Preclinical Evaluation of TriMix and Antigen mRNA-Based ... · mRNA encoding immunomodulating...

Microenvironment and Immunology

Preclinical Evaluation of TriMix and Antigen mRNA-BasedAntitumor Therapy

Sandra Van Lint1, Cleo Goyvaerts1, Sarah Maenhout1, Lode Goethals2, Aur�elie Disy1, Daphn�e Benteyn1,Joeri Pen1, Aude Bonehill1, Carlo Heirman1, Karine Breckpot1, and Kris Thielemans1

AbstractThe use of tumor-associated antigen (TAA)mRNA for therapeutic purposes is under active investigation. To be

effective, mRNA vaccines need to deliver activation stimuli in addition to TAAs to dendritic cells (DC). In thisstudy, we evaluated whether intranodal delivery of TAA mRNA together with TriMix, a mix of mRNA encodingCD40 ligand, constitutive active Toll-like receptor 4 andCD70, results in the in situmodification andmaturation ofDCs, hence, priming of TAA-specific T cells.We showed selective uptake and translation ofmRNA in vivo by lymphnode resident CD11cþ cells. This process was hampered by codelivery of classical maturation stimuli but not byTriMix mRNA. Importantly, TriMix mRNA induced a T-cell–attracting and stimulatory environment, includingrecruitment of antigen-specific CD4þ and CD8þ T cells and CTLs against various TAAs. In several mouse tumormodels, mRNA vaccination was as efficient in CTL induction and therapy response as vaccination with mRNA-electroporated DCs. Together, our findings suggest that intranodal administration of TAA mRNA together withmRNA encoding immunomodulating molecules is a promising vaccination strategy. Cancer Res; 72(7); 1661–71.�2012 AACR.

IntroductionThe immune system can mount immune responses against

tumor-associated antigens (TAAs). Such immune responses,mediated by CD4þ T-helper 1 (TH1) cells and CD8þ CTLs, canbe enhanced or induced de novo by immunotherapeutic stra-tegies using antigen-loaded dendritic cells (DCs refs. 1–3).Several strategies have been developed to deliver TAAs to DCs,including the use of mRNA (4–6). Autologous DCs loaded exvivo with TAA mRNA have been extensively tested in preclin-ical studies, showing their ability to induce functional TH1 cellsand CTLs (7–10). Moreover, clinical testing showed the induc-tion of antigen-specific immune responses byDC vaccines (11).However, the logistics of developing a specific vaccine for eachpatient may be prohibitive. Therefore, direct administrationof TAA mRNA has gained substantial interest (12–14). Thismethod offers a number of advantages, mRNA is not patient-specific, available at all times, safe, and easy to produce at lowcost (12–14).The success of mRNA vaccination depends on the engulf-

ment of mRNA by DCs and its potential to mature DCs.Consequently, the route of mRNA delivery and the modus ofDCmaturation are parameters thatwill critically impact on the

efficiency of the mRNA vaccine. It was recently showed thatintranodal delivery of mRNA results in the engulfment ofmRNA by DCs, as well as the activation of Toll-like receptors(TLR refs. 15–17). Nevertheless, it is suggested that nakedmRNA is insufficient to fully harness the stimulatory potentialof DCs (9, 18). Therefore, codelivery of additional stimuli, suchas lipopolysaccharide (LPS), CD40 ligand (CD40L), polyinosi-nic:polycytidylic acid (polyI:C), and protamine-complexedmRNA, has been evaluated (18, 19). However, defining theoptimal protocol for in vivoDCmaturation, without abrogatingthe uptake/translation of mRNA has proven to be challenging.The use of mRNA encoding immunomodulating proteinsmight be an attractive alternative to potentiate DCs in situ.

We previously showed that electroporation of human DCswith CD40L mRNA and mRNA encoding a constitutive activeform of TLR4 (caTLR4) induces DC maturation. We moreoverintroduced CD70 mRNA into these DCs to provide a costimu-latory signal to CD27þ T cells. We showed that DCs modifiedwith this so-called TriMix induce tumor-specific T-cellresponses in vitro as well as in vaccinated patients withmelanoma (20–23).

Here, we report on the delivery of TAA and TriMix mRNA insitu to generate T-cell–attracting and stimulating DCs, astrategy that was shown to be as efficient as vaccination within vitro electroporated DCs in terms of CTL induction andantitumor therapy.

Materials and MethodsMice

Female, 6- to 12-week-old C57BL/6, DBA/2, and BALB/cmice were purchased from Harlan. Transgenic mice were

Authors' Affiliations: 1Laboratory of Molecular and Cellular Therapy,Department of Immunology-Physiology; and 2In Vivo Cellular and Molec-ular Imaging Laboratory, Vrije Universiteit Brussel, Jette, Belgium

Corresponding Author:K. Breckpot, Laboratory of Molecular and CellularTherapy,Department of Immunology-Physiology, VrijeUniversiteit Brussel,Laarbeeklaan 103/E, Jette 1090, Belgium. Phone: 0032-2-4774565; Fax:0032-2-4774568; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-11-2957

�2012 American Association for Cancer Research.

