In contrast to donor T cells, natural killer (NK) cells are known to mediate anti-cancer effects without the risk of inducing graft-versus-host disease (GvHD). In order to improve cytotoxicity against resistant cancer cells, auspicious efforts have been made with chimeric antigen receptor (CAR) expressing T- and NK cells. These CAR-modified cells express antigen receptors against tumor-associated surface antigens, thus redirecting the effector cells and enhancing tumor-specific immunosurveillance. However, many cancer antigens are also expressed on healthy tissues, potentially leading to off tumor/on target toxicity by CAR-engineered cells. In order to control such potentially severe side effects, the insertion of suicide genes into CAR-modified effectors can provide a means for efficient depletion of these cells. While CAR-expressing T cells have entered successfully clinical trials, experience with CAR-engineered NK cells is mainly restricted to pre-clinical investigations and predominantly to NK cell lines. In this review we summarize the data on CAR expressing NK cells focusing on the possible advantage using these short-lived effector cells and discuss the necessity of suicide switches. Furthermore, we address the compliance of such modified NK cells with regulatory requirements as a new field in cellular immunotherapy.

BackgroundThe adoptive transfer of allogeneic antiviral T lymphocytes derived from seropositive donors can safely and effectively reduce or prevent the clinical manifestation of viral infections or reactivations in immunocompromised recipients after hematopoietic stem cell (HSCT) or solid organ transplantation (SOT). Allogeneic third party T-cell donors offer an alternative option for patients receiving an allogeneic cord blood transplant or a transplant from a virus-seronegative donor and since donor blood is generally not available for solid organ recipients. Therefore we established a registry of potential third-party T-cell donors (allogeneic cell registry, alloCELL) providing detailed data on the assessment of a specific individual memory T-cell repertoire in response to antigens of cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus (ADV), and human herpesvirus (HHV) 6.MethodsTo obtain a manufacturing license according to the German Medicinal Products Act, the enrichment of clinical-grade CMV-specific T cells from three healthy CMV-seropositive donors was performed aseptically under GMP conditions using the CliniMACS cytokine capture system (CCS) after restimulation with an overlapping peptide pool of the immunodominant CMVpp65 antigen. Potential T-cell donors were selected from alloCELL and defined as eligible for clinical-grade antiviral T-cell generation if the peripheral fraction of IFN-γ+ T cells exceeded 0.03% of CD3+ lymphocytes as determined by IFN-γ cytokine secretion assay.ResultsStarting with low concentration of IFN-γ+ T cells (0.07-1.11%) we achieved 81.2%, 19.2%, and 63.1% IFN-γ+CD3+ T cells (1.42 × 106, 0.05 × 106, and 1.15 × 106) after enrichment. Using the CMVpp65 peptide pool for restimulation resulted in the activation of more CMV-specific CD8+ than CD4+ memory T cells, both of which were effectively enriched to a total of 81.0% CD8+IFN-γ+ and 38.4% CD4+IFN-γ+ T cells. In addition to T cells and NKT cells, all preparations contained acceptably low percentages of contaminating B cells, granulocytes, monocytes, and NK cells. The enriched T-cell products were stable over 72 h with respect to viability and ratio of T lymphocytes.ConclusionsThe generation of antiviral CD4+ and CD8+ T cells by CliniMACS CCS can be extended to a broad spectrum of common pathogen-derived peptide pools in single or multiple applications to facilitate and enhance the efficacy of adoptive T-cell immunotherapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-014-0336-5) contains supplementary material, which is available to authorized users.

Multiple clinical studies have demonstrated that adaptive immunotherapy using redirected T cells against advanced cancer has led to promising results with improved patient survival. The continuously increasing interest in those advanced gene therapy medicinal products (GTMPs) leads to a manufacturing challenge regarding automation, process robustness, and cell storage. Therefore, this study addresses the proof of principle in clinical-scale selection, stimulation, transduction, and expansion of T cells using the automated closed CliniMACS® Prodigy system. Naïve and central memory T cells from apheresis products were first immunomagnetically enriched using anti-CD62L magnetic beads and further processed freshly (n = 3) or split for cryopreservation and processed after thawing (n = 1). Starting with 0.5 × 108 purified CD3+ T cells, three mock runs and one run including transduction with green fluorescent protein (GFP)-containing vector resulted in a median final cell product of 16 × 108 T cells (32-fold expansion) up to harvesting after 2 weeks. Expression of CD62L was downregulated on T cells after thawing, which led to the decision to purify CD62L+CD3+ T cells freshly with cryopreservation thereafter. Most important in the split product, a very similar expansion curve was reached comparing the overall freshly CD62L selected cells with those after thawing, which could be demonstrated in the T cell subpopulations as well by showing a nearly identical conversion of the CD4/CD8 ratio. In the GFP run, the transduction efficacy was 83%. In-process control also demonstrated sufficient glucose levels during automated feeding and medium removal. The robustness of the process and the constant quality of the final product in a closed and automated system give rise to improve harmonized manufacturing protocols for engineered T cells in future gene therapy studies.

Clinical studies using autologous CAR T cells have achieved spectacular remissions in refractory CD19+ B cell leukaemia, however some of the patient treatments with CAR T cells failed. Beside the heterogeneity of leukaemia, the distribution and senescence of the autologous cells from heavily pretreated patients might be further reasons for this. We performed six consecutive large-scale manufacturing processes for CD20 CAR T cells from healthy donor leukapheresis using the automated CliniMACS Prodigy® platform. Starting with a CD4/CD8-positive selection, a high purity of a median of 97% T cells with a median 65-fold cell expansion was achieved. Interestingly, the transduction rate was significantly higher for CD4+ compared to CD8+ T cells and reached in a median of 23%. CD20 CAR T cells showed a good specific IFN-γ secretion after cocultivation with CD20+ target cells which correlated with good cytotoxic activity. Most importantly, 3 out of 5 CAR T cell products showed an increase in telomere length during the manufacturing process, while telomere length remained consistent in one and decreased in another process. In conclusion, this shows for the first time that beside heterogeneity among healthy donors, CAR T cell products also differ regarding cell senescence, even for cells manufactured in a standardised automated process.

