Selection of adenovirus-specific and Epstein-Barr virus-specific T cells with major histocompatibility class I streptamers under Good Manufacturing Practice (GMP)-compliant conditions.

BACKGROUND AIMS: Despite antiviral drug therapies, human adenovirus (HAdV), cytomegalovirus (CMV) and Epstein-Barr virus (EBV) infections still contribute substantially to transplant-related death of patients after hematopoietic stem cell transplantation. Earlier clinical studies demonstrated successful adoptive transfer of magnetically selected CMV-specific T cells via removable, and thus Good Manufacturing Practice-compliant, major histocompatibility class I streptamers. Thus, the primary focus of the present study was the selection of HAdV-streptamer+ T cells, although in three experiments, EBV-streptamer+ T cells were also selected. METHODS: Cells from leukaphereses of healthy donors were prepared in large (1-6 × 10(9)) and small (25 × 10(6)) cell batches. Whereas the larger batch was directly labeled with streptamers to select HAdV- and/or EBV-specific T cells (large-scale), the smaller batch was used to generate in vitro virus-specific T-cell lines before streptamer labeling for streptamer selection (small-scale). Isolation of HAdV- and/or EBV-specific T cells was performed with the use of the CliniMACS device. RESULTS: The purity of HAdV- and EBV-streptamer+ T cells among CD3+ cells, obtained from large-scale selection, was up to 6.7% and 44%, respectively. If HAdV- and EBV-streptamers were applied simultaneously, the purity of antigen-specific T cells reached up to 50.7%. A further increase in purity reaching up to 98% was achieved by small-scale selection of HAdV-specific T cells. All final products fulfilled the microbiological and chemical release criteria. Interferon-γ-response indicating functional activity was seen in 6 of 9 HAdV and 2 of 3 EBV large-scale selections and in 2 of 3 HAdV small-scale selections. CONCLUSIONS: HAdV-streptamers were shown to be clinically feasible for few patients after the large-scale approach but for larger patient numbers if combined with EBV-streptamers or after the small-scale approach.

Rapid and sustained T cell-based immunotherapy against invasive fungal disease via a combined two step procedure.

Introduction: Aspergillus fumigatus (Asp) infections constitute a major cause of morbidity and mortality in patients following allogeneic hematopoietic stem cell transplantation (HSCT). In the context of insufficient host immunity, antifungal drugs show only limited efficacy. Faster and increased T-cell reconstitution correlated with a favorable outcome and a cell-based therapy approach strongly indicated successful clearance of fungal infections. Nevertheless, complex and cost- or time-intensive protocols hampered their implementation into clinical application. Methods: To facilitate the clinical-scale manufacturing process of Aspergillus fumigatus-specific T cells (ATCs) and to enable immediate (within 24 hours) and sustained (12 days later) treatment of patients with invasive aspergillosis (IA), we adapted and combined two complementary good manufacturing practice (GMP)-compliant approaches, i) the direct magnetic enrichment of Interferon-gamma (IFN-γ) secreting ATCs using the small-scale Cytokine Secretion Assay (CSA) and ii) a short-term in vitro T-cell culture expansion (STE), respectively. We further compared stimulation with two standardized and commercially available products: Asp-lysate and a pool of overlapping peptides derived from different Asp-proteins (PepMix). Results: For the fast CSA-based approach we detected IFN-γ+ ATCs after Asp-lysate- as well as PepMix-stimulation but with a significantly higher enrichment efficiency for stimulation with the Asp-lysate when compared to the PepMix. In contrast, the STE approach resulted in comparably high ATC expansion rates by using Asp-lysate or PepMix. Independent of the stimulus, predominantly CD4+ helper T cells with a central-memory phenotype were expanded while CD8+ T cells mainly showed an effector-memory phenotype. ATCs were highly functional and cytotoxic as determined by secretion of granzyme-B and IFN-γ. Discussion: For patients with IA, the immediate adoptive transfer of IFN-γ+ ATCs followed by the administration of short-term in vitro expanded ATCs from the same donor, might be a promising therapeutic option to improve the clinical outcome.

Robust multi-parameter single-platform quantification of myeloid and B-lymphoid CD34 progenitor cells in all clinical CD34 cell sources and in thawed PBSC.

