International Immunopharmacology 13 (2012) 61–68

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International Immunopharmacology

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Cryopreservation of adenovirus-transfected dendritic cells (DCs) for clinical use

D. Gülen a, S. Maas b, H. Julius b, P. Warkentin c, H. Britton d, I. Younos d, J. Senesac e,1,Samuel M. Pirruccello c, J.E. Talmadge d,⁎a Department of Medical Microbiology School of Health, Namik Kemal University, Takirdağ, Turkeyb Biologics Production Facility, The Nebraska Medical Center, Omaha, NE 68131, United Statesc 983135 University of Nebraska Medical Center, Omaha, NE 68198-3135, United Statesd 986495 University of Nebraska Medical Center, Omaha, NE 68198-6495, United Statese 20358 Seneca Meadows Parkway, Germantown, MD 20876, United States

⁎ Corresponding author at: Department of PathologyNebraska Medical Center, 986495 Nebraska Medical CeUnited States. Tel.: +1 402 559 5639; fax: +1 402 559

E-mail addresses: dumrulgulen@yahoo.com (D. Güle(S. Maas), hjulius@nebraskamed.com (H. Julius), pwarkehbritton@unmc.edu (H. Britton), ibrahimpharma1@hotmjoesene@gmail.com (J. Senesac), spirrucc@unmc.edu (Sjtalmadg@unmc.edu (J.E. Talmadge).

1 Formerly at Intrexon Corporation.

1567-5769/$ – see front matter © 2012 Elsevier B.V. Alldoi:10.1016/j.intimp.2012.03.009

a b s t r a c t

a r t i c l e i n f o

Article history:Received 14 March 2012Accepted 15 March 2012Available online 27 March 2012

Keywords:CryopreservationDendritic cellsAdenovirusStabilityViability

In this study, we examined the effects of cryoprotectant, freezing and thawing, and adenovirus (Adv) trans-duction on the viability, transgene expression, phenotype, and function of human dendritic cells (DCs). DCswere differentiated from cultured peripheral blood (PB) monocytes following Elutra isolation usinggranulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) for 6 days and thentransduced using an Adv vector with an IL-12 transgene. Fresh, cryopreserved, and thawed transduced im-mature DCs were examined for their: 1) cellular concentration and viability; 2) antigenicity using an alloge-neic mixed lymphocyte reaction (MLR); 3) phenotype (HLA-DR and CD11c) and activation (CD83); and4) transgene expression based on IL-12 secretion. Stability studies revealed that transduced DCs could beheld in cryoprotectant for as long as 75 min at 2–8 °C prior to freezing with little effect on their viabilityand cellularity. Further, cryopreservation, storage, and thawing reduced the viability of the transduced DCsby an average of 7.7%; and had no significant impact on DC phenotype and activation. In summary, cryopres-ervation, storage, and thawing had no significant effect on DC viability, function, and transgene expression byAdv-transduced DCs.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

DCs are potent antigen-presenting cells (APCs) and the only APCcapable of priming naïve T-cells [1]. In recent years, DC vaccineshave been developed to elicit tumor-specific T-cell responses [2,3],and clinical results from studies using DC-based vaccines have beenencouraging [3]. Nevertheless, many questions remain regarding clin-ical use and manufacture of DC-based therapeutics, including thenumber of DCs per injection, route of administration, the best DC sub-set to use, strategies for antigen loading (protein/peptide pulsing, ex-posure to whole tumor cells, or genetic modification), whichadjuvant, including genetic sequence, to use, and the most effectiveway to manufacture DC-based products. Results from animal andclinical studies have demonstrated that multiple cycles of administra-tion are necessary to overcome tolerance and to achieve clinically

& Microbiology, University ofnter, Omaha, NE 68198-6495,4990.n), smaas@nebraskamed.comnt@unmc.edu (P. Warkentin),ail.com (I. Younos),

.M. Pirruccello),

rights reserved.

relevant T-cell responses [4,5]. This suggests that multiple DC dosesare required, and a manufacturing strategy that results in multipledoses of DCs that can be cryopreserved until administered might beoptimal. The manufacture of DCs using good manufacturing practice(GMP) approaches is time intensive. The administration of fresh DCsrequires repeated manufacturing events and, potentially, multipleapheresis products [6]. One approach to address this is to cryopre-serve multiple aliquots from a single apheresis; however, this re-quires multiple manufacturing events. Thus, a single manufacturingevent and cryopreservation of multiple DC aliquots from a singleapheresis may increase the practicality of DC-based vaccination.

