Establishing the Production of Male Schistosoma mansoni Cercariae for a Controlled Human Infection Model.

To accelerate the development of novel vaccines for schistosomiasis, we set out to develop a human model for Schistosoma mansoni infection in healthy volunteers. During natural infections, female schistosomes produce eggs that give rise to morbidity. Therefore, we produced single-sex, male Schistosoma mansoni cercariae for human infection without egg production and associated pathology. Cercariae were produced in their intermediate snail hosts in accordance with the principles of good manufacturing practice (GMP). The application of GMP principles to an unconventional production process is a showcase for the controlled production of complex live challenge material in the European Union or under Food and Drug Administration guidance.

Understanding clinical development of chimeric antigen receptor T cell therapies.

BACKGROUND AIMS: In the past decade, many clinical trials with gene- and cell-based therapies (GCTs) have been performed. Increased interest in the development of these drug products by various stakeholders has become apparent. Despite this growth in clinical studies, the number of therapies receiving marketing authorization approval (MAA) is lagging behind. To enhance the success rate of GCT development, it is essential to better understand the clinical development of these products. Chimeric antigen receptor (CAR) T cells are a GCT product subtype with promising efficacy in cancer treatment which are tested in many clinical trials, but have not yet received MAA. METHODS: We generated an overview of the characteristics of CAR T-cell clinical development in the United States, Canada and Europe. Subsequently, the characteristics of clinical trials with CAR T-cell products that proceeded to a subsequent clinical trial, used as a proxy for success, were compared with those that did not proceed. RESULT: From the U.S. and European Union clinical trial databases, 106 CAR T-cell trials were selected, from which 49 were linked to a subsequent trial and 57 were not. The majority of the trials had an academic sponsor from which most did not proceed, whereas most commercially sponsored trials were followed by another clinical trial. Furthermore, trials with a subsequent trial more frequently recruited large patient cohorts and were more often multicenter compared with trials that were not followed up. DISCUSSION: These characteristics can be used by investigators to better design clinical trials with CAR T cells. We encourage sponsors to plan clinical development ahead for a higher efficiency of product development and thereby achieving a higher success rate of development towards MAA.

A review of factors explaining variability in fentanyl pharmacokinetics; focus on implications for cancer patients.

Fentanyl is a strong opioid that is available for various administration routes, and which is widely used to treat cancer-related pain. Many factors influence the fentanyl pharmacokinetics leading to a wide inter- and intrapatient variability. This systematic review summarizes multiple studied factors that potentially influence fentanyl pharmacokinetics with a focus on implications for cancer patients. The use of CYP3A4 inhibitors and inducers, impaired liver function, and heating of the patch potentially influence fentanyl pharmacokinetics in a clinically relevant way. In elderly patients, current data suggest that we should carefully dose fentanyl due to alterations in absorption and metabolism. The influence of BMI and gender on fentanyl pharmacokinetics is questionable, most probably due to a large heterogeneity in the published studies. Pharmacogenetics, e.g. the CYP3A5*3 gene polymorphism, may influence fentanyl pharmacokinetics as well, although further study is warranted. Several other factors have been studied but did not show significant and clinically relevant effects on fentanyl pharmacokinetics. Unfortunately, most of the published papers that studied factors influencing fentanyl pharmacokinetics describe healthy volunteers instead of cancer patients. Results from the studies in volunteers may not be simply extrapolated to cancer patients because of multiple confounding factors. To handle fentanyl treatment in a population of cancer patients, it is essential that physicians recognize factors that influence fentanyl pharmacokinetics, thereby preventing potential side-effects and increasing its efficacy.

Clinical development of gene- and cell-based therapies: overview of the European landscape.

