Somatic Cell Therapy Research Articles

Abstract CT028: Adoptive transfer of CMV- and EBV- specific peptide-stimulated T cells after allogeneic stem cell transplantation: A Phase I/IIa clinical trial

Abstract Allogeneic stem cell transplantation (ASCT) to date is the only permanent curative treatment for many hematological cancers. Besides the development of Graft versus Host disease (GvHD), infections are the major adverse effect of ASCT. Specifically, reactivation of viruses is highly problematic in the aftermath of ASCT. Reactivation of human CMV and EBV negatively impacts on outcome after ASCT. 40-50% of patients reactivate CMV following ASCT, while the only CMV specific antiviral therapy available is ganciclovir, with other drugs being used off-label. For the 20-30% of patients reactivating EBV, only rituximab is available to control EBV. Rituximab leads to long term B-cell depletion requiring frequent administration of immunoglobulins. To cover this unmet medical need of CMV and EBV control after ASCT, we investigate a somatic cell therapy approach by adoptive transfer of CMV- and EBV-specific peptide-stimulated T cells. We specifically are scrutinizing the effect of application of the cells to prevent virus reactivation before its onset or preemptively at the early stages of viral infection. We set up a prospective randomized controlled phase I/IIa multi-center clinical trial to evaluate the safety and efficacy of preventive and preemptive adoptive transfer of this ATMP in patients after ASCT (EudraCT number 2012-004240-30). The design of the trial allows to applicate low numbers of activated T cells, thereby reducing the potential risks of GvHD for the recipient of adoptively transferred T cells. The multi-center trial is currently recruiting, so far, 13 patients have been randomized. ASCT patients are randomly assigned to the intervention or control group. Subjects of the control group receive standard of care. For all subjects of the intervention group, a personalized cell product (ATMP) containing a standardized number of virus specific T cells is manufactured from an aliquot of the leukapheresis product previously used for ASCT and cryopreserved until being administered. Subjects receive the cell product in intervals of at least 30 days, starting with the first adoptive transfer 30 days after ASCT at the earliest. Cells are transferred as preventive, preemptive, or also as therapeutic treatment. Subjects are monitored for occurrence of GvHD, for viral load, as well as for immune reconstitution and composition of the TCR pool, especially of virus-specific T cells. Citation Format: Michael Aigner, Regina Gary, Andreas Moosmann, Stefanie Maas, Julian Strobl, Robert Zimmermann, Jürgen Zingsem, Anita Kremer, Andreas Mackensen, Armin Gerbitz. Adoptive transfer of CMV- and EBV- specific peptide-stimulated T cells after allogeneic stem cell transplantation: A Phase I/IIa clinical trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT028. doi:10.1158/1538-7445.AM2017-CT028

Advanced Therapy Medicinal Products for Rare Diseases: State of Play of Incentives Supporting Development in Europe.

In 2008, the European Union introduced the Advanced Medicines Regulation aiming to improve regulation of advanced therapy medicinal products (ATMPs). We applied the ATMPs classification definitions in this Regulation to understand the link of this emerging group of medicinal products and the use of the Orphan Regulation. A total of 185 products that can be classified as ATMPs based on this Regulation have been submitted for orphan designation. Prior to its introduction in 2008, 4.5% of the products submitted for orphan designation met these criteria. This percentage went up to 15% after 2008. We analyzed several parameters associated with active ATMP ODDs focusing on sponsor type and EU-Member State origin, therapeutic area targeted, and ATMP classification [i.e., somatic cell therapy medicinal product, tissue-engineered product (TEP), or gene therapy medicinal product (GTMP)] and the use of regulatory services linked to incentives such as the use of protocol assistance (PA) and other Committees [Committee for Advanced Therapies (CAT) and the Pediatric Committee]. The aim here was to gain insight on the use of different services. The UK submits the largest number of ATMPs for ODD representing ~30% of the total to date. Few submissions have been received from central and Eastern European Member States as well as some of the larger Member States such as Germany (3.6%). ATMPs ODDs were primarily GTMPs (48.7%) and SCTMPs (43.3%). TEPs only represented 8% of all submissions for this medicinal class. This is different from non-ODDs ATMPs where GTMPs make only 20% of ATMPs. A total of 11.7% of ATMP ODDs had received formal CAT classification. A total of 29.8% of all orphan drug (OD) ATMPs requested PA. A total of 71.8% did not have an agreed pediatric investigation plan (PIP). Four products (Glybera one PA; Zalmoxis two; Holoclar one; Strimvelis three) have received a marketing authorization (MAA) and a 10-year market exclusivity. Strimvelis also completed their PIP, which was compliant and received the additional 2-year extension to their 10-year market exclusivity. One OD ATMP (Cerepro) received a negative opinion for MAA. The use of services linked to incentives offered by different legislations for ATMP ODDs is low, indicating a need for increasing awareness.

