Comparative evaluation of techniques for the manufacturing of dendritic cell-based cancer vaccines

Alexander Michael Dohnal, Sebastian Graffi, Volker Witt, Christina Eichstill, Dagmar Wagner, Sidrah Ul-Haq, Doris Wimmer, Thomas Felzmann, Alexander Michael Dohnal, Sebastian Graffi, Volker Witt, Christina Eichstill, Dagmar Wagner, Sidrah Ul-Haq, Doris Wimmer, Thomas Felzmann

Abstract

Manufacturing procedures for cellular therapies are continuously improved with particular emphasis on product safety. We previously developed a dendritic cell (DC) cancer vaccine technology platform that uses clinical grade lipopolysaccharide (LPS) and interferon (IFN)-y for the maturation of monocyte derived DCs. DCs are frozen after 6 hrs exposure at a semi-mature stage (smDCs) retaining the capacity to secret interleukin (IL)-12 and thus support cytolytic T-cell responses, which is lost at full maturation. We compared closed systems for monocyte enrichment from leucocyte apheresis products from healthy individuals using plastic adherence, CD14 selection, or CD2/19 depletion with magnetic beads, or counter flow centrifugation (elutriation) using a clinical grade in comparison to a research grade culture medium for the following DC generation. We found that elutriation was superior compared to the other methods showing 36 +/- 4% recovery, which was approximately 5-fold higher as the most frequently used adherence protocol (8 +/- 1%), and a very good purity (92 +/- 5%) of smDCs. Immune phenotype and IL-12 secretion (adherence: 1.4 +/- 0.4; selection: 20 +/- 0.6; depletion: 1 +/-0.5; elutriation: 3.6 +/- 1.5 ng/ml) as well as the potency of all DCs to stimulate T cells in an allogeneic mixed leucocyte reaction did not show statistically significant differences. Research grade and clinical grade DC culture media were equally potent and freezing did not impair the functions of smDCs. Finally, we assessed the functional capacity of DC cancer vaccines manufactured for three patients using this optimized procedure thereby demonstrating the feasibility of manufacturing DC cancer vaccines that secret IL-12 (9.4 +/- 6.4 ng/ml). We conclude that significant steps were taken here towards clinical grade DC cancer vaccine manufacturing.