CancerResearch

www.aacrjournals.org 1661

on July 29, 2020. © 2012 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst February 15, 2012; DOI: 10.1158/0008-5472.CAN-11-2957

http://cancerres.aacrjournals.org/

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Van Lint et al.

Cancer Res; 72(7) April 1, 2012 Cancer Research1662




provided by B. Lambrecht (University of Ghent, Ghent,Belgium) and include OT-I mice that carry a transgenic CD8T-cell receptor (TCR) specific for the MHC I–restrictedovalbumin (OVA) peptide SIINFEKL, OT-II mice that carrya transgenic CD4 TCR specific for the MHC II–restrictedOVA peptide ISQAVHAAHAEINEAGR, and CD11c-diphthe-ria toxin receptor (DTR) mice in which CD11cþ cells aredepleted upon treatment with 4 ng diphtheria toxin (DT)/gmouse (Sigma). Where indicated mice received an intrave-nous hydrodynamic injection with 10 mg of a plasmidencoding Flt3 ligand (a gift from O. Leo, Universit�e Librede Bruxelles, Brussels, Belgium) in 0.9 NaCl in a final volumeequal to 10% of the mouse body weight. Animals were

treated according to the European guidelines for animalexperimentation. Experiments were reviewed by the Ethicalcommittee for use of laboratory animals of the Vrije Uni-versiteit Brussel (Jette, Belgium).

Mouse cell lines and DCsThe melanoma MO4, the T-cell lymphoma EG7-OVA, the

mastocytoma P815, and the myeloid leukemia C1498-WT1were obtained from the American Type Culture Collection,C. Uytttenhove (Universit�e Catholique de Louvain, Brussels,Belgium), and H.E. Kohrt (Stanford University Medical Cen-tre, Stanford, CA), respectively. No full authentication wascarried out. Cell lines were evaluated for the expression of

Figure 1. DCsmatured through electroporation of TriMix efficiently stimulate antigen-specific T cells. The histogramoverlays in (A) show the phenotype of DCselectroporatedwith tNGFRmRNAand left immature ormaturedby coelectroporation of TriMix or addition of LPS (n¼10). The graphs in (B) show thecytokinessecreted by these DCs (n ¼ 6). The graph in (C) depicts the incorporation of 3H thymidine by allogeneic spleen cells cultured with these DCs (n ¼ 3).D–F, mice were immunized intravenously with 5� 105 DCs electroporated with OVAmRNA andmatured by coelectroporation of TriMix mRNA or addition ofLPS. Five days later, the expansion of functional OVA-specific CD8þ T cells was assessed. The results of (D) the pentamer staining, (E) the in vivo cytotoxicityassay, and (F) the intracytoplasmatic staining of IFN-g on spleen cells restimulated with SIINFEKL-presenting DCs are shown (n ¼ 2). G, mice, immunizedwith Trp2-presenting DCs, were subjected to an in vivo cytotoxicity assay to evaluate the stimulation of Trp2-specific CD8þ T cells (n ¼ 2).

Figure 2. Formulation andpharmacokinetics of mRNA. A,mouse DCs were pulsed with FLucmRNA in the indicated buffer.Luminescence was measured 4hours later. The graph depicts thephoton emission (n ¼ 4). B and C,mice were injected intranodally withFLuc mRNA. B, in vivobioluminescence imaging wasconducted at the indicated timepoints (n ¼ 4). C, to evaluate thestability of FLucmRNA in vivo, lymphnodes were isolated 6, 12, and 24hours after injection and PCR carriedout on cDNA synthesized fromextracted mRNA (n ¼ 4). D, micereceived an intranodal injection ofeGFP mRNA formulated in 0.8 RL.Four hours later, the lymph node wasresected, a single-cell suspensionprepared and stained for CD11c. Thephotograph obtained byfluorescence microscopy showseGFP (green) expression by CD11cþ

cells (red, n ¼ 4). E, transgenicCD11c-DTR mice, which werepretreated with PBS or DT, receivedan intranodal injection with FLucmRNA. In vivo bioluminescenceimaging was conducted 4 hourslater. Single-cell suspensions wereprepared from the lymph nodes andanalyzed by flow cytometry for thepresence of CD11cþ cells (n ¼ 3). F,mice, of which the skin waspretreated with PBS or GM-CSF,were injected intradermallywith FLucmRNA. In vivo bioluminescenceimaging was conducted 6 hours later(n ¼ 3).

