Dendritic cells transfected with interleukin-12 and tumor ... - Nature

Gene Therapy (2007) 14, 366–375 & 2007 Nature Publishing Group All rights reserved 0969-7128/07 $30.00 www.nature.com/gt

ORIGINAL ARTICLE

Dendritic cells transfected with interleukin-12 and tumor-associated antigen messenger RNA induce high avidity cytotoxic T cells HJ Bontkes1, D Kramer1, JJ Ruizendaal1, EWM Kueter1, VFI van Tendeloo2, CJLM Meijer1 and E Hooijberg1 1

Department of Pathology, Vrije Universiteit Medical Center, Amsterdam, The Netherlands and 2Laboratory of Experimental Hematology, Faculty of Medicine, University of Antwerp, Antwerp University Hospital, Antwerp, Belgium

Dendritic cells (DC) transfected with messenger RNA (mRNA) encoding tumor-associated antigens (TAA) are able to induce potent tumor-specific T-cell responses directed to a broad spectrum of tumor-associated epitopes. The in vitro generation of DC possessing all the features crucial for the induction of type 1 immune responses, such as mature state, migratory potential and interleukin-12 (IL-12p70) production is complicated. Particularly migratory potential is inversely correlated with IL-12p70 production after maturation with prostaglandin E2 (PGE2), which is included in maturation cocktails currently used in most vaccination trials. Here, we show that transfection of PGE2 matured DC with a single mRNA strain encoding

for ubiquitin followed by a TAA which was linked to IL-12 by a self-cleaving 2A sequence, produced biological active IL-12p70 and were able to present the transfected TAA up to 72 h after transfection. Furthermore, use of the anti-reverse cap analog for in vitro transcription of the IL-12 mRNA enabled constitutive IL-12p70 production for up to 5 days. These transfected mature DC migrated efficiently towards lymph node derived chemokines. DCs constitutively expressing IL-12p70, generate TAA-specific cytotoxic T cells with an high functional avidity, independent of CD4+ T-cell help. Gene Therapy (2007) 14, 366–375. doi:10.1038/sj.gt.3302874; published online 12 October 2006

Keywords: dendritic cells; Interleukin-12; CTL; functional avidity; mRNA

Introduction Dendritic cells (DCs) loaded with tumor-associated antigen (TAA) are frequently applied as vaccines for cancer patients.1 These DCs should express high levels of co-stimulatory molecules2 and be able to migrate towards T-cell areas in the lymph nodes.3 Furthermore, the DCs should secrete high amounts of interleukin-12 (IL-12p70), which is a crucial third signal for induction of functional T helper (Th) cell 1 and cytotoxic T lymphocytes (CTL) responses.4 In addition, for clinical application DCs have to be generated under conditions free of animal sera. The current golden standard maturation cocktail (monocyte-conditioned medium (MCM) mimic) consists of IL-1b, tumor necrosis factor-a (TNFa), IL-6 and prostaglandin E2 (PGE2). This cocktail induces mature DC (mDC) expressing high levels of co-stimulatory molecules and CCR7.5 These DC migrate efficiently towards CCR7 ligands.6 However, whereas PGE2 is important for induction of migratory capacity of DCs, it significantly impairs IL-12p70 production.7 Recently an alternative maturation cocktail was proposed that Correspondence: Dr HJ Bontkes, Department of Pathology, Vrije Universiteit Medical Center, CCA room 2.22, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands. E-mail: [email protected] Received 19 June 2006; revised 17 August 2006; accepted 4 September 2006; published online 12 October 2006

includes polyinosinic:polycytidylic acid (p-I:C), interferon (IFN)-a and IFN-g. This cocktail generates mature DC (aDC) capable of producing IL-12p70 whereas retaining migratory capacity.8 Vaccination with DCs loaded with single CTL epitopes has lead to some therapeutic effects.9 However, it has become clear that the induction of both Th cell and CTL responses are necessary for an effective anti-tumor response. Delivery systems allowing DCs to present multiple epitopes presented by both major histocompatibility complex (MHC) class I and II are currently explored. Transfection of messenger RNA (mRNA) encoding tumor antigens is a safe and relatively easy method to accomplish this, and has already lead to clinical results.10 Here we show that in contrast to mDC, aDC do not express transgenes after mRNA transfection. In order to compensate for the reduced IL-12p70 production, PGE2-matured mDC were co-transfected with mRNA encoding IL-12 ensuring constitutive IL-12p70 production. CTL were induced with TAA mRNA-transfected DCs co-transfected with either green fluorescent protein (GFP) or IL-12 mRNA. In comparative experiments, higher numbers of melanoma antigen recognized by autologous T-cells (MART-1)-specific CTL with higher functional avidity were induced after stimulation with mDC transfected with MART-1 in combination with IL-12 mRNA. Thus, in contrast to aDC, MCM-mimic matured DC can be efficiently electroporated with mRNA. These DC express high levels of co-stimulatory molecules and are capable of

