FLT3 Ligand Enhances the Cancer Therapeutic Potency of ...Microenvironment and Immunology FLT3 Ligand Enhances the Cancer Therapeutic Potency of Naked RNA Vaccines Sebastian Kreiter1,

Microenvironment and Immunology

FLT3 Ligand Enhances the Cancer Therapeutic Potency ofNaked RNA Vaccines

Sebastian Kreiter1, Mustafa Diken1, Abderraouf Selmi1,2, Jan Diekmann1, Sebastian Attig2, Yves H€usemann1,Michael Koslowski1, Christoph Huber1, Özlem T€ureci4, and Ugur Sahin1,2,3

AbstractIntranodal immunization with antigen-encoding naked RNA may offer a simple and safe approach to induce

antitumor immunity. RNA taken up by nodal dendritic cells (DC) coactivates toll-like receptor (TLR) signalingthat will prime and expand antigen-specific T cells. In this study, we show that RNA vaccination can beoptimized by coadministration of the DC-activating Fms-like tyrosine kinase 3 (FLT3) ligand as an effectiveadjuvant. Systemic administration of FLT3 ligand prior to immunization enhanced priming and expansion ofantigen-specific CD8þ T cells in lymphoid organs, T-cell homing intomelanoma tumors, and therapeutic activityof the intranodal RNA. Unexpectedly, plasmacytoid DCs (pDC) were found to be essential for the adjuvant effectof FLT3 ligand and they were systemically expanded together with conventional DCs after treatment. Inresponse to FLT3 ligand, pDCs maintained an immature phenotype, internalized RNA, and presented the RNA-encoded antigen for efficient induction of antigen-specific CD8þ T-cell responses. Coadministration of FLT3ligand with RNA vaccination achieved remarkable cure rates and survival of mice with advanced melanoma. Ourfindings show how to improve the simple and safe strategy offered by RNA vaccines for cancer immunotherapy.Cancer Res; 71(19); 6132–42. �2011 AACR.

Introduction

Direct application of naked antigen-encoding RNA is formore than one decade under active investigation as a vaccineapproach against cancer (1). Compared with DNA, RNAimmunization lacks the risk of integration into the genomeand due to transient expression of the encoded antigensallows better control of immune responses. In contrast toviral recombinant vectors, RNA does not feature immunodo-minant viral antigens that interfere with the preferred antigen-specific immune response. Moreover, RNA-based compoundsnot only deliver the complete antigen but also have anintrinsic adjuvant activity. Recombinant RNA is easy to pro-duce by in vitro transcription in large amounts and high purity.The capability of RNA vaccines to confer antitumor immunityhas been shown in model systems (2–6) and promising resultsof early clinical testing have been reported recently (7–9).

A few years ago, we developed a strategy to generatepharmacologically optimized RNA with improved stability,translational performance, and presentation of the encodedantigen onMHC class I and II molecules (4, 10, 11). Injection ofsuch optimized RNA into lymph nodes is superior to any otherapplication route with regard to induction of potent antigen-specific T-cell immunity (5). The RNA propagates a proin-flammatory microenvironment in the lymph node, resulting inde novo priming and efficient expansion of antigen-specificCD8þ and CD4þ T cells (5). By immunizing tumor-bearingmice with this RNA vaccine, we achieved potent antitumorimmunity and remarkable survival benefit (5).

The objective of the current study was to further augmentthe potency of intranodally administered RNA by combinationwith a suitable adjuvant. Adjuvants are compounds thatenhance the magnitude, breadth, quality, and longevity ofspecific immune responses to antigens and have minimaltoxicity. For a long time, adjuvant research progressed at aslow pace and adjuvant design was largely empirical. In themeantime, as the development of immunotherapeutics isgaining speed, the need for rational selection strategies foradjuvant formulations based on sound immunologic princi-ples is becoming evident.

We recently revealed that RNA administered into the lymphnode is selectively internalized by resident immature dendriticcells (DC) and that the uptake is highly efficient and is drivenby macropinocytosis (12). Both the cell type and the uptakemechanism were shown to be functionally relevant for theobserved efficiency of intranodal RNA vaccination. Thesenotions were factored into the rationally guided search forsuitable adjuvants. With DCs being essential for vaccine

Authors' Affiliations: 1Institute for Translational Oncology and Immunol-ogy (TRON); 2Department of Internal Medicine III, Division of Translationaland Experimental Oncology, Johannes Gutenberg University; 3Ribologi-cal, Biontech AG; and 4Ganymed Pharmaceuticals AG, Mainz, Germany

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

S. Kreiter, M. Diken, and A. Selmi contributed equally to the first author-ship.

Corresponding Author: Ugur Sahin, Institute for Translational Oncologyand Immunology (TRON), Translational and Experimental Oncology,Johannes Gutenberg University, Langenbeckstr. 1, 55131 Mainz,Germany. Phone: 49-6131-178011; Fax: 49-6131-178059; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-11-0291

�2011 American Association for Cancer Research.

