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Agency: European Commission | Branch: FP7 | Program: CP-FP | Phase: HEALTH.2012.1.4-2 | Award Amount: 7.74M | Year: 2012

In BALANCE, the companies Hep-Art, Pharmacell and BioPredic join forces with academic partners Amsterdam Medical Centre and University of Edinburgh to offer Acute Liver Failure (ALF) patients a bioartificial liver-support system for bridging the waiting period for liver transplantation or recovery of the diseased liver. ALF is a highly lethal disorder and liver transplantation is the only life-saving therapy. However, limited availability of donor livers severely reduces its impact. A bioartificial liver (BAL) may support ALF patients by temporary and extracorporeal treatment of their plasma through a bioreactor with functional human liver cells. The central objective of BALANCE is to develop a HepaRG-BAL that executes the three key liver functions for a clinically relevant period in ALF and to reach proof of safety and feasibility. What makes the HepaRG-BAL extremely potent is the unique combination of an ideal liver cell ecosystem and the introduction of the HepaRG cell line which is the only human cell line in the world that approaches human liver in its functionality. A two-staged approach will be adopted. Stage I is designed for in-vitro optimisation of the BAL and the human cell line and stage II comprises of the ex-vivo activities in which the optimized and upscaled BAL is tested and validated in pigs (controlled study). In addition approvement of a Phase I/IIa in humans will be prepared. . BALANCE will give birth to four main results: an optimised and validated BAL, a large scale manufacturing process, proof of concept in a large animal model of ALF and basic information for regulatory approval for future clinical application. Hep-Art will further pursue clinical development of the BAL towards EMA registration and marketing together with a large industrial partner. BioPredic will further exploit the HepaRG cells. Last but not least, BALANCE will strengthen the European competitive advantage in the field of bioartificial organs.

Frings P.W.H.,Maastricht University | Van Elssen C.H.M.J.,PharmaCell | Wieten L.,Maastricht University | Matos C.,Maastricht University | And 4 more authors.
Breast Cancer Research and Treatment | Year: 2011

Metastatic breast cancer is currently incurable despite initial responsiveness, assumingly due to the presence of chemoresistant subpopulations that can be characterized as label retaining cells (LRC). In the 4T1 mouse breast cancer model, we previously achieved cure after Cyclophosphamide and Total Body Irradiation (CY + TBI) followed by haploidentical bone marrow and spleen transplantation (BMSPLT). CY + TBI without transplantation induced only transient impaired tumor growth indicating a critical role of donor immune cells. As it remained unknown if the 4T1 model resembles human disease with respect to the presence of subpopulations of chemoresistant LRC, we now demonstrate this is indeed the case. Chemoresistance of 4T1 LRC was demonstrated by in vitro co-incubation of fluorescently labeled 4T1 cells in limiting dilution with cyclophosphamide, doxorubicin or cisplatinum, after which only LRC containing colonies remained. LRC also remain in vivo after treatment with CY + TBI. Succeeding experiments set up to identify the haploidentical effector cell responsible for cure and, therefore, for the elimination of chemoresistant LRC designate donor NK cells crucial for the anti-tumor effect. NK cell depletion of the haploidentical graft fully abrogated the anti-tumor effect. Increased disease-free survival retained after transplantation of haploidentical bone marrow and NK cell-enriched spleen cell grafts, even in the absence of donor T-cells or of donor bone marrow. Tumor growth analysis indicates the anti-tumor effect being immediate (days). Based on these data, it is worthwhile to explore alloreactive adoptive NK cell therapy as consolidation for patients with metastasized breast cancer. © 2011 Springer Science+Business Media, LLC.

Meek B.,Maastricht University | Cloosen S.,Maastricht University | Borsotti C.,Institute for Research in Biomedicine | Van Elssen C.H.M.J.,Maastricht University | And 11 more authors.
Blood | Year: 2010

Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is a treatment option for patients with hematopoietic malignancies that is hampered by treatment-related morbidity and mortality, in part the result of opportunistic infections, a direct consequence of delayed T-cell recovery. Thymic output can be improved by facilitation of thymic immigration, known to require precommitment of CD34+ cells.We demonstrate that Delta-like ligand-mediated predifferentiation of mobilized CD34+ cells in vitro results in a population of thymocyte-like cells arrested at a T/natural killer (NK)-cell progenitor stage. On intrahepatic transfer to Rag2-/- γc -/- mice, these cells selectively home to the thymus and differentiate toward surface T-cell receptor- αβ+ mature T cells considerably faster than animals transplanted with noncultured CD34+ cells. This finding creates the opportunity to develop an early T-cell reconstitution therapy to combine with HSCT. © 2010 by The American Society of Hematology.