IL-12 mRNA-transfected DC induce high avidity CTL HJ Bontkes et al

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Figure 1 Modification of mRNA sequences and in vitro transcription. (a) MoDC were electroporated with a minigene of four repeats of the MART126–35L-modified epitope (MART4) mRNA or with MART4 mRNA preceded by ubiquitin (ubi(MART)4). These were used as APC in an intracellular IFNg assay, 3, 24 and 72 h after electroporation using a MART126–35-specific CTL clone (4.4TG). Peptide pulsed (2 h before the start of each IFNg assay) JY cells were used as positive control APC. One experiment out of two performed is shown. Percentages of IFNg-producing cells of the second experiment after 3, 24 and 72 h: MART(4) mRNA: 6, 0.6 and 0.3%; ubi(MART)4-2A mRNA: 74, 39 and 37%; MART1 peptide: 98, 95 and 90%. (b) MoDC were electroporated with IL-12, ubi(MART)4-2A or ubi(MART)4 mRNA linked with the 2A sequence to IL-12 mRNA (uMART2-A-IL12). These were used as APC in an intracellular IFNg assay 3 h after electroporation using another MART126–35-specific CTL clone (4TG-D8b). Peptide pulsed JY cells were used as positive control APC. Dot plots of one experiment out of three performed are shown. Numbers in the upper right quadrant represent the mean (s.d.) percentage of IFNg-positive cells of those three experiments. (c and d) MoDC were electroporated with IL-12 mRNA IVT using the conventional cap (CAP) or the modified cap (ARCA). MoDC electroporated with conventional capped GFP were used as a negative control. MoDC were resuspended to 2 10E5/ml and five replicate wells were set up. After 24 h supernatant was harvested, cells were washed extensively and cultured for another 24 h after which supernatant was collected. This was repeated for four consecutive days (n ¼ 3).

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Modifications of mRNA sequences and in vitro transcription Previously it has been described that proteosomal targeting of antigens transfected into antigen presenting cells (APC) using ubiquitin improves cell-surface MHC class I presentation.11 Antigen presentation after electroporation with in vitro transcribed (IVT) mRNA from the MART4 construct (a minigene encoding four repeats of the human leukocyte antigen (HLA)-A2.1 restricted MART1-derived altered peptide ligand MART126–35L), was compared with mRNA from a construct of the same minigene but preceded by ubiquitin (ubi(MART)4). Figure 1 shows that antigen presentation by DCs transfected with mRNA from the ubi(MART)4 construct to a specific CTL clone is not only increased but also prolonged compared to MART4 mRNA-transfected DCs. Over 75% of the CTL were positive for intracellular IFNg after stimulation with the peptide-loaded positive control APC, in comparison to 72% after stimulation with ubi(MART)4-transfected DC and 17% after stimulation with MART4-transfected DC (Figure 1a, top panel). In addition, possibly owing to this increased MART epitope expression, the antigen presentation was also prolonged, 24 and 72 h post-transfection. ubi(MART)4-transfected DC were still capable of inducing IFNg production by the specific CTL clone (51 and 23% of CTL-positive, respectively) at these time points whereas hardly or no IFNg-positive CTL were detected when stimulated with MART4-transfected DC (Figure 1a lower two panels).