A critical aspect of DC therapy includes antigen or cytokine deliveryto regulate T-cell immunity with the goal of inducing a therapeutic re-sponse [7,8]. Adv vectors, in addition to being an excellent for transgenedelivery, can also induce high levels of transgene expression [9,10] andhave been shown to be effective at stimulating a T-cell response. Advtransduction of DCs can also up-regulate membrane major histocom-patibility complex (MHC) and co-stimulatory molecule expression, aswell as, increase production of pro-inflammatory cytokines, such asIL-12 [10] and CD40 [11].

We report herein, studies using ex vivo differentiated tissue culture(TC)–DC products transduced with an Adv vector (Ad-RTS-hIL-12) in-corporating the human IL-12 transgene under control of the RheoS-witch Therapeutic System® (RTS®) whose activity can be induced

62 D. Gülen et al. / International Immunopharmacology 13 (2012) 61–68

with the activator drug INXN-1001, formerly RG-115932 [11]. This vec-torwas used to transduce DCs for 3 h at a viral particle:DC (VP:DC) ratioof 22,500:1, resulting in the transduction of 27.2±6.6% of DCs based onthe expression of intracellular IL-12 and the secretion of 62±43 ng/mlof IL-12 by 200,000 DCs. Further, their allo-antigen presenting functionwas not reduced by cryopreservation or thawing.

2. Materials and methods

2.1. Apheresis, monocyte enrichment, and DC differentiation

The use of human apheresis products was approved by the Institu-tional Review Board (IRB) of the University of Nebraska Medical Cen-ter. Following informed consent, healthy donors (males or females,ages 19–70) underwent mononuclear cell leukapheresis on a CobeSpectra (Gambro BCT, Lakewood, CO) apheresis cell separator. PBmonocytes were enriched directly from the leukapheresis productsusing the Elutra® (CaridianBCT, Lakewood, CO) cell separation sys-tem semi-automated method [12,13]. All manufacturing procedureswere performed in a class-10,000 clean room. DCs were differentiatedfrom monocytes using a closed system with CellGenix DC media(CellGenix, Antioch, IL) with recombinant human cytokines (IL-4,CellGenix, Antioch, IL) and GM-CSF (Leukine, Berlex, Seattle, WA) inVueLife® cell culture bags, (American Fluoroseal Corporation, Afc,Gaithersburg, MD) for use as a therapeutic TC–DC product [12].

2.2. Transduction with Adv-hIL-12

DCs were transduced with Ad-RTS-hIL-12 virus (Intrexon Corp.,Blacksburg, VA) that encodes hIL-12 under the control of the RheoS-witch® Therapeutic System (RTS) [10]. The transgene was inducedby the activator drug INXN-1001 (Intrexon Corp., Blacksburg, VA).The infection process by Adv for hematopoietic cells is dependentupon 3 parameters: time, VP number and concentration, and to an ex-tent, DC concentration [14]. Prior studies to optimize infection pa-rameters revealed that DCs were optimally transduced using aconcentration of 22,500:1 VP:DC and DCs cultured at 5×106 cells/ml [12]. Following a 3-hour co-culture, the infected cells were washedand concentrated using an Eppendorf 5810R centrifuge (Eppendorf,Westbury, NY) and/or a Cobe 2991 (Gambro BCT, Lakewood, CO),depending upon volume. The concentrated cells were then trans-ferred to 72 ml bags (2PF-0072, Afc) and centrifuged at 18 °C for10 min at 290×g. As previously described, a Fenwal Plasma Extractor(Fenwal, Inc., Lake Zurich, IL) was used to remove the supernatant[12]. Reflux of expressed supernatant was closely monitored andthe 2PF-0072 bag drained of as much media and virus as possible.The cells were then resuspended and washed twice with 30 ml ofPlasmaLyte A (Baxter Healthcare Corp., Deerfield, IL) plus 1% HSA(Buminate 5%, Baxter Healthcare Corp., Deerfield, IL). Cellularity wasassessed using an ABX Pentra 60 C+ hematology analyzer (HoribaABX, Inc., Irvine, CA), and the percentage of viable cells was deter-mined using a trypan blue assay. Samples were also taken for FACSanalyses, IL-12 production by IL-12 ELISA, intracellular IL-12 staining,and for MLR analysis.

2.3. Preparation of a TC–DC product for cryopresevation

Ad-RTS-hIL-12-infected cells were pelleted and adjusted to5×107/ml with chilled (2–8 °C) CryoStor5 (Biolife Solutions Inc.,Bothell, WA) added slowly by syringe [13]. The cells were transferredto 6 ml KyroSure® 6-F bags (Afc, Gaithersburg, MD) using a sterileconnecting device. The bags were maintained at 4 °C and transferredto a control-rate freezer in less than 45 min. They were cryopreservedby controlled-rate cooling of 1° per minute through−60 °C, then 5 °Cper minute to −100 °C. After freezing, the bags were stored in the

vapor phase of a continuously monitored liquid N2 chamber at−150 °C.

