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Development of a technology platform for large-scale clinical grade production of DC
Cytotherapy (2004) Vol. 6, No. 4, 363 /371
Development of a technology platform for largescale clinical grade production of DC L Adamson1, A Palmborg1, A Svensson2, A Lundqvist1, M Hansson2, R Kiessling1, G Masucci1, H Mellstedt1 and P Pisa1 1
Immune and Gene Therapy Laboratory, Department of Oncology & Pathology, Cancer Centrum Karolinska, and 2Immunohemotherapy Unit, Department of Clinical Immunology and Transfusion Medicine, Karolinska Hospital, Stockholm, Sweden
Background Clinical studies require protocols where a sufficient number of wellcharacterized highly immunogenic DC are produced according to good manufacturing practice (GMP) guidelines. Methods In the present study, using leukapheresis products from 10 cancer patients, we validated an elutriation technology for large-scale clinical grade production of monocyte-derived DC. Results The elutriation method gave a very high purity (mean9/SD) (86 9/5.3%) and recovery (669/10.4%) of monocytes. Specifically for the two monocyte-rich fractions (3 and 4,) the recovery was 429/13% of viable cells that could be further differentiated into immature DC in hydrophobic culture bags using GM-CSF and IL-4. The immature
Introduction DC have a unique ability to capture, process and present antigens, and thus possess the unique capacity to activate CD4 and CD8 naive T lymphocytes, leading to induction of primary immune responses [1,2]. DC derive their stimulatory potency from high constitutive and up-regulated expression of MHC class I, MHC class II and accessory molecules such as CD40, CD54, CD80, CD86 and T-cell activating cytokines [1,2]. These unique characteristics make DC a suitable candidate for immunotherapy against malignant and infectious diseases [3 /5]. Results obtained in animal studies and clinical trials confirm that DC are capable of inducing antigen-specific cytotoxic T-lymphocyte (CTL) responses, resulting in strong immunity to viruses and tumors and, in some
DC exhibited B/1% CD83 expression and /98% phagocytic activity. Maturation with TNF-a or poly I:C resulted in DC with expression of CD80 , CD86 and HLA-DR (/99%) and CD83 (809/11.9%), as well as producing IL-12p70 and lacking phagocytic activity ( B/5%). This cell product can be cryopreserved with cell viability /85% and cell recovery /80% after thawing. Discussion The elutriation procedure, when optimized and if the monocyte content of the starting material exceeds 5%, does not require further selection or depletion using affinity approaches. Keywords culture bags, elutriation, leukapheresis, maturation, monocytes, poly I:C.
instances, also objective regression of established disease [6]. DC exist in two functionally and phenotypically different stages, immature (im) and mature (m) DC [2]. imDC reside in peripheral tissue, where they capture antigens such as bacteria, viruses and damaged tissue. Upon exposure to pro-inflammatory cytokines or pathogen-derived products, DC mature, which is associated with a loss of phagocytic capacity and a gain in migratory potential to draining lymph nodes. mDC exhibit high antigenpresenting capability and T-cell stimulatory capacity. For clinical application, imDC are generally obtained after culture of monocytes for 5 /6 days in the presence of GM-CSF and IL-4 [7,8]. As imDC have a high phagocytic capacity, these cells are well suited for antigen loading
Correspondence to: Pavel Pisa, CCK R8:01, Karolinska Hospital, SE-171 76 Stockholm, Sweden. – 2004 ISCT
DOI: 10.1080/14653240410004934
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using various protocols. mDC are obtained after further exposure to pro-inflammatory cytokines, for example tumor necrosis factor (TNF-a), products from microbial or viral pathogens, such as double-stranded RNA, lipopolysaccharide (LPS) or CpG DNA binding to Toll-like receptors (TLR), and ligation to T cells via CD40L [9]. In this way ex vivo generated mDC are ideal tools for cancer vaccination therapy because of the high expression level of antigen-presenting and costimulatory molecules for T-cell stimulation [10]. Clinical studies require protocols where a sufficient number of well-characterized highly immunogenic DC is produced according to good manufacturing practice (GMP) guidelines. Several approaches are currently in use for this purpose, from CD34 hematopoietic stem cells after mobilization with G-CSF, from peripheral blood using density gradients or from PBMC [7,11]. CD34 -derived DC are generally obtained using positive selection. Monocyte-derived DC are cultured from monocytes obtained from PBMC enriched by plastic adherence, positive or negative immunoselection or elutriation. The principle of the elutriation method is based on the physical separation of cells using a counter flow centrifugation, where cells are separated depending on their size and density (reviewed in [12]). In the present study we describe and validate such a technology platform using elutriation technology for large-scale clinical grade production of DC. The elutriation method gives a very high recovery of monocytes that can be further differentiated into imDC and mDC suitable for vaccination protocols.
(COBE BCT Laboratories, Lakewood, CO) with software 7.1 was used and all patients were leukapheresed using peripheral veins. According to a previously described routine [13], a small sample, taken from the collect line early during the leukapheresis, was analyzed for degree of monocyte enrichment and monocyte concentration. The result of this sample was used to adjust the machine to obtain a maximal monocyte harvest ( /15%) and to decide the length of the apheresis procedure for collection of the targeted number of leukocytes (A Svensson et al ., manuscript submitted for publication). The amount of processed blood was [median (minimum /maximum)] 8.8 L (6.7 /10.4) L. None of the patients suffered any serious side-effects from the procedure.
Counterflow centrifugal elutriation (elutriation)
Ten patients treated for their malignancies at the Karolinska Hospital (Stockholm, Sweden; six with prostate cancer, three with melanoma and one with CLL), after informed consent, donated leukocytes using a leukapheresis procedure. The study was approved by the local ethics committee of the Karolinska Hospital, conforming to the guidelines of the Helsinki Protocol.