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When co-transfecting two separate mRNA transcripts, most of the cells become double positive for both genes (not shown). However, for clinical application it is preferable to be able to transfect with a single mRNA transcript if two or more genes need to be transfected. Therefore we employed the self-cleaving 2A peptide derived from the insect virus Thosea asigna.12 Through

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mRNA-transfected aDC fail to express the transfected gene Recently published data suggested that a-type-1 polarized DC (aDC) are superior to DCs matured with a cytokine mix that mimics MCM (mDC) owing to higher levels of IL-12p70 production by aDC.8 We set out to compare mDC and aDC for antigen-specific CD8 T-cell induction after mRNA electroporation. DC electroporated after maturation migrate more efficiently in vitro14,15 and are more effective in antigen presentation15 compared to DC electroporated before maturation. Furthermore, during the 48 h of in vitro maturation antigen presentation will be lost owing to loss of antigen expression (see Figure 1a). Electroporation of DC was therefore performed at the mature state. As previously described, both mDC and aDC are negative for CD14, but do express CD1a, CD83 and CCR7 as well as high levels of co-stimulatory molecules (data not shown).8 However, aDC did not express nerve growth factor receptor (NGFR) or GFP after transfection with the corresponding mRNA (not shown and Figure 2b), whereas mDC strongly express the transgene after mRNA electroporation (Figure 2a). Therefore, with the current protocols, mRNA transfection cannot be used for TAA loading of aDC. For optimal induction of T-cell responses, DCs need, next to a fully mature status, respond to lymph nodederived chemokines. It was shown that aDC migrate towards CCR7 ligands, be it with a lower efficiency compared to mDC.8 Here we confirm these results with DCs that were not electroporated (Figure 2c). Electroporation with GFP mRNA did not affect the migratory capacity of either mDC or aDC (Figure 2c). In contrast to Gene Therapy

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a ribosomal skip mechanism the 2A peptide impairs normal peptide bond formation between the last glycine of the 2A peptide and the proline which precedes the second protein, without negatively affecting translation of the second protein.13 Here we tested whether it is possible to generate a single IVT mRNA strain from a construct in which a TAA sequence is linked to the IL-12 sequence via a 2A peptide. Antigen presentation was comparable after DC transfection with IVT mRNA from the ubi(MART)4-2A-IL12 and ubi(MART)4-2A constructs (Figure 1b). IL-12p70 production over the first 6 h after transfection was comparable after transfection with either IL-12 or ubi(MART)4-2A-IL12 mRNA (204 and 352 pg/ml, respectively). Because IL-12p70 is secreted, constitutive expression mediated by mRNA transfection is expected to be rapidly lost in time. Therefore we investigated whether ARCA-capped mRNA results in prolonged IL-12p70 secretion by transfected DCs. Figure 1c shows that by using ARCA-capped mRNA, IL-12p70 production over the first 24 h is about twofold higher compared to conventionally capped mRNA. Over the next 5 days (extensively washed) DCs transfected with ARCA mRNA continued to produce higher levels of IL-12p70, but IL-12p70 production from DC transfected with conventionally capped mRNA produced much lower levels which had become undetectable by day 5. Although the IL-12p70 production wanes over 5 days after transfection, the IL-12p70 produced was stable during this period (Figure 1d).

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Figure 2 Transgene expression and migration of mature DC after electroporation. (a) mDC and (one experiment out of five performed is shown, mean % GFP (s.d.): 76% (4.3%) (b) aDC (one experiment out of three performed is shown, mean % GFP (s.d.): 1% (0.45%) were electroporated with mRNA encoding GFP. DCs were harvested 18 h after GFP electroporation and GFP expression was analyzed and (c) cells were used in an overnight migration assay (n ¼ 3); NEP: not electroporated (open bars), GFP: electroporated with GFP mRNA (black bars) migration towards 6Ckine, and migration of NEP DC towards medium (gray bars). (d) IL-12p70 production was measured over 24 h after electroporation with GFP or IL-12 mRNA with (+J558) or without (J558) CD40 ligation, by mDC or aDC (n ¼ 3).

a better migration by mDC, aDC are better equipped for IL-12p70 production,7,8 which is crucial for the induction of a strong type 1 cell-mediated immune response.4 This is also confirmed here, aDC already produce considerable amounts of IL-12p70 without CD40 ligation (J558), which is highly increased by stimulation with the CD40L-transfected cell line J558 (Figure 2d). Electroporation with IL-12 mRNA does not decrease, nor increase the IL-12p70 production by aDC, the latter is to be expected as mRNA-transfected aDC do not express the transgene (Figure 2b). As expected, mDC produced no IL-12p70 without CD40 ligation and relatively low levels after stimulation with J558. However, this lack of IL12p70 production could be remedied by transfection with IL-12 mRNA (Figure 2d). Figure 1c shows that this IL-12p70 production could even be boosted further by using ARCA-capped mRNA.