In the last decade, many clinical trials with gene- and cell-based therapies were performed and increasing interest in the development was established by (national) authorities, academic developers, and commercial companies. However, until now only eight products have received marketing authorization (MA) approval. In this study, a comprehensive overview of the clinical development of gene- and cell-based therapies in Europe is presented, with a strong focus on product-technical aspects. Public data regarding clinical trials with gene- and cell-based therapies, obtained from the European Union (EU) clinical trial database (EudraCT) between 2004 and 2014 were analyzed, including product-technical variables as potential determinants affecting development. 198 unique gene and cell therapy products were identified, which were studied in 278 clinical trials, mostly in phase 1/2 trials and with cell therapies as major group. Furthermore, most products were manufactured from autologous starting material mostly manufactured from stem cells. The majority of the trials were sponsored by academia, whereas phase 3 trials mostly by large companies. Academia dominated early-stage development by mainly using bone marrow derived products and stem cells. Conversely, commercial sponsors were more actively pursuing in vivo gene therapy medicinal product development, and cell therapies derived from differentiated tissue in later-stage development.

Potential use of lymph node-derived HPV-specific T cells for adoptive cell therapy of cervical cancer.

Adoptive transfer of tumor-specific T cells, expanded from tumor-infiltrating lymphocytes or from peripheral blood, is a promising immunotherapeutic approach for the treatment of cancer. Here, we studied whether the tumor-draining lymph nodes (TDLN) of patients with human papillomavirus (HPV)-induced cervical cancer can be used as a source for ACT. The objectives were to isolate lymph node mononuclear cells (LNMC) from TDLN and optimally expand HPV-specific CD4+ and CD8+ T cells under clinical grade conditions. TDLN were isolated from 11 patients with early-stage cervical cancer during radical surgery. Isolated lymphocytes were expanded in the presence of HPV16 E6 and E7 clinical grade synthetic long peptides and IL-2 for 22 days and then analyzed for HPV16 specificity by proliferation assay, multiparameter flow cytometry and cytokine analysis as well as for CD25 and FoxP3 expression. Stimulation of LNMC resulted in expansion of polyclonal HPV-specific T cells in all patients. On average a 36-fold expansion of a CD4+ and/or CD8+ HPV16-specific T cell population was observed, which maintained its capacity for secondary expansion. The T helper type 1 cytokine IFNγ was produced in all cell cultures and in some cases also the Th2 cytokines IL-10 and IL-5. The procedure was highly reproducible, as evidenced by complete repeats of the stimulation procedures under research and under full good manufacturing practice conditions. In conclusion, TDLN represent a rich source of polyclonal HPV16 E6- and E7-specific T cells, which can be expanded under clinical grade conditions for adoptive immunotherapy in patients with cervical cancer.

TLR2 ligand-synthetic long peptide conjugates effectively stimulate tumor-draining lymph node T cells of cervical cancer patients.

The potency of human papillomavirus type 16 (HPV16)-encoded synthetic long peptides (SLP), conjugated to an optimized Toll-like receptor 2 ligand (TLR2-L), was assessed in ex vivo activation of HPV16+ cancer patient-derived T cells. Two highly immunogenic SLP sequences derived from the oncogenic E6 protein of HPV16 were selected and conjugated to a Pam3CSK4-based TLR2-L under GMP conditions. Both conjugates were able to mature human DCs in vitro and to activate human skin-derived antigen-presenting cells (APCs) upon intradermal injection in an ex vivo skin model, associated with induction of a favorable chemokine profile to attract and activate T cells. The conjugated SLPs were efficiently processed by APCs, since HPV16-specific CD4+ and CD8+ T-cell clones isolated from HPV16+ cervical tumors proliferated in response to both conjugates. The TLR2-L SLP conjugates significantly enhanced ex vivo T helper type 1 T-cell activation in cell suspensions obtained from tumor-draining lymph nodes (LN) resected during hysterectomy of HPV16+ cervical cancer patients. These results show that TLR2-L SLP conjugates can activate circulating or LN-derived tumor-specific T cells, a promising outcome for studying these two conjugates in a phase I/II clinical safety and immunogenicity trial.

Hurdles in clinical implementation of academic advanced therapy medicinal products: A national evaluation.