2.4. MLR assay

The allo-MLR was performed by adding serial-diluted doses (rang-ing from 1:2 to 1:128) of irradiated (10,000 Rads) DCs in triplicate, to2×105 allogeneic, Ficoll-Hypaque-separated, normal PB mononuclearcells (PBMCs) as responders. The cells were cultured in 96-wellround-bottom plates (Techno Plastic Products, AG, Trasadingen, Swit-zerland) and co-incubated for 7 days in RPMI 1640 (Gibco, Invitrogen,Grand Island, NY) supplemented with 50 μg/ml gentamycin (Gibco,Invitrogen, Grand Island, NY), 1 μM glutamine (Gibco, Invitrogen,Grand Island, NY), and 10% heat-inactivated fetal bovine serum (AtlantaBiologicals, Lawrenceville, GA). The proliferation was assessed via theaddition of 1 μC3 H-Thymidine (Amersham Biosciences, Amersham,UK) in 50 μl during the last 18–24 h of culture. Cells were harvested(Harvester 96, Packard, Waltham, MA) and the extent of radioactivityincorporation measured in a TopCount scintillation counter (Packard,Waltham, MA). The responses are reported as a mean of triplicatecounts per minute.

2.5. Fluorescent flow cytometric cell (FCM) analysis

The DC phenotype-incorporated cells were gated on CD45+ andHLA-DR expression. The maturity of the DR+DCs was assessed byCD11c+ expression, and the extent of activation included an analysisof CD83 expression. All antibodies, unless otherwise identified, wereobtained from Becton Dickinson (BD) Bioscience, San Jose, CA. Thepanel relevant to this manuscript included: PE-labeled mouse anti-human CD11c (B-ly6), PE-Cy5-labeled mouse anti-human CD83(HB15e), ECD-labeled mouse anti-human HLA-DR (Beckman Coulter,HD237), and PE-Cy7-labeled mouse anti-human CD45 (J33).

Following Adv infection, we also assessed the frequency of DCsinfected with Ad-RTS-hIL-12. Special reagents to stain DCs for intracel-lular IL-12 staining included GolgiStop™ (BD Biosciences, San Jose, CA)and FIX & PERM® reagents (Invitrogen, Carlsbad, CA). Samples of DCswere cultured for 18 h with or without AL (38 nmol). Prior to harvest,the protein transport inhibitor, GolgiStop™, was added to the cell cul-ture, following the manufacturer's instructions. After harvest, mem-brane antigens were stained with antibodies to CD45 (PE-Cy5) andCD11c (APC). Control cells for background staining included isotypecontrols for each color IgG1 (PE-Cy5, PE, APC, and FITC). After staining,the cells were permeabilized using Caltag®, FIX & PERM® media Aand B (Invitrogen), and then stained using the Caltag® procedure for in-tracellular staining, “Detection of Intracellular Antigens,” using anti-bodies to intracellular IL-12 (PE, BD Bioscience) and/or unconjugatedAdv hexon protein (Abcam, Cambridge MA) followed by FITC goatanti-mouse (Abcam). We found it critical to use BD Stabilizing Fixative(BD Biosciences) to preserve tandem conjugate staining. The negativestaining control included cells stained with isotype control antibodiesper the kit's instructions.

2.6. Production of IL-12 from DCs cultured with AL

The DC concentration was adjusted to 2×105 cells/ml in CellGenixDC Media as soon as possible to ensure cell quality and viability.These cell culture studies had two conditions: Adv-hIL-12-DC aloneand Adv-hIL-12-DC with RG-AL. Each co-culture was established infive wells of a 96-well plate. Reagents were added to the wells begin-ning with the CellGenix media followed by cell suspension and, lastly,38 nmol of AL to obtain a total volume of 200 μl. The cell suspensionswere gently mixed and the plates incubated at 37 °C in 5% CO2 for18 h. Cells were harvested by pooling the well contents and obtainingsupernatants by centrifugation. The resulting supernatant was har-vested without disturbing the pellet, aliquoted into labeled cryovials,

63D. Gülen et al. / International Immunopharmacology 13 (2012) 61–68

and stored at −80 °C until assayed. The aliquots of transduced DCswere rapidly thawed in a 37 °C water bath, slowly diluted four foldswith culture media and held for 2 h to stabilize prior to analysis.

