Development of a clinical-scale method for generation of dendritic cells from PBMC for use in cancer immunotherapy

Cytotherapy (2001) Vol. 3, No. 1, 19–29

Development of a clinical-scale method for generation of dendritic cells from PBMC for use in cancer immunotherapy ECC Wong1, VE Maher2, K Hines1, J Lee1, CS Carter1, T Goletz3, W Kopp4, CL Mackall3, JA Berzofsky2 and EJ Read1 1

2

Department of Transfusion Medicine, Warren G. Magnuson Clinical Center, Metabolism Branch, National Cancer Institute, and 3Pediatric Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA and 4 SAIC, Frederick, Maryland, USA

Background

generated most consistently in RPMI 1640 supplemented with 10%

There is growing interest in the use of dendritic cells (DCs) for treatment of malignancy and infectious disease. Our goal was to develop a clinicalscale method to prepare autologous DCs for cancer clinical trials.

allogeneic AB serum or 10% autologous plasma. Cell yield was higher at Day 5 than Day 7, without detectable differences in phenotype or function. In pediatric sarcoma patients, autologous DCs had enhanced

Methods

function compared with monocytes from which they were generated. In this patient group, starting with 8.0 ± 3.7 3 108 fresh or cryopreserved autologous monocytes, DC yield was 2.1 ± 1.0 3 108 cells, or 29% of

PBMC were collected from normal donors or cancer patients by automated leukapheresis, purified by counterflow centrifugal elutriation and placed into culture in polystyrene flasks at 1 3 106 cells/mL for 5–7 days at 37oC, with 5% CO2, with IL-4 and GM-CSF. Conditions

the starting monocyte number.

investigated included media formulation, supplementation with heatinactivated allogeneic AB serum or autologous plasma and time to harvest (Day 5 or Day 7). DCs were evaluated for morphology, quantitative

In the optimized clinical-scale method, purified peripheral monocytes are cultured for 5 days in flasks at 1 3 106 cells/mL in RPMI 1640, 10% allogeneic AB serum or autologous plasma, IL-4 and GM-CSF.

yield, viability, phenotype and function, including mixed leukocyte response and recall response to tetanus toxoid and influenza virus.

This method avoids the use of FBS and results in immature DCs suitable for clinical trials.

Results

Keywords

DCs with a typical immature phenotype (CD14-negative, CD1a-positive, mannose receptor-positive, CD80-positive, CD83-negative) were

dendritic cells (DCs), peripheral blood monocytes, immunotherapy.

Introduction

There are currently two general approaches used for large-scale ex vivo culture of DCs for clinical use [5,6]. In the first, a DC population is generated by expansion and differentiation of a CD34+ monocyte precursor population from BM or peripheral blood, using granulocyte-macrophage colony-stimulating factor (GMCSF), tumor necrosis factor a (TNF-a) and other factors. If peripheral blood is used as the cell source, this method requires a selection step for isolation of the CD34+ cell

Dendritic cells (DCs) are migratory cells that capture, process and present foreign Ags to the immune system [1]. The primary activation of T-cells by DCs is a critical step in the development of specific T-cell cytotoxic responses [2,3]. In recent years, identification and in vitro culture of DCs have led to a large number of animal and human studies using DCs as immunological adjuvants in the treatment of cancer [4].

Discussion

Correspondence to: Elizabeth J. Read, Department of Transfusion Medicine, Warren G. Magnuson Clinical Center, National Institutes of Health, Bethesda, MD 20892, USA. © 2001 ISHAGE

