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

Dohnal AM, Graffi S, Witt V, Eichstill C, Wagner D, Ul-Haq S, Wimmer D, Felzmann T - J. Cell. Mol. Med. (2008)

Bottom Line: 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.We conclude that significant steps were taken here towards clinical grade DC cancer vaccine manufacturing.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Tumour Immunology, St Anna Children's Cancer Research Institute, Vienna, Austria.

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.

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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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fig03: 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.

Mentions: The percentage of monocytes in the leucocyte apheresis products from healthy donors before enrichment was similar for all enrichment procedures. Among the enrichment procedures, illustrated in Fig. 3, we isolated monocytes with a mean recovery for selection, depletion and elutriation of 59 ± 4%, 41 ± 3% and 87 ± 7%, respectively. The purity was found to be 96 ± 2% of leucocytes for selection, and 61 ± 4% and 82 ± 3% for depletion and elutriation, respectively. Product analysis could not be performed for monocytes enriched by adhesion without disrupting the culture.


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

Dohnal AM, Graffi S, Witt V, Eichstill C, Wagner D, Ul-Haq S, Wimmer D, Felzmann T - J. Cell. Mol. Med. (2008)

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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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3823041&req=5

fig03: 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.
Mentions: The percentage of monocytes in the leucocyte apheresis products from healthy donors before enrichment was similar for all enrichment procedures. Among the enrichment procedures, illustrated in Fig. 3, we isolated monocytes with a mean recovery for selection, depletion and elutriation of 59 ± 4%, 41 ± 3% and 87 ± 7%, respectively. The purity was found to be 96 ± 2% of leucocytes for selection, and 61 ± 4% and 82 ± 3% for depletion and elutriation, respectively. Product analysis could not be performed for monocytes enriched by adhesion without disrupting the culture.

Bottom Line: 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.We conclude that significant steps were taken here towards clinical grade DC cancer vaccine manufacturing.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Tumour Immunology, St Anna Children's Cancer Research Institute, Vienna, Austria.

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.

Show MeSH
Related in: MedlinePlus