DCs cultured without FCS have a fully mature phenotype and can be transfected efficiently with mRNA For clinical application it is necessary to generate DC under fetal calf serum (FCS)-free conditions. Previously it was shown that DC generated in X-vivo supplemented with 2% human AB serum (hAB) serum and matured in MCM were fully mature, migrated both in vivo and in vitro and induced T-cell activation.16 Here, we compared X-vivo cultured DC with DCs generated in Iscove’s modified Dulbecco’s medium (IMDM)/FCS and the medium specifically developed for the FCS-free generation of DC (CellGro-DC), with respect to phenotype, capacity to be transfected with mRNA and capability to migrate to 6Ckine after mRNA transfection. DCs generated in these specialized media supplemented with human serum were negative for CD14, but expressed lower levels of CD1a compared DCs generated in the presence of FCS. After maturation with MCM, mDC generated in all three media expressed high levels of CD40, CD80, CD83 and CCR7 (Figure 3a). FCS-free DC were efficiently transfected with mRNA (Figure 3b) and migrated equally well towards 6Ckine as DC generated in the presence of FCS (Figure 3c). Thus, mDC generated under FCS-free conditions can be efficiently transfected with mRNA, were fully mature and migrate towards CCR7 ligands. IL-12 and MART1 mRNA DC co-transfection leads to the efficient induction of CTL with high functional avidity It was investigated whether constitutive expression of IL-12p70 by mDC, achieved by transfection with IL-12 mRNA, could enhance the induction of antigen-specific CD8-positive T cells using the MART126–35L altered peptide ligand as a model antigen. Isolated CD8bpositive T cells were stimulated with mDC electroporated with ubi(MART)4 mRNA in combination with either GFP or IL-12 mRNA in multiple small-scale bulk cultures. After each stimulation, individual cultures were analyzed for the presence of specific T cells by Tm staining. Direct ex vivo analysis showed no MARTspecific CTL. After in vitro stimulation MART-specific CTL could be detected in all (8/8) bulk cultures (Figure 4a). Already after the first stimulation a significantly higher number of Tm-positive T cells could be detected in the bulk cultures stimulated with IL-12 mRNA cotransfected DC. This difference was sustained after a second stimulation, clearly indicating a quantitative difference. The generated T cells were functional as they specifically recognize target cells loaded with the altered peptide ligand in a cytotoxicity assay (Figure 4c). Furthermore, these CTL recognize wild type, endogenously processed antigen, as shown by specific IFNg production (Figure 4b) and cytolytic activity (Figure 4d) in response to the MART1 and HLA-A2.1-positive melanoma cell line Mel-AKR, with only low back-ground reactivity in response to the MART1-positive but HLAA2.1-negative melanoma cell line Mel-JKO. Apart from the quantitative difference between CTL induced with DCs transfected with or without IL-12 mRNA, these data also demonstrate a qualitative difference; the proportion of IFNg-producing CTL

increased massively within the Tm-positive cell population stimulated with IL-12 mRNA-transfected DCs (Figure 4b). In the cytotoxicity assays the number of effector cells used, was corrected for the percentage of Tm-positive T cells within the pooled bulk cultures, leading to equal Tm-positive specific T cell to target-cell ratio’s. Peptide pulsed JY and in particular Mel-AKR were more efficiently killed by T cells induced with IL-12 mRNA-transfected DC (Figure 3c and d). This qualitative difference was even more evident in peptide titration assays which showed an increased functional avidity of the CTL which were induced by DCs co-transfected with IL-12 mRNA. Intracellular IFNg production was analyzed in response to JY pulsed with 10-fold serial dilutions of the MART126–35L peptide. Only a maximum of 50% of the specific Tm-positive T cells induced with DC co-transfected with GFP mRNA responded to JY pulsed with 1 mM peptide. On the other-hand already 60% of the specific Tm-positive T cells induced by DC cotransfected with IL-12 mRNA responded to JY pulsed with a million fold lower peptide concentration (1 pM), whereas a maximum of 89% of the Tm-positive T-cells responding cells was reached after stimulation with JY loaded with 100 nM peptide (Figure 4e). These results clearly show that DCs overexpressing IL-12p70 not only induce higher numbers of specific T cells, but induce T cells with a higher functional avidity as well. CTL induced with IL-12 mRNA-transfected DC have a reduced expression of CD27 (P ¼ 0.06) and in particular reduced CD28 expression (P ¼ 0.03, Figure 5), which indicates an advanced differentiation into effector cells compared to CTL induced with control DCs. Other markers which are lost upon differentiation into effector cells such as CD62L and CCR7 were not expressed on any of the MART1-specific CTL whereas expression levels of CD45RA(+/) and CD45RO (+/) were similar on CTL induced with or without IL-12p70 (not shown).