Background and aimsThe infusion of enriched CMV-specific donor T-cells appears to be a suitable alternative for the treatment of drug-resistant CMV reactivation or de novo infection after both solid organ and hematopoietic stem cell transplantation. Antiviral lymphocytes can be selected from apheresis products using the CliniMACS Cytokine-Capture-System® either with the well-established CliniMACS® Plus (Plus) device or with its more versatile successor CliniMACS Prodigy® (Prodigy).MethodsManufacturing of CMV-specific T-cells was carried out with the Prodigy and Plus in parallel starting with 0.8–1 × 109 leukocytes collected by lymphapheresis (n = 3) and using the MACS GMP PepTivator® HCMVpp65 for antigenic restimulation. Target and non-target cells were quantified by a newly developed single-platform assessment and gating strategy using positive (CD3/CD4/CD8/CD45/IFN-γ), negative (CD14/CD19/CD56), and dead cell (7-AAD) discriminators.ResultsBoth devices produced largely similar results for target cell viabilities: 37.2–52.2% (Prodigy) vs. 51.1–62.1% (Plus) CD45+/7-AAD− cells. Absolute numbers of isolated target cells were 0.1–3.8 × 106 viable IFN-γ+ CD3+ T-cells. The corresponding proportions of IFN-γ+ CD3+ T-cells ranged between 19.2 and 95.1% among total CD3+ T-cells and represented recoveries of 41.9–87.6%. Within two parallel processes, predominantly IFN-γ+ CD3+CD8+ cytotoxic T-cells were enriched compared to one process that yielded a higher amount of IFN-γ+ CD3+CD4+ helper T lymphocytes. T-cell purity was higher for the Prodigies products that displayed a lower content of contaminating IFN-γ− T-cells (3.6–20.8%) compared to the Plus products (19.9–80.0%).ConclusionThe manufacturing process on the Prodigy saved both process and hands-on time due to its higher process integration and ability for unattended operation. Although the usage of both instruments yielded comparable results, the lower content of residual IFN-γ− T-cells in the target fractions produced with the Prodigy may allow for a higher dosage of CMV-specific donor T-cells without increasing the risk for graft-versus-host disease.

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Adenovirus (HAdV) infections confer a high risk of morbidity and mortality for immunocompromised patients after stem cell transplantation (SCT). Treatment with standard antiviral drugs is of limited efficacy and associated with a high rate of adverse effects. HAdV‐specific T cells are crucial for sustained viral elimination and the efficacy of adoptive T‐cell therapy with donor‐derived HAdV‐specific T cells has been reported by several investigators. Here, we report our experience with the transfer of HAdV‐specific T cells specific for penton, which was recently identified as an immunodominant target of T cells, and hexon in a 14‐year‐old boy after T‐cell‐depleted haploidentical SCT for myelodysplastic syndrome (MDS). He developed severe HAdV‐associated enteritis complicated by acute graft‐versus‐host disease (GvHD). The patient received ten infusions of allogeneic HAdV‐specific T cells manufactured from the haploidentical stem cell donor using the CliniMacs Interferon‐γ (IFN‐γ) cytokine capture and immunomagnetic selection. Initially, T cells were generated against the immunodominant target hexon and in subsequent transfers dual antigen‐specific T cells against hexon and penton were applied. T‐cell transfers were scheduled individually tailored to current immunosuppressive treatment. Each transfer was followed by reduction of HAdV load in peripheral blood and clinical improvement. Importantly, T‐cell responses to both penton and hexon pools emerged in patient blood after repetitive transfers. Unfortunately, the patient experienced bacterial sepsis, and in this context, severe GvHD requiring intensive immunosuppression followed by secondary progression of HAdV infection. The patient succumbed to multiorgan failure 283 days after SCT. This case demonstrates the feasibility of HAdV‐specific T‐cell transfer even in the presence of immunosuppressive treatment. Targeting of multiple immunodominant viral proteins may prove valuable in patients with complicated HAdV infections.

Academic institutions coin the ATMP landscape but do not possess an industry-like capacity to vigorously pursue the full developmental pathway to marketing authorization. At the same time, industry has fostered clinical trials with ATMPs, brought the first products to marketing authorization, and defined novel modes of interaction with academia. A regulatory niche for local manufacturing of ATMPs within an academic institution had been foreseen in Regulation (EU) 1394/2007 under the term “Hospital Exemption” but remained ill-defined. Manufacture in close proximity to the patient is difficult to accomplish, as “point of care” systems for the manufacture of ATMPs have encountered regulatory challenges hovering between process and product. The efforts and costs for the development of ATMPs continue to be dramatically underestimated, and few academic centers were persistent enough to invest in the GMP infrastructure needed and to recruit personnel trained in ATMP development. As a consequence, the contribution by hospitals to ATMP development has shifted from the finished ATMP toward the procurement of starting materials, selected manufacturing steps, storage of the product, clinical application, and participation in clinical trials. As the development and use of cell-based therapies and ATMPs continue to attract and challenge clinicians and scientists, this review aims to discuss logistical, financial, and regulatory issues that might contribute to the changing role of Academia in ATMP development, with an outlook into possible developments in the future and proposals for ways to reshape the academic environment under the auspices of what might truly have been meant by the hospital exemption clause.