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mRNA-Based Antitumor Vaccination

www.aacrjournals.org Cancer Res; 72(7) April 1, 2012 1663




MHC molecules and antigens (OVA, MO4 and EG7-OVA;P1A, P815; and WT1, C1498-WT1) by reverse transcriptasePCR (RT-PCR) or flow cytometry. Bone marrow–derived DCswere generated as described (9).

Messenger RNAThe vector, pST1 was provided by U. Sahin (Johannes-

Gutenberg University, Mainz, Germany). The vectors pGEM-Ii80tOVA, pST1-tyrosinase-DC-LAMP, pST1-sig-WT1-DNLS-DC-LAMP, pST1-caTLR4, and pGEM-tNGFR have beendescribed (Benteyn and colleagues; manuscript in preparation;refs. 9, 21, 24). The sequence encoding firefly luciferase (FLuc)was cloned into pST1 with minor modifications. The vectorpGEM-Ii80P1A was cloned analogous to the cloning of pGEM-Ii80tOVA. The codon-optimized cDNA encodingmouse CD40Lor CD70were obtained fromGeneart and cloned as a SpeI-XhoIfragment in the pST1 vector. A fragment of the mouse Trp2gene that encodes SVYDFFVWL was amplified with thefollowing primers: 50-GGGGATCCGGCCATCCTAAGACGG-30

and 30-GGGGGATCCGTGCACACGTCACACTCGTTC-50 andcloned as a BamHI fragment in the BamHI linearized andshrimp alkaline phosphatase–treated pST1-sig-DC-LAMP. Thesequence encoding enhanced GFP (eGFP) was isolated fromp-eGFP-N1 as a HindIII-NotI fragment and cloned into theHindIII-NotI digested pST1 vector. All enzymeswere purchasedfrom Fermentas.

Before in vitro transcription, pGEM and pST1 vectors werelinearized with SpeI and SapI, respectively. In vitro transcrip-

tion was carried out as described (9). ThemRNAwas dissolvedin PBS, Ca2þ-containing Hank's balanced salt solution (HBSS,Lonza), or 0.8 Ringer lactate (0.8 RL; Baxter).

Passive pulsing and electroporation of mRNATo pulse DCs with mRNA, 5 � 106 DCs were pelleted and

incubated for 15 minutes with 10 mg tNGFR or FLuc mRNAin 15 mL. Where indicated pulsing was carried out in thepresence of 1 ng/mL LPS from Escherichia coli serotype 055:B5 (Sigma-Aldrich), 10 mg/mL polyI:C (Sigma), or 100 ng/mLmonophosphoryl lipid A (MPL; GlaxoSmithKline). DCs werecultured in RPMI-1640 medium supplemented with 5% FCI(Harlan), 50 mmol/L b-mercaptoethanol, and 20 ng/mLmouse granulocyte macrophage colony-stimulating factor(GM-CSF; prepared in-house) at a cell density of 106 DCs permL. Four hours later, DCs were lysed using the reporter lysisbuffer from Promega. D-Luciferin (Xenogen) was added,luminescence measured with the Glomax 96-luminometer,and data analyzed with Glomax software (Promega). Elec-troporation of DCs with mRNA was carried out as described(9). Where indicated, DCs were activated for 4 hours with100 ng/mL LPS.

In situ delivery of mRNAFor intranodal delivery of mRNA, C57BL/6 mice were

anesthetized with ketamine (70 mg/kg; Ceva) and xylazine(10 mg/kg; Bayer). The inguinal lymph node was surgicallyexposed and injectedwith the indicated amount ofmRNA (and

tNGFR mRNA

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Figure 3. Intranodal delivery of TriMix generates an immunostimulatory environment. A and B, DCs were pulsed with FLucmRNA in the presence of activationstimuli after which uptake of mRNA and the DCs' phenotype was analyzed (n ¼ 4). The graph in (A) shows the photon emission as mean � SEM of 4experiments. Thehistogramoverlays in (B) show theexpressionofCD70,CD40,CD80, andCD86byDCspulsed in theabsenceof amaturation stimulus, in thepresence of LPS, poly[I:C], or TriMix. C, mice were injected intranodally with FLuc mRNA alone or combined with TriMix or LPS after which in vivobioluminescence imagingwas conducted (n¼5). D, activation ofDCs inmicepretreatedwith Flt3-L and injectedwith FLucmRNAaloneor combinedwith LPSor TriMix was evaluated by flow cytometry. The histograms depict the expression of CD40, CD80, and CD86 by CD11cþ cells obtained from lymph nodesinjected with FLuc mRNA alone or the latter together with TriMix mRNA or LPS (n ¼ 3).

Van Lint et al.





where indicated 1 ng LPS). Subsequently, the wound wasclosed. On 3 consecutive days before intradermal delivery ofmRNA, mice were injected intradermally with PBS or 20 ng ofmouse GM-CSF, after which the mRNA was administered.

