Abstract
Background and aims
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.
Key Words
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Article info
Publication history
Published online: May 13, 2014
Accepted:
February 27,
2014
Received:
November 20,
2013
Identification
Copyright
© 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.
