title: Generation of lentivirus-induced dendritic cells under GMP-compliant conditions for adaptive immune reconstitution against cytomegalovirus after stem cell transplantation.
abstract: BACKGROUND
  Reactivation of latent viruses such as human cytomegalovirus (HCMV) after allogeneic hematopoietic stem cell transplantation (HSCT) results in high morbidity and mortality. Effective immunization against HCMV shortly after allo-HSCT is an unmet clinical need due to delayed adaptive T cell development. Donor-derived dendritic cells (DCs) have a critical participation in stimulation of naïve T cells and immune reconstitution, and therefore adoptive DC therapy could be used to protect patients after HSCT. However, previous methods for ex vivo generation of adoptive donor-derived DCs were complex and inconsistent, particularly regarding cell viability and potency after thawing. We have previously demonstrated in humanized mouse models of HSCT the proof-of-concept of a novel modality of lentivirus-induced DCs ("SmyleDCpp65") that accelerated antigen-specific T cell development.
  
  
  METHODS
  Here we demonstrate the feasibility of good manufacturing practices (GMP) for production of donor-derived DCs consisting of monocytes from peripheral blood transduced with an integrase-defective lentiviral vector (IDLV, co-expressing GM-CSF, IFN-α and the cytomegalovirus antigen pp65) that were cryopreserved and thawed.
  
  
  RESULTS
  Upscaling and standardized production of one lot of IDLV and three lots of SmyleDCpp65 under GMP-compliant conditions were feasible. Analytical parameters for quality control of SmyleDCpp65 identity after thawing and potency after culture were defined. Cell recovery, uniformity, efficacy of gene transfer, purity and viability were high and consistent. SmyleDCpp65 showed only residual and polyclonal IDLV integration, unbiased to proto-oncogenic hot-spots. Stimulation of autologous T cells by GMP-grade SmyleDCpp65 was validated.
  