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effects of intranodal RNA, we came across Fms-like tyrosinekinase 3 (FLT3) ligand (13, 14), a naturally occurring glyco-protein stimulating early hematopoietic progenitors via theFLT3 receptor (15) and mobilizing them from the bonemarrow into the peripheral blood and secondary lymphaticorgans. FLT3 ligand is known to induce profound expansion ofDCs in the peripheral lymphoid organs (16–18), and FLT3ligand–induced DCs have an immature phenotype (19, 20).However, it is not clear whether these DCs are capable ofpresenting antigens encoded by RNA. In addition to expansionof DCs, various other effects of FLT3 ligand have beenreported, including stimulation of natural killer (NK), B,and T cells (21–24). These effects translated in improved T-cell immunity and antitumor potency in mouse models whenFLT3 ligand was used as an adjuvant in vaccinationapproaches (16–18, 25–27).In this study, we detail our efforts to evaluate FLT3 ligand in

combination with intranodally administered RNA and todissect the mechanisms underlying the strong synergy of bothcompounds. Surprisingly, we revealed that plasmacytoid DCs(pDC) are central to the observed adjuvant effects of FLT3ligand. Our findings not only open new paths for improvedRNA vaccination strategies but also give unexpected insightsinto the functional potency of pDCs.

Materials and Methods

AnimalsC57BL/6 mice were obtained from Jackson Laboratories.

TCR transgenic OT-I mice that recognize the H2-Kb-restrict-ed SIINFEKL epitope (OVA257-264) from chicken ovalbuminand C57BL/6-based IFN-a receptor (IFNAR)-deficient(IFNAR�/�) mice (28) were kindly provided by H.J. Schild(Mainz, Germany) and Paul-Ehrlich Institute (Langen,Germany), respectively. All mice were kept in accordancewith federal and state policies on animal research at theUniversity of Mainz.

CellsB16-F10 melanoma cell line expressing the chicken oval-

bumin (OVA) gene (B16-OVA, a gift of U. Hartwig, University ofMainz, Germany) was received in 2008 and subjected tomaster cell bank generation. Early (third and fourth) passagesof cells were used for tumor experiments. Cells were routinelytested for Mycoplasma. Reauthentification of cells has notbeen done since receipt. FLT3 ligand-conditioned bone mar-row–derived DCs (FLT3L-BMDC) were generated by 8-dayculture of bone marrow precursors with 200 ng/mL FLT3L-IgG4.

FLT3 ligand plasmid construction and proteinpurificationA cDNA representing the signal peptide and extracellular

domain of human FLT3 ligand (aa 1–185) was cloned into anexpression vector and carboxy-terminally fused to a 675-bpfragment coding for the hinge region and heavy chain con-stant regions 2 and 3 [(CH2 and CH3) of human IgG4], toobtain FLT3L-IgG4. As control, the 675-bp IgG4-Fc fragment

(IgG4) was cloned separately in the expression vector. HEK293cells were transfected with these constructs using polyetyle-nimine (PEI; Sigma-Aldrich) for production of FLT3L-IgG4 orIgG4 proteins, which subsequently were purified from culturesupernatants by affinity chromatography with Mab-SelectSuRe columns (GE Healthcare) and stored at �80�C. FLT3L-IgG4 or IgG4 fragments were administered intraperitoneally(i.p.). In some experiments, human IgG4 kappa antibody(Sigma-Aldrich) was used as a control.

RNA vaccines, in vitro transcription, and intranodalimmunization

All plasmids for in vitro transcription of naked antigen-encoding RNA encoding luciferase (Luc) are based on thepST1-2hBgUTR-A120 backbone (10), which feature a 30 humanb-globin untranslated region (hBgUTR) and a poly(A) tail of120 nucleotides and allow generation of pharmacologicallyimproved in vitro transcribed RNA. The MITD vectors pST1-sec-SIINFEKL-MITD-2hBgUTR-A120 (SIINFEKL; ref. 11) andpST1-sec-Influenza-HA-MITD-2hBgUTR-A120 (HA; ref. 11) al-low tagging of the respective protein with a secretion signal(Sec) and the transmembrane and cytosolic domains of theMHC class I (MITD) ensuring efficient presentation on MHCclass I and class II. The SIINFEKL construct contains aa 257 to264 of chicken OVA and the HA construct a codon-optimizedpartial sequence of influenza HA (aa 60–285 fused to aa 517–527; influenza strain A/PR/8/34) designed to combine majorimmunodominant MHC epitopes.