The Mechanism and Applications of CRISPR-Cas9

The recently developed CRISPR-Cas9 gene editing technology is transforming basic biomedical research, but it also may have therapeutic applications. This essay examines how the technology works, its possible applications in somatic and germline cell therapy, and the use of gene drives to control disease vectors like mosquito-borne illnesses. While potentially valuable, all of these applications present ethical problems, including the specific risks of unintentional mutations; pre-existing concerns over the relationship between biomedical technology, power, and procreation; and CRISPR’s unintended consequences for the environment.

Adoptive Transfer of CMV- and EBV- Specific Peptide-Stimulated T Cells after Allogeneic Stem Cell Transplantation: First Results of a Phase I/IIa Clinical Trial [Multivir-01

Background: Reactivation of CMV and EBV negatively impacts on outcome after allogeneic stem cell transplantation (aSCT). Specific antiviral therapy is only available for CMV. With the exception of ganciclovir all drugs are being used off-label. 40-50% of patients reactivate CMV following aSCT. For the 20-30% of patients reactivating EBV, only rituximab is available to control EBV. Rituximab leads to long term B-cell depletion requiring frequent administration of immunoglobulins. To cover the unmet medical need of CMV and EBV control after aSCT, we investigate a somatic cell therapy approach by means of CMV- and EBV-specific peptide-stimulated T cells. We have set up a prospective randomized controlled phase I/IIa multi-center clinical trial to evaluate the preventive and preemptive adoptive transfer of this ATMP in patients after aSCT (EudraCT number 2012-004240-30). The multi-center trial is currently recruiting.Methods: For manufacturing of the cell product two peptide pools (CMV and EBV) each covering 17 well-defined HLA class I and class II epitopes for stimulation of donor derived PBMC are used. PBMC collected by leukapheresis of mobilized or non-mobilized donors can be used as starting material. To avoid a second leukapheresis of the donor, CMV- and EBV-specific T cells are preferentially expanded from a small fraction of the stem cell graft. A strong expansion of virus-specific T cells could be observed in the cell products as determined by HLA class I multimer analysis. Reconstitution and cell counts of leukocytes after aSCT are monitored for both treatment and control group. To obtain further insights in the expansion of transferred T cells, the TCR beta (TCRb) repertoire of the T-cell product before and after adoptive transfer in the patient is monitored by high throughput sequencing. Specificities of TCRb sequences can be assigned by determining the repertoire of HLA/peptide-multimer-sorted CD8+ T cells. New virus-specific TCRb sequences can be identified thereby. Furthermore, TCR sequences within the T-cell product can be tracked in the patient.Study design: After recruitment patients are randomized in intervention or control group. Patients of the intervention group receive three applications of virus specific T cells (5x10e4/kg bodyweight) starting the first adoptive transfer 30 days after allogeneic stem cell transplantation. Cells are transferred as preventive, preemptive, or also as therapeutic treatment. Patients are monitored for occurrence of GvHD, for viral load, as well as for immune reconstitution, especially of virus-specific T cells.Results: So far, 10 patients have been randomized. The reconstitution of virus-specific T cells of treated patients looks encouraging after transfer so far. The immunomonitoring of six included patients is completed. New CMV- and EBV-specific TCRb sequences could be identified and tracked.Conclusion: Our first observations show promising results regarding feasibility and efficacy of our approach under clinical trial conditions. DisclosuresHennig:HS Diagnomics GmbH: Employment, Equity Ownership.