The leukapheresis product was separated in a blood centrifuge (Hettich Roto Silenta, Tuttingen, Germany) at 540 g for 6 min. After removing the platelet-rich plasma the leukapheresis product was diluted in PBS, pH 7.2 (CE0123; Baxter, Healthcare, Deerfield, IL), with 5 mM EDTA (Invitrogen, Carlsbad, CA), to approximately 5 /106 leukocytes/mL and subjected to elutriation using a Beckman-Coulter (Miami, FL) Avanti J20XPI centrifuge with JE-5.0 rotor and 40-mL chamber (#356940). Loading of cells was performed in a closed system at 2000 rpm (385 g ) at a flow rate of 53 mL/min, 208C. Cell fractions (approximately 1500 mL) were collected in bags (Teruflex transfer bag; Terumo, Tokyo, Japan) at a constant flow rate of 53 mL/min of PBS, pH 7.2 (3000-mL bags; Baxter), 2 mM EDTA (Invitrogen) using a peristaltic pump (Masterflex L/S; Cole-Parmer Instrument Company, Vernon Hills, IL). Elutriation of the different fractions was performed by changing the rotor speed as follows: fraction 1 was collected at 2000 rpm (385 g), fraction 2 at 1800 rpm (312 g), fraction 3 at 1700 rpm (278 g), fraction 4 at 1600 rpm (247 g) and fraction 5 rotor off (RO) at 0 rpm (247 /0 g ). After optimization of the elutriation procedure the use of EDTA in the fluid was proven unnecessary and therefore excluded from the preparation.
Leukapheresis
Chamber-cleaning procedure
Leukapheresis procedures were performed according to standard clinical routines at the Immunohemotherapy Unit, Department of Clinical Immunology and Transfusion Medicine, Karolinska Hospital. The Cobe Spectra
The rotor chamber was extensively cleaned with antimicrobial/antiviral substances, 70% ethanol and Virkon (CE0086; Antec Int., Sudbury, UK). Samples were taken before and after cleaning for CMV-PCR (Department of
Methods Human subjects
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Clinical Microbiology, Karolinska Hospital). After assembly of the rotor to a closed system, sterilization and equilibration, samples were taken for pyrogene analysis (Limulus test; Karolinska Hospital Pharmacy). All cell fractions were tested for sterility and the final DC product also tested by Limulus before cell banking.
CD45-FITC and CD14-PE. Monocytes and mature DC were assayed for phenotype using CD80-FITC, CD83-PE, CD86-PE, CD1a-PC5, ILT3-PC5 and HLA-DR-FITC. MAb and corresponding isotype-labeled Ig controls were all from Beckman-Coulter. 7AAD-positive (non-viable) cells were excluded from FCM analysis.
Cell culture
Mixed leukocyte reaction
The elutriated fractions 3 and 4 were centrifuged (5700 g for 5 min) and, after the supernatant was removed, the cells transferred from the transfer bags into cell culture bags by sterile welding (welding set; Tamuro, Tokyo, Japan). Resuspended cells were cultured in 85 cm2 /180 cm2 cell culture bags (Lifecell OptiCyte Cell Culture containers; Baxter) at a cell density of 2.5 /5 /106 cells/mL, in CellGro DC serum-free medium (Cell Genix, Freiburg, Germany) supplemented with 100 ng/mL GM-CSF (Leukine; Immunex, Seattle, WA) and 20 ng/mL IL-4 (Schering-Plough, Kennilworth, NJ). Fresh medium, half the volume, including twice the concentration of cytokines, was added after 2 days. After 5 days of culture cells were resuspended in new medium, CellGro DC supplemented with GM-CSF, IL-4 and 50 ng/mL TNF-a (Chiron, Emeryville, CA), or GM-CSF, IL-4 and 50 mg/mL poly I:C (Sigma-Aldrich, St Louis, MO), for an additional 48-h maturation. On day 7 cell viability and recovery were determined by trypan blue dye exclusion and 7AAD staining (Beckman-Coulter). Cells were cryopreserved at 5/10 /106 cells/mL ampoules (Nunc, Roskilde, Denmark), in 90% autologous plasma and 10% DMSO (Sigma-Aldrich). The plateletrich autologous plasma separated from the leukapheresis product was centrifuged at 5700 g and platelets removed by transferring of the plasma to another bag using a plasma extractor. The plasma was frozen, heat-inactivated at 568C for 60 min and centrifuged at 5700 g for 5 min (Hettich Roto Silenta) and transferred into another bag using a plasma extractor.
Primary allogeneic MLR was performed in serum-free CellGro medium, with the non-adherent fraction of Ficoll-separated PBMC from a buffy-coat from a healthy donor at 105 cells/well in 96-well U-bottomed plates at different responder/stimulator ratios, ranging between 10:1 and 1250:1. After 5 days, 1 mCi of H3-thymidine (Amersham Biosciences, Amersham, UK) was added to each well and incubated for 18 h. Cells were harvested and measured in a scintillation counter (1450 MicroBetaTM; Wallac, Turku, Finland).
Flow cytometry analysis Flow cytometry (FCM) acquisition and analysis was performed using EPICS XL (Beckman-Coulter) with Coulter System II software. Whole blood and the leukapheresis material were analyzed to identify cell distribution between lymphocytes, monocytes, granulocytes, T cells, B -cells and natural killer (NK) cells, using directly conjugated MAB for CD3-PC5, CD19-ECD, CD56-PE,
FITC dextran uptake /
imDC and mDC were plated in separate wells in 24-well plates at 106 cells/mL in 500 ml volume/well. After 48 h FITC /dextran (Sigma-Aldrich) was added to a final concentration of 1 mg/mL and incubated for 1 h at / 378C or at /48C. Harvested cells were washed with icecold PBS, fixed in 4% formaldehyde and stained with antiCD83-PC5 (Beckman-Coulter). FITC/dextran uptake and CD83 expression were analyzed by FCM using a FACS Calibur (Becton-Dickinson, Franklin Lakes, NJ).
Cytokine production assay Supernatants from DC cultures were collected 48 h after addition of maturation agent and analyzed for IL-12p70 by ELISA (Mabtech AB, Stockholm, Sweden).
Results The aim of this work was to validate a technology platform for generation of autologous DC from patients with different cancer diagnoses that were suitable for further ex vivo manipulation to produce clinical grade cancer vaccines (Figure 1). The starting material was nonmobilized leukapheresis products from 10 cancer patients (six prostate, three melanoma and one CLL) that was elutriated, cultured ex vivo for 7 days, frozen and thawed. All cell product manipulation was performed in a closed system, to conform to the current European regulatory policy.
