title: Rapid generation of clinical-grade antiviral T cells: selection of suitable T-cell donors and GMP-compliant manufacturing of antiviral T cells.
abstract: BACKGROUND
  The adoptive transfer of allogeneic antiviral T lymphocytes derived from seropositive donors can safely and effectively reduce or prevent the clinical manifestation of viral infections or reactivations in immunocompromised recipients after hematopoietic stem cell (HSCT) or solid organ transplantation (SOT). Allogeneic third party T-cell donors offer an alternative option for patients receiving an allogeneic cord blood transplant or a transplant from a virus-seronegative donor and since donor blood is generally not available for solid organ recipients. Therefore we established a registry of potential third-party T-cell donors (allogeneic cell registry, alloCELL) providing detailed data on the assessment of a specific individual memory T-cell repertoire in response to antigens of cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus (ADV), and human herpesvirus (HHV) 6.
  
  
  METHODS
  To obtain a manufacturing license according to the German Medicinal Products Act, the enrichment of clinical-grade CMV-specific T cells from three healthy CMV-seropositive donors was performed aseptically under GMP conditions using the CliniMACS cytokine capture system (CCS) after restimulation with an overlapping peptide pool of the immunodominant CMVpp65 antigen. Potential T-cell donors were selected from alloCELL and defined as eligible for clinical-grade antiviral T-cell generation if the peripheral fraction of IFN-γ(+) T cells exceeded 0.03% of CD3(+) lymphocytes as determined by IFN-γ cytokine secretion assay.
  
  
  RESULTS
  Starting with low concentration of IFN-γ(+) T cells (0.07-1.11%) we achieved 81.2%, 19.2%, and 63.1% IFN-γ(+)CD3(+) T cells (1.42 × 10(6), 0.05 × 10(6), and 1.15 × 10(6)) after enrichment. Using the CMVpp65 peptide pool for restimulation resulted in the activation of more CMV-specific CD8(+) than CD4(+) memory T cells, both of which were effectively enriched to a total of 81.0% CD8(+)IFN-γ(+) and 38.4% CD4(+)IFN-γ(+) T cells. In addition to T cells and NKT cells, all preparations contained acceptably low percentages of contaminating B cells, granulocytes, monocytes, and NK cells. The enriched T-cell products were stable over 72 h with respect to viability and ratio of T lymphocytes.
  