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CD4+ T-cell help increases numbers but not functional avidity of specific CTL CD4-positive T cells provide help during the induction of antigen-specific CD8+ T cells by DCs, also by inducing IL-12p70 production by DCs. To investigate whether CD4+ T cells could still further enhance the induction of antigen-specific CTL by mature DCs transfected with IL-12 mRNA, MART-specific CTL were induced in the presence or absence or irradiated autologous CD4+ T cells with ubi(MART)4-transfected DC co-transfected with either GFP or IL-12 mRNA. Indeed, despite the constitutive high expression of IL-12p70, addition of irradiated CD4+ T cells further increased the number of antigen-specific CTL (Figure 6a), but CD4 help did not support an increase in functional avidity (Figure 6b).

Discussion DCs are the most powerful APC able to induce primary T cells and maintain previously induced immune responses. Methodologies to generate DCs from circulating precursors in vitro have boosted the adoptive transfer of antigen-loaded DCs as adjuvant therapy for various types of cancers. Multiple clinical trials have shown induction of limited vaccine-specific immune responses. As then numerous studies have addressed optimizing Gene Therapy


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Figure 3 High expression levels of co-stimulatory molecules and efficient transfection with mRNA of mDCs generated without animal serum. (a) Phenotype of MoDC generated different media (as indicated) were matured with MCM mimic for 48 h. The open histograms indicate the fluorescence intensity of cells stained with isotype controls, the closed histograms indicate the fluorescence intensity of the indicated markers. Plots of one experiment out of three performed are shown. Numbers in dot plots indicate the mean (s.d.) percentage of CD1a and CD14-positive cells, the numbers in the histograms indicate the mean fluorescence index (s.d.) which was calculated as described in the Materials and methods, of the three experiments performed. (b) GFP expression 18 h after transfection with GFP mRNA, of MCM mimic matured MoDC generated different media (as indicated). One experiment out of three performed is shown, mean % GFP (s.d.) of those three experiments: IMDM/FCS: 78(16); X-vivo/hAB: 76(4) and CellGro/hAB: 95(2). (c) MoDC generated different media (as indicated) were matured with MCM mimic for 48 h and used in an overnight migration assay towards medium or 6Ckine. Two out of two experiments performed are shown.

maturation and antigen loading procedures of DCs. Transfection with mRNA coding for TAA has multiple advantages above peptide loading, DNA-vector transfection or use of recombinant viruses. mRNA transfection is safe, highly efficient, leads to high levels of protein expression of which a broad spectrum of epitopes over various HLA restrictions can be presented to T cells. Clinical studies have shown that adoptive transfer of mRNA-transfected DCs is safe and induces TAA-specific T-cell responses10,17–19 However, the in vitro generation of DC possessing all the features crucial for the induction of a type 1 immune response, such as mature state, migratory potential and IL-12p70 production is complicated. In particular PGE2, which is essential for efficient DC migration,20 inhibits IL-12p70 production. An alternative cytokine cocktail without PGE2 but including IFNa and poly I:C was proposed by Mailliard et al.8 Gene Therapy

These so-called aDC were still able to migrate towards CCR7 ligands, be it with lower efficiency than PGE2 matured DC, whereas still producing high levels of IL-12p70 after CD40 ligation. Here, we have confirmed these findings. However, we found that after transfection with mRNA, aDC do not express the transgene, aDC are therefore not suitable for antigen loading with mRNA. IL-12p70 enhances natural killer cell cytotoxicity and IFNg production (reviewed by Trinchieri21). Furthermore, murine studies have shown that for naı¨ve CD8+ T cells to differentiate into lytic effector cells a third signal is needed apart from T-cell receptor (TCR) engagement and co-stimulation through CD80/86 and CD28 interactions, which can be provided by IL12p70.22,23 Human DC, transduced with adenoviral vectors expressing the IL-12 gene, effectively stimulate specific type 1 CD4+ T cells from melanoma patients


in vitro.24 Moreover, we have recently shown that IL12p70 enhances proliferation and IFN-g production of human antigen-specific effector memory CTL as well.25 These studies underscore the importance of IL-12p70 in the induction and maintenance of antigen-specific CTL. Therefore we co-transfected IL-12 mRNA together with TAA mRNA to compensate for the lack of IL-12p70