PHP44 - Risk of Discontinuation of Advanced Therapy Medicinal Products Clinical Trials

Advanced Therapy medicinal products (ATMPs) constitute a class of innovative products that encompasses: gene therapy, somatic cell therapy and tissue engineered products (TEP). There is an increased investment of commercial and non-commercial sponsors in this field. Randomized clinical trials (RCT) generate data to prove the efficacy of a new therapy, but the discontinuation of RCTs wastes scarce resources. Our objective is to identify the number and characteristics of discontinued ATMPs trials in order to evaluate the rate of discontinuation. We searched for ATMPs trials conducted between 1999 to June 2015 using three databases: Clinicaltrials.gov, the International Clinical Trials Registry Platform (ICTRP) and the EudraCT. We selected the ATMPs trials after elimination of duplicates. We identified the disease areas and the sponsors as commercial or non-commercial organization. We classified ATMPs by type and trial status: ongoing, completed, terminated, discontinued, and prematurely ended. Then we calculated the rate of discontinuation. 143 withdrawn, terminated or prematurely ended ATMPs clinical trials were identified between 1999 and June 2015 and 474 ongoing and completed clinical trials. Therefore, the rate of discontinuation of ATMPs trials is 23.17% similar to non-ATMPs drugs in development. The probability of discontinuation is respectively 27.35%, 16.28%, and 16.34% for cell therapies, gene therapies, and TEP. The highest discontinuation rate is for oncology (44%) followed by cardiology and immunology/inflammation (22.2%). It is almost the same for commercial and non-commercial sponsors (24%); Suggesting discontinuation may not be financially driven. No failure risk rate per development phase is available for ATMPs. Discontinuation rate may prove helpful when assessing expected net present value to support portfolio arbitration. These results carry limitation as the reason for discontinuation is unknown. Further research about the reasons of discontinuation and the risk of negative results is needed to inform manufacturers and investors decisions.

Hitachi builds cell plant in Japan

Hitachi Chemical will spend $19 million to build a custom manufacturing facility producing live regenerative-medicine cells in Yokohama, Japan. The cells, produced in vitro at the facility, could be used for organ restoration or to repair immune functions damaged by injury or disease. Other applications are in cancer immunotherapy treatments and somatic stem cell therapy. The plant will make use of know-how and technology supplied by PCT, a subsidiary of Caladrius BioSciences in which Hitachi acquired a 20% stake for close to $20 million in March.

The expanding role of the clinical haematologist in the new world of advanced therapy medicinal products.

Advanced therapy medicinal products (ATMPs) represent the current pinnacle of 'patient-specific medicines' and will change the nature of medicine in the near future. They fall into three categories; somatic cell-therapy products, gene therapy products and cells or tissues for regenerative medicine, which are termed 'tissue engineered' products. The term also incorporates 'combination products' where a human cell or tissue is combined with a medical device. Plainly, many of these new medicines share similarities with conventional haematological stem cell transplant products and donor lymphocyte infusions as well as solid organ grafts and yet ATMPs are regulated as medicines and their development has remained predominantly in academic settings and within specialist centres. However, with the advent of commercialisation of dendritic cell vaccines, chimeric antigen receptor (CAR)-T cells and genetically modified autologous haematopoietic stem cells to cure single gene-defects in β-thalassaemia and haemophilia, the widespread availability of these therapies needs to be accommodated. Uniquely to ATMPs, the patient or an allogeneic donor is regularly part of the manufacturing process. All of the examples given above require procurement of blood, bone marrow or an apheresate from a patient as a starting material for manufacture. This can only occur in a clinical facility licensed for the procurement of human cells for therapeutic use and this is likely to fall to haematology departments, either as stem cell transplant programmes or as blood transfusion departments, to provide under a contract with the company that will manufacture and supply the final medicine. The resource implications associated with this can impact on all haematology departments, not just stem cell transplant units, and should not be under-estimated.