RNA isolation, cDNA synthesis, and RT-PCRRNAwas extracted using the SV Total RNA Isolation System

(Promega) and converted to cDNA by the RevertAid H-MinusFirst Strand cDNA Synthesis Kit (Fermentas). The sequenceencoding FLuc was amplified with 50-AAGGTGTGGCCC-TTCC-30 and 50-CCAAGAATGAAAATAGGGTTG-30, whereasthe sequence encoding b-actin was amplified with 50-TGCTATCCAGGCTGTGCTAT-30 and 50-GATGGAGTTGAAG-GTAGTTT-30 using the following PCR program: 94�C 50, 45�(94�C 30", 52�C 30", 72�C 30"), 72�C 100, hold 4�C.

Immune arrayRNA of lymph nodes injected with 0.8 RL, 10 mg anti-

gen mRNA supplemented with 20 mg tNGFR mRNA or TriMix(10 mg per component) was extracted and converted to cDNA.Quantitative RT-PCR by the TaqManmouse immune responsearray (Applied Biosystems) and analysis was conductedaccording to the manufacturer's instructions.

Flow cytometryAllophycocyanin-conjugated anti-CD11c (HL3), -CCR7

(2H4), and phycoerythrin-conjugated anti-CD40L (MR1) and-CD70 (FR70) antibodies were purchased from Pharmingen.The antibodies against CD40 (FGK45), CD80 (16-10A1), andCD86 (GL-1) were prepared in-house. Nonreactive isotypematched antibodies served as controls (Pharmingen). Labelingof DCs was carried out as described (9). Data were collectedusing the FACSCanto Flow Cytometer (Becton Dickinson)and analyzed with FACSDiva or FlowJo software.

Allogeneic mixed lymphocyte reactionThe ability of electroporated DCs to stimulate allogeneic

CD90 purified (Miltenyi Biotec) T cells was assessed in amixedlymphocyte reaction (25).

ELISASupernatants were screened in a sandwich ELISA for the

presence of interleukin (IL)-6, IL-12p70, TNF-a, or IFN-g(eBioscience).

In vivo bioluminescence imagingIn vivo bioluminescence imaging was conducted as

described (26).

Fluorescence microscopyLymph nodes were injected with 10 mg eGFP mRNA, 1 day

before isolation. Single-cell suspensions were prepared andstained with a phycoerythrin-conjugated anti-CD11c antibody.Expression of CD11c and eGFP was evaluated with the Evosfl

fluorescence microscope.

Immunization of miceMice were immunized intravenously with 5 � 105 antigen-

presentingDCs activatedwith TriMix or LPS, or intranodally or

intradermally with 10 mg antigenmRNA supplemented with 30mg tNGFR mRNA or TriMix (10 mg per component). Immuni-zation with DCs electroporated with tNGFR mRNA or withtNGFR mRNA as such served as a control. For assessment oftherapeutic efficacy, 5 � 105 tumor cells were administeredsubcutaneously in the lower back, 7 days before immunization.

Intracytoplasmatic staining of IFN-gSpleen cells of immunizedmicewere stimulated for 24 hours

with DCs pulsed for 2 hours with 5 mmol/L SIINFEKL peptideand matured with LPS. GolgiPlug was added 24 hours beforeintracytoplasmatic staining of IFN-g .

Pentamer stainingThe staining of CD8þ T cells with H2-Kb/SIINFEKL penta-

mers (Immunosource) was carried out as described (25).

In vivo cytotoxicity assaySpleen cells from syngeneic mice were labeled with

10 mmol/L carboxyfluorescein diacetate succinimidyl ester(CFSE) as described (9). These were pulsed with the peptideSIINFEKL (OVA) or SVYDFFVWL (Trp2; Thermo ElectronCooperation) or a set of overlapping peptides covering WT1(kind gift from V. Van Tendeloo, University of Antwerp,Edegem, Belgium) or tyrosinase (EMC microcultures) at 5mmol/L for 2 hours. Peptide-pulsed cells were mixed at a 1:1ratio with nonpulsed cells, labeled with 0.5 mmol/L CFSE.

Table 1. Intranodal delivery of TriMix mRNAgenerates an immunostimulatory environment

AntigenmRNA

TriMixmRNA

Antigen-presenting moleculesMHC II 6.2 � 2.3 27.9 � 6.5

Proinflammatory cytokinesIL-6 3.7 � 1.3 9.0 � 3.0IL-15 5.9 � 0.8 16.1 � 1.5IFN-g 2.3 � 0.1 5.1 � 0.1

T-cell–attracting moleculesMCP-1 1.9 � 0.2 6.1 � 1.1IP-10 10.3 � 2.3 35.9 � 5.1

Signaling moleculesSOCS1 2.5 � 0.6 7.1 � 1.9STAT1 2.8 � 0.7 4.3 � 0.1

OthersGranzyme B 9.2 � 1.7 24.4 � 1.8

NOTE:Mice receivedan intranodal injectionof 0.8RL, antigenmRNA combined with tNGFR mRNA, or with TriMix. Lymphnodes were removed 8 hours later, RNA extracted, cDNAsynthesized, and quantitative RT-PCR carried out. It sum-marizes the molecules of which the upregulation was at least2-fold higher when TriMix was coadministered when com-pared with antigen mRNA alone. The data show the relativeupregulation compared with injection with 0.8 RL alone. Theresults are shown as mean � SEM of 3 experiments.