In vitro transcription and purification of RNA were previ-ously described (29). HA-RNA was labeled with Cy5-UTP (Cy5-RNA) according to the manufacturer's instructions (Amer-sham). Purified RNA was assessed by spectrophotometry, gelelectrophoresis, and BioAnalyzer (Agilent).

As described previously (5), for intranodal immunization ofmice with naked RNA, the inguinal lymph node of anaesthe-tized mice was surgically exposed, 20 mg RNA in 10 mL RNase-free PBS (Ambion) were injected intranodally, and the woundwas closed.

Flow cytometry and tetramer stainingAll monoclonal antibodies (mAb) were from BD Pharmin-

gen, except anti-PDCA1 (Miltenyi Biotec). Hypotonically lysedblood and splenocyte samples were incubated at 4�C withmAbs. Lymph node cells were obtained by digestion withcollagenase (1 mg/mL; Roche). Direct ex vivo quantification ofSIINFEKL-specific CD8þ cells with H-2 Kb/SIINFEKL tetramer(Beckman-Coulter) without in vitro stimulation (10) and in-tracellular cytokine staining (29) were previously described.Absolute quantification of NK cells in blood was carried outwith Trucount tubes (BD Biosciences) by adding 50 mLheparinized blood to the Trucount tube, staining with anti-bodies against CD45, CD3, and NK1.1, and quantification ofcells after lysis of erythrocytes with fluorescence-activatedcell-sorting (FACS) lysing solution (BD Biosciences). Regula-tory T cells (Treg) were analyzed with the FoxP3 Staining Kit(eBioscience). Conventional DCs (cDC; (CD11cþPDCA1�) andpDCs (CD11cþPDCA1þ) were sorted by FACSAria (BD Bios-ciences). Quantification of H-2 Kb/SIINFEKL complexes by

FLT3 Ligand Enhances Therapeutic Potency of RNA Vaccine

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25-D1.16 antibody was carried out as previously described(10). Flow cytometric data were acquired on a FACSCaliburand FACSCanto analytic flow cytometers (BD Biosciences)and analyzed with FlowJo (Tree Star) software.

In vivo bioluminescence imagingUptake and translation of Luc-RNA were evaluated by

in vivo bioluminescence imaging (BLI) using the IVIS Luminaimaging system (Caliper Life Sciences; ref. 4). Briefly, anaqueous solution of D-luciferin (150 mg/kg body weight; BDBiosciences) was injected intraperitoneally 24 hours afteradministration of Luc-RNA. After 15minutes, emitted photonswere quantified (integration time of 1 minute). In vivo biolu-minescence in regions of interest (ROI) were quantified astotal flux (photons per seconds) using IVIS Living Image 3.0Software.

Quantitative real time PCRExtraction of total cellular RNA, first-strand cDNA synthe-

sis, real-time PCR (RT-PCR) using TaqMan Gene ExpressionAssays (Applied Biosystems), and normalization to the house-keeping gene Hprt1 were described previously (30).

Tumor experimentsTumor vaccination protocols were previously described (5).

Briefly, 2 � 105 B16-OVA melanoma cells were inoculatedsubcutaneously into the flanks of C57BL/6 mice. FLT3L-IgG4(10 mg) or human IgG4 control antibody were administeredintraperitoneally on day 3, day 7, day 14, and day 18 aftertumor inoculation. Four intranodal immunizations of SIIN-FEKL-RNA (20 mg) in 3- to 4-day intervals were initiated11 days after tumor cell inoculation (diameter of tumors 2mm). The tumor sizes were measured every 3 days. Mice weresacrificed when tumor diameter reached 15 mm.

Tumor-infiltrating cells (TIC) were obtained by mechanicaldisruption of the tumor tissue and phenotyping: myeloid-derived suppressor cells (MDSC; CD11bþ, Gr1þ), Tregs(CD4þ, CD25þFoxP3þ), NK cells (NK1.1þ, CD3�), B cells(CD19þ, CD3�), SIINFEKLþ CD8þ T cells (CD3þ, CD8þ,tetramerþ), CD8þ T cells (CD3þ, CD8þ, CD4�), CD4þ T cells(CD3þ, CD4þCD8�), pDC (CD11cþ, PDCA1þ, NK1.1�), andcDC (CD11cþ, PDCA1�, NK1.1�).

In vivo depletion of pDCsC57BL/6 mice received 500 mg of anti-PDCA1 (Miltenyi

Biotec) mAb or a monoclonal rat IgG1 mAb (Jackson Immu-noResearch) as a control 2 times in 6-day intervals.

Enzyme-linked immunospot assayEnzyme-linked immunospot (ELISPOT) assay was carried

out as described before (5). Briefly, 1 � 104 cDCs or pDCssorted from SIINFEKL-RNA or control RNA (HA-RNA) elec-troporated FLT3L-BMDCs were coincubated with 1 � 105

magnetic-associated cell-sorted CD8þ OT-I T cells in a mi-crotiter plate coated with anti-IFN-g antibody (10 mg/mL,clone AN18; Mabtech). After 18 hours at 37�C, cytokinesecretion was detected with an anti-IFN-g antibody (cloneR4-6A2; Mabtech).