Quantitative proteome profiling of dystrophic dog skeletal muscle reveals a stabilized muscular architecture and protection against oxidative stress after systemic delivery of MuStem cells.

Proteomic profiling plays a decisive role in the elucidation of molecular signatures representative of a specific clinical context. MuStem cell based therapy represents a promising approach for clinical applications to cure Duchenne muscular dystrophy (DMD). To expand our previous studies collected in the clinically relevant DMD animal model, we decided to investigate the skeletal muscle proteome 4 months after systemic delivery of allogenic MuStem cells. Quantitative proteomics with isotope-coded protein labeling was used to compile quantitative changes in the protein expression profiles of muscle in transplanted Golden Retriever muscular dystrophy (GRMD) dogs as compared to Golden Retriever muscular dystrophy dogs. A total of 492 proteins were quantified, including 25 that were overrepresented and 46 that were underrepresented after MuStem cell transplantation. Interestingly, this study demonstrates that somatic stem cell therapy impacts on the structural integrity of the muscle fascicle by acting on fibers and its connections with the extracellular matrix. We also show that cell infusion promotes protective mechanisms against oxidative stress and favors the initial phase of muscle repair. This study allows us to identify putative candidates for tissue markers that might be of great value in objectively exploring the clinical benefits resulting from our cell-based therapy for DMD. All MS data have been deposited in the ProteomeXchange with identifier PXD001768 (http://proteomecentral.proteomexchange.org/dataset/PXD001768).

Advanced therapy medicinal products: current and future perspectives

BackgroundAdvanced therapy medicinal products (ATMPs) are innovative therapies that encompass gene therapy, somatic cell therapy, and tissue-engineered products. These therapies are expected to bring important health benefits, but also to substantially impact the pharmaceuticals budget.ObjectiveThe aim of this study was to characterise the ATMPs in development and discuss future implications in terms of market access.MethodsClinical trials were searched in the following databases: EudraCT (EU Drug Regulating Authorities Clinical Trials), ClinicalTrials.gov, and ICTRP (International Clinical Trials Registry Platform of the World Health Organization). Trials were classified by category of ATMP as defined by European regulation EC No. 1394/2007, as well as by development phase and disease area.ResultsThe database search identified 939 clinical trials investigating ATMPs (85% ongoing, 15% completed). The majority of trials were in the early stages (Phase I, I/II: 64.3%, Phase II, II/III: 27.9%, Phase 3: 6.9%). Per category of ATMP, we identified 53.6% of trials for somatic cell therapies, 22.8% for tissue-engineered products, 22.4% for gene therapies, and 1.2% for combined products (incorporating a medical device). Disease areas included cancer (24.8%), cardiovascular diseases (19.4%), musculoskeletal (10.5%), immune system and inflammation (11.5%), neurology (9.1%), and others. Of the trials, 47.2% enrolled fewer than 25 patients. Due to the complexity and specificity of ATMPs, new clinical trial methodologies are being considered (e.g., small sample size, non-randomised trials, single-arm trials, surrogate endpoints, integrated protocols, and adaptive designs). Evidence generation post-launch will become unavoidable to address payers’ expectations.ConclusionATMPs represent a fast-growing field of interest. Although most of the products are in an early development phase, the combined trial phase and the potential to cure severe chronic conditions suggest that ATMPs may reach the market earlier than standard therapies. Targeted therapies have opened the way for new trial methodologies, from which ATMPs could benefit to get early access. ATMPs may be the next source of major impact on payers’ drug budgets.