Van Elssen C.H.M.J.,Maastricht University | Van Elssen C.H.M.J.,PharmaCell | Vanderlocht J.,Maastricht University | Frings P.W.H.,Maastricht University | And 8 more authors.
European Journal of Immunology | Year: 2010

Besides their role in destruction of altered self-cells, NK cells have been shown to potentiate T-cell responses by interacting with DC. To take advantage of NK-DC crosstalk in therapeutic DC-based vaccination for infectious diseases and cancer, it is essential to understand the biology of this crosstalk. We aimed to elucidate the in vitro mechanisms responsible for NK-cell recruitment and activation by DC during infection. To mimic bacterial infection, DC were exposed to a membrane fraction of Klebsiella pneumoniae, which triggers TLR2/4. DC matured with these bacterial fragments can actively recruit NK cells in a CCR5-dependent manner. An additional mechanism of DC-induced NK-cell recruitment is characterized by the induction of CCR7 expression on CD56 dimCD16 + NK cells after physical contact with membrane fraction of K. pneumoniae-matured DC, resulting in an enhanced migratory responsiveness to the lymph node-associated chemokine CCL19. Bacterial fragment-matured DC do not only mediate NK-cell migration but also meet the prerequisites needed for augmentation of NK-cell cytotoxicity and IFN-γ production, the latter of which contributes to Th1 polarization. © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Van Elssen C.H.M.J.,Maastricht University | Van Elssen C.H.M.J.,PharmaCell | Vanderlocht J.,Maastricht University | Oth T.,Maastricht University | And 3 more authors.
Blood | Year: 2011

Among prostaglandins (PGs), PGE2 is abundantly expressed in various malignancies and is probably one of many factors promoting tumor growth by inhibiting tumor immune surveillance. In the current study, we report on a novel mechanism by which PGE2 inhibits in vitro natural killer-dendritic cell (NK-DC) cross-talk and thereby innate and adaptive immune responses via its effect on NK-DC crosstalk. The presence of PGE2 during IFN-γ/membrane fraction of Klebsiella pneumoniae DC maturation inhibits the production of chemokines (CCL5, CCL19, and CXCL10) and cytokines (IL-12 and IL-18), which is cAMP-dependent and imprinted during DC maturation. As a consequence, these DCs fail to attract NK cells and show a decreased capacity to trigger NK cell IFN-γ production, which in turn leads to reduced T-helper 1 polarization. In addition, the presence of PGE2 during DC maturation impairs DC-mediated augmentation of NK-cell cytotoxicity. Opposed to their inhibitory effects on peripheral blood-derived NK cells, PGE2 matured DCs induce IL-22 secretion of inflammation constraining NKp44+ NK cells present in mucosa-associated lymphoid tissue. The inhibition of NK-DC interaction is a novel regulatory property of PGE2 that is of possible relevance in dampening immune responses in vivo. © 2011 by The American Society of Hematology.

Van Elssen C.H.M.J.,Maastricht University | Van Elssen C.H.M.J.,PharmaCell | Frings P.W.H.,Maastricht University | Bot F.J.,Maastricht University | And 6 more authors.
Histopathology | Year: 2010

Aims: Mucin 1 (MUC1) is an important tumour-associated antigen (TAA), both overexpressed and aberrantly glycosylated in adenocarcinomas. The aim of this study was to examine the MUC1-glycosylation status of primary ovarian adenocarcinomas and metastatic lesions.Methods and results: Paraffin-embedded tissue sections of 37 primary ovarian adenocarcinomas representing all histotypes (22 serous, five mucinous, two clear-cell, eight endometrioid), four serous borderline tumours with intraepithelial carcinoma, seven sections of ovarian endometriosis and 13 metastatic lesions were analysed by immunohistochemistry. Non-neoplastic ovarian surface epithelium and serous cystadenomas were used as controls. All epithelia expressed MUC1 protein. Of primary tumours, 76% expressed the differentiation-dependent glycoform and 84% the cancer-associated glycoform (Tn/Sialyl-Tn-epitopes). In metastatic lesions this was 77% and 85%, respectively. Notably, in 57% of ovarian endometriosis and 75% of intraepithelial lesions, the cancer-associated MUC1 epitopes were expressed, whereas normal ovarian surface epithelium and serous cystadenomas did not express these epitopes.Conclusions: The underglycosylated MUC1 epitopes are expressed by all histotypes of primary ovarian adenocarcinomas, by the vast majority of metastatic lesions and by possible ovarian cancer precursor lesions, but not by normal ovarian tissue. These results indicate that MUC1-associated Tn/STn-epitopes are important targets for immunotherapy and diagnostic imaging in ovarian cancer patients. © 2010 Blackwell Publishing Limited.