BACKGROUND: Since the implementation of the European Union (EU) regulation for advanced therapy medicinal products (ATMPs) in 2009, only six ATMPs achieved marketing authorization approval in the EU. Recognizing the major developments in the ATMP field, starting mostly in academic institutions, we investigated which hurdles were experienced in the whole pathway of ATMP development towards clinical care. METHODS: Quality interviews were executed with different stakeholders in The Netherlands involved in the ATMP development field, e.g. academic research groups, national authorities and patient organizations. Based on the hurdles mentioned in the interviews, questionnaires were subsequently sent to the academic principal investigators (PIs) and ATMP good manufacturing practice (GMP) facility managers to quantify these hurdles. RESULTS: Besides the familiar regulatory routes of marketing authorization (MA) and hospital exemption (HE), a part of the academic PIs perceived that ATMPs should become available by the Tissues and Cells Directive or did not anticipate on the next development steps towards implementation of their ATMP towards regular clinical care. The main hurdles identified were: inadequate financial support, rapidly evolving field, study-related problems, lacking regulatory knowledge, lack of collaborations and responsibility issues. DISCUSSION: Creating an academic environment stimulating and planning ATMP development and licensing as well as investing in expanding the relevant regulatory knowledge in academic institutions seems a prerequisite to develop ATMPs from bench to patient.

Development of cell therapy medicinal products by academic institutes.

In the rapidly evolving fields of cellular immunotherapy, gene therapy and regenerative medicine, a wide range of promising cell therapy medicinal products are in clinical development. Most products originate from academic research and are explored in early exploratory clinical trials. However, the success rate toward approval for regular patient care is disappointingly low. In this paper, we define strengths and hurdles applying to the development of cell therapy medicinal products in academic institutes, and analyze why only a few promising cell therapies have reached late-stage clinical development. Subsequently, we provide recommendations to stakeholders involved in development of cell therapies to exploit their potential clinical benefit.

Safety of allogeneic bone marrow derived mesenchymal stromal cell therapy in renal transplant recipients: the neptune study.

BACKGROUND: Mesenchymal stromal cells (MSC) may serve as an attractive therapy in renal transplantation due to their immunosuppressive and reparative properties. While most studies have used autologous MSCs, allogeneic MSCs offer the advantage of immediate availability for clinical use. This is of major importance for indications where instant treatment is needed, for example allograft rejection or calcineurin inhibitor toxicity. Clinical studies using allogeneic MSCs are limited in number. Although these studies showed no adverse reactions, allogeneic MSCs could possibly elicit an anti-donor immune response, which may increase the incidence of rejection and impact the allograft survival in the long term. These safety issues should be addressed before further studies are planned with allogeneic MSCs in the solid organ transplant setting. METHODS/DESIGN: 10 renal allograft recipients, 18-75 years old, will be included in this clinical phase Ib, open label, single center study. Patients will receive two doses of 1.5 × 10(6) per/kg body weight allogeneic bone marrow derived MSCs intravenously, at 25 and 26 weeks after transplantation, when immune suppression levels are reduced. The primary end point of this study is safety by assessing biopsy proven acute rejection (BPAR)/graft loss after MSC treatment. Secondary end points, all measured before and after MSC infusions, include: comparison of fibrosis in renal biopsy by quantitative Sirius Red scoring; de novo HLA antibody development and extensive immune monitoring; renal function measured by cGFR and iohexol clearance; CMV and BK infection and other opportunistic infections. DISCUSSION: This study will provide information on the safety of allogeneic MSC infusion and its effect on the incidence of BPAR/graft loss. TRIAL REGISTRATION: NCT02387151.

Monitoring antigen-specific biologics: current knowledge and future prospects.

An increasing number of antigen-specific biologics have been introduced into clinical practice, and the ones that arrived first have already reached the end of their patented life span. Despite the use of these agents for over a decade, individualized dosing is not standard practice. Most patients are treated according to treatment protocols, with a fixed dose administered at fixed time intervals. Although the between-subject variability in the volume of distribution is small, there is a moderate to high between-subject variability in the clearance of these biologics. The formation of neutralizing antidrug antibodies (ADAs) further contributes to the variability in the pharmacokinetics and pharmacodynamics. After the development of assays to detect biologic drug serum concentrations and ADA titers, the first clinical studies in immune-mediated diseases such as rheumatology, gastroenterology, and dermatology have now shown clear concentration-effect relationships. By monitoring the serum trough concentrations of biologics, patients with high drug exposure could be identified and dose reductions in those patients may lead to improved safety and substantial cost savings. Low biologic drug serum concentrations may be due to the development of ADAs, and if these are detected, a switch to an alternative treatment is indicated. We envision a vast expansion of therapeutic drug monitoring to support the use of biologics, and we urge the International Association of Therapeutic Drug Monitoring and Clinical Toxicology to embark on initiatives to investigate the contribution of therapeutic drug monitoring to this field.