Immunohistochemistry and immunofluorescenceCryosections (4–6 mm) frommouse lymph nodes were fixed

with cold acetone and blocked with PBS-containing 5%mouseserum.

For indirect staining, rat-anti-PDCA1 (JF05-1C2.4.1; Milte-nyi), rat-anti-F4/80 (BM8; eBioscience) or rat-anti-NK1.1(PK136; BD Biosciences) as primary antibodies were com-bined with Cy3-anti-rat-IgG (N418; Molecular Probes). Fordirect staining, reticular fibroblasts and fibers were visualizedwith Alexa488-anti-ER-TR7 (BMA), sections were mounted inVectashield mounting medium (VECTORLabs), analyzed witha Zeiss immunofluorecence microscope (Carl Zeiss Micro-Imaging GmbH), and digital images were prepared usingPhotoshop CS4 (Adobe).

StatisticsGraphPad Prism software using unpaired 2-tailed Student's

t test or ANOVA with Tukey's multiple comparison test wasused. Differences in survival were analyzed by the log-ranktest. Values of P < 0.05 were considered statistically significant.

Results

FLT3 ligand treatment expands DCs and NK cells in thelymph node and spleen

In an attempt to test compounds expanding the DCpopulation in combination with intranodal RNA, we cameacross FLT3 ligand. As the half-life of FLT3 ligand in serumis very short (31), we engineered a recombinant fusionprotein (Fig. 1A) linking the extracellular domain of humanFLT3 ligand to the heavy chain constant regions 2 and 3(CH2-CH3 domain) of human IgG4 to increase the serumhalf-life (data not shown). It was already shown that humanFLT3 ligand can cross-react with mouse FLT3 (32),which enabled the use of human FLT3 ligand in the murinesetting. Two intraperitoneal applications of this FLT3ligand IgG4 fusion protein (referred to as FLT3L-IgG4)resulted in significant increase of cellularity in lymphnodes and spleen as compared with controls [PBS or theIgG4-Fc protein fragment lacking the FLT3 ligand moiety(referred to as IgG4; Fig. 1B; Supplementary Fig. S1A)]. AsDCs resident in lymphoid organs are of major relevance forthe mechanism of action of intranodally administered RNA,we analyzed these cells in more detail. Subpopulations ofcDCs and pDCs were profoundly expanded in both com-partments of FLT3 ligand–treated mice as compared withcontrols (Fig. 1C; Supplementary Fig. S1B). As revealed inlymph node sections, pDCs accumulated in the T-cell zones(Fig. 1D). Noteworthy, neither lymph node cDCs nor pDCsof FLT3 ligand–treated mice showed molecular signs ofmaturation (Fig. 1E).

As described recently (22, 33), CD3�NK1.1þ NK cellswere significantly expanded in lymph nodes and spleenof mice treated with FLT3 ligand (Fig. 1D and F) and weremost prominent around day 7 in blood (SupplementaryFig. S2). In line with reports on FLT3 ligand application inrats (34), we found no expansion of macrophages in mice(Fig. 1D).

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Cancer Res; 71(19) October 1, 2011 Cancer Research6134




FLT3 ligand and intranodal RNA vaccination synergizein inducing a Th1 lymph node milieu and in activationof DCs and NK cellsNext, we investigated the fate and effects of intranodally

injected RNA when combined with FLT3 ligand as adjuvant.We found that Cy5-labeled HA-RNA injected into lymph nodesof mice preconditioned with 2 treatment cycles of FLT3 ligandwas internalized by both pDCs and cDCs with high efficiency(Fig. 2A). Moreover, as shown by in vivo BLI, translation of Luc-RNA injected into lymph nodes of these mice was comparablewith that of control mice (Fig. 2B).

Most interestingly, when FLT3 ligand–treated mice re-ceived RNA intranodally, maturation of pDCs was induced asdocumented by increase of markers CD80, CD86, CD40, andMHC II (Fig. 2C). This is in analogy to what we recentlyreported for cDCs consecutively to intranodal RNA admin-istration (5). DCs started secretion of high levels of inter-leukin (IL)-12 (Fig. 2D, left). Most interestingly, pDCs werepreferentially expanded and activated by RNA so that theirabsolute number per lymph node surpassed that of cDCs(Fig. 2D, right). Expression profiling of pDCs and cDCs fromlymph nodes by quantitative RT-PCR revealed profound