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Figure 5 Induced MART26–35-specific CTL have an effector T-cell phenotype. Expression of CD27 and CD28 on TmMART126–35Lpositive CD8+ T cells was measured on CTL induced with DC co-transfected with either GFP or IL-12 mRNA (n ¼ 5). Mean fluorescence index is shown, line indicates the mean. MF indices were compared using a Wilcoxon-signed rank test. An example of a CD27 and a CD28 staining is shown: thin lines, CTL induced with DC co-electroporated with GFP mRNA; bold lines, CTL induced with DC co-electroporated with IL-12 mRNA. The horizontal bars represent the isotype histograms.

avidity of CD8+ T cells is dependent on IL-12p70.30 Furthermore, the reduced expression of CD27 and CD28 on CTL induced with IL-12 mRNA-transfected DC indicates an advanced differentiation into effector T cells.31,32 Initial experiments were all performed in the presence of autologous irradiated CD4+ T cells. However, as we used fully mature DC expressing high levels of IL-12p70, it may be expected that CD4 help was redundant. However, we show here that CD4+ T cells provided additional help in inducing higher numbers of CTL, but without increasing the functional avidity. This could be due to a further elevation of IL-12 levels, however Figure 2d shows that CD40 ligation only induces a minor increase in IL-12 levels of IL-12 mRNA-transfected DCs. What other factors may be involved in this extra help is currently under investigation. Various groups have investigated co-transfection of (co-)stimulatory molecules such as, OX40L,33 4-1BBL34 and polyI:C35 or used CD40L encoding adenoviruses to transduce DC.36 Co-transfection or -transduction of these agents with tumor antigens into DCs, increased antigenspecific CTL induction, with differential effects on IL12p70 expression and DC migration. However, none of these studies addressed the duration of the increased expression of IL-12p70 and/or the transfected costimulatory molecule. Furthermore, it is not clear from those studies what the quality, that is, the functional avidity of the induced CTL, is. Comparative studies are warranted to elucidate which approach or combination will be the most efficient in inducing strong tumorspecific immune responses both in vitro and in vivo. In conclusion, we describe here a potent DC-based vaccine consisting of PGE2 matured DC transfected with Gene Therapy

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peptide loading concentration Figure 6 CD4+ T-cell help induces higher frequencies of specific CTL without affecting the functional avidity. (a) Isolated CD8b were stimulated with mDC electroporated with ubi(MART)4 mRNA in combination with GFP or with ubi(MART)4-2A-IL-12 mRNA, either in the absence or presence of irradiated autologous CD4+ T cells. After the second stimulation the number of MART1-specific CTL was determined using Tm staining. The lines indicate the mean. One representative donor of three tested is shown. Differences between stimulation conditions were compared using a two-sided student’s t-test. (b) Functional avidity analysis of pooled bulk cultures as determined by intracellular IFNg/TmMART126–35L staining using HLA-A2.1-positive JY cells as target, loaded with serial 10-fold dilutions of the MART126–35L peptide. The percentage of IFNg-positive cells within the CD8+ TmMART126–35L+ population is plotted. Open squares (&), T cells stimulated with ubi(MART)4 plus IL-12 mRNA-transfected DC in the absence of CD4+ T cells; closed squares (’) T cells stimulated with ubi(MART)4 plus IL-12 mRNA-transfected DC in the presence of CD4+ T cells. One representative experiment of three performed is shown.

ARCA-capped mRNA, encoding TAA preceded by ubiquitin and linked to IL-12 by a 2A peptide. This method provides DC with high responsiveness to secondary lymphoid organ chemokines, which secrete IL-12p70 and present antigen for at least 72 h, and which induce high avidity tumor antigen-specific CTL with, but also without, CD4 T-cell help. Our approach offers a DCbased vaccine, generated under conditions without FCS, possessing all the features considered necessary for induction of optimal tumor reactive Th1 immune responses.