Effective treatment of refractory CMV reactivation after allogeneic stem cell transplantation with in vitro-generated CMV pp65-specific CD8+ T-cell lines.

To treat patients with refractory cytomegalovirus (CMV) reactivation after allogeneic stem cell transplantation, a phase I/II clinical study on adoptive transfer of in vitro-generated donor-derived or patient-derived CMV pp65-specific CD8* T-cell lines was performed. Peripheral blood mononuclear cells from CMV seropositive donors or patients were stimulated with HLA-A*0201-restricted and/or HLA-B*0702-restricted CMV pp65 peptides (NLV/TPR) and 1 day after stimulation interferon-γ)-producing cells were enriched using the CliniMACS Cytokine Capture System (interferon-γ), and cultured with autologous feeders and low-dose interluekin-2. After 7-14 days of culture, quality controls were performed and the CMV-specific T-cell lines were administered or cryopreserved. The T-cell lines generated contained 0.6-17 × 10(6) cells, comprising 54%-96% CMV pp65-specific CD8 T cells, and showed CMV-specific lysis of target cells. Fifteen CMV-specific T-cell lines were generated of which 8 were administered to patients with refractory CMV reactivation. After administration, no acute adverse events and no graft versus host disease were observed and CMV load disappeared. In several patients, a direct relation between administration of the T-cell line and the in vivo appearance of CMV pp65-specific T cells could be documented. In conclusion, administration of CMV pp65-specific CD8* T-cell lines was found to be feasible and safe, and enduring efficacy of administered CMV pp65-specific CD8* T-cell lines could be demonstrated.

Efficiency and mechanism of antigen-specific CD8+ T-cell activation using synthetic long peptides.

Synthetic long peptides that contain immunogenic T-cell epitopes have been used to induce activation of antigen-specific CD8 T cells in vitro for immune monitoring or adoptive transfer, or in vivo after peptide vaccination. However, the efficiency and mechanisms of presentation of exogenous long peptides in human leukocyte antigen (HLA) class I remain to be elucidated. In this study, we demonstrated that the efficiency of antigen-specific CD8 T-cell activation using extended peptide variants of common viral epitopes is variable. We demonstrated that processing and HLA class I presentation of the long peptides were not dependent on the proteasome and transporter associated with antigen processing, illustrating that the classic route of HLA class I presentation was not required for activation of specific CD8 T cells by exogenous synthetic long peptides. Although long peptides were shown to bind to the relevant HLA class I molecules, peptide trimming was likely to be essential for optimal HLA class I presentation and T-cell activation. As the proteasome was not required for processing of exogenous peptides, it is very likely that peptide trimming was mediated by peptidases, which may be located extracellularly at the cell surface, in the cytosol, endoplasmic reticulum, or in endosomal and lysosomal compartments. Furthermore, the results suggested that processing of the correct minimal peptides was facilitated by binding in HLA class I molecules. This mechanism of HLA-guided processing may be important in HLA class I presentation of exogenous long peptides to induce activation of specific CD8 T cells.

Simultaneous isolation of CD8(+) and CD4(+) T cells specific for multiple viruses for broad antiviral immune reconstitution after allogeneic stem cell transplantation.