2.7. IL-12 ELISA procedure

To determine IL-12 concentrations released by the cultured DCs,we used an Opt EIA kit for human IL-12 (p70) (BD Biosciences, SanJose, CA). Following the manufacturer's instructions, the microwellswere coated with capture antibody, the plate sealed, and incubatedat 4 °C overnight. Afterward, the wells were washed and nonspeci-fic binding blocked. The wells were then washed, and standards andsample dilutions were pipetted into appropriate wells. The platewas sealed and incubated for 2 h at room temperature. Followingincubation, the wells were washed, and a detection antibody andStreptavidin (SAv)-HRP reagent (BD Biosciences, San Jose, CA)was added to each well. The plate was sealed and incubated for1 h and washed. Freshly made substrate solution was added toeach well, incubated for 30 min at room temperature, and thenstop solution was added. The absorbance was assessed at 450 nmwithin 30 min of stopping the reaction using an Epoch microplatereader (BioTek, Winooski, VT) using 570 nm absorbance for wave-length correction.

2.8. Statistics

SPSS 16.0 for Windows was used for statistical analyses, and apaired student T-test was used to analyze the data.

3. Results

3.1. Effect of cryopreservation and thawing on cellularity and viability ofAdv-infected DCs

In these studies, we examined the stability of Ad-RTS-hIL-12-infected DCs to cryopreservation, storage, and thawing using prod-ucts frozen at a cellularity of 3×107 to 7×107 cells/ml, resulting inan average cell count of 4.8±0.7×107 cells/ml. The average cellcount after freezing, storage, and thawing (5.8±1.1×107 cells) didnot significantly differ from that measured pre-freezing (Table 1). Incontrast, cell viability (trypan blue) (92.9±2.6%) before freezing(Table 1) was significantly decreased following freezing, storage inliquid N2, and rapid thawing (85.2±6.6%) resulting in a 7.7%decrease in viability of Ad-RTS-hIL-12-infected DCs.

Table 1Cellularity (A) and viability (B) of the Ad-RTS-hIL-12-transduced DCs before freezingand after cryopreservation, storage, and thawing. Cell counts used an automatedblood analyzer, and viability was assessed by trypan blue staining. Paired student Ttest results for cell count (p=0.003) and for viability (p=0.013) at 15 min, signifi-cance is taken as p≤0.05. N=8.

Before freezing After thawing

Exp # Cell count ×107 % viability Cell count ×107 % viability

1 3.00 92.6 2.98 86.52 3.00 98.4 2.76 92.63 7.00 90.8 7.0 774 5.00 94.0 7.0 86.45 5.00 89.9 6.57 92.16 5.00 91.0 4.96 907 5.00 92.7 5.71 75.38 5.00 93.4 5.24 82Avg SEM 92.9±2.6 85.2±6.6

3.2. Effect of cryopreservation medium on cellularity and viability ofAd-RTS-hIL-12 transduced DCs

During the manufacture of DC products, a delay can occur be-tween the addition of cryoprotectant and critical-point freezing.Thus, in these studies, we assessed the stability of the manufacturedand transduced DCs in cryoprotectant. We examined the cellularityand viability of Ad-RTS-hIL-12-transduced DCs in CryoStor5 [15]cryoprotectant held at 2–8 °C over 75-minutes representing whatwe believe would be a worst possible delay in freezing. This assess-ment used cell aliquots to which CryoStor5 was added and the cellssampled every 15 min. The average viable cell count at the time ofCryoStor5 addition was 5×107 cells/ml, and at the initial 15-minutesampling point, we observed a significant decrease in the average vi-able cell count (3.35±0.3×107 cells/ml). This viable cellularityremained relatively constant throughout the remainder of the studywith 3.31±0.2×107 cells/ml observed at 75min (Fig. 1A). The prod-uct viability was similarly little affected by holding the Ad-RTS-hIL-12-infected DCs in CryoStor5 at 2–8 °C (Fig. 1B). Following manufac-ture and Adv transduction, at the time of CryoStor5 cryoprotectantaddition, average cellular viability was 91.2±0.8% for these products.Fifteen minutes after adding the CryoStor5 medium and storage at 2–8 °C, we observed a significant drop in viability to 77.7±2.3%. This vi-ability remained stable (non-significant differences) through the 75-minute sample point when the average viability was 81.7±3.2%.We conclude from these studies that Ad-RTS-hIL-12-transduced DCscan be held in CryoStor5 cryoprotectant at 2–8 °C for at least 75 minprior to freezing.

Fig. 1. Cellularity (A) and trypan blue viability (B) of Ad-RTS-hIL-12-transduced DCs inCryostor5 at 2–8 °C held for up to 75 min. Samples were taken at time zero and every15 min, and the mean±SEM for 5 studies are shown.