Specific lysis of target cells was analyzed 18 hours later byflow cytometry. The percentage of killing was calculated asdescribed (27).

In vivo proliferation assayOne day before immunization, 106 purified and CFSE-

labeled CD8þOT-I or CD4þOT-II spleen cells were transferredto mice by intravenous injection. Five days postimmunization,proliferation of T cells was analyzed in peripheral blood,spleen, and lymph nodes (27).

Statistical analysesA one-way ANOVA followed by the Bonferroni multiple

comparison test was conducted. Sample sizes and numberof times experiments were repeated are indicated in the

figure legends. Number of asterisks in the figures indicatesthe level of statistical significance as follows: �, P < 0.05;�� , P < 0.01; ��, P < 0.001. The results are shown in a scatterplot in which each mouse is depicted as a dot and the meanas a horizontal line or in a column graph or table as themean � SEM. Survival was visualized in a Kaplan–Meierplot. Differences in survival were analyzed by the log-ranktest.

ResultsDCs matured through electroporation with TriMixmRNA efficiently stimulate antigen-specific T cells

We recently showed that the T-cell stimulatory capacityof human DCs electroporated with TAA mRNA is

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Figure 4. Intranodal delivery of TriMix but not LPS together withOVAmRNA results in stimulation of OVA-specificCD4þ andCD8þ T cells. CFSE-labeledCD4þ

OT-II or CD8þ OT-I cells were adoptively transferred 1 day before immunization of mice with tNGFR mRNA, OVA mRNA alone, or combined withTriMix or LPS. The amount ofmRNAwas kept constant by addition of tNGFRmRNA. Five days postimmunization, stimulation of T cells within the lymph nodewas analyzed. A, proliferation of CD4þ OT-II cells was analyzed by flow cytometry (n ¼ 3). B and C, stimulation of CD8þ OT-I cells was analyzed by (B)pentamer staining (n ¼ 5) and (C) in vivo cytotoxicity assay (n ¼ 3). D, stimulation of CTLs after immunization with OVA and TriMix mRNA eitherdelivered intradermally in mice pretreated with GM-CSF or intranodally was analyzed by in vivo cytotoxicity assay (n ¼ 2).

Van Lint et al.





considerably increased by simultaneous coelectroporationwith TriMix (20). As we wanted to investigate the use ofTriMix for the in situ modification of mouse DCs, weevaluated whether electroporation of mouse DCs withTriMix results in immunogenic DCs. We showed thatTriMix-electroporated DCs displayed a phenotype (Fig.1A), cytokine secretion profile (Fig. 1B), and allogeneic T-cell stimulatory capacity (Fig. 1C) comparable with that ofLPS-activated DCs. Importantly, we showed that TriMix-matured DCs were superior to LPS-matured DCsin stimulation of functional antigen-specific CD8þ T cellsin vivo. This was shown for OVA (Fig. 1D–F) and the TAATrp2 (Fig. 1G).

Formulation and pharmacokinetics of mRNA forvaccination purposes

It was previously shown that cellular uptake of mRNAcan be influenced by the composition of the injectionsolution (28). Therefore, we evaluated which buffer is bestsuited to deliver mRNA to DCs. FLuc mRNA was dissolvedin PBS, Ca2þ-containing HBSS, or 0.8 RL. Luminescenceanalysis of passively pulsed DCs showed high FLuc expres-sion when the mRNA was dissolved in 0.8 RL or HBSS (Fig.2A). Next, we administered FLuc mRNA intranodally. In vivobioluminescence imaging showed short-term FLuc expres-sion when mRNA was formulated in PBS when comparedwith high and long FLuc expression when mRNA wasformulated in HBSS or 0.8 RL (Fig. 2B). The latter wasunexpected as naked mRNA is believed to have a shortextracellular half-life (29). To analyze the stability of mRNAin vivo upon delivery in 0.8 RL, we resected lymph nodesinjected with FLuc mRNA 6, 12, and 24 hours after injection.RT-PCR showed the presence of FLuc mRNA up to 12 hoursafter injection. No FLuc mRNA was detectable at later timepoints (Fig. 2C).