Figures

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Dendritic cell (DC) manufacturing. (A) Flow chart of the standard operating procedure (SOP) for the manufacturing of a cancer vaccine. From patients undergoing tumour surgery a piece of tumour tissue is delivered to the manufacturing facility. The tumour tissue is disrupted mechanically, and the tumour cells are lysed to enrich soluble protein containing tumour antigens. After recovery from surgery, leucocyte apheresis is performed to collect peripheral blood mononuclear cells (PBMCs) from the patients, the monocytes are enriched and cultivated for 6 days in the presence of interleukin (IL)-4 and granulocyte-macrophage colony-stimulation factor (GM-CSF) in order to obtain iDCs. The iDCs are charged with tumour antigens, exposed to LPS/IFN-γ to trigger maturation and cryopreserved until treatment. An aliquot of the DC cancer vaccine is subjected to quality control, a potency assay and sterility control. If all criteria are met the DC cancer vaccine is released for treatment. (B) Flow chart of DC manufacturing using different monocyte enrichment protocols. Monocytes are isolated from PBMCs of a healthy donor or cancer patient using leucocyte apheresis. The PBMCs are further subjected to monocyte enrichment using adherence, or semi-automated elutriation, CD14 selection, or CD2/19 depletion. The differentiation into DCs is done in the same way independently of the enrichment procedure used (see Fig. 1A).
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Cell composition of leucocyte apheresis products. The leucocyte apheresis product as well as peripheral blood (PB) before and immediately after leucocyte apheresis from healthy individuals (n= 11) and patients (n= 3) was analysed for the leucocyte number and distribution (monocytes, lymphocytes, granulocytes), erythrocyte number, haematocrit and platelet number. Percentage and cell number are given as mean ± SD. Comparisons of whole leucocyte apheresis products as well as peripheral blood before and after leucocyte apheresis from healthy and patient donors show no statistically significant differences.
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Recovery and Purity of LPS/IFN-γ activated-DCs manufactured by using different monocyte enrichment protocols. Six hours (smDCs) and 48 hrs (mDCs) LPS/IFN-γ-activated iDCs were generated using monocytes from healthy individuals. Monocytes were enriched from leucocyte apheresis products by plastic adherence, CD14 selection, CD2/CD19 depletion or elutriation (as indicated) using AIM V/Octaplas or clinical grade CellGro DC Medium. Upper: The mean percentage ± SEM of recovery of monocytes after enrichment, smDCs or mDCs, as indicated, is given relative to monocytes in the leucocyte apheresis product. Lower: The mean percentage ± SEM of monocytes before and after enrichment, smDCs and mDCs, as indicated, is given relative to the total number of leucocytes. Number of independent DC preparations in AIM-V/Octaplas medium: adherence, n= 10; selection, n= 5; depletion, n= 9; elutriation, n= 7; and CellGro DC medium: adherence, n= 5, selection, n= 3; depletion, n= 2; elutriation, n= 10. na, no data available; p, P < 0.01.
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DC quality control. Monocytes from healthy individuals were enriched from leucocyte apheresis products by different enrichment procedures, as indicated, differentiated into DCs followed by activation with LPS/IFN-γ. DC preparations using AIM-V/Octaplas or CellGro DC medium are combined. (A) The left-hand panel shows the ratio of increase in the expression density ± SEM of the indicated DC maturation markers measured 6 hrs (smDCs) and 48 hrs (mDC) after activation. DCs generated from elutriated monocytes (number of preparations, n= 35) are analysed. The right-hand panel compares the expression density mean fluorescence intensity (MFI) ± SEM of maturation markers measured on mDCs that are generated from monocytes isolated by the indicated enrichment procedures. MFIs of the isotype controls are below 5 (data not shown). (B) Secretion of IL-12 ± SEM secreted from mDCs analysed in (A) is illustrated. (C) Proliferation of allogeneic PBMCs in co-cultures with the DCs analysed in (A) is given relative to PBMCs stimulated with the super-antigen staphylococcal enterotoxin A/B (SEA/SEB) (normalized to 100%) and the background proliferation of unstimulated PBMCs (normalized to 0%). Number of preparations in (B) and (C): adherence, n= 10; selection, n= 8; depletion, n= 11, elutriation, n= 35. Comparisons of the enrichment procedures in (A) to (C) show no statistically significant differences.
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Optimization of DC maturation. Left-hand panels: Expression density (MFI) of the indicated DC membrane molecules and viability of mDCs as well as IL-12 secretion during the differentiation of smDCs into mDCs is shown after exposure to increasing concentrations of lipopolysaccharide (LPS) at constant IFN-γ concentration. Data from two healthy donors are shown. Right-hand panel: LPS at a concentration of 30 ng/ml was applied for 6 hrs (+) or the DCs were left immature (–) in the presence of IFN-β. According to the DC-manufacturing protocol for cancer vaccines, DC cultures were frozen after 6 hrs in liquid nitrogen, recovered from freezing and re-cultured; or DCs were continuously cultured until hour = 48 as indicated. * Below the detection limit of the IL-12 ELISA.
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Validation of the DC manufacturing process for three patients. DC quality control of cancer vaccines from three cancer patients using monocytes enriched by elutriation was performed according to the flow chart shown in Fig. 1A. For DC activation, 30 ng/ml LPS was used. (A) Purity, yield and viability ± SEM was assessed for monocytes, smDCs and mDCs as indicated. (B) Mean ± SEM of IL-12 secreted from three vaccines. (C) Immune phenotype measuring the expression density of the depicted DC membrane molecules for three patients. (D) AlloMLR using CFSE dilution was performed as potency assay for the stimulatory capacity of smDCs at the indicated DC/PBMC ratios. The dot plots illustrate the gating for proliferating CD3 expressing T lymphocytes that lost CFSE due to proliferation as shown in the histograms. As a negative control we used un-stimulated PBMCs, as positive control PBMCs exposed to the super-antigen SEA/SEB (one representative experiment of three is given). The bar graph shows the mean ± SEM of the percentage of proliferating allogeneic T lymphocytes co-cultured with DCs from three patients.

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