Risk of discontinuation of Advanced Therapy Medicinal Products clinical trials

ObjectiveAdvanced therapy medicinal products (ATMPs) constitute a class of innovative products that encompasses gene therapy, somatic cell therapy, and tissue-engineered products (TEP). There is an increased investment of commercial and non-commercial sponsors in this field and a growing number of ATMPs randomized clinical trials (RCT) and patients enrolled in such trials. RCT generate data to prove the efficacy of a new therapy, but the discontinuation of RCTs wastes scarce resources. Our objective is to identify the number and characteristics of discontinued ATMPs trials in order to evaluate the rate of discontinuation.MethodsWe searched for ATMPs trials conducted between 1999 to June 2015 using three databases, which are Clinicaltrials.gov, the International Clinical Trials Registry Platform (ICTRP), and the EU Drug Regulating Authorities Clinical Trials (EudraCT). We selected the ATMPs trials after elimination of the duplicates. We identified the disease areas and the sponsors as commercial or non-commercial organizations. We classified ATMPs by type and trial status, that is, ongoing, completed, terminated, discontinued, and prematurely ended. Then, we calculated the rate of discontinuation.ResultsBetween 1999 and June 2015, 143 withdrawn, terminated, or prematurely ended ATMPs clinical trials were identified. Between 1999 and June 2013, 474 ongoing and completed clinical trials were identified. Therefore, the rate of discontinuation of ATMPs trials is 23.18%, similar to that for non-ATMPs drugs in development. The probability of discontinuation is, respectively, 27.35, 16.28, and 16.34% for cell therapies, gene therapies, and TEP. The highest discontinuation rate is for oncology (43%), followed by cardiology (19.2%). It is almost the same for commercial and non-commercial sponsors; therefore, the discontinuation reason may not be financially driven.ConclusionNo failure risk rate per development phase is available for ATMPs. The discontinuation rate may prove helpful when assessing the expected net present value to support portfolio arbitration and may be considered by patients and potential investigators in their decisions to participate in ATMP trials. These results carry limitation because the rationale for discontinuation is unknown. Further research about the reasons of discontinuation and the risk of negative results is needed to inform stakeholders.

An ASGCT Perspective on the National Academies Genome Editing Summit.

The armamentarium of modern genetics and gene therapy has recently expanded with the rapid development of the tools of “genome editing,” including zinc-finger nucleases, TALENs, and CRISPR/Cas9 techniques that allow exquisitely targeted sequence modifications. An alluring application of such technology is somatic cell genome editing for the prevention and therapy of genetic disease. Most scientific, ethics, and policy agencies support somatic cell applications of genome editing; the gene therapy community has understandably taken deep interest because we represent the major biomedical conduit through which the technology will eventually be translated to clinical application.

Fabrication of Large Size Ex Vivo-Produced Oral Mucosal Equivalents for Clinical Application.

The soft tissue reconstruction of significant avulsed and/or surgically created tissue defects requires the ability to manufacture substantial soft tissue constructs for repair of the resulting wounds. In this study, we detail the issues that need to be addressed in upsizing the manufacture of larger tissue-engineered devices (ex vivo-produced oral mucosa equivalent [EVPOME]) in vitro from a methodology previously used for smaller constructs. The larger-sized EVPOME, consisting of autologous human oral keratinocytes and a dermal substitute, AlloDerm(®), was fabricated for the purpose of reconstructing large clinical defects. Regulated as an autologous somatic cell therapy product, the fabrication process abided by current Good Manufacturing Practices and current Good Tissue Practices as required by the Center for Biologics Evaluation and Research (CBER) of the United States Food and Drug Administration (FDA). Successful fabrication of large EVPOMEs utilized a higher cell seeding density (5.3×10(5) cells/cm(2)) with a relatively thinner AlloDerm, ranging from 356.6 to 508.0 μm in thickness. During the air-liquid interface culture, the thickness of the scaffold affected the medium diffusion rate, which, in turn, resulted in changes of epithelial stratification. Histologically, keratinocyte progenitor (p63), proliferation (Ki-67), and late differentiation marker (filaggrin) expression showed differences correlating with the expression of glucose transporter-1 (GLUT1) in the EVPOMEs from the thickest (550-1020 μm) to the thinnest (228.6-330.2 μm) AlloDerm scaffold. Glucose consumption and 2-deoxyglucose (2DG) uptake showed direct correlation with scaffold thickness. The scaffold size and thickness have an impact on the cellular phenotype and epithelial maturation in the manufacturing process of the EVPOME due to the glucose accessibility influenced by the diffusion rate. These outcomes provide basic strategies to manufacture a large-sized, healthy EVPOME graft for reconstructing large mucosa defects.

