Author:

Keywords:

Science & Technology, Life Sciences & Biomedicine, Cell & Tissue Engineering, Biotechnology & Applied Microbiology, Cell Biology, Hematology, Medicine, Research & Experimental, Research & Experimental Medicine, cancer vaccines, dendritic cells, GMP production, high-grade glioma, CLINICAL-SCALE GENERATION, RELAPSED MALIGNANT GLIOMA, CD8(+) T-CELLS, CD14(+) MONOCYTES, VACCINATION, PLATFORM, IMMUNE, IMMUNOTHERAPY, MATURATION, IL-12, CD8-Positive T-Lymphocytes, Cancer Vaccines, Cell Culture Techniques, Cell- and Tissue-Based Therapy, Dendritic Cells, Glioma, Granulocyte-Macrophage Colony-Stimulating Factor, Humans, Leukapheresis, Monocytes, Vaccination, 1103 Clinical Sciences, Immunology, 3204 Immunology, 3206 Medical biotechnology

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.