  
  CONCLUSIONS
  These results underscore further developments of this individualized donor-derived cell vaccine to accelerate immune reconstitution against HCMV after HSCT in clinical trials.
authors.affiliation: REBIRTH, Regenerative Immune Therapies Applied, Hannover Medical School, OE6862, Hans Borst Zentrum, Carl Neuberg Strasse 1, 30625, Hannover, Germany. sundarasetty.balasai@mh-hannover.de.|Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, OE6862, Hans Borst Zentrum, Carl Neuberg Strasse 1, 30625, Hannover, Germany. sundarasetty.balasai@mh-hannover.de.
authors.forename: Bala Sai
authors.initials: BS
authors.lastname: Sundarasetty
authors.affiliation: Institute of Cellular Therapeutics and GMP Core Facility IFB-Tx, Hannover Medical School, Hannover, Germany. kloess.stephan@mh-hannover.de.
authors.forename: Stephan
authors.initials: S
authors.lastname: Kloess
authors.affiliation: Institute of Cellular Therapeutics and GMP Core Facility IFB-Tx, Hannover Medical School, Hannover, Germany. oberschmidt.olaf@mh-hannover.de.
authors.forename: Olaf
authors.initials: O
authors.lastname: Oberschmidt
authors.affiliation: EUFETS GmbH, Idar-Oberstein, Germany. sonja.naundorf@eufets.com.
authors.forename: Sonja
authors.initials: S
authors.lastname: Naundorf
authors.affiliation: EUFETS GmbH, Idar-Oberstein, Germany. klaus.kuehlcke@eufets.com.
authors.forename: Klaus
authors.initials: K
authors.lastname: Kuehlcke
authors.affiliation: REBIRTH, Regenerative Immune Therapies Applied, Hannover Medical School, OE6862, Hans Borst Zentrum, Carl Neuberg Strasse 1, 30625, Hannover, Germany. anusara.daen@gmail.com.|Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, OE6862, Hans Borst Zentrum, Carl Neuberg Strasse 1, 30625, Hannover, Germany. anusara.daen@gmail.com.
authors.forename: Anusara
authors.initials: A
authors.lastname: Daenthanasanmak
authors.affiliation: REBIRTH, Regenerative Immune Therapies Applied, Hannover Medical School, OE6862, Hans Borst Zentrum, Carl Neuberg Strasse 1, 30625, Hannover, Germany. gerasch.laura@mh-hannover.de.|Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, OE6862, Hans Borst Zentrum, Carl Neuberg Strasse 1, 30625, Hannover, Germany. gerasch.laura@mh-hannover.de.
authors.forename: Laura
authors.initials: L
authors.lastname: Gerasch
authors.affiliation: REBIRTH, Tolerogenic Cell Therapy, Department of Transfusion Medicine, Hannover Medical School, Hannover, Germany. figueiredo.constanca@mh-hannover.de.
authors.forename: Constanca
authors.initials: C
authors.lastname: Figueiredo
authors.affiliation: REBIRTH, Tolerogenic Cell Therapy, Department of Transfusion Medicine, Hannover Medical School, Hannover, Germany. blasczyk.rainer@mh-hannover.de.
authors.forename: Rainer
authors.initials: R
authors.lastname: Blasczyk
authors.affiliation: Division of Translational Oncology, National Center for Tumor Diseases, Heidelberg, Germany. eliana.ruggiero@nct-heidelberg.de.
authors.forename: Eliana
authors.initials: E
authors.lastname: Ruggiero
authors.affiliation: Division of Translational Oncology, National Center for Tumor Diseases, Heidelberg, Germany. raffaele.fronza@nct-heidelberg.de.
authors.forename: Raffaele
authors.initials: R
authors.lastname: Fronza
authors.affiliation: Division of Translational Oncology, National Center for Tumor Diseases, Heidelberg, Germany. manfred.schmidt@nct-heidelberg.de.
authors.forename: Manfred
authors.initials: M
authors.lastname: Schmidt
authors.affiliation: Division of Translational Oncology, National Center for Tumor Diseases, Heidelberg, Germany. christof.kalle@nct-heidelberg.de.
authors.forename: Christof
authors.initials: C
authors.lastname: von Kalle
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany. rothe.michael@mh-hannover.de.
authors.forename: Michael
authors.initials: M
authors.lastname: Rothe
authors.affiliation: Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, OE6862, Hans Borst Zentrum, Carl Neuberg Strasse 1, 30625, Hannover, Germany. ganser.arnold@mh-hannover.de.
authors.forename: Arnold
authors.initials: A
authors.lastname: Ganser
authors.affiliation: Institute of Cellular Therapeutics and GMP Core Facility IFB-Tx, Hannover Medical School, Hannover, Germany. koehl.ulrike@mh-hannover.de.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Koehl
authors.affiliation: REBIRTH, Regenerative Immune Therapies Applied, Hannover Medical School, OE6862, Hans Borst Zentrum, Carl Neuberg Strasse 1, 30625, Hannover, Germany. stripecke.renata@mh-hannover.de.|Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, OE6862, Hans Borst Zentrum, Carl Neuberg Strasse 1, 30625, Hannover, Germany. stripecke.renata@mh-hannover.de.
authors.forename: Renata
authors.initials: R
authors.lastname: Stripecke
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002470:Cell Survival; D015925:Cryopreservation; D003587:Cytomegalovirus; D003586:Cytomegalovirus Infections / Q000276:immunology* / Q000517:prevention & control; D003713:Dendritic Cells / Q000166:cytology* / Q000821:virology; D005434:Flow Cytometry; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism; D057809:HEK293 Cells; D018380:Hematopoietic Stem Cell Transplantation; D006801:Humans; D016898:Interferon-alpha / Q000378:metabolism; D016086:Lentivirus; D007963:Leukocytes, Mononuclear / Q000166:cytology; D010750:Phosphoproteins / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D033581:Stem Cell Transplantation / Q000379:methods*; D019076:Transgenes; D014763:Viral Matrix Proteins / Q000378:metabolism
chemical_list: D016898:Interferon-alpha; D010750:Phosphoproteins; D014763:Viral Matrix Proteins; C065418:cytomegalovirus matrix protein 65kDa; D016178:Granulocyte-Macrophage Colony-Stimulating Factor