64 D. Gülen et al. / International Immunopharmacology 13 (2012) 61–68

3.3. DC phenotype following cryopreservation

Freezing and thawing had no significant effect on the phenotypeof Ad-RTS-hIL-12-transduced DCs. Stable HLA-DR expression was ob-served with Ad-RTS-hIL-12-transduced DCs that had a frequencyranging from 95.4% to 98.8% (mean 97.3±0.4%) (Fig. 2A). Flow cyto-metric analysis also revealed that before freezing, the Ad-RTS-hIL-12-transduced DCs had a frequency of CD11c expression ranging from85.7% to 97.3% (mean 91.1±1.4%) (Fig. 2B). After freezing, storagefor a minimum of 14 days, and thawing, the frequency of CD11c+ ex-pressing cells was not significantly different and ranged from 85.6% to96.7% (mean 93.3±0.4%) (Fig. 2B). We also examined Adv-transduced DC activation based on CD83 expression (Fig. 3A and B).Before freezing, the frequency of CD83 expression by the transducedDCs ranged from 2.63% to 13.31% (mean of 8.29±2.4%) demonstrat-ing that approximately 3 h following initiation of Adv infection, fewDCs were activated. After freezing, storage, and thawing, the frequen-cy of HLA-DR+CD11c+CD83+ cells ranged from 2.13% to 12.04%(mean of 7.43±2.3%) suggesting that freezing and thawing did littleto activate the DCs despite having been transduced by Ad-RTS-hIL-12.It is noted that although Adv transduction can activate DCs, it requireslonger than 3 h for this to occur. Thus, based on cellular phenotype,Ad-RTS-hIL-12-transduced DCs remain stable during freezing, stor-age, and thawing with few cells expressing an activated phenotype.

A representative flow cytometric dot plot showing CD11c andHLA-DR expression by DCs immediately following culture and a 3-hour transduction by Ad-RTS-hIL-12 is shown in Fig. 3A. Three cellpopulations were observed including DCs with a mature phenotype(50.7%) expressing CD11cbrightHLA-DRbright. In this study, 41.5% ofthe cells were immature DCs with an HLA-DRdullCD11cdull phenotype.In addition, there was a small subset (1.34%) of HLA-DRbrightCD11cdull

Fig. 2. Flow cytometric analysis of products manufactured from normal donor aphere-ses following transduction with Ad-RTS-hIL-12 (solid bars) and 1–4 products cryopre-served in Cryostor5 using 6 ml cryostorage bags from Afc for storage and thawing. Nosignificant difference was observed in HLA-DR frequency (A) and the frequency ofCD11c (B) expression between products prior to cryopreservation (solid bars) and fol-lowing cryopreservation, storage, and thawing (hatched bars).

cells, which were, presumably, DCs transiting from an immature to amature phenotype. DC activation was assessed as CD83 expression asshown on a CD83 and CD11c dot plot (Fig. 3B) for the cells fromFig. 3A. Three subpopulations were again observed based on the ex-pression of CD83 (bright or dull) and CD11c+ including 38.8% thatwere CD11cbrightCD83dull described as mature, non-activated DCs. Inaddition, a population of CD11cdullCD83− DCs (32.5%) were observed,which were non-activated, immature DCs. Lastly, 12.5% of the cellswere CD11cbrightCD83bright, which were mature, activated DCs. Fur-ther, following a 20-hour co-culture, CD83 expression was signifi-cantly increased (results not shown).

3.4. T-cell stimulatory properties of transduced DCs

We also examined the influence of freezing and thawing on allo-antigen presentation by Ad-RTS-hIL-12-transduced DCs using a one-way allogeneic MLR assay that was performed before and after cryo-preservation, storage, and thawing. The average stimulation index(SI) of the Ad-RTS-hIL-12-transduced DCs for allogeneic T-cells beforefreezing was 12.7±2.3, and after freezing, storage, and thawing itwas not significantly changed (p=0.496 and SI of 10.3±1.0). Theseresults suggest that freezing, storage, and thawing of the transducedDCs had little effect on alloantigen presentation by the Ad-RTS-hIL-12-transduced DCs (Fig. 4).

3.5. Transgene expression of transduced DCs before and aftercryopreservation

The ability of the Ad-RTS-hIL-12 vector to transduce DCs wasassessed based on intracellular expression of IL-12 and hexon, a lateAdv structural protein by transduced DCs. These studies also allowedthe assessment of DC activation by Adv transduction. Before freezing,7.2±1.7% of the Ad-RTS-hIL-12-transduced DCs expressed intracellularIL-12+, when cultured for 20 h without AL (Table 2). Following freez-ing, storage and thawing, Ad-RTS-hIL-12-transduced DCs had a signifi-cant increase in DC frequency with an average intracellular IL-12+

expression of 9.5±2.3% when cultured for 20 h without AL. In both in-stances, this represents the frequency of endogenous DC activation byAd-RTS-hIL-12 transduction. However, when the same transducedDCs were co-cultured with 38 nmol AL, they had a significantly higherfrequency of intracellular IL-12 expression, ranging from 10.9% to46.1% with a mean of 27.2±6.6 DCs expressing IL-12 (Table 2).Freezing, storage, and thawing had no effect on intracellular IL-12 ex-pression by DCs cultured with AL, resulting in a frequency of IL-12+