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population from a cytokine-mobilized leukapheresis collection. In the second approach, DCs are generated in cultures containing IL-4 and GM-CSF [7] starting with PBMC containing monocytes, which may be either unseparated or purified. This latter approach was chosen because of: n The ready availability in our institution of established methods for isolating large numbers of relatively pure monocytes by countercurrent centrifugal elutriation of leukapheresis collections. n Our interest in isolating and retaining the lymphocyte population from the same collection. n Access to the cytokines IL-4 and GM-CSF in clinicalgrade forms. The purpose of this work was to develop a method for generating autologous DCs from cancer patients that could be subjected to further manipulation, i.e. peptide pulsing, prior to i.v. infusion. A major emphasis was to identify clinical-grade media and reagents, including human serum protein sources, that could be used as effective substitutes for the FBS commonly used to generate DCs in research laboratories. Although our ultimate goal is to design closed clinical-scale systems, the primary strategy in the current study was to mimic the small-scale culture system as closely as possible; therefore we employed flasks rather than bags. Furthermore, although this initial development was aimed at generating DC suspensions without a priori specification of the maturity of the cell populations, we did anticipate that, without the use of agents such as CD40 ligand or TNF-a, the DCs generated would most likely have an immature phenotype, as defined in recent scientific literature [1].

Methods Human subjects Normal healthy donors and patients with pediatric sarcoma gave informed consent to leukapheresis procedures done as specified in clinical protocols approved by the Institutional Review Board of the National Cancer Institute.

Leukapheresis and counterflow centrifugal elutriation Leukapheresis procedures were done using the CS3000 Plus (Fenwal Division, Baxter, Deerfield, IL) which processed a 7.5 L blood volume to collect a total of 5–10

3 109 MNC. MNC were suspended in HBSS without magnesium, calcium and phenol red (BioWhittaker, Walkersville, MD), and subjected to counterflow centrifugal elutriation using a Beckman J-6M centrifuge equipped with a JE 5.0 rotor (Beckman Instruments, Palo Alto, CA) operating at 1725 g at 20°C. Cell fractions (450–550 mL each) were collected at flow rates of 120, 140 and 190 mL/min during centrifugation, and at 190 mL/min with the rotor off (RO). The last two fractions have been previously shown in our laboratory to be enriched for CD14+ monocytes, with a mean purity of 70% and a mean recovery of 80%, and have also been shown to be enriched for cells with an immature dendritic cell phenotype [8]. Monocyte-enriched fractions (30 and 100 3 106/mL cells) were either placed directly into culture or were cryopreserved using controlled-rate freezing in 10% DMSO (Cryoserv, Research Industries, Salt Lake City, UT), 15 U/mL heparin (Fujisawa USA, Deerfield, IL), 10 µg/mL DNAase (Genentech, Emeryville, CA), and 50% allogeneic AB serum from a single normal donor or 50% autologous plasma. The lymphocyteenriched fractions (120 and 140 mL/min) were also cryopreserved by the same method. Autologous plasma collected into a blood bag during the leukapheresis procedure was heat-inactivated at 55oC for 60 min, centrifuged at 4650 g for 6.5 min, expressed into another bag using a plasma extractor, filtered, and stored at –30oC. In a similar manner, heat inactivation was carried out on allogeneic AB serum, which had been obtained either from a pooled donor commercial source (BioWhittaker, Walkersville, MD) or from the plasma of a single normal donor that had been converted to serum using 100 units of human thrombin (Jones Pharma, Inc., St Louis, MO) and 1 mL of 10% calcium chloride (Fujisawa USA, Inc., Deerfield, IL) per 100 mL plasma.

Cell culture Thawed or fresh normal donor monocytes were cultured in 75 cm2 polystyrene Falcon flasks (Becton Dickinson Labware, Franklin Lakes, NJ). After completing the pilot work on normal donor cells, thawed or fresh monocytes from sarcoma patients were cultured in T-162 polystyrene flasks (Sarstedt, Newton, NC), which have a larger volume and surface area; these flasks can accommodate up to 150 mL of cell suspension. Cultures were supplemented with 10 µg/mL gentamicin (GIBCO Laboratories, Grand Island NY) and clinical grade