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Figure 1. A flow chart of the technological platform.
fraction 5 at 0 rpm was heavily enriched in granulocytes, 759/25%. Starting with 21.19/7.1 /108 monocytes, the yield of monocytes after elutriation was 14.09/4.5 /108 cells. The total recovery of elutriated viable cells was 849/6.8% and the viability exceeded 95%, as measured by FCM and 7AAD staining (Figure 3a). The recovery of the monocyte population was 669/10.4%, with viability greater than 90%. The yield of the two main monocyte-rich fractions 3 and 4 was 8.89/2.8 /108 monocytes, resulting in a practically usable yield of 429/13%. The maximum cell number in the starting material loaded, giving similar results, was 4 /1010 cells.
Elutriation The time of the elutriation procedure was 135 min. The different cell populations were collected in five different fractions in a volume of 1500 mL per transfer bag and analyzed by FCM for CD14 cells vs. CD45 and CD45 vs. side scatter characteristics (SSC). Although the differential blood cell count varied among the patients (data not shown) and therefore the proportions of the cell populations also differed in the starting material, the individual fractions after elutriation exhibited little variation (Figure 2). The fractions with the highest monocyte content were fraction 4 at 1600 rpm, and fraction 3 at 1700 rpm, containing (mean9/SD) 869/9.4% and 809/11.9%, respectively. The low density fraction 1 at 2000 rpm consisted of pure lymphocytes, 969/5.5%, while the final
Cell culture After 5 days of differentiation of fractions 3 and 4, the recovery of imDC was 759/9.6%, and after TNF-a maturation for an additional 48 h, 539/12.3% (Figure 3b). The overall recovery of practically usable (from fractions 3 and 4) imDC and mDC was 32.09/9.6% and 22.09/12.3%, respectively. Viability of cultured cells was 959/4% and 94%9/6%, for imDC and mDC, respectively. Recovery and viability from cultures matured with poly I:C were similar to those obtained with TNF-a (data not shown). In initial experiments, X-VIVO 15 (BioWhittaker, Rockland, ME) was used. However, the viability and recovery were higher using CellGro DC (data not shown)
Figure 2. Cell populations after elutriation detected by flow cytometry for CD45 cells vs. SSC. Results (mean9/SD) from 10 cancer patients (six prostate, three melanoma, one CLL). (The same monocyte profile was obtained using CD14 /CD45 ).
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present on monocytes, disappeared on imDC and appeared again after 48 h maturation into mDC. The mDC exhibited better stimulatory capacity in all MLR than their immature counterparts (Figure 5e). IL-12p70 is considered to be one of the key T helper-1 (Th-1) polarizing cytokines [14]. IL-12p70 was produced to very low extent by imDC15, or in presence of TNF-a, and increased five-fold upon culture in poly I:C containing medium (Figure 4b).
Freeze thawing of cells /
Figure 3. Recovery (mean9/SD) (open bar) and viability (black bar) prior and post culture (n /10). (a) Analysis of elutriated total (Tot) cells and monocytes (Mo). (b) Cells cultured for 5 days (immature) and after addition of TNF-a (mature), in total and in practically usable fractions 3 and 4 (F3/4).
and therefore the results reported here were all performed with CellGro DC. Contaminating agents or pyrogens have not been detected in any elutriated material (data not shown) assayed by standard routines and controls used at Karolinska Hospital and by the Limulus clotting test according to the European Pharmacopoeia [14].
Phenotype of DC Kinetics of surface antigen expression were analyzed for CD80, CD83, CD86, CD1a, ILT3 and HLA-DR (Figure 4a). The phenotypic variability between DC from different cancer patients was subtle. Expression of CD80 and CD1a increased during differentiation and maturation with TNF-a and poly I:C, while CD83 was absent on monocytes and on day 7 imDC and appeared only if a maturation signal was delivered. The majority of imDC and mDC exhibited CD86 and HLA-DR. Expression of the ILT3 molecule appeared to be bi-phasic, as it was
Cell viability after freezing and thawing exceeded 85%, and recovery 80%. The functionality of frozen and thawed cells was tested by endocytosis of FITC/dextran, which demonstrated phagocytic activity in more than 95% of imDC (B/1% CD83 positive; Figure 5b) compared with less that 5% in mDC (65% CD83 positive with poly I:C; Figure 5d). The freezing and thawing procedure did not affect the phenotype of the cells, including IL-12p70 production (data not shown), nor their stimulatory capacity in MLR (Figure 5e) or endocytotic capacity assayed by FITC /dextran (Figure 5a /d). Preliminary data demonstrated induction of a HLA-A2-restricted peptide-specific IFN-g T-cell response, when stimulated with DC after thawing (assayed by Elispot; data not shown).
Discussion The current enthusiasm in clinical application of immunotherapy with DC-based vaccines for cancer has been inspired by the recent clinical successes of early phase trials with DC [6,16 /18]. The methodological aspects of these studies in terms of source of cells, technologies of preparation, culture conditions, antigen loading and degree of maturation vary significantly and it is therefore difficult to find a general explanation for why only 10 /30% of enrolled patients respond to this therapy [19]. Based on the past few years of development in the field, it has been proposed that a prerequisite for clinical grade DC preparation should be not only a GMP facility but also quality control of the DC preparation, including the DC isolation procedures, in vitro growth conditions, DC yield, purity and viability, cryopreservation, as well as phenotypic and functional characteristics [20]. Obstacles in translating research protocols for cellular therapy into clinical application are numerous. The obvious objective is to maintain the biological properties
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Figure 4. Phenotype and IL-12p70 production. (a) Phenotype of cells before and after maturation with TNF-a or poly I:C detected by flow cytometry (n /10). (b) IL-12p70 levels measured by solid-phase ELISA (n /3).