DCs ranging from 10.8% to 42.5% (mean of 27.6±4.9). A representativeflow cytometry dot plot of transduced DCs culturedwith AL is shown inFig. 3C demonstrating intracellular IL-12 expression by 16% of CD11c+

cells. In several studies, we also stained for the late Adv structural pro-tein hexon and found that 100% of the cells were hexon positive(Fig. 3D). In the experiment shown in Fig. 3D, consistentwith the resultsshown in Fig. 3C, 15.4% of the cells were IL-12+. The difference in thefrequency of IL-12 transgene expression, as opposed to hexon staining,may be due to the presence of empty capsids in the vector preparation[16] resulting in hexon-positive DCs after immunostaining and flowanalysis. Alternatively, it could be due directly to pinocytosis of de-tached hexons. We did not detect hexon expression by DC controlsthat were not co-cultured with Ad-RTS-hIL-12 suggesting that thiswas not a false-positive assessment.

We also assessed the levels of IL-12 secreted by the transduced DCsduring a 20-hour co-culture with or without AL. These studiesrevealed that, in the absence of AL, no detectable levels of IL-12 (lessthan 10 pg/ml) were observed following co-culture. In contrast,when cultured with 38 nmol AL, IL-12 levels were significantly in-creased to 62,496±43,210 pg/ml, relative to that observed in the ab-sence of AL (Table 2). There was also a significant increase in IL-12levels following co-culture with AL post freeze/thaw of the transduced

Fig. 3. Flow cytometric dot plots of Ad-RTS-hIL-12-transduced DCs following 3 h of incubation with vector, washing (A and B), and 18–20 hour incubation with AL (C and D). In A,the dot plots, both using FlowJo analysis, are HLA-DR by CD11c, and in B they are CD83 by CD11c. Figure C is following 18–20 hour co-culture of CD11c using intracellular IL-12staining and CD11c expression, and D shows CD11c+-gated cells reflecting intracellular IL-12 and hexon expression.

Fig. 4. The function of Ad-RTS-hIL-12-transduced DCs was assessed in an allogeneicMLR assay using allogeneic PB lymphocytes from normal donors. The solid bars arethe stimulation index obtained with DCs at a 1:2 stimulator-to-responder ratio priorto cryopreservation. The hatch bars represent 1–4 bags of transduced DCs, whichhave been cryopreserved, thawed, and their allo-stimulatory properties assessed.These studies revealed no significant difference in the average T-cell allo-stimulationbetween products prior to cryopreservation and following freezing, storage, and thaw-ing, although, there was considerable assay-to-assay difference.

65D. Gülen et al. / International Immunopharmacology 13 (2012) 61–68

DCs resulting in an average IL-12 concentration of 27,397±5464 pg/ml.These results did not significantly differ between pre-freezing and post-freezing/thawing based on the co-culture secretion of IL-12 by ELISAassay or the frequency of IL-12+ DCs by intracellular staining. It isnoted that following freezing and thawing, IL-12 levels significantly in-creased in the absence of AL suggesting that freezing and thawing mayhave slightly activated the DCs (Table 2). In some studies, multipleproduct bags were frozen, stored, and thawed, and Fig. 5 shows thatthe observed significant increase in IL-12 was consistent.

Several studies were used to assess if there was a correlation be-tween IL-12 secretion levels and frequency of IL-12+ DCs using re-sults from DC products from normal donors cultured with AL. Acorrelation analysis using log-transformed data for the IL-12 levelsas assessed by ELISA, revealed a significant correlation with the fre-quency of IL-12 intracellular-positive DCs (R=0.368 and p=0.035by the Pearson's correlation analysis). This linear regression analysisincorporated both post-manufacturing and post-freeze/thaw samples(Fig. 6).

4. Discussion

Themanufacture of sufficient numbers of functional DCs for multipletherapeutic cycles is critical for successful DC-based immunotherapy

Table 2Ad-RTS-hIL-12-transduced DCs were co-cultured in complete DC media for 18 h at 200,000 cells/well (200 μl media), and the frequency of cells expressing cytoplasmic IL-12 byflow cytometry and levels of IL-12 secreted in the media expressed as pg/ml by ELISA analysis are reported. These studies were undertaken in the presence or absence of38 nmol of AL. The average±SEM is also shown. These studies used Ad-RTS-hIL-12-transduced DCs just prior to adding cryopreservative and following cryopreservation, storage,and thawing. Paired student T-test, *=significant difference from culture before freezing (p≤0.05); # significantly different from culture without AL.

