Development and validation of a fully GMP-compliant production process of autologous,tumor-lysate-pulsed dendritic cells |
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| Institution: | 1. Stem Cell Laboratory, University Children''s Hospital Würzburg, University of Würzburg, Germany;2. Department of Pediatric Neurosurgery, University Medical Center, Würzburg, Germany;3. Laboratory of Pediatric Immunology, Department of Microbiology and Immunology, KU Leuven, Belgium (on behalf of the HGG-Immuno Network);4. Institute of Mathematics, University of Würzburg, Germany;5. Institute of Virology and Immunobiology, University of Würzburg, Germany;6. Laboratory of Experimental Neurosurgery and Neuroanatomy, Department of Neurosciences, KU Leuven, Belgium;1. San Antonio Military Medical Center, 3551 Roger Brooke Dr., Ft. Sam Houston, TX 78234, United States;2. Womack Army Medical Center, 2817 Reilly Rd, Ft. Bragg, NC 28310, United States;3. Madigan Army Medical Center, 9040 Jackson Ave, Ft. Lewis, WA 98431, United States;4. Orbis Health Solutions, 111 Smith Hines Rd, Greenville, SC 29607, United States;5. Clemson University Biological Sciences Dept, 101 Barre Hall, Clemson, SC 29634, United States;6. Cancer Vaccine Development Program, 110 E. Houston St, San Antonio, TX 78205, United States;1. Division of Cardiology, Department of Medicine, George Washington University Hospital, Washington, DC;2. Division of Cardiac Surgery, Department of Surgery, George Washington University Hospital, Washington, DC;1. Biotherapy Center, the General Hospital of Beijing Military Command, Beijing, China;2. School of Cellular and Molecular Medicine, University of Bristol, University Walk, Bristol, UK;1. Department of Molecular Oncology, Cancer Institute (WIA), India;2. Department of Radiation Oncology, Cancer Institute (WIA), India |
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| Abstract: | Background and aimsOne 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.MethodsAll production steps (lysate generation, monocyte selection, DC culture and cryopreservation) were standardized and validated.ResultsTumor 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.ConclusionsOur 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. |
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| Keywords: | cancer vaccines dendritic cells GMP production high-grade glioma |
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