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Efficient generation of clinical-grade genetically modified dendritic cells for presentation of multiple tumor-associated proteins

Authors

  • Henk S.P. Garritsen,

    1. From the Institute for Clinical Transfusion Medicine and the Department of Hematology/Oncology Städtisches Klinikum Braunschweig gGmbH, Braunschweig; and the Department of Molecular Biotechnology and the Department of Cell- and Immunobiology, Helmholtz Centre for Infection Research (HZI), Braunschweig, Germany.
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  • Lars Macke,

    1. From the Institute for Clinical Transfusion Medicine and the Department of Hematology/Oncology Städtisches Klinikum Braunschweig gGmbH, Braunschweig; and the Department of Molecular Biotechnology and the Department of Cell- and Immunobiology, Helmholtz Centre for Infection Research (HZI), Braunschweig, Germany.
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  • Wilhelm Meyring,

    1. From the Institute for Clinical Transfusion Medicine and the Department of Hematology/Oncology Städtisches Klinikum Braunschweig gGmbH, Braunschweig; and the Department of Molecular Biotechnology and the Department of Cell- and Immunobiology, Helmholtz Centre for Infection Research (HZI), Braunschweig, Germany.
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  • Horst Hannig,

    1. From the Institute for Clinical Transfusion Medicine and the Department of Hematology/Oncology Städtisches Klinikum Braunschweig gGmbH, Braunschweig; and the Department of Molecular Biotechnology and the Department of Cell- and Immunobiology, Helmholtz Centre for Infection Research (HZI), Braunschweig, Germany.
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  • Ute Pägelow,

    1. From the Institute for Clinical Transfusion Medicine and the Department of Hematology/Oncology Städtisches Klinikum Braunschweig gGmbH, Braunschweig; and the Department of Molecular Biotechnology and the Department of Cell- and Immunobiology, Helmholtz Centre for Infection Research (HZI), Braunschweig, Germany.
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  • Bernhard Wörmann,

    1. From the Institute for Clinical Transfusion Medicine and the Department of Hematology/Oncology Städtisches Klinikum Braunschweig gGmbH, Braunschweig; and the Department of Molecular Biotechnology and the Department of Cell- and Immunobiology, Helmholtz Centre for Infection Research (HZI), Braunschweig, Germany.
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  • Robert Geffers,

    1. From the Institute for Clinical Transfusion Medicine and the Department of Hematology/Oncology Städtisches Klinikum Braunschweig gGmbH, Braunschweig; and the Department of Molecular Biotechnology and the Department of Cell- and Immunobiology, Helmholtz Centre for Infection Research (HZI), Braunschweig, Germany.
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  • Kurt E.J. Dittmar,

    1. From the Institute for Clinical Transfusion Medicine and the Department of Hematology/Oncology Städtisches Klinikum Braunschweig gGmbH, Braunschweig; and the Department of Molecular Biotechnology and the Department of Cell- and Immunobiology, Helmholtz Centre for Infection Research (HZI), Braunschweig, Germany.
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  • Werner Lindenmaier

    1. From the Institute for Clinical Transfusion Medicine and the Department of Hematology/Oncology Städtisches Klinikum Braunschweig gGmbH, Braunschweig; and the Department of Molecular Biotechnology and the Department of Cell- and Immunobiology, Helmholtz Centre for Infection Research (HZI), Braunschweig, Germany.
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  • The first two authors contributed equally to this work.

  • This work was supported by grants from the Federal Ministry of Economics and Technology (Innonet project 16IN170) and the EC (Clinigene-NoE, LSHB-CT-2006-018933).

Dr Werner Lindenmaier, Department of Molecular Biotechnology, Helmholtz Centre for Infection Research (HZI), Inhoffenstrasse 7, D-38124 Braunschweig, Germany; e-mail: werner.lindenmaier@helmholtz-hzi.de.

Abstract

BACKGROUND: Dendritic cells (DCs) play a central role in the initiation and regulation of immune responses. DCs for clinical applications can be generated with high yield from leukapheresis products. Using adenoviral transduction we genetically modified human DCs to produce and present melanoma-associated antigens. Coexpression of green fluorescent protein and epitope tags were used to monitor genetic modification. Generation, genetic modification, and cryoconservation of gene modified human DCs on a clinical scale in a closed system is feasible.

STUDY DESIGN AND METHODS: CD14-positive monomuclear cells were isolated from leukapheresis products of HLA-A* 0201 positive voluntary blood donors using immunomagnetic beads. Selected cells were cultivated for 7 days. Adenovirus transduction was optimal on Day 4. Maturation was induced on Day 5. Mature DC were aliquoted and cryoconserved on Day 7. Quality control was performed using flow cytometry, expression profiling, and functional assays (ELISPOT, CBA).

RESULTS: We were able to generate sufficient genetically modified mature DCs in serum-free cultures that could be stored by cryopreservation. The use of a closed system facilitated development of methods for standardized production of clinically applicable genetically modified DCs. The adenoviral transduction system allowed simultaneous and flexible expression of tumor-associated antigens for prolonged presentation of multiple epitopes.

CONCLUSION: The feasibility of a closed-bag system for the cultivation of genetically modified human DCs is shown. The immature DCs were genetically modified by recombinant replication-deficient adenoviruses to express multiple epitopes of tumor-associated proteins and then differentiated to mature antigen-presenting DCs.

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