Specific requirements for somatic cell therapy medicinal products and tissue engineered products

Advanced-therapymedicinal products (ATMPs) are medicines for human use that are based on genes (gene therapy), cells (somatic-cell therapy) or tissues (tissue engineering) and include autologous, allogeneic, or xenogenic origin products. They are new therapeutic strategies and their development will contribute to providing opportunities for some diseases that so far lack effective treatments. The main objective of the pharmaceutical industry is to introduce on the market effective medications that increase hope and quality of life of the population, not to mention the safety criteria. To do this, it is important to know well the existing advanced therapies regulation allowing you to more easily perform the translation of basic research carried out in research laboratories and that decision making is quality. Definitions from a point of view regulatory of these medications are in the Regulation (EC) N◦ 1394/2007 of the European Parliament and of the Council of 13 November 2007 on advanced therapy (Chapter 1, article 2) drugs and the Directive 2001/83/CE of the European Parliament and of the Council of 6 November 2001 by establishing a community relating to medicinal products for human use code (annex I part IV, according to its last modification).

Creating Conditions for the Success of The French Industrial Advanced Therapy Sector

Although the European Union merely followed the initiatives of the United States and Japan by introducing special regimes for orphan medicinal products, it has introduced a special status for a new category of biological medicinal products, advanced therapy medicinal products (ATMPs), adopting specific associated regulations. European Regulation (which constitutes the highest legal instrument in the hierarchy of European law texts) [EC] No. 1394/2007, published in 2007, uses this term to define somatic cell therapy medicinal products, tissue-engineered products, and gene therapy medicinal products, possibly combined with medical devices. The stated objective was two-fold: both to promote their industrialization and market access, while guaranteeing a high level of health protection for patients. Since publication of the regulation, few marketing authorizations have been granted in Europe, and these have not been accompanied by commercial success. However, certain recent studies show that this is a growing sector and that France remains the leading European nation in terms of clinical trials. This round table brought together a panel of representatives of French public and private protagonists from the advanced therapy sector. The discussions focused on the conditions to ensure the success of translational research and, more generally, the French advanced therapy sector. These enabled a number of obstacles to be identified, which once lifted, by means of recommendations, would facilitate the development and success of this sector.

THE PROBLEMATIC ASPECTS OF THE HOSPITAL EXEMPTION REGULATION FOR ADVANCED THERAPY MEDICINAL PRODUCTS

Advanced therapy medicinal products – new category of biological pharmaceutical products, which covers somatic cell therapy, gene therapy, tissue-engineered products and combined advanced therapy medicinal products. Regulation (EC) No 1394/2007 defined advanced therapy medicinal products and set up special rules for advanced therapy medicinal products intended to be placed on the market in Member States and prepared industrially or manufactured by a method involving an industrial process. The scope of Regulation (EC) No 1394/2007 is not applicable for advanced therapy medicinal products which are prescribed under the exclusive professional responsibility of a medical practitioner, prepared on a non-routine basis according to specific quality standards and used within the same Member State in a hospital, but at the same time it is stressed that Comunity rules related to quality and safety should not be breaked for these products. For this purpose Member States should provide

Ectopic HOXB4 Expression Expands the Pool of Hemogenic Endothelium-Initiating Progenitor Cells during Pluripotent Stem Cell Differentiation