Opportunistic viral infections can cause serious morbidity and mortality in immunocompromised patients after allogeneic stem cell transplantation. Clinical studies have shown that adoptive transfer of donor-derived T cells specific for cytomegalovirus (CMV), Epstein-Barr virus (EBV), or human adenovirus (HAdV) can be a safe and effective treatment of infections with these major viral pathogens. The aim of this study was to develop a method for the simultaneous isolation of coordinated CD8(+) and CD4(+) memory T-cell responses against a broad repertoire of viral epitopes. To ensure that the method was applicable to a wide variety of virus-specific T cells that may differ in phenotypic and functional properties, we focused on T cells specific for the persistent viruses, CMV and EBV, and T cells specific for HAdV and influenza (FLU), which are not repetitively activated in vivo after initial viral clearance. Following in vitro activation, nearly all T cells specific for these viruses produced interferon γ (IFN-γ) and tumor necrosis factor α, and expressed CD137, whereas the populations varied in the production of interleukin-2, degranulation, and expression of phenotypic markers. Different kinetics of IFN-γ production were observed in CMV/EBV-specific T cells and HAdV/FLU-specific T cells. However, after the stimulation of peripheral blood from seropositive donors with viral protein-spanning peptide pools, the activated virus-specific CD8(+) and CD4(+) T cells could be simultaneously isolated by either IFN-γ-based or CD137-based enrichment. This study provides an efficient and widely applicable strategy for the isolation of virus-specific T cells, which may be used for the reconstitution of virus-specific immunity in allogeneic stem cell transplantation recipients.

Combined CD8+ and CD4+ adenovirus hexon-specific T cells associated with viral clearance after stem cell transplantation as treatment for adenovirus infection.

BACKGROUND: Human adenovirus can cause morbidity and mortality in immunocompromised patients after allogeneic stem cell transplantation. Reconstitution of adenovirus-specific CD4(+) T cells has been reported to be associated with sustained protection from adenovirus disease, but epitope specificity of these responses has not been characterized. Since mainly CD4(+) T cells and no CD8(+) T cells specific for adenovirus have been detected after allogeneic stem cell transplantation, the relative contribution of adenovirus-specific CD4(+) and CD8(+) T cells in protection from adenovirus disease remains to be elucidated. DESIGN AND METHODS: The presence of human adenovirus hexon-specific T cells was investigated in peripheral blood of pediatric and adult allogeneic stem cell transplant recipients, who showed spontaneous resolution of disseminated adenovirus infection. Subsequently, a clinical grade method was developed for rapid generation of adenovirus-specific T-cell lines for adoptive immunotherapy. RESULTS: Clearance of human adenovirus viremia coincided with emergence of a coordinated CD8(+) and CD4(+) T-cell response against adenovirus hexon epitopes in patients after allogeneic stem cell transplantation. Activation of adenovirus hexon-specific CD8(+) and CD4(+) T cells with a hexon protein-spanning peptide pool followed by interferon-γ-based isolation allowed rapid expansion of highly specific T-cell lines from healthy adults, including donors with very low frequencies of adenovirus hexon-specific T cells. Adenovirus-specific T-cell lines recognized multiple MHC class I and II restricted epitopes, including known and novel epitopes, and efficiently lysed human adenovirus-infected target cells. CONCLUSIONS: This study provides a rationale and strategy for the adoptive transfer of donor-derived human adenovirus hexon-specific CD8(+) and CD4(+) T cells for the treatment of disseminated adenovirus infection after allogeneic stem cell transplantation.

Co-ordinated isolation of CD8(+) and CD4(+) T cells recognizing a broad repertoire of cytomegalovirus pp65 and IE1 epitopes for highly specific adoptive immunotherapy.