Development of a clinical-scale method for generation of DCs from PBMC for use in cancer immunotherapy

recombinant cytokines, human IL-4 and GM-CSF (kindly provided by Schering-Plough, Kenilworth NJ). Culture media evaluated included RPMI 1640 supplemented with L-glutamine, XVIVO 10 and XVIVO 20 (BioWhittaker, Walkersville, MD), and AIM-V (GIBCO Laboratories, Grand Island, NY). Cytokines were added every 2–3 days without changing the culture medium. After 5–7 days at 37oC in a 5% CO2 incubator, cells were harvested by pipetting medium over loosely-adherent cells and gently agitating the container. After centrifugation and resuspension in HBSS (BioWhittaker, Walkersville, MD), cells were aliquotted for cytospin preparation and Wright-Giemsa staining, flow cytometry and functional assays. Tissue culture supernatants were assayed for pH using an Orion Research digital ionalyzer (Orion Research, Beverly, MA), and for glucose and lactate using the ACA Star (Dade International, Newark, DE). Automated cell counts were obtained from the CellDyn 3500 (Abbott Diagnostics, Santa Clara, CA) using impedance gating. Viability was determined by Trypan blue dye exclusion. Percent DC yield was calculated as the number of viable DC harvested divided by the initial number of viable monocytes placed into culture 3 100%. After pilot experiments evaluating culturing at different cell concentrations, all cultures, unless otherwise stated, were seeded at 1 3 106/mL in the presence of 2000 units/mL IL-4 and 2000 units/mL GM-CSF.

Flow cytometric analysis Samples of 100 µL containing 100 000 to 200 000 cells were incubated for 10 min with 100 µL of an ice-cold 1:5 dilution of reconstituted IVIgG (Sandoglobulin, Sandoz Pharmaceuticals Corp, East Hanover, NJ) in HBSS. Cells were then vortexed and stained with fluorochromelabeled Abs to the following cell-surface markers: MHC Class I, MHC Class II, CD14, CD54, CD83, CD86, IgG2b, IgG2a (Caltag Laboratories, Burlingame CA), IgG1, mannose receptor (Pharmingen, San Diego, CA), CD1a (Immunotech, Marseille, France), CD80 (Becton Dickinson Immunocytometry Systems, San Jose, CA). 7AAD (Pharmingen, San Diego, CA) was used to identify non-viable cells. Flow cytometry acquisition and analysis were performed using a FACSort with CellQuest software (Becton Dickinson, Mountain View, CA). Cellsurface marker analysis was performed by identifying and gating on a cell population with high forward and side

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scatter that had high intrinsic autofluorescence and excluded 7AAD, and then quantitating the number of cells in the gated population that expressed the single marker. The percentage of cells expressing that marker was calculated as the number of cells expressing the marker divided by the total number of cells in the gated population 3 100%.

Functional assays Allogeneic mixed leukocyte response (MLR): After cell harvest, a sample of the normal donor or pediatric sarcoma patient DC was centrifuged and resuspended in MLR medium, which consisted of RPMI 1640 supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, non-essential amino acids, penicillin, streptomycin and 10% pooled human AB serum. The cell suspension was irradited (3000 Rad) and seeded in 96-well, flat bottom Costar plates (Corning, Corning, NY) at densities of 20 000 to 625 cells in the presence of 200 000 allogeneic mononuclear cells. Thawed monocytes from the same donor were used as controls. Mononuclear responder cells were obtained from density-gradient separation of buffy coats and each DC preparation was tested against responder cells from two different donors. After 5 days in a 37oC 5% CO2 incubator, cultures were pulsed with 1 µCi/well with methyl 3H-thymidine (248 GBq/mmol, New England Nuclear Life Science Products, Boston, MA) and harvested after 18 h using an automated cell harvester. Incorporated 3H-thymidine counts were determined by liquid scintillation counting using a MicroBeta TriLux liquid scintillation counter (Wallac, Gaithersburg, MD). Autologous lymphocyte recall response to specific antigens: Thawed autologous lymphocytes, 100 µL adjusted to 2 3 106/ml in MLR medium, were added to 96-well, flat bottom plates containing 100 µL of MLR medium with 20 000 to 625 previously irradiated monocytes or dendritic cells. 20 µL of influenza virus (strain JAP, J. Berzofsky) diluted 1:1000 (1:10 000 final concentration) or 40 LF units/mL tetanus toxoid (4 LF units/mL final; Connaught Laboratories, Toronto, Canada) were added to each well. Controls for the background response of the responder cells to the recall Ag included incubating Ag with irradiated DC alone. Culture, harvest and determination of incorporated 3Hthymidine counts were performed as in the allogeneic MLR. Relative potencies of the allogeneic MLR and

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autologous lymphocyte recall responses were estimated by graphic examination of the differences in the stimulator:responder ratios between the monocyte and DC curves, in the parallel areas of the curves, at the same levels of tritiated thymidine incorporation.