of the cell product, while incorporating the consistency, reproducibility and safety of the procedure required not only by the regulatory authorities but also by the treating physician. Furthermore, cell therapeutics are still at a stage of experimental development and therefore the production system needs a great deal of flexibility to permit different approaches, and the economical aspects of the procedure are equally important. Centers worldwide using immunotherapeutic concepts differ in their potential to use open separation and culture systems, such as adherence and tissue culture flasks, or closed systems, such as immunomagnetic separation and permeable culture bags. Affinity-based separation systems for clinical grade monocyte separation are currently commercially available only outside the USA and only
for positive CD14 selection (Miltenyi Biotec, Bergisch Gladbach, Germany). As the CD14 molecule possesses some important activation signaling capacities, this approach may disturb the further differentiation process [21]. For this reason many centers introduced the elutriation procedure, despite of the fact that it is a semi-closed system, because it requires assembly and sterilization prior to each procedure [22 /24]. We describe the validation of this relatively inexpensive, GMP friendly and rapid technology platform, which when optimized both at the leukapheresis step and elutriation step yields large-scale, high-purity DC that are suitable for further manipulation, using different methods of antigen loading and maturation, for use in cancer vaccination protocols. An additional aspect of the flexibility of the system is that the 95% pure
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Figure 5. Phagocytosis and stimulatory capacity in MLR. After thawing imDC were incubated for 1 h at /48C (a) and at /378C (b) with FITC /dextran (FL1-H) and stained for CD83 (FL3-H). mDC were incubated for 1 h at /378C with FITC/dextran and stained for CD83, after maturation with either TNF-a (c) or poly I:C (d). Numbers depict percentage of cells in the four quadrants. Results are representative of three separate experiments. (e) MLR with 105/well allogeneic PBMC as responders, stimulated with graded numbers of mature and immature DC (ratios ranging between 10:1 and 1250:1). DC only showed B/200 cpm.
lymphocyte fraction may be used further in adoptive immunotherapeutic protocols. We have not accounted for the necessity of further depletion of certain cell populations in the elutriated cell fraction, as sometimes suggested [25]. In fact, even patients with CLL, with a peripheral differential blood cell count of 8% monocytes, were successfully elutriated, with 88% monocyte purity in fraction 4 at 1600 rpm (a patient in this study). However, when in another CLL patient the starting material consisted of 98% lymphocytes and 1 /2% monocytes the final yield in fraction 4 at 1600 rpm was only 62% monocytes. The limit of this technology is reached if the cell type of interest constitutes 5/5% of the starting material.
An important aspect of clinical DC based vaccine therapy is the number of vaccination courses feasible from one leukapheresis procedure. Although the optimal number of ex vivo generated DC required for a functional vaccine is not established and probably will differ depending on the type of antigen used, nevertheless it is of interest to appreciate the absolute number of the product obtained using this platform. Starting from leukapheresis material comprising 2.0 /109 monocytes and taking into account the losses due to elutriation, differentiation, maturation and freeze/thaw the overall recovery was 3.6 /108 mDC. Initially, the platform was developed for culture in X-VIVO 15 serum-free medium. However, the presented
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results were obtained using CellGro DC serum-free medium, which in this culture system yields DC with superior properties in terms of phenotype, cell viability and recovery, especially after freeze/thawing (data not shown). The use of culture bags compared with open flasks has been discussed by others and reported as yielding superior [23] or equivalent quality of DC [25]. Our observation has been that bags minimize the surface activation of DC, resulting in truly imDC with 1% CD83 and /95% phagocytic DC by day 5. Two different substances for maturation were used, TNF-a (50 ng/mL) and poly I:C (50 mg/mL), supplemented for 48 h to cultured cells (day 6 /day 7). The latter was unavailable in GMP quality and therefore validated as pyrogen-free. At the end of day 7 of culture, cells matured with TNF-a expressed 100%, 90%, 100% and 62% CD80, CD86, HLA-DR and CD83, respectively. Expression of all these molecules was equal or even higher for CD86 and CD83 when poly I:C was used as the maturation signal, with sustained high viability. Compared with TNFa, poly I:C signaling induced significantly higher IL-12p70 production, which is functionally important for the skewing of the cellular response toward the Th-1 phenotype. mDC also showed higher stimulatory capacity and low phagocytic activity and, according to current knowledge, is well suited for vaccination purposes [10,19]. In summary, when optimized, the combination of leukapheresis and elutriation creates a flexible platform yielding large-scale high-grade /90% pure cell populations of monocytes and lymphocytes. The monocyteenriched fraction is well suited for further transfer and manipulation in culture bags by means of sterile docking and sealing devices that are available in blood banking units and approved for clinical use.
Acknowledgements This work was supported in part by the Cancer Society in Stockholm and the Swedish Cancer Society. Kia Heimersson and Volkan Ozenci are thanked for their technical assistance.
References 1 2
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3 Nestle FO. Dendritic cell vaccination for cancer therapy. Oncogene 2000;19:6673 /9. 4 Fong L, Engleman EG. Dendritic cells in cancer immunotherapy. Annu Rev Immunol 2000;18:245 /73. 5 Lundqvist A, Pisa P. Gene modified dendritic cells for immunotherapy against cancer. Med Oncol 2002;19:197 /211. 6 Kono K, Takahashi A, Sugai H et al . Dendritic cells pulsed with HER-2/neu-derived peptides can induce specific T-cell responses in patients with gastric cancer. Clin Cancer Res 2002;8:3394 /400. 7 Romani N, Gruner S, Brang D et al . Proliferating dendritic cell progenitors in human blood. J Exp Med 1994;180:83 /93. 8 Romani N, Reider D, Heuer M et al . Generation of mature dendritic cells from human blood. An improved method with special regard to clinical applicability. J Immunol Methods 1996;196:137 /51. 9 Morse MA, Mosca PJ, Clay TM et al . Dendritic cell maturation in active immunotherapy strategies. Expert Opin Biol Ther 2002;2:35 /43. 10 Jonuleit H, Giesecke-Tuettenberg A, Tuting T et al . A comparison of two types of dendritic cell as adjuvants for the induction of melanoma-specific T-cell responses in humans following intranodal injection. Int J Cancer 2001;93:243 /51. 11 Bernhard H, Disis ML, Heimfeld S et al . Generation of immunostimulatory dendritic cells from human CD34/hematopoietic progenitor cells of the bone marrow and peripheral blood. Cancer Res 1995;55:1099 /104. 12 Pretlow TG II, Pretlow TP. Centrifugal elutriation (counterstreaming centrifugation) of cells. Cell Biophys 1979;1:195 /210. 13 Svensson A, Engervall P, Soderstrom T et al . PBSC harvests individually optimized by using pre-collection CD34(/) values and on-line flow cytometric analysis of the mononuclear cell enrichment. Cytotherapy 1999;1:165 /74. 14 European Pharmacopoeia, 4th Edition 2002;140 /7. 15 Moser M, Murphy KM. Dendritic cell regulation of TH1-TH2 development. Nat Immunol 2000;1:199 /205. 16 Nestle FO, Alijagic S, Gilliet M et al . Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells [see comments]. Nature Med 1998;4:328 /32. 17 Murphy G, Tjoa B, Ragde H et al . Phase I clinical trial: T-cell therapy for prostate cancer using autologous dendritic cells pulsed with HLA-A0201-specific peptides from prostate-specific membrane antigen. Prostate 1996;29:371 /80. 18 Heiser A, Coleman D, Dannull J et al . Autologous dendritic cells transfected with prostate-specific antigen RNA stimulate CTL responses against metastatic prostate tumors. J Clin Invest 2002;109:409 /17. 19 Svane IM, Soot ML, Buus S et al . Clinical application of dendritic cells in cancer vaccination therapy. Apmis 2003;111:818 /34. 20 Nestle FO, Banchereau J, Hart D. Dendritic cells: on the move from bench to bedside. Nat Med 2001;7:761 /5. 21 Devitt A, Moffatt OD, Raykundalia C et al . Human CD14 mediates recognition and phagocytosis of apoptotic cells. Nature 1998;392:505 /9.
