title: Clinical-scale isolation of the total Aspergillus fumigatus-reactive T-helper cell repertoire for adoptive transfer.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Cellular Immunology, Clinic for Rheumatology and Clinical Immunology, Charité, University Medicine Berlin, Germany.
authors.forename: Petra
authors.initials: P
authors.lastname: Bacher
authors.affiliation: Institute for Transfusion Medicine and Immunohematology, Department of Translational Development of Cellular Therapeutics (GMP), Goethe University, Frankfurt, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Jochheim-Richter
authors.affiliation: Miltenyi Biotec, Bergisch-Gladbach, Germany.
authors.forename: Nadine
authors.initials: N
authors.lastname: Mockel-Tenbrink
authors.affiliation: Integrated Research and Treatment Center, Center for Sepsis Control and Care (CSCC), University Hospital, Jena, Germany.
authors.forename: Olaf
authors.initials: O
authors.lastname: Kniemeyer
authors.affiliation: Institute for Transfusion Medicine and Immunohematology, Department of Translational Development of Cellular Therapeutics (GMP), Goethe University, Frankfurt, Germany.
authors.forename: Eva
authors.initials: E
authors.lastname: Wingenfeld
authors.affiliation: Miltenyi Biotec, Bergisch-Gladbach, Germany.
authors.forename: Regina
authors.initials: R
authors.lastname: Alex
authors.affiliation: Institute for Transfusion Medicine and Immunohematology, Department of Translational Development of Cellular Therapeutics (GMP), Goethe University, Frankfurt, Germany.
authors.forename: Alice
authors.initials: A
authors.lastname: Ortigao
authors.affiliation: Institute for Transfusion Medicine and Immunohematology, Department of Translational Development of Cellular Therapeutics (GMP), Goethe University, Frankfurt, Germany.
authors.forename: Darja
authors.initials: D
authors.lastname: Karpova
authors.affiliation: Pediatric Hematology and Oncology, Children's Hospital III, Johann Wolfgang Goethe University, Frankfurt, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Lehrnbecher
authors.affiliation: Division of Infectious Diseases, Department of Internal Medicine II, University Medical Center, Würzburg, Germany.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Ullmann
authors.affiliation: 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.
authors.forename: Axel
authors.initials: A
authors.lastname: Hamprecht
authors.affiliation: Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Cologne, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Cornely
authors.affiliation: 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.
authors.forename: Axel A
authors.initials: AA
authors.lastname: Brakhage
authors.affiliation: Miltenyi Biotec, Bergisch-Gladbach, Germany.
authors.forename: Mario
authors.initials: M
authors.lastname: Assenmacher
authors.affiliation: 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.
authors.forename: Halvard
authors.initials: H
authors.lastname: Bonig
authors.affiliation: Department of Cellular Immunology, Clinic for Rheumatology and Clinical Immunology, Charité, University Medicine Berlin, Germany; German Rheumatism Research Center, Berlin, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Scheffold
mesh_terms: D000946:Antigens, Fungal / Q000276:immunology; D001228:Aspergillosis / Q000276:immunology / Q000628:therapy*; D001232:Aspergillus fumigatus / Q000276:immunology*; D002176:Candida albicans / Q000276:immunology; D002469:Cell Separation / Q000379:methods*; D016207:Cytokines / Q000276:immunology; D018380:Hematopoietic Stem Cell Transplantation / Q000009:adverse effects; D006801:Humans; D016219:Immunotherapy, Adoptive / Q000379:methods*; D007963:Leukocytes, Mononuclear / Q000276:immunology; D008213:Lymphocyte Activation / Q000276:immunology*; D008212:Lymphocyte Depletion / Q000379:methods; D006377:T-Lymphocytes, Helper-Inducer / Q000276:immunology / Q000637:transplantation*; D053261:Tumor Necrosis Factor Receptor Superfamily, Member 9 / Q000378:metabolism
chemical_list: D000946:Antigens, Fungal; D016207:Cytokines; D053261:Tumor Necrosis Factor Receptor Superfamily, Member 9
keywords: GMP-compliant protocol; T-helper cells; aspergillosis; cell therapy; prospective isolation