Next, we evaluated the role of DCs in the uptake of mRNAin vivo. Lymph nodes were injected with eGFP mRNA 24hours before their isolation. Single-cell suspensions wereprepared and stained for CD11c. Fluorescence microscopyshowed a small number of eGFPþ cells. Importantly, alleGFPþ cells were CD11cþ, showing uptake and translationof mRNA by DCs (Fig. 2D). To further evidence a role forDCs, we used CD11c-DTR transgenic mice in which admin-istration of DT results in the depletion of CD11cþ cells. Invivo bioluminescence imaging showed the absence of FLucexpression in mice that were treated with DT beforeintranodal administration of FLuc mRNA. Mice treatedwith PBS served as a control (Fig. 2E). Flow cytometricanalysis of the lymph nodes of these mice confirmed thatthe absence of luminescence was correlated with the deple-tion of DCs (Fig. 2E). As delivery of mRNA into the inguinallymph node is technically challenging, we finally examined

tNGFR mRNA

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C

Figure 5. Inclusion of TriMix in the mRNA vaccine enhances the inductionof TAA-specific CTLs. An in vivo cytotoxicity assay was conducted to

evaluate the induction of CTLs in mice immunized intranodally with TAAmRNAaloneor combinedwith TriMix. The graphs depict the specific lysisof target cells upon immunization against (A) Trp2 (n¼ 2), (B) WT1 (n¼ 3),and (C) tyrosinase (n ¼ 2).






100

80

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A B C

D

E

F

G

H

2,000

1,500

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0

2,500

2,000

1,500

1,000

500

0

OVA

DC

Days after MO4 tumor inoculation Days after MO4 tumor inoculation

Days after MO4 tumor inoculation

Days after EG7-OVA tumor inoculation Days after EG7-OVA tumor inoculation

Days after C1498-WT1 tumor inoculation Days after C1498-WT1 tumor inoculation

Days after P815 tumor inoculation Days after P815 tumor inoculation

Days after MO4 tumor inoculation

9 11 14 16 18 0 15 20 30 40 50 60

20100 30 40

20100 30 40

200 40 60

50

20100 30 40 50

11 14 16

7 10 13 15

13 15 18 20

7 10 12 14

18 21

mRNA

P = 0.0015

P = 0.2428

P = 0.1032

P < 0.0001

P = 0.0012

P = 0.0037

P < 0.0001

P < 0.0001

P < 0.0001

P = 0.0037

P = 0.0006

P = 0.0025 P = 0.0120

Vaccine

DC mRNA

Vaccine

DC mRNA

Vaccine

Trp2 WT1

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DC or mRNA - tNGFRDC - OVA TriMixmRNA - OVA TriMix

DC or mRNA - tNGFRDC - Trp2 TriMixmRNA - Trp2 TriMix

DC or mRNA - tNGFRDC - OVA TriMixmRNA - OVA TriMix

DC or mRNA - tNGFRDC - WT1 TriMixmRNA - WT1 TriMix

DC or mRNA - tNGFRDC - P1A TriMixmRNA - P1A TriMix

% s

peci

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** ** *

Figure 6. Immunization with antigen mRNA and TriMix is as efficient in stimulation of CTLs and in therapy as immunization with ex vivo–modified DCs. A–C,C57BL/6 mice were immunized intravenously with antigen and TriMix mRNA–modified DCs or intranodally with antigen and TriMix mRNA. The in vivocytotoxicity assaywas conducted 5 days later. The graphs show the specific lysis of target cells in peripheral blood upon immunization against (A)OVA (n¼ 2),(B) Trp2 (n ¼ 2), or (C) WT1 (n ¼ 2). D–H, mice bearing palpable tumors (10 mice per group) were immunized by intravenous injection of antigen andTriMix mRNA–electroporated DCs or by intranodal injection with antigen and TriMix mRNA. The graphs show the tumor growth (left) and survival (right) in theMO4 model after immunization with the antigen OVA (D) or the TAA Trp2 (E), in the EG7-OVA model after immunization with OVA (F), in the C1498-WT1model after immunization with the TAA WT1 (G) all in C57BL/6 mice, and in the P815 model after immunization with the TAA P1A (H) in DBA-2 mice.

Van Lint et al.





the feasibility of delivering mRNA intradermally. Becausewe showed in the former experiment that CD11cþ cells areresponsible for the DC uptake, we pretreated the mice withan intradermal injection of PBS or GM-CSF on 3 consecu-tive days before the intradermal injection of FLuc mRNA. Invivo bioluminescence imaging, conducted 6 hours later,showed FLuc expression only in mice pretreated withGM-CSF (Fig. 2F).