Generation of hematopoietic stem cells (HSCs) from pluripotent stem cells, in vitro, holds great promise for future somatic gene and cell therapy. So far, HSCs capable of long-term multilineage reconstitution in mice have only been obtained when the homeodomain transcription factor HOXB4 was ectopically expressed during pluripotent stem cell differentiation (Kyba et al. Cell 109(1): 29-37, 2002; Pilat et al. Proc Natl Acad Sci USA 102(34): 12101-12106, 2005; Lesinski et al. Stem Cells Transl Med 1(8): 581-591, 2012). However, the primary "target" cell of HOXB4 during hematopoietic development, in vitro, is not yet known. Its identification is a prerequisite for unambiguously identifying the molecular circuits driving HSC development, at least in vitro.To pin down this cell, we retrovirally expressed HOXB4 or a Tamoxifen-inducible HOXB4-ERT2 fusion protein in different reporter and knock-out mouse embryonic stem cell (ESC) lines. For these experiments, ESCs were differentiated for 6 days as embryoid bodies (EBs), dissociated and subsequently cocultured on OP9 stroma cells in medium supplemented with 100 ng/ml mSCF, 40 ng/ml mTPO, 100 ng/ml hFlt3L and 40 ng/ml hVEGF (STFV) for further 3 days. Use of a Runx1(-/-) ESC-line containing a doxycycline-inducible Runx1 coding sequence (“iRunx1”; kindly provided by G. Lacaud, Manchester) uncovered that HOXB4 acts during formation of the hemogenic endothelium (HE) from which HSCs arise. Without Runx1 induction, which arrests hematopoietic development at the HE-stage, ectopic HOXB4 expression mediated an approximately 30-fold increase in the number of circular endothelial, bona fide HE-sheets being Flk1+VE-Cadherin+Tie2+ (mean values: control: 11+/-4.8 n=7; HOXB4: 301+/-47 n=7; P<0.0001, unpaired, 2-sided Student´s t-test) and expressing Sox17 and Lmo2. This observation suggested an expansion of HE progenitors, detectable from day 5 of EB differentiation on. Determination of their frequencies within the VE-Cadherin+ population revealed a HOXB4-mediated increase from 1:360 cells (control) up to 1:15 cells (HOXB4; 95% C.I. = 1:12-1:21). After additional Runx1 induction, the endothelial cells morphologically underwent an Endothelial-to-Hematopoietic Transition (EHT) as verified at the single cell level by time-lapse microscopy. Concomitantly, they upregulated transcription of Gfi1, Gfi1b and Pu.1, initiated surface expression of the pan-hematopoietic marker CD45 and generated hematopoietic colony forming cells (CFC), thus proving their identity as real hemogenic endothelial cells.Taken together, our results strongly suggest that HOXB4 first and foremost promotes hematopoiesis by substantially increasing the number of hemogenic endothelium progenitors during mouse pluripotent stem cell differentiation. DisclosuresNo relevant conflicts of interest to declare.

A High Content Screening Assay for Evaluation of Biomaterial‐Mediated Cell Fusion Processes

SummaryBiomaterials are of increasing importance in regenerative medicine and entail delivery systems in somatic cell therapies, matrices for tissue engineering or tissue regeneration. The evaluation of biomaterial induced biological effects remains a key issue in clinical application. Cell‐based assays for potential cytotoxic and immunological responses have been developed but are often inadequate to address cell‐type specific responses to biomaterials. To quantitatively monitor attachment, survival, proliferation and fusion‐controlled differentiation of osteoclasts (bone resorbing cells), a High Content Screening (HCS) assay has been developed based on osteoclast differentiation of the murine monocytic cell line RAW 264.7. This assay was applied to investigate the influence of degradation products of polymers from gelatin and lysine diisocyanate, which display tailorable mechanical properties and have potential as biomaterials. The data show that the degradation products inhibit formation of multinuclear osteoclasts and suggest a potential support of bone regeneration by suppression of bone resorption.

Vascular endothelia growth factor targeted therapy may improve the effect of dendritic cell-based cancer immune therapy

Glioblastoma (GBM) is the most com-mon and most lethal subtype of glioma and, despite advances, therapy prognosis re-mains poor with a median survival of ~15 months and a 2-year survival rate of 26% [1]. Standard therapy consists of tumor re-section, radiation and chemotherapy and a majority of patients receive antiangioge-netic therapy with the humanized anti-vas -cular endothelial growth factor monoclonal antibody bevacizumab as second-line ther-apy. In a randomized Phase II clinical trial we use an individualized cancer immune therapy concept based on dendritic cells (DC-CIT) as add on to first-line therapy [2]. Our cancer vaccine AV0113 is comprised of two main components: Type 1 dendritic cells, that are characterized by the secretion of the immune regulatory molecule IL-12 to polarize the immune system towards a cy-tolytic immune response, are derived from the patient’s monocytes and are pulsed with tumor antigens, that are extracted from the patient’s autologous tumor tissue. AV0113 represents a fully individualized somatic cell therapy.