Figure 1. FLT3 ligand treatmentresults in expansion of DCpopulations and NK cells in lymphnodes and spleen. A, structure ofthe human FLT3 ligand IgG4-Fcfusion protein (Flt3L-IgG4). B–E,C57BL/6 mice (n¼ 3–5) received 2intraperitoneal injections of Flt3L-IgG4 (10 mg; day 0 and day 3) orPBS (control) in 4 independentexperiments and were subjectedto analysis on day 10. B, cellularitywas assessed in lymph node andspleen. C, DC subpopulationswere quantified both in lymphnode (left) and in spleen (right).D, fluorescence microscopy ofinguinal lymph nodes was carriedout for visualization of pDCs(PDCA1), NK cells (NK1.1),macrophages (F4/80), andreticular fibroblasts (ER-TR7).Scale bars, 100 mm. E, activationstatus of cDCs (CD11cþPDCA�)and pDCs (CD11cþPDCAþ) wasassessed by flow cytometry.F, absolute numbers of NK cells(NK1.1þCD3�) were determinedby flow cytometry after Flt3L-IgG4application (as in Fig. 1B). Data(mean � SEM) are from 1 of 4(B, C, E, and F) independentexperiments with similar results.*, P < 0.05; **, P < 0.01 [Student'st test (B, C, E, and F)].

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alteration of the lymph node microenvironment of micereceiving the combination of FLT3 ligand and RNA ascompared with mice receiving FLT3 ligand alone (Fig. 2E).NK cells and Th1 lymphocytes attracting chemokinesCXCL10 (IP10) and CXCL9 (35, 36) were induced up to 2logs. IL-6, which supports expansion and survival of primedT cells (37) and CD40 receptor, a key mediator of DCactivation by CD4þ T cells, were upregulated, the latterparticularly in pDCs. DC recruiting chemokines CCL3(MIP-1a) and CCL4 (38) were modestly elevated, whereasIFN-a expression did not change.Intranodal administration of naked RNA to FLT3 ligand–

treated mice also had a major impact on NK cells. Thefrequency and absolute number of lymph node NK cells(Fig. 2F) as well as their intracellular IFN-g levels (Supple-mentary Fig. S3A) increased significantly and they exhibitedan activated CD69þ phenotype (Fig. 2G). Moreover, absolutenumber and frequency of lymph node NK cells secreting highamounts of IFN-g , when treated with PMA in vitro, were clearlyhigher in RNA treated group (Supplementary Fig. S3B), show-ing enhanced susceptibility of NK cells for inflammatorystimuli.These findings underline that the combination of FLT3

ligand and intranodal RNA administration induces an inflam-matory Th1 milieu, favorable for the efficient priming andexpansion of antigen-specific T cells, and leads to functionalactivation of both antigen presenters as well as NK cells, thusintegrating all factors required for an efficient immuneresponse.

pDCs are essential for augmentation of RNA-inducedantigen-specific T-cell response by FLT3 ligandpreconditioningWe then characterized T-cell responses induced by

combining FLT3 ligand with intranodal RNA vaccination.Mice received FLT3 ligand at day 0 and day 3 followed byintranodal SIINFEKL-RNA immunization at day 7 and day10. Frequencies of SIINFEKL-specific CD8þ T cells mea-sured 5 days after the last immunization were profoundlyincreased in blood, spleen, and lymph nodes of FLT3ligand preconditioned mice (Fig. 3A), and these cellssecreted IFN-g upon antigen encounter (SupplementaryFig. S4). To determine the relevance of pDCs for theobserved adjuvant effect, pDC-depleting antibody anti-

PDCA1 was administered to mice preconditioned withFLT3 ligand prior to immunization with SIINFEKL-RNA,resulting in reduction of the pDC subpopulation in thelymph node to half of normal levels and nearly completeabrogation in the spleen (Fig. 3B), whereas cDC and NKcell numbers were not altered (data not shown). Frequen-cies of SIINFEKL-specific CD8þ T cells achieved by thecombination of FLT3 ligand and intranodal SIINFEKL-RNA administration were significantly lower in pDC-de-pleted mice than in mice with normal pDC frequencies(Fig. 3C). pDC-depleted mice treated with FLT3 ligandand SIINFEKL-RNA had T-cell frequencies comparablewith those of mice that were immunized with SIIN-FEKL-RNA alone, suggesting that the increment in T-cellresponse achieved by FLT3 ligand preconditioning issignificantly mediated by pDCs.

To elucidate the mechanism by which pDCs mediate theFLT3 ligand adjuvant effect, we first tested their capability toact as antigen presenters. BMDCs were differentiated in vitroin FLT3 ligand–supplemented medium that resulted in amixed population of cDCs and pDCs (FLT3L-BMDCs).FLT3L-BMDCs were transfected with SIINFEKL- or con-trol-RNA, and SIINFEKL peptide/MHC molecule complexeswere quantified with the 25-D1.16 antibody (Fig. 3D). Mostinterestingly, we found efficient processing and presentationof the peptide by pDCs (factor 3.0 vs. 3.7 as compared withcDCs). When these peptide-presenting cDCs and pDCs weresorted and tested in ELISPOT for recognition by TCR trans-genic OT-I CD8þ T cells (Fig. 3E), pDCs strongly inducedIFN-g secretion, indicating their capability to act as efficientantigen-presenting cells.