Materials and methods Media and reagents and cell lines TNFa (50 ng/ml), IL-6 (100 ng/ml), IL-7 (5 ng/ml), IL-1b (25 ng/ml) and IL-2 (10 IU/ml) were purchased from


Strathmann Biotech, Hanover, Germany. Granulocyte macrophage-colony stimulating factor (GM-CSF, Schering-Plough, Kenilworth, NJ, USA) was used at 100 ng/ ml; IL-4 (R&D systems, Abingdon, UK) was used at 10 ng/ml; PGE2 (Sigma-Aldrich, St Louis, MO, USA) was used at 1 mg/ml; IFNg (Biosource, Camarillo, CA, USA) was used at 1000 U/ml; IFNa (Peprotech, London, UK) was used at 3000 U/ml and pI:C (Sigma-Aldrich) was used at 20 mg/ml. IMDM (Cambrex, Verviers, Belgium) was supplemented with 10% FCS (Perbio, Helsingborg, Sweden). X-vivo-15 (Cambrex) and CellGro-DC (CellGenix, Freiburg, Germany) were both supplemented with 2% hAB (ICN Biomedicals, Zoetermeer, The Netherlands). All media were supplemented with 100 IU/ml sodium penicillin (Yamanouchi Pharma, Leiderdorp, The Netherlands), 100 mg/ml streptomycin sulfate (Radiumfarma-Fisiopharma, Naples, Italy), 2.0 mM L-glutamine (Invitrogen, Breda, Netherlands) and 0.01 mM 2-mercapo-ethanol (Merck, Darmstadt, Germany). The melanoma cell lines Mel-JKO and MelAKR and the EBV-LCL JY were cultured in IMDM/10% FCS. The HLA-A2.1 restricted melanoma-(MART-1(27– 35)) specific CTL clones (4-TG-D8b and 4.4TG) were generated and propagated with feeder mix as described previously.37 Fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)- or APC-labeled isotype controls and mouse mAbs to CD1a, CD86, CD40 (Pharmingen, San Diego, CA, USA), CD80 and CD14 (BD Biosciences, Heidelberg, Germany), CD83 (Immunotech, Marseille, France), CCR7 (R&D, Minneapolis, MN, USA) and NGFR (Chromaprobe, Maryland Heights, MO, USA) were used to determine the phenotype and transfection efficiency by fluorescence-activated cell sorting (FACS) analysis. Mean fluorescence index was calculated as follows: MFIndex ¼ mean fluorescence intensity marker/ fluorescence intensity isotype.

mRNA in vitro transcription Enhanced GFP (EGFP) was replaced by the various genes of interest in the pGEM4Z/EGFP/A64 vector, which was used for in vitro mRNA transcription (IVT). The IL-12elasti cassette containing the p35 and p40 subunits of IL-12 joined together by a flexible linker (InvivoGen, San Diego, CA, USA) was used. This construct guarantees equal expression of both subunits and prevents over-expression of p40 and the creation of p40 homodimers which behave as an antagonist of IL-12p70.38 A codon-optimized minigene was constructed containing four repeats of the altered peptide ligand MART126–35A27L39 (MART4) in a string of beads manner (GeneArt, Regensburg, Germany). For proteosomal targeting, the MART sequence is preceded by the ubiquitin open-reading frame. To facilitate the translation of two separate proteins from one mRNA message, we made use of the T. asigna virus derived 2A sequence.12 The sequence of ubi(MART)4-2A with the ubiquitin sequence (bold and italic), the four MART epitopes (bold) and the 2A sequence (bold underlined): MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPP DQQRLIFAGKQLEGRTLSDYNIQKESTLHLVLRLRGV VNSEFKHEELAGIGILTVAEFKSEELAGIGILTVAEEEL AGIGILTVAEEELAGIGILTVAEEVNRAEGRGSLLTC GDVEENPGPMGSMRGCGR. Additional sequences served as spacer or cloning sites. IVT was carried out using the T7 mMessage-mMachine kit (Ambion, Hun-

tingdon, Cambridgeshire, UK) to generate m7G(50 )pppGcapped IVT mRNA (CAP) or the T7 MessageMachine Ultra kit (Ambion) using the anti-reverse cap analog to generate 30 -O-methyl-m7G(50 )pppG-capped IVT mRNA (ARCA) as described.40 In contrast to the conventional CAP, ARCA has only one 30 -OH group because the second 30 -OH group is replaced with OCH3. Because of this substitution, the RNA polymerase can only initiate transcription with the remaining hydroxyl group thus forcing ARCA incorporation in the forward orientation. As a result, unlike transcripts synthesized with the conventional CAP analog, 100% of the transcripts synthesized with ARCA at the 50 -end are translatable, leading to a strong stimulatory effect on translation.29 0.1 U pyrophosphatase (PPase; Frementas life sciences, St Leon-Rot, Germany) per IVT reaction of 20 ml was added. As previously described,41 addition of PPase increased the yield of IVT mRNA (1557 (SD 593) versus 1033 (SD 556) ng/ml; P ¼ 0.04, paired student’s t-test). mRNA quality was checked by agarose gel electrophoresis. RNA concentration was assayed by spectrophotometrical analysis at OD260. RNA was stored at 801C in small aliquots (1 mg/ml).