Regulation of advanced therapy medicinal products in Europe and the role of academia

Background aimsAdvanced therapy medicinal products (ATMP) are gene therapy, somatic cell therapy or tissue-engineered products regulated under (EC) No. 1394/2007 to ensure their free movement within the European Union while guaranteeing the highest level of health protection for patients. Academic good manufacturing practice (GMP) centers are major contributors in the development of ATMPs and this study assessed the impact of regulations on them. MethodsEuropean academic and non-industrial facilities (n = 747) were contacted, and a representative sample of 50 replied to a detailed questionnaire. Experienced centres were further selected in every Member State (MS) for semi-structured interviews. Indicators of ATMP production and development success were statistically assessed, and opinions about directive implementation were documented. ResultsFacilities experienced in manufacturing cell therapy transplant products are the most successful in developing ATMPs. New centres lacking this background struggle to enter the field, and there remains a shortage of facilities in academia participating in translational research. This is compounded by heterogeneous implementation of the regulations across MS. ConclusionsGMP facilities successfully developing ATMPs are present in all MS. However, the implementation of regulations is heterogeneous between MS, with substantial differences in the definition of ATMPs and in the approved manufacturing environment. The cost of GMP compliance is underestimated by research funding bodies. This is detrimental to development of new ATMPs and commercialization of any that are successful in early clinical trials. Academic GMP practitioners should strengthen their political visibility and contribute to the development of functional and effective European Union legislation in this field.

The Evolution of Nonclinical Regulatory Science: Advanced Therapy Medicinal Products as a Paradigm

Advanced therapy medicinal products (ATMPs) comprise gene therapy medicinal products (GTMPs), somatic cell therapy medicinal products (CTMPs), and tissue-engineered products (TEPs). So-called combined ATMPs incorporate one or more medical devices as an integral part of the product. Because of their complexity and innovative nature, ATMPs pose new scientific and regulatory challenges to both developers and regulators.1 Many developers of such products request scientific advice from the Committee for Medicinal Products for Human Use (CHMP) at the European Medicines Agency (EMA). We have repeatedly been asked by stakeholders to analyze the scientific advice provided by the CHMP with regard to the identification of common principles that might be useful to share with product developers. Here we comprehensively analyze the scientific advice provided by the CHMP following implementation of the 2009 legislation on ATMPs, in which the Committee for Advanced Therapies (CAT) plays an integral role. This analysis allowed us to conclude that, for these innovative medicines, a classic guideline-based approach is insufficient owing to the complexity of the products. Regulators must employ a more tailored approach that includes principles of risk identification and mitigation. In the current analysis, we focus on the translational aspects of ATMPs (i.e., nonclinical issues) because these are a direct measure of developmental complexity of innovative treatments and often constitute a bottleneck for ATMP development. ATMPs are pharmaceuticals with a high level of complexity linked to their composition, development, manufacturing, characterization, and administration. Such products can serve as a paradigm for any innovative medicinal product or personalized medicine because of their high specificity, particularly with regard to how regulators provide guidance to accommodate the needs of stakeholders. The current regulatory framework was fully implemented in the European Community in 2009.2 Central to this regulatory framework for ATMPs was the establishment of a multidisciplinary expert scientific committee, the CAT, which is tightly integrated into the EMA procedure for CHMP scientific advice. In addition to the well-established procedures for the EMA's scientific advice and protocol assistance,3,4 classification and certification procedures have been established5,6,7,8 to facilitate discussions with developers. The CAT has published several guidelines for ATMPs to facilitate development of this complex class of therapy.9 However, the broad spectrum of ATMPs with regard to structure and mechanism of action means that such general guidance can be difficult to translate into product-specific requirements. These difficulties can be substantial for sponsors developing ATMPs, considering that the major stakeholders in Europe are academia, charities, and small companies, which often have limited financial resources or regulatory expertise when compared with large pharmaceutical companies.10