Results Identification and characterization of DC by morphology and flow cytometry Identification of DC relied on inverted phase microscopy, conventional microscopy of Wright–Giemsa stained cytospin preparations and flow cytometric analysis. Initial studies were done on DC grown in RPMI 1640 at 1 3 106/mL in 10% autologous plasma supplemented every 2–3 days with 2000 units/mL IL-4 and 2000 units/mL GM-CSF. Under these conditions, conversion of adherent monocytes to cells with typical DC morphology, i.e. greatly increased size and multiple dendritic projections, could be seen by Day 5 by inverted phase microscopy (Figures 1a and 1b) and on Wright–Giemsa stained cytospins (Figures 2a and 2b). These morphologic findings were accompanied by the development, on flow cytometry, of a high forward and side-scatter population that excluded 7AAD; this population was used to define a DC gate for further phenotypic analysis. The

a

high degree of autofluorescence of the cultured DC population was also found to be useful for identifying DC on flow cytometric analysis. Flow cytometric scattergrams illustrating identification of thawed monocytes and cultured DC are presented in Figure 3. Compared with uncultured monocytes, Day 7 DC had a marked decrease in CD14 expression, a marked increase in mannosereceptor expression, and increased expression of the costimulatory molecules B7.1 (CD80) and B7.2 (CD86), I-CAM 1 (CD54), MHC Class II, and CD1a (Figure 4). Expression of MHC Class I and CD83 were not substantially changed compared with monocytes.

Effect of different media formulations and protein supplementation on DC phenotype in normal donor cultures RPMI 1640 with 2% or 10% autologous plasma was most effective in generating a CD1a-positive, CD14-negative DC population. AIM V and XVIVO 20 also generated increased CD1a expression, but were associated with relatively higher expression of CD14 compared to RPMI 1640. Culture in XVIVO 10 failed to increase expression of CD1a and, like AIM V and XVIVO 20, resulted in relatively high CD14 expression. Using a second donor, a cell concentration of 2 3 106/mL, and allogeneic serum, we

b

Figure 1. Inverted phase microscopy showing (a) freshly thawed monocytes placed into culture medium (RPMI 1640, 10% autologous plasma, and cytokines IL-4 and GM-CSF) in polystyrene Falcon flasks for 4 h, and (b) 7 days later, DCs cultured from the same monocytes in the same medium in polystyrene Falcon flasks, with IL-4 and GM-CSF added every 2–3 days.

Development of a clinical-scale method for generation of DCs from PBMC for use in cancer immunotherapy

a

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b

Figure 2. Wright–Giemsa stained cytospin preparations showing (a) freshly thawed monocytes, and (b) 7 days later, DCs harvested from flask culture of monocytes in RPMI 1640, 10% autologous plasma, IL-4 and GM-CSF. obtained similar surface marker expression patterns with the various media formulations. Based on these findings, RPMI 1640 was chosen as the culture medium. Although use of higher concentrations (10% versus 2%) of autologous plasma tended to result in higher recovery of viable dendritic cells at Day 7 (mean ± SD = 25.4 ± 8.1% versus 12.6 ± 8.4%), these differences were not statistically significant by Student’s t testing. We also observed a fair amount of variability in the time-course of surfacemarker expression for mannose receptor, CD1a, CD80, CD83, CD86 and MHC Class II receptor among the different sources of human protein tested (pooled AB serum, single donor AB serum and autologous plasma). However, this variability was relatively low by Day 7, and was not accompanied by any differences between AB serum and autologous plasma in functional assays (MLR and recall response to Ags).