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22 Bernard J, Ittelet D, Christoph A et al . Adherent-free generation of functional dendritic cells from purified blood monocytes in view of potential clinical use. Hematol Cell Ther 1998;40:17 /26. 23 Guyre CA, Fisher JL, Waugh MG et al . Advantages of hydrophobic culture bags over flasks for the generation of monocyte-derived dendritic cells for clinical applications. J Immunol Methods 2002;262:85 /94.
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Development of a technology platform for largescale clinical grade production of DC L Adamson1, A Palmborg1, A Svensson2, A Lundqvist1, M Hansson2, R Kiessling1, G Masucci1, H Mellstedt1 and P Pisa1 1
Immune and Gene Therapy Laboratory, Department of Oncology & Pathology, Cancer Centrum Karolinska, and 2Immunohemotherapy Unit, Department of Clinical Immunology and Transfusion Medicine, Karolinska Hospital, Stockholm, Sweden
Background Clinical studies require protocols where a sufficient number of wellcharacterized highly immunogenic DC are produced according to good manufacturing practice (GMP) guidelines. Methods In the present study, using leukapheresis products from 10 cancer patients, we validated an elutriation technology for large-scale clinical grade production of monocyte-derived DC. Results The elutriation method gave a very high purity (mean9/SD) (86 9/5.3%) and recovery (669/10.4%) of monocytes. Specifically for the two monocyte-rich fractions (3 and 4,) the recovery was 429/13% of viable cells that could be further differentiated into immature DC in hydrophobic culture bags using GM-CSF and IL-4. The immature
Introduction DC have a unique ability to capture, process and present antigens, and thus possess the unique capacity to activate CD4 and CD8 naive T lymphocytes, leading to induction of primary immune responses [1,2]. DC derive their stimulatory potency from high constitutive and up-regulated expression of MHC class I, MHC class II and accessory molecules such as CD40, CD54, CD80, CD86 and T-cell activating cytokines [1,2]. These unique characteristics make DC a suitable candidate for immunotherapy against malignant and infectious diseases [3 /5]. Results obtained in animal studies and clinical trials confirm that DC are capable of inducing antigen-specific cytotoxic T-lymphocyte (CTL) responses, resulting in strong immunity to viruses and tumors and, in some
DC exhibited B/1% CD83 expression and /98% phagocytic activity. Maturation with TNF-a or poly I:C resulted in DC with expression of CD80 , CD86 and HLA-DR (/99%) and CD83 (809/11.9%), as well as producing IL-12p70 and lacking phagocytic activity ( B/5%). This cell product can be cryopreserved with cell viability /85% and cell recovery /80% after thawing. Discussion The elutriation procedure, when optimized and if the monocyte content of the starting material exceeds 5%, does not require further selection or depletion using affinity approaches. Keywords culture bags, elutriation, leukapheresis, maturation, monocytes, poly I:C.
instances, also objective regression of established disease [6]. DC exist in two functionally and phenotypically different stages, immature (im) and mature (m) DC [2]. imDC reside in peripheral tissue, where they capture antigens such as bacteria, viruses and damaged tissue. Upon exposure to pro-inflammatory cytokines or pathogen-derived products, DC mature, which is associated with a loss of phagocytic capacity and a gain in migratory potential to draining lymph nodes. mDC exhibit high antigenpresenting capability and T-cell stimulatory capacity. For clinical application, imDC are generally obtained after culture of monocytes for 5 /6 days in the presence of GM-CSF and IL-4 [7,8]. As imDC have a high phagocytic capacity, these cells are well suited for antigen loading
Correspondence to: Pavel Pisa, CCK R8:01, Karolinska Hospital, SE-171 76 Stockholm, Sweden. – 2004 ISCT
DOI: 10.1080/14653240410004934
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L Adamson et al .
using various protocols. mDC are obtained after further exposure to pro-inflammatory cytokines, for example tumor necrosis factor (TNF-a), products from microbial or viral pathogens, such as double-stranded RNA, lipopolysaccharide (LPS) or CpG DNA binding to Toll-like receptors (TLR), and ligation to T cells via CD40L [9]. In this way ex vivo generated mDC are ideal tools for cancer vaccination therapy because of the high expression level of antigen-presenting and costimulatory molecules for T-cell stimulation [10]. Clinical studies require protocols where a sufficient number of well-characterized highly immunogenic DC is produced according to good manufacturing practice (GMP) guidelines. Several approaches are currently in use for this purpose, from CD34 hematopoietic stem cells after mobilization with G-CSF, from peripheral blood using density gradients or from PBMC [7,11]. CD34 -derived DC are generally obtained using positive selection. Monocyte-derived DC are cultured from monocytes obtained from PBMC enriched by plastic adherence, positive or negative immunoselection or elutriation. The principle of the elutriation method is based on the physical separation of cells using a counter flow centrifugation, where cells are separated depending on their size and density (reviewed in [12]). In the present study we describe and validate such a technology platform using elutriation technology for large-scale clinical grade production of DC. The elutriation method gives a very high recovery of monocytes that can be further differentiated into imDC and mDC suitable for vaccination protocols.