To investigate whether IFN-a secretion, one of the mostprominent constitutive functions of pDCs, plays a role in theadjuvant effect of FLT3 ligand, we treated IFNAR�/� micewith FLT3 ligand or PBS as control followed by vaccinationwith SIINFEKL-RNA. The increased frequency of SIINFEKL-specific CD8þ T cells in FLT3 ligand–treated mice comparedwith control mice showed that IFN-a secretion is not key forthe FLT3 ligand adjuvant effect (Fig 3F).

FLT3 ligand improves antitumoral therapeuticimmunity mediated by intranodal RNA vaccination

Next, we wanted to know whether the profound thera-peutic benefit of intranodal RNA immunization in animal

Figure 2. FLT3 ligand and intranodal RNA vaccination synergize in induction of a Th1-favoring lymph node milieu and activation of DCs and NK cells.C57BL/6 mice (n ¼ 3–12) were treated with 2 cycles of Flt3L-IgG4 (10 mg i.p., day 0 and day 3) followed by intranodal vaccination with RNA (20 mg;day 7; HA-RNA, if not indicated otherwise) or control (in vitro transcription reaction mixture lacking RNA polymerase) in several independent experiments. If notindicated otherwise, analysis was done 24 hours after RNA injection. A, HA-RNA administered intranodally to Flt3L-IgG4–treated C57BL/6 mice (n ¼ 4)was Cy5-labeled (Cy5-nucleotides as control). One hour after RNA injection, uptake in DCs was quantified. B, Luc-RNA (20 mg) was injected into lymph nodesof C57BL/6 mice (n ¼ 8), and BLI was carried out 24 hours later (left; red lines, mean values). A representative image is shown (right). C, maturationmarkers of pDCs from inguinal lymph nodes were analyzed by flow cytometry. D, 20 hours after vaccination, frequencies of IL-12–secreting pDCs(CD11cþPDCA1þ) and cDC (CD11cþPDCA1�) in injected lymph nodes were quantified by intracellular cytokine staining after in vitro culture for 6 hours in thepresence of brefeldin A (BFA) to allow intracellular cytokine accumulation before staining. Representative dot plots (left) show gating strategy andpercentage of IL-12þ DC subpopulations (right). The bar chart shows the absolute number of IL-12–secreting DCs per lymph node. E, quantitative RT-PCRanalysis of immunomodulatory molecules expressed on DC subpopulations sorted from pooled inguinal lymph nodes of treated and control mice (n¼ 2� 12)8 hours after intranodal injection of HA-RNA. Bars indicate fold change in expression level compared with respective controls. F, the frequency (left)and absolute number (right) as well as (G) the activation status of NK cells in the inguinal lymph node were analyzed. Data (mean� SEM; C, D, and F) are eachrepresentative for 2 independent experiments with similar results. *, P < 0.05; **, P < 0.01; ***, P < 0.001 [Student's t test (B, C, D, and F)].






models can be topped by the addition of FLT3 ligand. For anadvanced B16-OVA melanoma model, tumor cells wereinoculated (day 0) and mice were conditioned with 4 admin-istrations of FLT3 ligand. Tumors were grown to macro-scopic visibility, and 4 cycles of SIINFEKL-RNA vaccinationwere initiated 11 days after tumor inoculation (Fig. 4A). Assingle agents, intranodal RNA immunization cured only 30%of mice and FLT3 ligand alone had a very moderate effect onsurvival. By combining the RNA vaccine and FLT3 ligand,however, 70% of the treated mice were cured from theircancer and survived or became tumor-free. When we char-acterized the TICs in the treatment groups, we foundsignificant differences. Although FLT3 ligand as single agentdid not alter the composition of immune cell infiltrations inthe tumor nodule, intranodal administration of SIINFEKL-RNA alone expanded nearly all tested subpopulations, inparticular of pDCs, cDCs, NK cells, T cells, MDSCs, and Tregs(Fig. 4B). The combination of both agents had a minorincremental effect on cellularity as compared with RNAalone. Interestingly, the percentage of antigen-specificCD8þ T cells was profoundly increased (Fig. 4B). As aconsequence, in animals treated with the combination,Tregs were outnumbered by cytotoxic antigen-specificCD8þ effector T cells (Fig. 4C).