Before freezing After thawing Before freezing After thawing

DC alone Avg %CD45+ IL-12+

DC+AL Avg %CD45+ IL-12+

DC alone Avg %CD45+ IL-12+

DC+AL Avg %CD45+ IL-12+

DC alone Avg IL-12concentration(pg/ml)

DC+AL Avg IL-12concentration(pg/ml)

DC alone Avg IL-12concentration(pg/ml)

DC+AL Avg IL-12concentration(pg/ml)

Exp 1 6.2 45.7 8.7 22.3 b10 28,842 b10 40,191Exp 2 2.5 10.9 10.3 10.8 b10 25,572 36 16,849Exp 3 6.7 13.2 20.3 19.2 b10 11,556 b10 6475Exp 4 10 15.8 4.4 33.8 b10 21,446 131 27,158Exp 5 14 46.1 5.6 42.5 b10 9571 11 35,394Exp 6 3.5 31.3 7.7 37.2 b10 277,988 b10 38,317Average+SEM 7.2±1.7 27.2±6.6# 9.5±2.3* 27.6±4.9# b10 62,496±43,210# 34.7±19.7* 27,397±5464#

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protocols. DC production for clinical use is a time- and resource-intensive procedure, and if “fresh” DCs are used, multiple apheresisproducts are required. One approach to this problem is to freeze multi-ple aliquots of a single apheresis for use in multiple manufacturingevents. Studies by Makino and Baba [17] describing a cryopreservationmethod for human PBMCs to produce DCs, found that freezing/thawingdid not impair the ability of PBMCs to differentiate into DCs. They alsoreported that DCs from frozen PBMCs did not differ substantially fromDCs generated from fresh PBMCs in terms of viability, cell yield, pheno-type, and functional properties. However, with this approach, mononu-clear cells must be thawed and differentiated for each product-manufacturing event, and the characteristics of the DCs generated bymultiple manufacturing events may vary. Alternatively, one apheresis-manufacturing event, which provides enoughDCs formultiple DC injec-tions, can be cryopreserved as DC aliquots and thawed for each sched-uled administration. Thus, from a practical standpoint, it may beadvantageous to manufacture multiple DC doses simultaneously andcryopreserve them for clinical use.

The use of antigen-preloaded, cryopreserved DCs has obvious ad-vantages for clinical applications. Although satisfactory and clinicallyapplicable cryopreservation methods for DCs have been described[18–21], methods to cryopreserve products transduced with Adv vec-tors have been little studied [22]. In most clinical trials reported todate, DCs were freshly prepared for each administration either fromfresh blood [23–27], leukapheresis products [23,28,29], or from fro-zen aliquots of PBMCs obtained by leukapheresis [27].

Fig. 5. Ad-RTS-hIL-12-transduced DCs were cultured for 18–20 h in complete DC mediafrom CellGenix and 38 nmol AL and the cells pelleted and supernatants removed for as-sessment of IL-12 levels in culture supernatants expressed as pg/ml. In these studies,solid bars reflect IL-12 levels secreted prior to preservation, and the hatch bars showthe results of 2–4 bags of cells cryopreserved using CryoStor5. These studies revealeda significant increase in IL-12 secretion following cryopreservation. (p=.001 by thepaired student T-test).

Given the history of successful freezing of hematopoietic cells forsubsequent clinical use, our primary focus in this study was on the ef-fect of Adv transduction on the response of DCs to cryopreservationand thawing based on viability, number, phenotype, antigen presen-tation, and transgene expression. Most cryopreservation studiesusing DCs employ cells that are not transduced with Adv vectors. Inthese studies; the viability and functionality of the cryopreservedDCs and their ability to induce antigen-specific immune responsesappeared little changed by freezing [22,30,31]. In the present studies,it was considered critical to assess the stability of transduced DCs aswe considered it possible that Adv-transduced DCs might have an in-creased sensitivity to injury by cryopreservation [32]. In our study, weused a closed-culture system to mature DCs and then transducedthem with Ad-RTS-hIL-12. These studies used the GMP-grade cryo-preservant CryoStor5 as the cryoprotectant and KyroSure® 6-F bagsto freeze the cells. These unique, 6 ml, cryopreservation bags werevalidated as robust and allow sterile docking and closed-system fill-ing and storage with a septum for therapeutic withdrawal (Fig. 7).We found that cryopreservation with CryoStor5 had minimal effecton Ad-RTS-hIL-12-transduced DC viability, cellularity, phenotype,and function as was reported for non-transfected DCs [30]. Our ap-proach differed somewhat from a recent study by Garritsen et al.,that used a multi-cistronic Adv vector to transfect DCs to allow theexpression of multiple tumor-associated antigens [32]. These prod-ucts were cryopreserved in 9% HSA and 5% DMSO containing CellGro

Fig. 6. Correlation between the frequency of intracellular IL-12 positive DCs and super-natant IL-12 levels from Ad-RTS-hIL-12-transduced DCs. This was following a 3-hourco-incubation with the vector, washing, and culture for an additional 18–20 h in com-plete DC media (CellGenix) and 38 nmol AL. Results are shown as a linear regression ofthe cytoplasmic IL-12 expression and secretion of Il-12 in the culture media resultingin a significant (R=0.368 and p=0.035) correlation (Pearson's correlation analysis)between these two measurements.