(COBE BCT Laboratories, Lakewood, CO) with software 7.1 was used and all patients were leukapheresed using peripheral veins. According to a previously described routine [13], a small sample, taken from the collect line early during the leukapheresis, was analyzed for degree of monocyte enrichment and monocyte concentration. The result of this sample was used to adjust the machine to obtain a maximal monocyte harvest ( /15%) and to decide the length of the apheresis procedure for collection of the targeted number of leukocytes (A Svensson et al ., manuscript submitted for publication). The amount of processed blood was [median (minimum /maximum)] 8.8 L (6.7 /10.4) L. None of the patients suffered any serious side-effects from the procedure.
Counterflow centrifugal elutriation (elutriation)
Ten patients treated for their malignancies at the Karolinska Hospital (Stockholm, Sweden; six with prostate cancer, three with melanoma and one with CLL), after informed consent, donated leukocytes using a leukapheresis procedure. The study was approved by the local ethics committee of the Karolinska Hospital, conforming to the guidelines of the Helsinki Protocol.
The leukapheresis product was separated in a blood centrifuge (Hettich Roto Silenta, Tuttingen, Germany) at 540 g for 6 min. After removing the platelet-rich plasma the leukapheresis product was diluted in PBS, pH 7.2 (CE0123; Baxter, Healthcare, Deerfield, IL), with 5 mM EDTA (Invitrogen, Carlsbad, CA), to approximately 5 /106 leukocytes/mL and subjected to elutriation using a Beckman-Coulter (Miami, FL) Avanti J20XPI centrifuge with JE-5.0 rotor and 40-mL chamber (#356940). Loading of cells was performed in a closed system at 2000 rpm (385 g ) at a flow rate of 53 mL/min, 208C. Cell fractions (approximately 1500 mL) were collected in bags (Teruflex transfer bag; Terumo, Tokyo, Japan) at a constant flow rate of 53 mL/min of PBS, pH 7.2 (3000-mL bags; Baxter), 2 mM EDTA (Invitrogen) using a peristaltic pump (Masterflex L/S; Cole-Parmer Instrument Company, Vernon Hills, IL). Elutriation of the different fractions was performed by changing the rotor speed as follows: fraction 1 was collected at 2000 rpm (385 g), fraction 2 at 1800 rpm (312 g), fraction 3 at 1700 rpm (278 g), fraction 4 at 1600 rpm (247 g) and fraction 5 rotor off (RO) at 0 rpm (247 /0 g ). After optimization of the elutriation procedure the use of EDTA in the fluid was proven unnecessary and therefore excluded from the preparation.
Leukapheresis
Chamber-cleaning procedure
Leukapheresis procedures were performed according to standard clinical routines at the Immunohemotherapy Unit, Department of Clinical Immunology and Transfusion Medicine, Karolinska Hospital. The Cobe Spectra
The rotor chamber was extensively cleaned with antimicrobial/antiviral substances, 70% ethanol and Virkon (CE0086; Antec Int., Sudbury, UK). Samples were taken before and after cleaning for CMV-PCR (Department of
Methods Human subjects
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Clinical Microbiology, Karolinska Hospital). After assembly of the rotor to a closed system, sterilization and equilibration, samples were taken for pyrogene analysis (Limulus test; Karolinska Hospital Pharmacy). All cell fractions were tested for sterility and the final DC product also tested by Limulus before cell banking.
CD45-FITC and CD14-PE. Monocytes and mature DC were assayed for phenotype using CD80-FITC, CD83-PE, CD86-PE, CD1a-PC5, ILT3-PC5 and HLA-DR-FITC. MAb and corresponding isotype-labeled Ig controls were all from Beckman-Coulter. 7AAD-positive (non-viable) cells were excluded from FCM analysis.
Cell culture
Mixed leukocyte reaction
The elutriated fractions 3 and 4 were centrifuged (5700 g for 5 min) and, after the supernatant was removed, the cells transferred from the transfer bags into cell culture bags by sterile welding (welding set; Tamuro, Tokyo, Japan). Resuspended cells were cultured in 85 cm2 /180 cm2 cell culture bags (Lifecell OptiCyte Cell Culture containers; Baxter) at a cell density of 2.5 /5 /106 cells/mL, in CellGro DC serum-free medium (Cell Genix, Freiburg, Germany) supplemented with 100 ng/mL GM-CSF (Leukine; Immunex, Seattle, WA) and 20 ng/mL IL-4 (Schering-Plough, Kennilworth, NJ). Fresh medium, half the volume, including twice the concentration of cytokines, was added after 2 days. After 5 days of culture cells were resuspended in new medium, CellGro DC supplemented with GM-CSF, IL-4 and 50 ng/mL TNF-a (Chiron, Emeryville, CA), or GM-CSF, IL-4 and 50 mg/mL poly I:C (Sigma-Aldrich, St Louis, MO), for an additional 48-h maturation. On day 7 cell viability and recovery were determined by trypan blue dye exclusion and 7AAD staining (Beckman-Coulter). Cells were cryopreserved at 5/10 /106 cells/mL ampoules (Nunc, Roskilde, Denmark), in 90% autologous plasma and 10% DMSO (Sigma-Aldrich). The plateletrich autologous plasma separated from the leukapheresis product was centrifuged at 5700 g and platelets removed by transferring of the plasma to another bag using a plasma extractor. The plasma was frozen, heat-inactivated at 568C for 60 min and centrifuged at 5700 g for 5 min (Hettich Roto Silenta) and transferred into another bag using a plasma extractor.
Primary allogeneic MLR was performed in serum-free CellGro medium, with the non-adherent fraction of Ficoll-separated PBMC from a buffy-coat from a healthy donor at 105 cells/well in 96-well U-bottomed plates at different responder/stimulator ratios, ranging between 10:1 and 1250:1. After 5 days, 1 mCi of H3-thymidine (Amersham Biosciences, Amersham, UK) was added to each well and incubated for 18 h. Cells were harvested and measured in a scintillation counter (1450 MicroBetaTM; Wallac, Turku, Finland).
