy), and five had stable disease as overall best response per mRECIST. The median time to progression was 5.5 months, and overall survival ranged from 1.6 to 21.4 months. Our study confirms the safety of ilixadencel as single agent or in combination with sorafenib and indicates tumor-specific immunological responses in advanced HCC. Clinical Trial Registration: www.ClinicalTrials.gov, identifier: NCT01974661.

Ilixadencel - an Allogeneic Cell-Based Anticancer Immune Primer for Intratumoral Administration.

Intratumoral administration of an immune primer is a therapeutic vaccine strategy aimed to trigger dendritic cell (DC)-mediated cross-presentation of cell-associated tumor antigens to cytotoxic CD8+ T cells without the need for tumor antigen characterization. The prevailing view is that these cross-presenting DCs have to be directly activated by pathogen-associated molecular patterns (PAMPS), including Toll-like receptor ligands or live microbial agents like oncolytic viruses. Emerging data are however challenging this view, indicating that the cross-presenting machinery in DCs is suboptimally activated by direct PAMP recognition, and that endogenous inflammatory factors are the main drivers of DC-mediated cross-presentation within the tumor. Here we present preclinical mode of action data, CMC and regulatory data, as well as initial clinical data on ilixadencel. This cell-based drug product is an off-the-shelf immune primer, consisting of pro-inflammatory allogeneic DCs secreting high amounts of pro-inflammatory chemokines and cytokines at the time of intratumoral administration. The mechanism of action of ilixadencel is to induce recruitment and activation of endogenous immune cells, including NK cells that subsequently promotes cross-presentation of cell-associated tumor antigens by co-recruited DCs.

Future perspectives in melanoma research. Meeting report from the "Melanoma Bridge. Napoli, December 2nd-4th 2012".

Recent insights into the genetic and somatic aberrations have initiated a new era of rapidly evolving targeted and immune-based treatments for melanoma. After decades of unsuccessful attempts to finding a more effective cure in the treatment of melanoma now we have several drugs active in melanoma. The possibility to use these drugs in combination to improve responses to overcome the resistance, to potentiate the action of immune system with the new immunomodulating antibodies, and identification of biomarkers that can predict the response to a particular therapy represent new concepts and approaches in the clinical management of melanoma. The third "Melanoma Research: "A bridge from Naples to the World" meeting, shortened as "Bridge Melanoma Meeting" took place in Naples, December 2 to 4th, 2012. The four topics of discussion at this meeting were: advances in molecular profiling and novel biomarkers, combination therapies, novel concepts toward integrating biomarkers and therapies into contemporary clinical management of patients with melanoma across the entire spectrum of disease stage, and the knowledge gained from the biology of tumor microenvironment across different tumors as a bridge to impact on prognosis and response to therapy in melanoma. This international congress gathered more than 30 international faculty members who in an interactive atmosphere which stimulated discussion and exchange of their experience regarding the most recent advances in research and clinical management of melanoma patients.

Effects of T cell-induced colonic inflammation on epithelial barrier function.

BACKGROUND: Epithelial barrier disturbance is thought to contribute to the pathogenesis of inflammatory bowel diseases; however, it remains unclear whether it is a primary defect participating to the onset of inflammation or only a consequence of sustained inflammation. METHODS: A time course study of epithelial barrier functions and immune mediators was performed in the CD4(+)CD45RB(hi) T cell transfer model of colitis using Ussing chambers. RESULTS: In nonreconstituted severe combined immunodeficiency (SCID) mice, no epithelial dysfunction was observed. However, after transfer of CD4(+)CD45RB(hi) T cells or total CD4(+) T cells, colon of SCID mice displayed a decreased epithelial resistance, even before overt microscopic inflammation had occurred. Sustained colitis of CD4(+)CD45RB(hi) T cell reconstituted mice was also associated with enhanced subepithelial resistance, enhanced paracellular permeability, and decreased net ion transport. All these reflect a disturbance of barrier function and may contribute to diarrhea. Epithelial resistance was positively correlated with interleukin 10 (IL-10) and transforming growth factor beta (TGF-beta) levels and net ion transport inversely correlated with tumor necrosis factor alpha (TNF-alpha) levels, pointing to the protective effect of IL-10 and TGF-beta and to a damaging effect of TNF-alpha. Indomethacin, a nonselective COX inhibitor, decreased epithelial resistance independent of T cells and inflammation, but its effect was more pronounced in inflamed colon. CONCLUSIONS: Induction of colitis by transfer of CD4(+)CD45RB(hi) T cells in SCID mice leads to changes in the colonic epithelium before colitis develops. Decreased epithelium resistance might contribute to the development of colitis; however, it is not sufficient to lead to chronic inflammation.

Pouchitis, similar to active ulcerative colitis, is associated with impaired butyrate oxidation by intestinal mucosa.