Intranodal delivery of TriMix generates an immunestimulatory environmentInduction of antitumor immune responses requires antigen-

presentation by mature DCs (1–3). To evaluate the effect ofTriMix and classical maturation stimuli on the engulfment ofmRNA and the induction of an immune stimulatory environ-ment, we first passively pulsed DCs in vitro with FLuc mRNAand these maturation stimuli, showing a reduction in FLucexpression after pulsing of DCs with FLuc mRNA in thepresence of LPS, MPL, or polyI:C. This reduction in proteinexpression was less pronounced when TriMix was codelivered(Fig. 3A). In addition, DCs pulsed with TriMix mRNA showed ahigher expression of CD40, CD70, CD80, and CD86 than theDCs pulsed with MPL (data not shown), LPS, or polyI:C (Fig.3B).Next, we evaluated the uptake of FLuc mRNA when

delivered as such or together with LPS or TriMix in vivo.We showed that codelivery of TriMix had a lesser impact onthe uptake of mRNA than its codelivery with LPS (Fig. 3C).To increase the number of DCs that can be recovered fromthe injected lymph node for analysis, we pretreated themice with a hydrodynamic injection of a plasmid encodingFlt3 ligand. In analogy with the data described by Kreiterand colleagues (30), FLuc mRNA injected into these miceresulted in increased luminescence reflecting the specificuptake by the DCs (data not shown). Flow cytometryshowed that DCs (CD11cþ) from lymph nodes coinjectedwith TriMix displayed the highest expression of CD40,CD80, and CD86 than DCs isolated from lymph nodesinjected with FLuc mRNA alone or combined with LPS(Fig. 3D).These findings prompted us to analyze, whether codelivery

of TriMix promotes a T-cell–attracting and activating envi-ronment, by profiling the expression levels of maturation-associated markers by quantitative RT-PCR. We observedupregulation of several markers in lymph nodes injected withFLuc and tNGFR mRNA when compared with lymph nodesinjected with 0.8 RL. Importantly, the upregulation of thefollowing markers: MHC II, IL-6, IL-15, IFN-g , MCP-1, IP-10,granzyme B, SOCS1, and STAT1was at least 2-fold higher whenTriMix was codelivered (Table 1).

Intranodal delivery of TriMix but not LPS together withOVA mRNA results in expansion of OVA-specific CD4þ

and CD8þ T cells with potent effector functionActivation of CD4þ T cells is critical for the induction of

long-lasting antitumor immunity (31). Therefore, we eval-uated the expansion of OVA-specific CD4þ T cells uponintranodal delivery of tNGFR mRNA, OVA mRNA, or com-

bined with TriMix or LPS. Proliferation of CFSE-labeledCD4þ OT-II cells was evaluated by flow cytometry, showingenhanced proliferation of OT-II cells in mice receiving OVAand TriMix mRNA. Of note, transferred T cells hardlyproliferated when LPS was coinjected with OVA mRNA(Fig. 4A). Similar results were obtained with CD8þ OT-Icells (data not shown). To further evaluate the expansionand function of OVA-specific CD8þ T cells, mice wereimmunized 1 day after adoptive transfer of CD8þ OT-Icells. Five days postimmunization, we carried out anH2-kb/SIINFEKL pentamer staining or an in vivo cytotox-icity assay. Both assays showed the enhanced stimulation ofOVA-specific CD8þ T cells when mice were immunized withOVA mRNA and TriMix when compared with mice immu-nized with OVA mRNA alone or combined with LPS (Fig. 4Band C).

Using the model antigen OVA, we finally compared intra-dermal delivery of OVA and TriMix mRNA in mice pretreatedwith GM-CSF to its intranodal delivery. Using the in vivocytotoxicity assay we showed that the lysis of target cellswas the highest when the mRNA was delivered intranodally(Fig. 4D).

Inclusion of TriMix in the mRNA-based antitumorvaccine enhances the induction of TAA-specific cytotoxicT cells

Next, we assessed whether the results obtained with theantigen OVA are representative for other TAAs. Mice wereimmunized with Trp2, WT1, or tyrosinase mRNA alone orcombined with TriMix. The in vivo cytotoxicity assay showedenhanced lysis of target cells when TriMix was included in theimmunization regimen (Fig. 5A–C).

Immunization with antigen mRNA and TriMix is asefficient in stimulating cytotoxic T cells and in therapy asimmunization with ex vivo–modified DCs

Therapeutic immunization with human DCs electropo-rated with TAA and TriMix mRNA has shown promise inclinical evaluation (23). Therefore, we compared the efficacyof DC- to mRNA-based immunization, evaluating the induc-tion of antigen-specific CTLs in vivo. We showed thatimmunization with antigen and TriMix mRNA was asefficient as immunization with antigen and TriMixmRNA–electroporated DCs for the antigen OVA and theTAAs, Trp2, and WT1 (Fig. 6A–C). We next evaluated thetherapeutic efficacy of such vaccines. First, mice bearingMO4 tumors were treated with antigen and TriMix mRNA–modified DCs or antigen and TriMix mRNA as such. Similarresults were obtained upon immunization with OVA (Fig.6D) or Trp2 (Fig. 6E) as an antigen. Mice treated withtNGFR-electroporated DCs or tNGFR mRNA as such servedas controls. Mice from control groups showed rapid tumorgrowth, whereas mice immunized with a single intravenousinjection of DCs electroporated with antigen and TriMixmRNA or an intranodal injection of antigen and TriMixmRNA showed a reduced tumor growth, hence, prolongedsurvival. These data were extended to the mouse T-celllymphoma EG7-OVA, the myeloid leukaemia C1498-WT1 in






C57BL/6 mice, and the mastocytoma P815 in DBA-2 miceusing OVA, WT1, and P1A as the antigen applied forimmunization, respectively (Fig. 6F–H).