Effect of culture duration on cell recovery, pH, glucose and lactate in normal donor cultures Figure 5 shows the effect of culture duration on cell recovery, pH, glucose and L-lactate in DC cultures containing either 10% allogeneic AB serum or 10% autologous plasma. Over the 7 day culture period, there were consistent decreases in percent recovery of viable cells, pH, and glucose, and an increase in L-lactate. By

Day 7, for AB serum and autologous plasma-containing cultures, there was a viable cell recovery of 7.8–24.1%, a 0.15–0.22 unit decrease in pH, a 34–44% decrease in glucose, and a 5.4–7.1 mmol/L increase in L-lactate. The changes over the last 48 h of culture were used as the basis for deciding that the optimal time of harvest should be 5 days: between Day 5 and Day 7, there were marked decrements in viable cell recovery, from 26.1 ± 8.6% to 13.2 ± 4.4% (mean ± SD).

Generation of autologous DC from monocytes of pediatric sarcoma patients using allogeneic AB serum or autologous plasma Prior to clinical implementation of large-scale culture of autologous DCs from pediatric sarcoma patients, monocytes from two patients with extensive disease were thawed and placed into culture for 5 days in the presence of 10% allogeneic AB serum or 10% autologous plasma. Regardless of protein source, Day 5 cultures showed typical DC morphology by phase microscopy and Wright–Giemsa stained cytopsins and flow cytometric analysis showed appearance of a cell population with low expression of CD14 and increased expression of CD1a, CD54, CD80, CD86, mannose receptor and MHC Class II receptor. Figure 6 shows representative results of MLR and recall Ag responses to influenza virus and tetanus

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Figure 3. Flow cytometric identification of monocytes and DCs, using forward scatter (FSC), side scatter (SSC), and the viability stain 7AAD. Panel (a) shows FSC and SSC characteristics of an ungated population of freshly-thawed purified peripheral blood monocytes. Panel (b) shows a marked increase in FSC and SSC in the ungated population of Day 7 DCs cultured from the monocytes in panel (a). In panel (c), within the ungated population of Day 7 dendritic cells, a gate (R1) is drawn around a population with high FSC and low 7AAD uptake. Panel (d) shows the FSC and SSC characteristics of the DC population gated in R1 (panel (c)); this population was further evaluated for expression of single cell surface markers (see Figure 4). toxoid for the autologous DCs and monocytes from one patient treated on the clinical trial. For all three functional measures, the dendritic cells were 8 to 16-fold more active than the monocytes, based on the number of cells required to achieve the same response.

Application of large-scale method to Phase I/II clinical trial in pediatric sarcoma patients Table 1 summarizes the quantitative cell recovery and viability data from our experience with large-scale generation of autologous DC from PBMC, for an ongoing Phase I/II clinical trial in pediatric sarcoma patients. To date, a total of 20 ex vivo culture procedures, 11 with

autologous plasma and nine with allogeneic AB serum, have been performed for 10 different patients. In all but two cultures, the monocytes underwent cryopreservation and thawing before culture. The total cell content in the starting monocyte aliquots ranged from 1.7 –13.4 (mean 8.0) 3 108 cells, and the number available for culture after thawing was 1.0–9.8 (mean 5.8) 3 108 cells, which represented a mean post-thaw recovery of 71%. Harvest of the cultures occurred on Day 5 for 18 and on Day 6 for two of the cultures. The final DC yield was 0.3–3.7 (mean 2.1) 3 108 cells, representing an overall yield from the original monocyte aliquot to DC harvest of 10–64% (mean 29%). Flow cytometric analysis at the time of harvest

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Figure 4. (left) Representative single-cell surface-marker histograms (shown in magenta, with isotype controls shown in blue) from flow cytometric analysis of a normal donor’s DCs and the monocytes from which they were cultured for 7 days. Compared to the monocyte population (top row), the DC population (bottom row) showed increased autofluorescence, loss of CD14 expression, and increased expression of CD1a, CD54, CD80, CD86, MHC Class II, and mannose receptor. CD83 expression was only slightly enhanced and there was no change in expression of MHC Class 1.

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Development of a clinical-scale method for generation of DCs from PBMC for use in cancer immunotherapy

Figure 5. Viable cell yield, pH, glucose and L-lactate were determined on Days 0, 3, 5 and 7 on DC cultures from four different normal donors. Over the 7 day culture period, there were consistent decreases in viable-cell yield, glucose, and pH, and increases in L-lactate. The decrement in viable-cell yield between Day 5 and Day 7 was the basis for deciding to perform the cell harvest after 5 days in the final large-scale procedure.