Flow cytometry analysis Flow cytometry (FCM) acquisition and analysis was performed using EPICS XL (Beckman-Coulter) with Coulter System II software. Whole blood and the leukapheresis material were analyzed to identify cell distribution between lymphocytes, monocytes, granulocytes, T cells, B -cells and natural killer (NK) cells, using directly conjugated MAB for CD3-PC5, CD19-ECD, CD56-PE,
FITC dextran uptake /
imDC and mDC were plated in separate wells in 24-well plates at 106 cells/mL in 500 ml volume/well. After 48 h FITC /dextran (Sigma-Aldrich) was added to a final concentration of 1 mg/mL and incubated for 1 h at / 378C or at /48C. Harvested cells were washed with icecold PBS, fixed in 4% formaldehyde and stained with antiCD83-PC5 (Beckman-Coulter). FITC/dextran uptake and CD83 expression were analyzed by FCM using a FACS Calibur (Becton-Dickinson, Franklin Lakes, NJ).
Cytokine production assay Supernatants from DC cultures were collected 48 h after addition of maturation agent and analyzed for IL-12p70 by ELISA (Mabtech AB, Stockholm, Sweden).
Results The aim of this work was to validate a technology platform for generation of autologous DC from patients with different cancer diagnoses that were suitable for further ex vivo manipulation to produce clinical grade cancer vaccines (Figure 1). The starting material was nonmobilized leukapheresis products from 10 cancer patients (six prostate, three melanoma and one CLL) that was elutriated, cultured ex vivo for 7 days, frozen and thawed. All cell product manipulation was performed in a closed system, to conform to the current European regulatory policy.
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Figure 1. A flow chart of the technological platform.
fraction 5 at 0 rpm was heavily enriched in granulocytes, 759/25%. Starting with 21.19/7.1 /108 monocytes, the yield of monocytes after elutriation was 14.09/4.5 /108 cells. The total recovery of elutriated viable cells was 849/6.8% and the viability exceeded 95%, as measured by FCM and 7AAD staining (Figure 3a). The recovery of the monocyte population was 669/10.4%, with viability greater than 90%. The yield of the two main monocyte-rich fractions 3 and 4 was 8.89/2.8 /108 monocytes, resulting in a practically usable yield of 429/13%. The maximum cell number in the starting material loaded, giving similar results, was 4 /1010 cells.
Elutriation The time of the elutriation procedure was 135 min. The different cell populations were collected in five different fractions in a volume of 1500 mL per transfer bag and analyzed by FCM for CD14 cells vs. CD45 and CD45 vs. side scatter characteristics (SSC). Although the differential blood cell count varied among the patients (data not shown) and therefore the proportions of the cell populations also differed in the starting material, the individual fractions after elutriation exhibited little variation (Figure 2). The fractions with the highest monocyte content were fraction 4 at 1600 rpm, and fraction 3 at 1700 rpm, containing (mean9/SD) 869/9.4% and 809/11.9%, respectively. The low density fraction 1 at 2000 rpm consisted of pure lymphocytes, 969/5.5%, while the final
Cell culture After 5 days of differentiation of fractions 3 and 4, the recovery of imDC was 759/9.6%, and after TNF-a maturation for an additional 48 h, 539/12.3% (Figure 3b). The overall recovery of practically usable (from fractions 3 and 4) imDC and mDC was 32.09/9.6% and 22.09/12.3%, respectively. Viability of cultured cells was 959/4% and 94%9/6%, for imDC and mDC, respectively. Recovery and viability from cultures matured with poly I:C were similar to those obtained with TNF-a (data not shown). In initial experiments, X-VIVO 15 (BioWhittaker, Rockland, ME) was used. However, the viability and recovery were higher using CellGro DC (data not shown)
Figure 2. Cell populations after elutriation detected by flow cytometry for CD45 cells vs. SSC. Results (mean9/SD) from 10 cancer patients (six prostate, three melanoma, one CLL). (The same monocyte profile was obtained using CD14 /CD45 ).
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present on monocytes, disappeared on imDC and appeared again after 48 h maturation into mDC. The mDC exhibited better stimulatory capacity in all MLR than their immature counterparts (Figure 5e). IL-12p70 is considered to be one of the key T helper-1 (Th-1) polarizing cytokines [14]. IL-12p70 was produced to very low extent by imDC15, or in presence of TNF-a, and increased five-fold upon culture in poly I:C containing medium (Figure 4b).
Freeze thawing of cells /
Figure 3. Recovery (mean9/SD) (open bar) and viability (black bar) prior and post culture (n /10). (a) Analysis of elutriated total (Tot) cells and monocytes (Mo). (b) Cells cultured for 5 days (immature) and after addition of TNF-a (mature), in total and in practically usable fractions 3 and 4 (F3/4).
and therefore the results reported here were all performed with CellGro DC. Contaminating agents or pyrogens have not been detected in any elutriated material (data not shown) assayed by standard routines and controls used at Karolinska Hospital and by the Limulus clotting test according to the European Pharmacopoeia [14].
Phenotype of DC Kinetics of surface antigen expression were analyzed for CD80, CD83, CD86, CD1a, ILT3 and HLA-DR (Figure 4a). The phenotypic variability between DC from different cancer patients was subtle. Expression of CD80 and CD1a increased during differentiation and maturation with TNF-a and poly I:C, while CD83 was absent on monocytes and on day 7 imDC and appeared only if a maturation signal was delivered. The majority of imDC and mDC exhibited CD86 and HLA-DR. Expression of the ILT3 molecule appeared to be bi-phasic, as it was
Cell viability after freezing and thawing exceeded 85%, and recovery 80%. The functionality of frozen and thawed cells was tested by endocytosis of FITC/dextran, which demonstrated phagocytic activity in more than 95% of imDC (B/1% CD83 positive; Figure 5b) compared with less that 5% in mDC (65% CD83 positive with poly I:C; Figure 5d). The freezing and thawing procedure did not affect the phenotype of the cells, including IL-12p70 production (data not shown), nor their stimulatory capacity in MLR (Figure 5e) or endocytotic capacity assayed by FITC /dextran (Figure 5a /d). Preliminary data demonstrated induction of a HLA-A2-restricted peptide-specific IFN-g T-cell response, when stimulated with DC after thawing (assayed by Elispot; data not shown).