In summary, these data imply that FLT3 ligand precondi-tioning further improves the survival benefit achieved byintranodal vaccination with antigen-encoding RNA and thatthis is associated with an increase of antigen-specific cytotoxicT cells infiltrating the tumor lesion.

Discussion

The use of antigen-encoding RNA for cancer vaccinationconfers several advantages. These can be fully exploited whenRNA is administered intranodally and unfolds its dual poten-tial as a template for efficient translation as well as immu-nostimulatory ligand for Toll-like receptors (TLR).

This study was undertaken to evaluate FLT3 ligand as asystemic adjuvant in conjunction with intranodal RNA vac-cination. This report shows for the first time that FLT3ligand potentiates immunogenicity of intranodal RNA vac-cination. We reveal that several mechanisms contribute tothis synergy.

First, FLT3 ligand expands DC and NK populations indifferent compartments including the lymph node and thusmobilizes beforehand a higher number of antigen presentersand effectors to the site the antigen will be delivered to. Ourfindings verify our primary hypothesis that the number ofantigen presenters in the lymph node may be rate limitingfor efficacy of RNA vaccines and should be considered whenselecting candidate compounds to combine with RNA. Sur-prisingly, we found that within the DC population, pDCshave the most dominant impact on the magnitude of in-duced immune responses and that in pDC-depleted miceadjuvant effects of FLT3 ligand are abrogated. According toour data, this can be attributed to the fact that in combi-nation with antigen-encoding RNA, pDCs act as efficient

antigen presenters, which is in analogy to effects of RNA oncDCs (5). As we show for the first time, pDCs are able toefficiently internalize RNA, are thereby activated to secreteIL-12, and process as well as present the encoded antigenwith high efficiency.

Another mechanism is that FLT3 ligand not as single agentbut together with RNA activates both cell types to develop aphenotype optimal for their cross-talk and further augmentsthe Th1-skewing effect of RNA resulting in a pronouncedproinflammatory lymph node microenvironment.

Moreover, in accordance with previous studies (19, 20), weshow that systemic FLT3 ligand administration does not leadto DC maturation; hence, FLT3 ligand–expanded DCs retaintheir immature phenotype, thereby enabling efficient inter-nalization of vaccine RNA by immature DCs. Many adjuvantsmediate maturation of antigen-presenting cells and this effectis associated with efficient induction of sustained immuneresponses and therefore highly desired. It is already knownthat macropinocytic activity of DCs is abolished by matura-tion stimuli (39, 40). Accordingly, we previously observed thatpreincubation of DCs with maturation-inducing adjuvantssuch as LPS, CD40L, or dsRNA abrogated the capability ofDCs to engulf RNA (12). The concomitant impairment ofmacropinocytosis is less relevant for extracellularly stablevaccine formats (e.g., protein or DNA-based vaccines). ForRNA vaccines, in contrast, which have a short extracellularhalf-life, antigen salvage into DCs by macropinocytosis is acritical requirement.

In summary, these mechanisms result in a significantlyhigher frequency of antigen-specific T cells in the peripheryand in tumor lesions of B16 melanoma. Higher CD8/Tregratios within tumors are reached, which are known to beindicative for effective vaccination and in fact result in acompelling cure rate in mice suffering from advanced B16melanoma (41, 42).

These observations imply that an adjuvant should be cho-sen on the basis of complementarity of its mode of action withthat of the vaccine format it will be combined with. Thesuperior immunopharmaceutical performance of RNA admin-istered intranodally depends on its selective uptake via im-mature DCs and creation of a Th1-type microenvironment.These prerequisites are easy to meet for FLT3 ligand, as it doesnot counteract, but rather enhances these 2 advantageousfeatures.

Our data also convey a better understanding of adjuvantfeatures of FLT3 ligand. It is known that daily FLT3 ligandinjections alter cellular composition in blood and secondarylymphatic organs of mice including induction of a marked DChyperplasia (16–18). In contrast to the reported regimensbased usually on administering 10 to 20 mg FLT3 ligand for7 to 9 days (18, 43), we achieved even superior antitumoraleffects in vivo with only 2 injections of 10 mg (day 0 and day 3)most likely owing to the high cooperative complementarity ofnaked RNA and FLT3 ligand. Most interestingly, amongthe different vaccine formats with which FLT3 ligand havebeen combined in the past, DNA vaccines have yielded thebest results (27, 44, 45). Neither uptake mechanisms of DNA

Kreiter et al.