  
  CONCLUSIONS
  The generation of antiviral CD4(+) and CD8(+) T cells by CliniMACS CCS can be extended to a broad spectrum of common pathogen-derived peptide pools in single or multiple applications to facilitate and enhance the efficacy of adoptive T-cell immunotherapy.
authors.affiliation: Institute for Transfusion Medicine, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625, Hannover, Germany. tischer.sabine@mh-hannover.de.|Integrated Research and Treatment Center (IFB-Tx), Hannover Medical School, Carl-Neuberg-Strasse 1, 30625, Hannover, Germany. tischer.sabine@mh-hannover.de.
authors.forename: Sabine
authors.initials: S
authors.lastname: Tischer
authors.affiliation: Integrated Research and Treatment Center (IFB-Tx), Hannover Medical School, Carl-Neuberg-Strasse 1, 30625, Hannover, Germany. priesner.christoph@mh-hannover.de.|Institute of Cellular Therapeutics, Hannover Medical School, Feodor-Lynen Strasse 21, 30625, Hannover, Germany. priesner.christoph@mh-hannover.de.
authors.forename: Christoph
authors.initials: C
authors.lastname: Priesner
authors.affiliation: Institute for Transfusion Medicine, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625, Hannover, Germany. heuft.hans-gert@mh-hannover.de.
authors.forename: Hans-Gert
authors.initials: HG
authors.lastname: Heuft
authors.affiliation: Institute for Transfusion Medicine, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625, Hannover, Germany. goudeva.lilia@mh-hannover.de.
authors.forename: Lilia
authors.initials: L
authors.lastname: Goudeva
authors.affiliation: Institute for Transfusion Medicine, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625, Hannover, Germany. mende.wolfgang@mh-hannover.de.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Mende
authors.affiliation: Staff office for Quality Management in Clinical Research, Hannover Medical School, Feodor-Lynen Strasse 21, 30625, Hannover, Germany. barthold.marc@mh-hannover.de.
authors.forename: Marc
authors.initials: M
authors.lastname: Barthold
authors.affiliation: Integrated Research and Treatment Center (IFB-Tx), Hannover Medical School, Carl-Neuberg-Strasse 1, 30625, Hannover, Germany. kloess.stephan@mh-hannover.de.|Institute of Cellular Therapeutics, Hannover Medical School, Feodor-Lynen Strasse 21, 30625, Hannover, Germany. kloess.stephan@mh-hannover.de.
authors.forename: Stephan
authors.initials: S
authors.lastname: Kloeß
authors.affiliation: Integrated Research and Treatment Center (IFB-Tx), Hannover Medical School, Carl-Neuberg-Strasse 1, 30625, Hannover, Germany. arseniev.lubomir@mh-hannover.de.|Institute of Cellular Therapeutics, Hannover Medical School, Feodor-Lynen Strasse 21, 30625, Hannover, Germany. arseniev.lubomir@mh-hannover.de.
authors.forename: Lubomir
authors.initials: L
authors.lastname: Arseniev
authors.affiliation: Institute of Cellular Therapeutics, Hannover Medical School, Feodor-Lynen Strasse 21, 30625, Hannover, Germany. aleksandrova.krasimira@mh-hannover.de.
authors.forename: Krasimira
authors.initials: K
authors.lastname: Aleksandrova
authors.affiliation: Integrated Research and Treatment Center (IFB-Tx), Hannover Medical School, Carl-Neuberg-Strasse 1, 30625, Hannover, Germany. maecker.britta@mh-hannover.de.|Department of Paediatric Haematology and Oncology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625, Hannover, Germany. maecker.britta@mh-hannover.de.
authors.forename: Britta
authors.initials: B
authors.lastname: Maecker-Kolhoff
authors.affiliation: Institute for Transfusion Medicine, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625, Hannover, Germany. blasczyk.rainer@mh-hannover.de.|Integrated Research and Treatment Center (IFB-Tx), Hannover Medical School, Carl-Neuberg-Strasse 1, 30625, Hannover, Germany. blasczyk.rainer@mh-hannover.de.
authors.forename: Rainer
authors.initials: R
authors.lastname: Blasczyk
authors.affiliation: Integrated Research and Treatment Center (IFB-Tx), Hannover Medical School, Carl-Neuberg-Strasse 1, 30625, Hannover, Germany. koehl.ulrike@mh-hannover.de.|Institute of Cellular Therapeutics, Hannover Medical School, Feodor-Lynen Strasse 21, 30625, Hannover, Germany. koehl.ulrike@mh-hannover.de.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Koehl
authors.affiliation: Institute for Transfusion Medicine, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625, Hannover, Germany. eiz-vesper.britta@mh-hannover.de.|Integrated Research and Treatment Center (IFB-Tx), Hannover Medical School, Carl-Neuberg-Strasse 1, 30625, Hannover, Germany. eiz-vesper.britta@mh-hannover.de.
authors.forename: Britta
authors.initials: B
authors.lastname: Eiz-Vesper
mesh_terms: D000256:Adenoviridae / Q000276:immunology; D001782:Blood Donors; D017690:Cell Transplantation; D003587:Cytomegalovirus / Q000276:immunology; D004345:Drug Industry / Q000592:standards*; D004854:Herpesvirus 4, Human / Q000276:immunology; D015654:Herpesvirus 6, Human / Q000276:immunology; D006801:Humans; D007167:Immunotherapy; D011786:Quality Control; D013601:T-Lymphocytes / Q000276:immunology*; D014777:Virus Diseases / Q000276:immunology / Q000628:therapy* / Q000821:virology