Brandao R.D.,Maastricht University | Veeck J.,Maastricht University | Veeck J.,RWTH Aachen | Van de Vijver K.K.,Maastricht University | And 10 more authors.
Breast Cancer Research | Year: 2013

Introduction: Cyclooxygenase-2 (COX-2) is frequently over-expressed in primary breast cancer. In transgenic breast cancer models, over-expression of COX-2 leads to tumour formation while COX-2 inhibition exerts anti-tumour effects in breast cancer cell lines. To further determine the effect of COX-2 inhibition in primary breast cancer, we aimed to identify transcriptional changes in breast cancer tissues of patients treated with the selective COX-2 inhibitor celecoxib.Methods: In a single-centre double-blind phase II study, thirty-seven breast cancer patients were randomised to receive either pre-operative celecoxib (400 mg) twice daily for two to three weeks (n = 22) or a placebo according to the same schedule (n = 15). Gene expression in fresh-frozen pre-surgical biopsies (before treatment) and surgical excision specimens (after treatment) was profiled by using Affymetrix arrays. Differentially expressed genes and altered pathways were bioinformatically identified. Expression of selected genes was validated by quantitative PCR (qPCR). Immunohistochemical protein expression analyses of the proliferation marker Ki-67, the apoptosis marker cleaved caspase-3 and the neo-angiogenesis marker CD34 served to evaluate biological response.Results: We identified 972 and 586 significantly up- and down-regulated genes, respectively, in celecoxib-treated specimens. Significant expression changes in six out of eight genes could be validated by qPCR. Pathway analyses revealed over-representation of deregulated genes in the networks of proliferation, cell cycle, extracellular matrix biology, and inflammatory immune response. The Ki-67 mean change relative to baseline was -29.1% (P = 0.019) and -8.2% (P = 0.384) in the treatment and control arm, respectively. Between treatment groups, the change in Ki-67 was statistically significant (P = 0.029). Cleaved caspase-3 and CD34 expression were not significantly different between the celecoxib-treated and placebo-treated groups.Conclusions: Short-term COX-2 inhibition by celecoxib induces transcriptional programs supporting anti-tumour activity in primary breast cancer tissue. The impact on proliferation-associated genes is reflected by a reduction of Ki-67 positive cells. Therefore, COX-2 inhibition should be considered as a treatment strategy for further clinical testing in primary breast cancer.Trial registration: NCT01695226. © 2013 Brandão et al.; licensee BioMed Central Ltd.

Agency: European Commission | Branch: FP7 | Program: CSA-SA | Phase: HEALTH.2013.4.1-2 | Award Amount: 554.97K | Year: 2013

We propose an academic & industrial partnership to facilitate safe and effective delivery of new advanced therapy medicines within the framework of the relevant EU regulations which our previous FP7 funded project have identified as creating unanticipated barriers to development of this field. Over 80% of advanced therapy medicines (ATMPs) trials in the EU are academic investigator led and sponsored. Many ATMPs are not making it to first-in-man trials due to the difficulties of academic manufacture to GMP compliance and the variation of application of the Directives between EU MS. We will deliver a supporting programme of resources to academic trialists to streamline their development programmes, enhance compliance with GMP requirements, develop industrial partnerships for improved manufacture and scale-up and establish the first university certified training programmes in cell/tissue engineering to address the critical shortage in scientific/technical/regulatory staff in this newly expanding field across the EU. The future supply of successful ATMPs to EU citizens will require partnerships between academic GMP facilities, contract ATMP manufacturers (CMOs) and the biopharma industry and this project will deliver the necessary support and training structure to create this partnership. The need for this project has been identified from the impact assessment part of our current FP7 project, Academic GMP and the culmination of the project will be a scientific analysis of the effect of the programme on the impact of the legislation relevant to ATMP development and provision across the EU.

Agency: European Commission | Branch: H2020 | Program: RIA | Phase: PHC-16-2015 | Award Amount: 5.12M | Year: 2016

Self-renew and multilineage potential characterize stem cells. We have recently described that pancreas progenitor cells extracted from adult donors can be expanded long-term in vitro into 3D structures, which we have termed organoids. Pancreas organoids reproduce in vitro all the features of pancreas ductal epithelia, and have a limitless expansion potential. Thus, pancreas organoids promise to boost cell therapy of type 1 diabetes. We have recently observed that progenitor cells organoids preserve their genetic stability over a long time in culture. That represents an advantage, when compared to iPS or hES derived approaches, where genetic instability raises concerns for their future therapeutic applications. While progenitor organoids are promising for the future of cell therapy, bringing stem cell-based therapies to patients requires a reliable characterization (knowing what the cells do and how they do it, i.e. a phenotypic and molecular biology characterization), chemically well-defined culture media, and the capacity of mass-production under GLP/GMP conditions. The LSFM4LIFE consortium aims to the mass production of pancreas organoids for the cellular therapy of type 1 diabetes. The goals of the project are: (1) optimize growth and differentiation of human pancreas stem-cell organoids by employing phenotypic and molecular high-throughput screening (2) standardize the growth and differentiation of the organoids under well-defined biochemical conditions, and (3) achieve GLP/GMP-production of the human organoids for preclinical studies and phase I clinical studies. The close collaboration in the consortium between academic researchers and industry, as well as its cross-disciplinary composition, are essential to realize the goals of the project. The work packages of the project will have a technological impact in the form of patents and first market replication.

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