Petra Bacher1, Andrea Jochheim-Richter2, Nadine Mockel-Tenbrink3, Olaf Kniemeyer4, Eva Wingenfeld2, Regina Alex3, Alice Ortigao2, Darja Karpova2, Thomas Lehrnbecher5, Andrew J Ullmann6, Axel Hamprecht7, Oliver Cornely8, Axel A Brakhage9, Mario Assenmacher3, Halvard Bonig10, Alexander Scheffold11. 1. Department of Cellular Immunology, Clinic for Rheumatology and Clinical Immunology, Charité, University Medicine Berlin, Germany. 2. Institute for Transfusion Medicine and Immunohematology, Department of Translational Development of Cellular Therapeutics (GMP), Goethe University, Frankfurt, Germany. 3. Miltenyi Biotec, Bergisch-Gladbach, Germany. 4. Integrated Research and Treatment Center, Center for Sepsis Control and Care (CSCC), University Hospital, Jena, Germany. 5. Pediatric Hematology and Oncology, Children's Hospital III, Johann Wolfgang Goethe University, Frankfurt, Germany. 6. Division of Infectious Diseases, Department of Internal Medicine II, University Medical Center, Würzburg, Germany. 7. Department I of Internal Medicine, University Hospital of Cologne, Germany; Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD); German Centre for Infection Research (DZIF), Partner Site Bonn-Cologne, Cologne, Germany. 8. Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Cologne, Germany. 9. Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knoell Institute (HKI) Jena and Institute of Microbiology, Friedrich Schiller University Jena, Jena, Germany. 10. Institute for Transfusion Medicine and Immunohematology, Department of Translational Development of Cellular Therapeutics (GMP), Goethe University, Frankfurt, Germany; German Red Cross Blood Service Baden-Württemberg-Hessen, Institute Frankfurt, Germany; Division of Hematology, Department of Medicine, University of Washington, Seattle, Washington, USA. Electronic address: h.boenig@blutspende.de. 11. Department of Cellular Immunology, Clinic for Rheumatology and Clinical Immunology, Charité, University Medicine Berlin, Germany; German Rheumatism Research Center, Berlin, Germany.
Abstract
BACKGROUND AIMS: Evidence of the criticality of the adaptive immune response for controlling invasive aspergillosis has been provided. This observation is supported by the fact that invasive aspergillosis, a grave complication of allogeneic stem cell transplantation, occurs long after myeloid reconstitution in patients with low T-cell engraftment and/or on immunosuppressants. Adoptive T-cell transfer might be beneficial, but idiosyncrasies of Aspergillus fumigatus and the anti-Aspergillus immune response render established selection technologies ineffective. METHODS: We developed a Good Manufacturing Practice (GMP)-compliant protocol for preparation of A. fumigatus-specific CD4+ cells by sequentially depleting regulatory and cytotoxic T cells, activating A. fumigatus-specific T-helper cells with GMP-grade A. fumigatus lysate, and immuno-magnetically isolating them via the transiently up-regulated activation marker, CD137. RESULTS: In 13 full-scale runs, we demonstrate robustness and feasibility of the approach. From 2 × 10(9) peripheral blood mononuclear cells, we isolated 27 × 10(3)-318 × 10(3)Aspergillus-specific T-helper cells. Frequency among total T cells was increased, on average, by 200-fold. Specific studies indicate specificity and functionality: After non-specific in vitro expansion and re-stimulation with different antigens, we observed strong cytokine responses to A. fumigatus and some other fungi including Candida albicans, but none to unrelated antigens. DISCUSSION: Our technology isolates naturally occurring Aspergillus-specific T-helper cells within 2 days of identifying the clinical indication. Rapid adoptive transfer of Aspergillus-specific T cells may be quite feasible; the clinical benefit remains to be demonstrated. A manufacturing license as an advanced-therapy medicinal product was received and a clinical trial in post-transplantation invasive aspergillosis patients approved. The product is dosed at 5 × 10E3/kg T cells (single intravenous injection), of which at least 10% must be A. fumigatus-specific.
BACKGROUND AIMS: Evidence of the criticality of the adaptive immune response for controlling invasive aspergillosis has been provided. This observation is supported by the fact that invasive aspergillosis, a grave complication of allogeneic stem cell transplantation, occurs long after myeloid reconstitution in patients with low T-cell engraftment and/or on immunosuppressants. Adoptive T-cell transfer might be beneficial, but idiosyncrasies of Aspergillus fumigatus and the anti-Aspergillus immune response render established selection technologies ineffective. METHODS: We developed a Good Manufacturing Practice (GMP)-compliant protocol for preparation of A. fumigatus-specific CD4+ cells by sequentially depleting regulatory and cytotoxic T cells, activating A. fumigatus-specific T-helper cells with GMP-grade A. fumigatus lysate, and immuno-magnetically isolating them via the transiently up-regulated activation marker, CD137. RESULTS: In 13 full-scale runs, we demonstrate robustness and feasibility of the approach. From 2 × 10(9) peripheral blood mononuclear cells, we isolated 27 × 10(3)-318 × 10(3)Aspergillus-specific T-helper cells. Frequency among total T cells was increased, on average, by 200-fold. Specific studies indicate specificity and functionality: After non-specific in vitro expansion and re-stimulation with different antigens, we observed strong cytokine responses to A. fumigatus and some other fungi including Candida albicans, but none to unrelated antigens. DISCUSSION: Our technology isolates naturally occurring Aspergillus-specific T-helper cells within 2 days of identifying the clinical indication. Rapid adoptive transfer of Aspergillus-specific T cells may be quite feasible; the clinical benefit remains to be demonstrated. A manufacturing license as an advanced-therapy medicinal product was received and a clinical trial in post-transplantation invasive aspergillosispatients approved. The product is dosed at 5 × 10E3/kg T cells (single intravenous injection), of which at least 10% must be A. fumigatus-specific.
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