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Figure 3. FLT3 ligand acts as adjuvant for intranodal RNA vaccination via its effect on pDCs. A, C57BL/6 mice (n ¼ 4–5) received either Flt3L-IgG4 (10 mg;day 0 and day 3) or PBS (control) intraperitoneally, followed by 2 intranodal immunizations (day 7 and day 10) with SIINFEKL-RNA (20 mg) in several independentexperiments. SIINFEKL-specific CD8þ T cells were quantified in different compartments by tetramer staining carried out ex vivo (left). Bar chart shows data(mean� SEM) from 2 independent experiments. Representative dot plots show the percentage of SIINFEKL-specific CD8þ T cells in all CD8þ cells 5 days afterthe last immunization (right). B andC,mice were treated as described above. After Flt3L-IgG4 preconditioning,mice received anti-PDCA1 antibody (500 mg i.p.)or a control antibody (rat-IgG1) 1 day prior to each immunization at day 6 and day 9. B, dot plots provide percentages of pDCs within all CD11cþ DCs in lymphnodes and spleen. C, percentages (mean � SEM) of SIINFEKL-specific T cells within the CD8þ population in different treatment groups were determined bytetramer staining. D, BMDCs generated with Flt3L-IgG4 (Flt3L-BMDCs) were eletroporated with either SIINFEKL-RNA (30 mg) or HA-RNA (30 mg) ascontrol together with eGFP-RNA (10 mg). Surface densities of SIINFEKL/H2-Kb complexes on eGFPþ cDCs and pDCs were assessed after 18 hours by flowcytometry with the 25-D1.16 antibody. Numbers in parentheses represent mean fluorescence intensity of 25-D1.16. Data are representative for 2 independentexperiments. E, Flt3L-BMDCs electroporated with either SIINFEKL-RNA (30 mg) or HA-RNA (30 mg) as control were sorted into cDCs and pDCs (1� 104), whichwere then coincubated with CD8þ OT-I T cells (1 � 105) and SIINFEKL-specific CD8 T-cell response (mean � SEM) was analyzed in an IFN-g ELISPOTassay 18 hours later. F, IFNAR�/� mice (n ¼ 4) received either Flt3L-IgG4 (10 mg; day 0 and day 3) or PBS (control) intraperitoneally, followed by 2 intranodalimmunizations (day 7 and day 10) with SIINFEKL-RNA (20 mg). Percentages (mean � SEM) of SIINFEKL-specific CD8þ T cells were quantified in blood bytetramer staining 5 days after the last immunization. *, P < 0.05; ***, P < 0.001 [Student's t test (A, E, and F); ANOVA with Tukey's multiple comparison test (C)].






vaccines nor the reason for synergizing with FLT3 ligand isknown, but it is well conceivable that our findings for RNAmay apply for nucleic acids in general.

Safety and tolerability of FLT3 ligand have been shown inhealthy human donors (46, 47) and cancer patients (48–51).Two clinical trials in cancer patients combined peptidevaccination with FLT3 ligand as adjuvant but failed toaugment antigen-specific T-cell immunity, which was at-tributed to the nonactivated state of FLT3 ligand–mobilizedDCs (50, 52). By addition of the TLR7 ligand imiquimod to apeptide vaccine plus FLT3 ligand, the lack of peptide-specificT-cell responses could be overcome in a melanoma trial (53).Similar data have been obtained when DNA vaccines werecombined with FLT3 ligand (44). Future studies have toshow whether the property of RNA to trigger TLR signalingpathways in conjunction with the adjuvant effect of FLT3ligand may further improve vaccine potency in the clinicalsetting as well.

In summary, these unexpected findings not only are of highrelevance for design of future RNA-based cancer vaccineprotocols but also contribute to a better conceptual under-standing of adjuvant strategies.

Disclosure of Potential Conflicts of Interest

U. Sahin (founder and chief executive officer) and C. Huber (founder) areassociated with Ribological, BioNTech AG (Mainz, Germany), a company thatdevelops RNA-based cancer vaccines in the indicated functions. U. Sahin, S.Kreiter, Ö. T€ureci, and A. Selmi are inventors on a patent application, in whichparts of this article are covered. The other authors disclosed no potentialconflicts of interest.

Acknowledgments

We thank Paul-Ehrlich Institute (Langen, Germany) for providing IFNAR�/�

mice and U. Kalinke (Twincore, Hannover, Germany) for scientific advice. Wealso thank M. Holzmann, M. C. Rethagen, and M. Brkic for excellent technicalassistance.

Grant Support

This work was supported by the by the GO-Bio program of the FederalMinistry of Education and Research.

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received January 25, 2011; revised July 29, 2011; accepted July 30, 2011;published OnlineFirst August 4, 2011.

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**

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2011;71:6132-6142. Published OnlineFirst August 4, 2011.Cancer Res Sebastian Kreiter, Mustafa Diken, Abderraouf Selmi, et al. RNA VaccinesFLT3 Ligand Enhances the Cancer Therapeutic Potency of Naked

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http://cancerres.aacrjournals.org/content/71/19/6132.full#ref-list-1

http://cancerres.aacrjournals.org/content/71/19/6132.full#related-urls

http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://cancerres.aacrjournals.org/content/71/19/6132


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