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serum AP AP AP serum serum AP AP AP serum AP AP serum AP AP serum serum serum serum AP

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Table 1. Cell yield and viability during clinical-scale autologous dendritic cell preparation in pediatric sarcoma patients*

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% DC Yield from monocytes to DC harvest

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Development of a clinical-scale method for generation of DCs from PBMC for use in cancer immunotherapy

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substantially higher than those of CD14-positive monocytes, which typically express these markers at levels < 4%. CD1a expression was the most highly variable, ranging from 0.4–63.7%, with a mean of 22.0%. MLR was assayed in a total of 11 products from seven patients, using MNCs from 2–3 allogeneic donors. In the MLR, compared with monocytes, DCs were 2–4 times more active in two products, 4–8 times more active in three products and 16 to > 32 times more active in six products, with activity based on the number of cells required to achieve the same MLR response. Recall Ag responses of autologous lymphocytes were measured in DCs and monocytes from seven patients. DCs were at least as potent and up to eight times more potent than monocytes in recall responses to influenza virus, and at least as potent and up to 32 times as potent as monocytes in recall responses to tetanus toxoid. As shown in Table 1, for each of the four patient/donors (1, 4, 6, and 9) for whom both allogeneic AB serum and autologous plasma were used for DC generation, the % DC yields were comparable between cultures, regardless of whether allogeneic serum or autologous plasma was used. In data not shown, the flow cytometric phenotype and in vitro functional assays were also comparable.

Discussion

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1:10 1:20 1:40 1:80 1:160 1:320 Stimulator:Responder

Figure 6. Representative functional assays on autologous monocytes and DCs generated from autologous monocytes of one pediatric sarcoma patient, including (a) MLR, (b) recall Ag response to influenza virus (Flu), and (c) recall Ag response to tetanus toxoid (TT). For all three functional assays, the dendritic cells were 8–16-fold more active than monocytes, based on the number of cells required to achieve the same response. showed the development of a cell population with high forward and side scatter that was 62–98% (mean 86%) CD14-negative. Phenotyping of the cultured CD14-negative population showed markedly increased expression of mannose receptor (mean 75%, range 50.5–94.4%), moderately increased expression of CD80 (mean 37.4%, range 15.7–64.1%), and only slightly increased expression of CD83 (mean 15.3%, range 2.9–53.8). These values are

In this study we defined optimal conditions for generating DCs in ex vivo flask culture, starting with PBMC collected by leukaphereis and purified by countercurrent centrifugal elutriation. The resulting large-scale method uses RPMI 1640, the cytokines IL-4 and GM-CSF, and protein supplementation with either heat-inactivated allogeneic AB serum or autologous plasma. While other investigators have used automated cell-separation methods to collect and PBMC for subsequent DC generation [9–11], our goal was to design a process that not only took advantage of these separation methods, but also incorporated only reagents suitable for use in human clinical trials. The use of human serum or plasma has two distinct advantages over the use of FBS, which had previously been regarded as a necessary ingredient for successful DC culture. First, substitution of human serum or plasma for FBS eliminates the risk of bovine protein-induced immune responses, such as delayed hypersensitivity and anaphylaxis, which have been reported in recipients of cellular products processed with FBS as a reagent [12,13].

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ECC Wong et al.