BACKGROUND AIMS: Adoptive transfer of cytomegalovirus (CMV)-specific memory T cells can be used for treatment of CMV reactivation after allogeneic stem cell transplantation. As co-ordinated CD8(+) and CD4(+) T cells specific for a broad repertoire of CMV epitopes may be most effective for adoptive immunotherapy, the aim of this study was to isolate these cells from peripheral blood of CMV seropositive donors, irrespective of their HLA type. METHODS: Activation of CMV-specific CD8(+) and CD4(+) T cells was compared after stimulation of donor peripheral blood with minimal epitope peptides, pools of overlapping 15-mer peptides or full-length protein. Furthermore, the kinetics of interferon (IFN)-γ production after stimulation was analyzed to determine the optimal time-point for IFN-γ-based isolation of CMV-specific T cells. The specificity, phenotype and functionality of generated T-cell lines were analyzed. RESULTS: CMV protein-spanning 15-mer peptide pools induced simultaneous activation of both CD8(+) and CD4(+) CMV-specific T cells, while full-length CMV protein only efficiently activated CD4(+) CMV-specific T cells. Isolation of IFN-γ-secreting cells at the peak of the IFN-γ response after 4-h stimulation with CMV pp65 and IE1 peptide pools resulted in efficient enrichment of CMV-specific T cells. The T-cell lines contained high frequencies of CD8(+) and CD4(+) T cells recognizing multiple CMV pp65 and IE1 epitopes, and produced IFN-γ and tumor necrosis factor (TNF)-α upon specific restimulation. CONCLUSIONS: This study provides a feasible strategy for the rapid generation of clinical-grade CD8(+) and CD4(+) T-cell lines with high specificity for multiple CMV pp65 and IE1 epitopes, which may be used for effective adoptive immunotherapy.

Detailed analysis of IFNg response upon activation permits efficient isolation of cytomegalovirus-specific CD8+ T cells for adoptive immunotherapy.

Adoptive transfer of donor-derived cytomegalovirus (CMV)-specific T cells may provide long-lived protection from CMV disease after allogeneic stem cell transplantation. Isolation of IFNg-secreting cells after CMV peptide stimulation can be performed by IFNg capture assay to generate highly specific T-cell lines without the need for extensive culture, which may hamper their in vivo efficacy. To exploit the full potential of this approach, we analyzed the IFNg response of CMV-specific CD8+ T cells in detail. Kinetic studies showed that T-cell receptor down-regulation coincided with the induction of IFNg production upon activation, which rapidly declined thereafter despite the continued presence of specific peptide. By varying the strength of stimulation we observed that overstimulation can result in profound T-cell receptor down-regulation, more rapid decline of IFNg production and reduced expansion. On the basis of these findings, we defined optimal conditions for IFNg-based isolation of CMV-specific CD8+ T cells with maximal potential for clinical application. These data stress the importance of analyses of the kinetics of cytokine production for isolation of T cells specific for other infectious or malignant antigens to exploit the full potential of cytokine capture isolation of antigen-specific T cells.

Distinct effects of amlodipine treatment on vascular elastocalcinosis and stiffness in a rat model of isolated systolic hypertension.

OBJECTIVE: Medial elastocalcinosis (MEC) contributes to the development of large artery stiffness and isolated systolic hypertension. Since endothelin receptor antagonists can prevent and regress elastocalcinosis, our aim was to determine whether amlodipine, a calcium channel blocker that inhibits endothelin signaling, could likewise influence MEC, or reduce pressure mainly through its vasorelaxing properties. METHODS: Control male Wistar rats were compared with rats receiving warfarin (20 mg/kg per day) with vitamin K1 (15 mg/kg per day) alone (WVK) or in association with amlodipine (15 mg/kg per day) for 4 weeks or during the last week or last 4 weeks of an 8-week WVK treatment (two regression groups). RESULTS: Inactivation of matrix Gla protein by WVK for 4 or 8 weeks increased the calcium content 10-fold in the aorta, inducing a significant elevation of pulse wave velocity and pulse pressure by selective augmentation of systolic blood pressure. Amlodipine prevented aortic MEC, pulse wave velocity and pulse pressure elevation, but reversed only MEC and pulse pressure when administered for 4 weeks. One week of amlodipine administered after 7 weeks of WVK partially decreased pulse pressure without modifying aortic MEC. Amlodipine did not reduce the fibrosis associated with calcified areas in the WVK model during the regression protocols. CONCLUSION: The clinical efficacy of amlodipine in improving hemodynamic variables and reducing cardiovascular events in isolated systolic hypertension could be explained by its beneficial effect on vascular calcification. Amlodipine's lack of effect on pulse wave velocity and collagen deposition, however, suggests that it may reduce pulse pressure by means other than improving arterial stiffness.