Christine Freimüller1, Julia Stemberger1, Michaela Artwohl2, Lothar Germeroth3, Volker Witt4, Gottfried Fischer5, Sabine Tischer6, Britta Eiz-Vesper6, Ilka Knippertz7, Jan Dörrie8, Niels Schaft8, Thomas Lion9, Gerhard Fritsch10, René Geyeregger11. 1. Department of Clinical Cell Biology and FACS Core Unit, Children's Cancer Research Institute (CCRI), Vienna, Austria. 2. Department of Quality Management, CCRI, Vienna, Austria. 3. Stage Cell Therapeutics, Göttingen, Germany. 4. St. Anna Children's Hospital, Vienna, Austria. 5. Department of Blood Group Serology and Transfusion Medicine, Medical University of Vienna, Vienna, Austria. 6. Institute of Transfusion Medicine, Hannover Medical School, Hannover, Germany. 7. Department of Immune Modulation at the Department of Dermatology, Universitätsklinikum Erlangen, Germany. 8. Department of Dermatology, Universitätsklinikum Erlangen, Erlangen, Germany. 9. Department Pediatrics, Medical University of Vienna, Vienna, Austria; Department of Molecular Microbiology, CCRI, Vienna, Austria. 10. Department of Clinical Cell Biology and FACS Core Unit, Children's Cancer Research Institute (CCRI), Vienna, Austria; Department Pediatrics, Medical University of Vienna, Vienna, Austria. 11. Department of Clinical Cell Biology and FACS Core Unit, Children's Cancer Research Institute (CCRI), Vienna, Austria. Electronic address: rene.geyeregger@ccri.at.
Abstract
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.
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.
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