Fig. 7. Photograph of a KyroSure® 6-F Afc bag designed for freezing DC products. Note:these 6 ml bags have a pigtail at one end for sterile docking, space to heat seal the en-tire bag, and an enclosed rubber septum to withdraw the product after thawing.

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DC medium. After thawing, DC viability was reported to be 70%, aminimal level for regulatory release. This contrasts with our resultswhere transduced DC viability after thawing averaged 85.2±6.6%.As in our findings, Garritsen's results showed that the frozen DC prod-uct retained antigen-presenting function [33]. While CryoStor10 canalso be used successfully to cryopreserve DCs (results not shown),we found similar post-thaw viability and cellularity with CryoStor5frozen cells, and as CryoStor5 results in the injection of less DMSO,we preferred to use this. We also examined if a delay between the ad-dition of cryopreservation media and critical-point freezing would af-fect cell viability. Based on our studies, we concluded that Adv-transfected DCs could be held for as long as 75 min at 2–8 °C fortransport and freezing with little effect on viability. Further, followingfreezing, storage, and rapid thaw, and in the presence of CryoStor5,DCs remained viable and their phenotype stable for at least 30 min(results not shown).

The flow cytometric-determined phenotype of DCs before and aftercryopreservation and thawing did not vary. Similarly, T-cell alloantigenpresentation by TC–DCs remained stable, as assessed by aMLR, demon-strated no significant decrease in the stimulatory capacity. We alsofound that cultured DCs, transduced with Ad-RTS-hIL-12 for 3 h, havean immature phenotype, and following an additional 18 h of culturefor transgenic secretion, they secrete high levels of IL-12. Further, theonly observed difference between transduced DCs pre-freeze andpost-cryopreservation and thaw, was a slight increase in an activationphenotype based on IL-12 secretion and CD83 (activation marker)expression.

The vectors used in these studies are similar to those used byKomita et al. [10] where they transduced mouse DCs with an rAd.-cIL12 Adv vector. They found that BM-derived DCs infected withRheol-IL-12 conditionally produce high levels of IL-12 when treatedwith AL, and that the intratumoral administration of DC.cIL-12 orDC.Rheol IL-12, combined with the i.p. administration of AL, promot-ed the regression of established subcutaneous B16 melanoma lesions[10]. In our studies, following a 3-hour co-incubation with the Advvector and 20-hour incubation for transgene expression, an averageof 27.2±6.6% DCs expressed intracellular IL-12. We also observedthat 100% of the DCs expressed hexon, a late Adv protein; however,as this replication-defective vector has a deletion in E1, it is not tran-scribed. Thus, the difference between IL-12 transgene expression andhexon (~100%) suggests that most DCs are infected, but a limitednumber are transduced and secrete IL-12. As such, the definitive anal-ysis for the transduction frequency in our studies is intracellular IL-12

expression. Alternatively, the difference between hexon and IL-12 ex-pressionmay be due to the pinocytosis of detached hexons in the vectorproduct or to non-specific binding of the virus to the DCs. In our studies,transduced DCs had an intracellular IL-12 expression frequency of 7.2±1.7% in the absence of AL, suggesting a slightDC activation by Adv trans-duction. The supernatants from the co-cultures with AL also had signif-icant IL-12 levels suggesting high levels of IL-12 secretion per cell,supported by a significant correlation between the frequency of IL-12intracellular-positive DCs and the culture level of IL-12.

In summary, CryoStor5, a GMP-grade cryoprotectant, and Kyro-Sure® 6-F bags are suitable for closed-system production freezingof Ad-RTS-hIL-12 transfected DCs. Further, cultured DCs transducedwith Ad-RTS-hIL-12 for 3 h are phenotypically immature DCs, but,following an additional 18 h of culture with AL, secrete high levelsof IL-12. The only difference observed between transduced DCsprior to freezing and post cryopreservation and thawing was a slightincrease in activation based on data from antigen-presentationstudies (MLR). Minimal differences in cellularity and viability werealso observed, including storage of transduced DCs in CryoStor5 at2–8 °C for 75 min, and that frozen and thawed DCs remained stable(viability and phenotypically) for 30 min under similar conditions.

5. Support and financial disclosure

This research was funded through the Nebraska Research Initiative.None of the contributing authors has any financial conflict of interest.

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