Development and validation of a fully GMP-compliant production process of autologous, tumor-lysate-pulsed dendritic cells.
OBJECTIVE
One of the major challenges of dendritic cell (DC) vaccination is the establishment of harmonized DC production protocols. Here, we report the transfer and validation of a successfully used open DC manufacturing method into a closed system, good manufacturing practice (GMP)-compatible protocol.


METHODS
All production steps (lysate generation, monocyte selection, DC culture and cryopreservation) were standardized and validated.


RESULTS
Tumor lysate was characterized by histology, mechanically homogenized and avitalized. This preparation yielded a median of 58 ± 21 μg protein per milligram of tumor tissue. Avitality was determined by trypan blue staining and confirmed in an adenosine triphosphate release assay. Patient monocytes were isolated by elutriation or CD14 selection, which yielded equivalent results. DCs were subsequently differentiated in Teflon bags for an optimum of 7 days in CellGro medium supplemented with interleukin (IL)-4 and granulocyte macrophage colony stimulating factor and then matured for 48 h in tumor necrosis factor-α and IL-1ß after pulsing with tumor lysate. This protocol resulted in robust and reproducible upregulation of DC maturation markers such as cluster of differentiation (CD)80, CD83, CD86, human leukocyte antigen-DR and DC-SIGN. Functionality of these DCs was shown by directed migration toward C-C motif chemokine ligand 19/21, positive T-cell stimulatory capacity and the ability to prime antigen-specific T cells from naive CD8(+) T cells. Phenotype stability, vitality and functionality of DCs after cryopreservation, thawing and washing showed no significant loss of function. Comparison of clinical data from 146 patients having received vaccinations with plate-adherence versus GMP-grade DCs showed no inferiority of the latter.


CONCLUSIONS
Our robust, validated and approved protocol for DC manufacturing forms the basis for a harmonized procedure to produce cancer vaccines, which paves the way for larger multi-center clinical trials.
Stem Cell Laboratory, University Children's Hospital Würzburg, University of Würzburg, Germany. Electronic address: eyrich_m@ukw.de.
Matthias
M
Eyrich
Stem Cell Laboratory, University Children's Hospital Würzburg, University of Würzburg, Germany.
Susanne C
SC
Schreiber
Stem Cell Laboratory, University Children's Hospital Würzburg, University of Würzburg, Germany.
Johannes
J
Rachor
Department of Pediatric Neurosurgery, University Medical Center, Würzburg, Germany.
Jürgen
J
Krauss
Laboratory of Pediatric Immunology, Department of Microbiology and Immunology, KU Leuven, Belgium (on behalf of the HGG-Immuno Network).
Femke
F
Pauwels
Institute of Mathematics, University of Würzburg, Germany.
Johannes
J
Hain
Stem Cell Laboratory, University Children's Hospital Würzburg, University of Würzburg, Germany.
Matthias
M
Wölfl
Institute of Virology and Immunobiology, University of Würzburg, Germany.
Manfred B
MB
Lutz
Laboratory of Experimental Neurosurgery and Neuroanatomy, Department of Neurosciences, KU Leuven, Belgium.
Steven
S
de Vleeschouwer
Stem Cell Laboratory, University Children's Hospital Würzburg, University of Würzburg, Germany.
Paul G
PG
Schlegel
Laboratory of Pediatric Immunology, Department of Microbiology and Immunology, KU Leuven, Belgium (on behalf of the HGG-Immuno Network).
Stefaan W
SW
Van Gool
D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D019496:Cancer Vaccines / Q000276:immunology / Q000378:metabolism; D018929:Cell Culture Techniques; D064987:Cell- and Tissue-Based Therapy; D003713:Dendritic Cells / Q000276:immunology* / Q000473:pathology; D005910:Glioma / Q000276:immunology / Q000473:pathology / Q000628:therapy*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism; D006801:Humans; D007937:Leukapheresis; D009000:Monocytes; D014611:Vaccination
D019496:Cancer Vaccines; D016178:Granulocyte-Macrophage Colony-Stimulating Factor
GMP production; cancer vaccines; dendritic cells; high-grade glioma