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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 × 109) and small (25 × 106) 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.

Mesenchymal Stromal Cells (MSCs) have achieved a great potential in Regenerative Medicine, although limited information exists about bioprocess and production on Good Manufacture Practices (GMP)-grade, standardization and quality controls. The present work describes our experience on the validation and GMP production of 25 consecutive bone marrow (BM)-derived MSCs batches for clinical use. BM was obtained from posterior iliac crests of patients. Manufacturing included isolation by density-gradient centrifugation and plating, trypsinization and secondary expansion cultures and, harvesting and formulation of a suspension containing 40 10E6 viable MSCs. Quality controls included cell counting and viability assessment; immunophenotyping; Sterility Test, Mycoplasma detection and Endotoxin Tests conducted according to European Pharmacopeia (Eur. Ph.) and, finally, Gram Staining. All products were shipped according to current EU Regulations. After validation, 25 MSCs batches for clinical application were manufactured. BM harvested volumes ranged from 98 to 163 mL (median 132,1mL) containing 8,38E8 to 4,29E9 viable Nucleated Cells (NCs) (median 2,53E9). Cell concentrations ranged from 5.74E6 to 3.56E7 NCs/mL (median 2.34E7). Final product volume was 10,0 mL (4-10,5 mL) containing a median of 4.02E7 (3.21E7-4.87E7) viable MSCs. Antigen expression was consistent with MSCs phenotype as described by ISCT. In a minority of the batches produced, we observed an increase of HLA-DR expression compatible with the activation on the MSCs. Sterility, Mycoplasma, Gram Staining and Endotoxin tests of the finished products were always negative. We have established a GMP protocol, using human sera as supplement, confirming its safety and feasibility. It is consistent and reproducible and the bioprocess developed demonstrated to be robust, safe and reliable. This work was cofounded by MEDCEL and FACTOCEL projects from the Spanish government (Ministerio de Ciencia e Innovacion).