BACKGROUND: Healthy colonic mucosa uses butyrate as the major energy source. In ulcerative colitis (UC) butyrate oxidation has been shown to be disturbed, but it remains unclear whether this is a primary defect. The aim of this study was to measure mucosal butyrate oxidation in UC (involved and noninvolved colon) and in pouchitis and to study the relationship with endoscopic as well as histological disease activity. METHODS: Butyrate oxidation was measured in 73 UC patients, 22 pouchitis patients, and 112 controls (95 colon, 17 ileum) by incubating biopsies with 1 mM 14C-labeled Na-butyrate and measuring the released 14CO2. RESULTS: Compared with that in normal colon, butyrate oxidation was significantly impaired in endoscopically active but not in quiescent disease or uninvolved colon segments. The severity of the metabolic defect was related to histological disease activity and decreased epithelial cell height. In active pouchitis, butyrate oxidation was significantly decreased compared with that in normal ileum and excluded pouches without inflammation. The histological pouchitis score correlated significantly with butyrate oxidation. CONCLUSIONS: Active UC and pouchitis show the same inflammation-related metabolic defect. Our data suggest that the defect is a consequence of inflammation and that pouchitis is metabolically similar to active UC.

Hyperresponsiveness of the mucosal barrier in Crohn's disease is not tumor necrosis factor-dependent.

BACKGROUND: Crohn's disease (CD) is associated with gut barrier dysfunction. Besides the baseline barrier defect, a subgroup of patients also expresses an intestinal barrier hyperresponsiveness to nonsteroidal anti-inflammatory drugs. We studied whether reducing inflammation and restoring gut barrier dysfunction with anti-tumor necrosis factor (TNF) antibody treatment also antagonizes the permeability increase by oral nonsteroidal anti-inflammatory drug intake in patients with CD. METHODS: Thirty-one healthy control subjects and 25 patients with active CD were studied. The 31 controls performed intestinal permeability testing for Cr-EDTA before (baseline) and after oral intake of indomethacin (50 + 75 mg). Twenty-five patients carried out a baseline and indomethacin-mediated permeability test before infliximab infusion. The patients repeated either the indomethacin test (12/25) or baseline and indomethacin tests (13/25), 1 month after this treatment. Intestinal permeability was studied by measurement of urinary excretion of Cr-EDTA after oral intake. RESULTS: Increased whole gut permeation before treatment (3.16%; interquartile range [IQR], 2.92-5.72) was restored to normal values (2.47%; IQR, 1.97-2.78) by anti-TNF treatment. Indomethacin increased whole gut permeability significantly more in patients with CD (before anti-TNF: 6.50%; IQR, 4.84-10.38; after anti-TNF: 5.50%; IQR, 3.97-10.09) compared with the healthy subjects (4.66%; IQR, 3.51-5.64). Eleven of 25 patients (44%) had an abnormal whole gut permeability response to indomethacin before anti-TNF, and 9 of them remained hyperresponsive after infusion, despite clinical remission. CONCLUSIONS: Although anti-TNF treatment suppresses inflammation and restores gut barrier function in patients with CD, it does not antagonize the barrier hyperresponsiveness to indomethacin. These data support the notion of an underlying intestinal mucosal barrier hyperresponsiveness in a subset of patients with CD, independent of inflammation.

Anti-tumor necrosis factor treatment restores the gut barrier in Crohn's disease.

OBJECTIVES: A primary defect of the tight junctions and, hence, increased intestinal epithelial permeability has been proposed as a basic pathogenic event in Crohn's disease. Challenge of the mucosal immune system by the commensal gut flora would then result in chronic inflammation. Alternatively, increased permeability could be the result of inflammation. Our aim was to study intestinal permeability in refractory Crohn's disease before and after treatment with monoclonal chimeric antibodies directed against tumor necrosis factor (TNF) to investigate whether the abnormal permeability persists after control of inflammation. METHODS: Twenty-three patients with active Crohn's disease were evaluated before and 4 wk after a single infusion of 5 mg/kg infliximab. Intestinal permeability was studied by measurement of urinary excretion of 51Cr-EDTA after oral intake. RESULTS: The increased permeation of 51Cr-EDTA through the small intestine (1.63% interquartile range [IQR] 1.06-2.07) and the overall permeation (3.27% IQR 2.40-4.38) before therapy decreased significantly after infliximab infusion to values (1.04% IQR 0.74-1.54 and 2.42% IQR 2.03-2.80, respectively) in the range of those found in normal volunteers (1.12% IQR 0.85-1.58 and 2.28% IQR 1.88-2.86, respectively). CONCLUSION: Inhibiting the proinflammatory cytokine tumor necrosis factor dramatically reduces gut inflammation and largely restores the gut barrier in Crohn's disease. Our data confirm the central role of TNF in gut barrier modulation in inflammatory conditions in vivo.