Second, elimination of FBS decreases the risk of transmission of bovine infectious agents, such as the prion associated with bovine spongiform encephalopathy. Our method has now been applied successfully to large-scale generation of autologous DCs in an ongoing Phase I/II clinical trial in pediatric sarcoma. Starting with monocytes obtained from a single 7.5 L leukapheresis procedure, we have been able to isolate purified monocytes in numbers sufficient for cryopreservation in at least three aliquots for subsequent thawing and culture. Although it is still unclear how many DC are needed to produce a specific immunotherapeutic response, these cell numbers allow the possibility of subsequent ex vivo manipulation, such as peptide pulsing, and are clearly adequate for initial Phase I/II clinical testing. We were also able to demonstrate that, despite relatively large tumor burdens, circulating factors in autologous plasma did not prevent the development of immature DC from monocytes in these cancer patients. One unexpected observation during our methods development was the variability associated with different media formulations in the cell-surface expression of CD14 and CD1a, an MHC-like molecule known to be highly expressed on DC when cultured in 5% FBS and RPMI 1640. The exact composition of each of these commercial media is unknown; it is therefore uncertain what ingredient or property of the media led to this variability. However, of interest is a recent report that DCs cultured at pH 7.0 are not as mature as those cultured at pH 7.4 [14], suggesting that a feature such as pH or the buffering capacity of the medium may affect expression of certain cell-surface markers. Another observation of particular interest was that the use of higher concentrations (10% rather than 2%) of autologous plasma tended to result in higher DC recovery in these cultures. While this was not surprising, we also noted that there was a moderate degree of variability in the time course of cell-surface expression of CD1a, CD80, CD83, CD86, mannose receptor and the MHC Class II receptor among the different human-protein sources tested. However, the variability was no longer present after 7 days of culture, and corresponding functional assays (MLR and recall response to antigens) for allogeneic AB serum versus autologous plasma were not different. This suggests that expression of certain surface markers may be affected by differences in protein sources, but the biological significance of these changes is unclear.

The selection of serum protein source is of special concern in the setting of autologous immunotherapy for cancer because of recent reports that cytokines and growth factors produced by tumors (IL-10 and VEGF) can affect ex vivo DC maturation [15, 16]. However, our experience to date suggests that, although there is some variability in flow cytometric phenotyping of DC surface markers, the use of autologous plasma at this dilution has no detrimental effect on the quantitative recovery or function of cultured DCs in the pediatric sarcoma-patient group. The advantages of using autologous plasma over allogeneic AB serum are that plasma is available for collection during the same apheresis procedure used to collect the MNC, and that autologous products do not carry the risks of immunogenicity and infectious disease transmission that are associated with allogeneic products. The relative maturity of DC generated by a given method has implications for the efficacy of Ag presentation, a feature of particular interest for clinical immunotherapy. In vivo, one would expect immature DC to be more effective at capturing, processing and presenting Ag, whereas mature DC would be more potent at stimulation of T cells. According to Steinman and colleagues, the phenotypic characteristics of a more mature DC population are complete loss of CD14, increased expression of CD1a, MHC Class II receptor, the costimulatory molecules CD80 and CD86, and ICAM-1 (CD54), and expression of mannose receptor that is increased but lower than on more immature DCs [1]. Therefore, DCs generated by our method, which had low expression of CD14, high expression of mannose receptor and moderate expression of CD80, CD1a and CD83, would be considered relatively immature compared with DC generated by methods that use TNF-a or CD40 ligand [17,18]. Whether or not the relative maturity of the DC assessed by flow cytometric phenotype will translate into different clinical effects remains to be seen. Although the current method using flasks was designed to minimize excessive handling and transfer of cells between containers, future development in our laboratory will be focused on improving the closed system, i.e. adapting the method to culture containers that can undergo sterile connection. Ongoing studies are also aimed at generating DCs in defined ‘serum-free’ media that would completely avoid the use of serum or plasma and produce DCs with a more mature phenotype; this approach has been previously demonstrated, starting with

Development of a clinical-scale method for generation of DCs from PBMC for use in cancer immunotherapy

CD34+ cells [19]. Ultimately, well-designed clinical trials will define the role of these ex vivo-generated cells in immunotherapy for cancer and other diseases.

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Acknowledgements The authors thank T. Bui and A. Cooper in the Hematology Section, Clinical Pathology Department, National Institutes of Health Clinical Center, for staining of cytospin preps; and T. Anderson in the Clinical Chemistry Section, Department of Clinical Pathology, National Institutes of Health Clinical Center, for performing glucose and lactate measurements on cultures. The functional assays (MLR and autologous lymphocyte response to TT and Flu recall antigens) were funded in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. NO1-CO-56000.

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Disclaimer The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government.

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