Discussion The current enthusiasm in clinical application of immunotherapy with DC-based vaccines for cancer has been inspired by the recent clinical successes of early phase trials with DC [6,16 /18]. The methodological aspects of these studies in terms of source of cells, technologies of preparation, culture conditions, antigen loading and degree of maturation vary significantly and it is therefore difficult to find a general explanation for why only 10 /30% of enrolled patients respond to this therapy [19]. Based on the past few years of development in the field, it has been proposed that a prerequisite for clinical grade DC preparation should be not only a GMP facility but also quality control of the DC preparation, including the DC isolation procedures, in vitro growth conditions, DC yield, purity and viability, cryopreservation, as well as phenotypic and functional characteristics [20]. Obstacles in translating research protocols for cellular therapy into clinical application are numerous. The obvious objective is to maintain the biological properties
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Figure 4. Phenotype and IL-12p70 production. (a) Phenotype of cells before and after maturation with TNF-a or poly I:C detected by flow cytometry (n /10). (b) IL-12p70 levels measured by solid-phase ELISA (n /3).
of the cell product, while incorporating the consistency, reproducibility and safety of the procedure required not only by the regulatory authorities but also by the treating physician. Furthermore, cell therapeutics are still at a stage of experimental development and therefore the production system needs a great deal of flexibility to permit different approaches, and the economical aspects of the procedure are equally important. Centers worldwide using immunotherapeutic concepts differ in their potential to use open separation and culture systems, such as adherence and tissue culture flasks, or closed systems, such as immunomagnetic separation and permeable culture bags. Affinity-based separation systems for clinical grade monocyte separation are currently commercially available only outside the USA and only
for positive CD14 selection (Miltenyi Biotec, Bergisch Gladbach, Germany). As the CD14 molecule possesses some important activation signaling capacities, this approach may disturb the further differentiation process [21]. For this reason many centers introduced the elutriation procedure, despite of the fact that it is a semi-closed system, because it requires assembly and sterilization prior to each procedure [22 /24]. We describe the validation of this relatively inexpensive, GMP friendly and rapid technology platform, which when optimized both at the leukapheresis step and elutriation step yields large-scale, high-purity DC that are suitable for further manipulation, using different methods of antigen loading and maturation, for use in cancer vaccination protocols. An additional aspect of the flexibility of the system is that the 95% pure
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Figure 5. Phagocytosis and stimulatory capacity in MLR. After thawing imDC were incubated for 1 h at /48C (a) and at /378C (b) with FITC /dextran (FL1-H) and stained for CD83 (FL3-H). mDC were incubated for 1 h at /378C with FITC/dextran and stained for CD83, after maturation with either TNF-a (c) or poly I:C (d). Numbers depict percentage of cells in the four quadrants. Results are representative of three separate experiments. (e) MLR with 105/well allogeneic PBMC as responders, stimulated with graded numbers of mature and immature DC (ratios ranging between 10:1 and 1250:1). DC only showed B/200 cpm.
lymphocyte fraction may be used further in adoptive immunotherapeutic protocols. We have not accounted for the necessity of further depletion of certain cell populations in the elutriated cell fraction, as sometimes suggested [25]. In fact, even patients with CLL, with a peripheral differential blood cell count of 8% monocytes, were successfully elutriated, with 88% monocyte purity in fraction 4 at 1600 rpm (a patient in this study). However, when in another CLL patient the starting material consisted of 98% lymphocytes and 1 /2% monocytes the final yield in fraction 4 at 1600 rpm was only 62% monocytes. The limit of this technology is reached if the cell type of interest constitutes 5/5% of the starting material.
An important aspect of clinical DC based vaccine therapy is the number of vaccination courses feasible from one leukapheresis procedure. Although the optimal number of ex vivo generated DC required for a functional vaccine is not established and probably will differ depending on the type of antigen used, nevertheless it is of interest to appreciate the absolute number of the product obtained using this platform. Starting from leukapheresis material comprising 2.0 /109 monocytes and taking into account the losses due to elutriation, differentiation, maturation and freeze/thaw the overall recovery was 3.6 /108 mDC. Initially, the platform was developed for culture in X-VIVO 15 serum-free medium. However, the presented
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results were obtained using CellGro DC serum-free medium, which in this culture system yields DC with superior properties in terms of phenotype, cell viability and recovery, especially after freeze/thawing (data not shown). The use of culture bags compared with open flasks has been discussed by others and reported as yielding superior [23] or equivalent quality of DC [25]. Our observation has been that bags minimize the surface activation of DC, resulting in truly imDC with 1% CD83 and /95% phagocytic DC by day 5. Two different substances for maturation were used, TNF-a (50 ng/mL) and poly I:C (50 mg/mL), supplemented for 48 h to cultured cells (day 6 /day 7). The latter was unavailable in GMP quality and therefore validated as pyrogen-free. At the end of day 7 of culture, cells matured with TNF-a expressed 100%, 90%, 100% and 62% CD80, CD86, HLA-DR and CD83, respectively. Expression of all these molecules was equal or even higher for CD86 and CD83 when poly I:C was used as the maturation signal, with sustained high viability. Compared with TNFa, poly I:C signaling induced significantly higher IL-12p70 production, which is functionally important for the skewing of the cellular response toward the Th-1 phenotype. mDC also showed higher stimulatory capacity and low phagocytic activity and, according to current knowledge, is well suited for vaccination purposes [10,19]. In summary, when optimized, the combination of leukapheresis and elutriation creates a flexible platform yielding large-scale high-grade /90% pure cell populations of monocytes and lymphocytes. The monocyteenriched fraction is well suited for further transfer and manipulation in culture bags by means of sterile docking and sealing devices that are available in blood banking units and approved for clinical use.
Acknowledgements This work was supported in part by the Cancer Society in Stockholm and the Swedish Cancer Society. Kia Heimersson and Volkan Ozenci are thanked for their technical assistance.
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