As B-lymphoid progenitor cells do not give rise to in vitro colony formation and are unlikely to support myeloid engraftment, we validated a five-color extension of the single platform Stem Cell Enumeration (SCE) kit, to routinely quantify myeloid and B-lymphoid progenitor cells. Fresh samples (n > 20 each) of granulocyte colony stimulating factor mobilized blood (peripheral blood (PB)), cord blood (CB), bone marrow (BM), and apheresis products (APs) were stained in TruCOUNT™ tubes and the results were compared with those from the two-color CD45/CD34 reagent combination and the three-color SCE kit. To address repeatability, 10 samples from one AP were prepared by four technicians. Aliquots (n = 15) of four frozen AP were analyzed after thawing. Excellent correlations were observed between the three kits (R(2) > 0.99), for the quantification of white blood cells and total CD34. The extended kit showed considerable amounts of B-lymphoid progenitors in all CD34 sources (0-20% of all CD34 in PB, AP, and CB; 3-90% in BM). Very similar results were obtained when the same sample was prepared by different technicians. After thawing of frozen AP, the recovery of viable cells varied depending on the freezing medium employed, but the results from the different quantification methods were identical. Most non-viable cells were clearly identified with 7 Aminoactinomycin D (7AAD) but an additional gate in the forward scatter/side scatter was necessary to address dead cells negative for 7AAD. The extended SCE kit allows rapid and exact quantification of viable B-lymphoid and myeloid CD34(+) cells in all cell sources and in thawed stem cell harvests, and may thus improve the correlation between CD34 number and engraftment kinetics.

Novel single-platform multiparameter FCM analysis of apoptosis: Significant differences between wash and no-wash procedure.

FCM is a generally accepted tool to analyze apoptosis. Unfortunately, the cell preparation of all commercial kits available includes cell washing known to cause cell loss which is most likely to affect apoptotic cells in particular. To address this, we developed a seven-color single-platform no-wash analysis technique and compared the results with those from an analogous procedure including cell washing. A five-color mAb cocktail was employed to address target cells by surface labeling, Yo-PRO-1® and DAPI were used to discriminate apoptotic and necrotic from viable cells. Cells were quantified on the basis of internal-standard fluorescent beads. Jurkat cells ACC 282 treated with camptothecin were employed to establish the staining procedure, which was then applied to blood cells collected by extracorporeal apheresis and treated with UV irradiation. Data evaluation showed that although each method by itself was highly reproducible (R(2) = 0.973), the numbers of apoptotic cells detected with the no-wash procedure were significantly higher than those obtained after cell washing (P = 6.6 E(-5), Wilcoxon Test). In addition, the observed differences increased with higher cell numbers (Bland and Altmann). We conclude that the described test is a feasible and reliable tool for apoptosis measurement and it provides results that are definitely closer to the truth than those obtained from kits that require cell washing.

First-in-man clinical results with good manufacturing practice (GMP)-compliant polypeptide-expanded adenovirus-specific T cells after haploidentical hematopoietic stem cell transplantation.

Adoptive immunotherapy against viral infections is a promising treatment option for patients after hematopoietic stem cell transplantation. However, the generation of virus-specific T cells is either cost-intensive or time-consuming. We developed the first GMP-compliant protocol to generate donor-derived adenovirus (HAdV), cytomegalovirus, and Epstein-Barr virus-specific T-cell lines (TCLs) within 12 days by the use of overlapping polypeptides derived from different viruses in combination with IL-15. Two patients after undergoing haploidentical hematopoietic stem cell transplantation with HAdV viremia displaying rising viral loads despite treatment with cidofovir received 1×10 donor-derived short-term expanded HAdV-specific TCLs per kg body weight. In both patients, HAdV-specific T cells could be detected by IFN-γ-ELISpot 30 and 22 days postinfusion, and resulted in complete clearance or >1.5 log reduction of viral load within 15 and 18 days, respectively. This protocol facilitates rapid and cost-effective generation of virus-specific TCLs, which appear to provide an effective treatment option.

Short-term in-vitro expansion improves monitoring and allows affordable generation of virus-specific T-cells against several viruses for a broad clinical application.

Adenoviral infections are a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT) in pediatric patients. Adoptive transfer of donor-derived human adenovirus (HAdV)-specific T-cells represents a promising treatment option. However, the difficulty in identifying and selecting rare HAdV-specific T-cells, and the short time span between patients at high risk for invasive infection and viremia are major limitations. We therefore developed an IL-15-driven 6 to 12 day short-term protocol for in vitro detection of HAdV-specific T cells, as revealed by known MHC class I multimers and a newly identified adenoviral CD8 T-cell epitope derived from the E1A protein for the frequent HLA-type A*02∶01 and IFN-γ. Using this novel and improved diagnostic approach we observed a correlation between adenoviral load and reconstitution of CD8(+) and CD4(+) HAdV-specific T-cells including central memory cells in HSCT-patients. Adaption of the 12-day protocol to good manufacturing practice conditions resulted in a 2.6-log (mean) expansion of HAdV-specific T-cells displaying high cytolytic activity (4-fold) compared to controls and low or absent alloreactivity. Similar protocols successfully identified and rapidly expanded CMV-, EBV-, and BKV-specific T-cells. Our approach provides a powerful clinical-grade convertible tool for rapid and cost-effective detection and enrichment of multiple virus-specific T-cells that may facilitate broad clinical application.