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DC generation and transfection Peripheral blood mononuclear cells were isolated from buffy coats of healthy donors by density gradient centrifugation using Lymphoprep (Nycomed, Oslo, Norway). Monocyte-derived DC (MoDC) were generated using GM-CSF and IL-4 in IMDM, X-vivo-15 or CellGroDC as described.42 DC maturation was induced by an additional 2-day culture with MCM mimic (IL-1b, TNFa, IL-6 and PGE2),5 25% v/v MCM (generated as described43) supplemented with TNFa or a combination of IL-1b, TNFa, IFNg, IFNa and pI:C.8 Electroporation (300 V, 150 mF) was carried out with 10 mg of IVT mRNA as described.40 For most experiments DCs were generated in IMDM/10% FCS and matured with MCM supplemented with TNFa unless specified otherwise. DCs transfected with IL-12 or GFP mRNA were resuspended at 2 10E5 cells/ml and incubated with or without CD40L-transfected cells (2 10E5/ml J558),42 IL12p70 concentration was measured in 24 h supernatants by enzyme-linked immunosorbent assay.44 Chemotaxis of electroporated DCs was tested 24 h after electroporation in an overnight migration assay as described,6 using 250 ng/ml 6Ckine (Biosource). Quantification of migrated cells was by flow cytometry using a FACScalibur and True-count fluorospheres (Beckman Coulter, Mijdrecht, The Netherlands) according to the manufacturer’s instructions. Induction of MART126–35A27L peptide-specific CD8+ T cells CD8b-positive CTL precursors from buffy coats of HLAA2.1-positive healthy donors were isolated by positive selection on an automated magnetic sorting device (autoMACS; Miltenyi Biotec, Bergisch Gladbach, Germany) as described.45 Mature DC were transfected with ubi(MART)4 mRNA in combination with either GFP or IL-12 mRNA or with ubi(MART)4-2A-IL12 mRNA. Four hours after electroporation DCs were washed and multiple bulk cultures containing 0.5–1 106 CD8b T cells, 0.5–1 105 mRNA-transfected DCs and 0.25– Gene Therapy


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0.5 106 irradiated autologous CD4+ T cells were set up in Yssel’s medium supplemented with 1% hAB.45 The next day IL-7 was added. After 10 days, T cells were analyzed for specificity using PE- and/or APC-labeled HLA-A*0201 tetramers (Tm) presenting the MART126–35A27L epitope. Tm staining was performed as described.45 On day 10 the bulk cultures were restimulated with mRNAtransfected DCs and the next day IL-2 was added. Tm staining was performed after 7 days and the bulk cultures were restimulated as on day 10.

Functional assays Eight to 10 days after the last (DC) stimulation the T-cell bulk cultures or CTL clones were harvested and the bulk cultures were pooled per condition, washed and incubated with target cells. Intracellular IFNg staining was performed using the BD cytofix/cytoperm plus kit (BD Biosciences) according to the manufacturer’s instructions. One hour after the start of the stimulation, GolgiPlug was added to each well (0,1% v/v). After 5 h cells were washed, stained with APC-labeled tetramers and PE labeled CD8 followed by intracellular staining with FITC-labeled anti-IFNg. Cytolytic activity of the pooled bulk cultures was determined using a standard chromium release assay as described45 in the presence of a 50 times excess of unlabeled K562.

Acknowledgements We thank A Stam for technical assistance, and the Maurits and Anna de Kock Foundation for financial support in the purchase of an HPLC. We thank Dr ML Kapsenberg for providing IL-12 antibodies and Dr S Hallez for providing the ubiquitin open-reading frame. We are grateful to Drs KJB Scholten and Dr MWJ Schreurs for fruitful discussions. This work was financially supported by Dutch Cancer Society (KWF) Grant VU2002-2627. VFIVT is a postdoctoral fellow of the fund for Scientific Research-Flanders (FWO-Vlaanderen).

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