DiscussionDelivery of TAA mRNA to DCs for cancer therapy offers

many advantages, which can be fully exploitedwhen themRNAis administered intranodally (15). It is proposed that mRNAfunctions as a template for translation as well as a ligand forTLRs (32). It is not clear, however, whether the intrinsicadjuvant effect of mRNA is sufficient to fully exploit theimmunostimulatory capacity of DCs (33).

Therefore, we evaluated the local delivery ofmRNAencodingCD40L, CD70, and caTLR4 (referred to as TriMix) as anadjuvant in conjunctionwith intranodal TAARNA vaccination.We show that TriMix but not classical maturation stimulipotentiates the immunogenicity of intranodal mRNA vaccina-tion. We moreover showed that the strength of TriMix is dual:low impact on antigen mRNA immunobioavailability andsimultaneous delivery of stimuli that act synergistic in termsof activation of T-cell responses.

It has been suggested that the immunobioavailability ofantigen mRNA is a critical success-limiting factor in view ofcancer therapy (34). First, we showed high antigen expressionwhen mRNA was delivered in Ca2þ-containing HBSS or theclinically applied 0.8 RL, confirming the Ca2þ dependency forefficient uptake of mRNA (29). It was previously shown thatseveral adjuvants hamper mRNA uptake, as it is criticallydependent on macropinocytosis, a process that is rapidlydownregulated upon DC activation (35). Therefore, we nextevaluated the engulfment of antigen mRNA when codeliveredwith TriMix or LPS. We confirmed the severe reduction inantigen expression when LPS was coadministered. However,this phenomenon was less pronounced when TriMix wascodelivered and might be explained by the timing of DCactivation, which most likely is initiated after the uptake andtranslation of the TriMix mRNA.

Recently, Diken and colleagues (35) hypothesized thatsimultaneous delivery of classical activation stimuli mightresult in imperilment of the induction of an immuneresponse. We now show that the codelivery of LPS but notTriMix indeed completely abrogates the stimulation of anti-gen-specific T cells. In contrast, we showed that the code-livery of TriMix mRNA significantly enhanced the inductionof antigen-specific T cells. The latter can be explained in partby the fact that the intranodal delivery of TriMix mRNAresulted in phenotypically more mature DCs and created anenvironment that is even better suited to recruit and acti-vate T cells than with the use of antigen mRNA alone.However, this cannot be the only explanation as codeliveryof LPS also resulted in highly mature DCs. The explanationfor the differences in T-cell stimulation in mice immunizedwith antigen mRNA or the latter combined with TriMix orLPS might be found in the levels of MHC/peptide complexeson DCs, which are determined by the availability of theantigen. It was shown that a certain threshold antigen doseis required for T cells to decide to participate in immuneresponses (36). We hypothesize that this threshold is not

met when classical adjuvants are codelivered with antigenmRNA, as these almost completely abrogate the engulfmentof mRNA. We showed that codelivery of TriMix mRNA withantigen mRNA resulted in a 2-fold higher antigen expressionthan with the delivery in the presence of LPS. This amountmight surpass the required threshold for T-cell recognitionand engagement. Although the delivery of mRNA aloneresulted in the highest availability of the antigen mRNA andactivated the DCs to a certain extent, we observed that thecodelivery of TriMix resulted in enhanced T-cell responses.The latter might be partially explained by the observationthat lower antigen doses that exceed the above-mentionedthreshold are correlated with enhanced T-cell activation andfunctionality (36, 37).

Because we showed that codelivery of TriMix allows theuptake of antigen mRNA and has an added benefit in termsof activation of adaptive T-cell responses, we next evaluatedits therapeutic efficacy. Because we described the inductionof antigen-specific T cells both in vitro (20, 21) and invaccinated patients with melanoma (22) by TAA and TriMixmRNA–electroporated human DCs, we decided to compareDC to mRNA immunization. We showed that the thera-peutic efficacy of antigen and TriMix mRNA is comparablewith that of DCs electroporated with this mRNA. As suchwe here highlight the feasibility and potency of the TriMixand antigen mRNA-based immunization strategy.

It was recently implied that an adjuvant should be chosenon the basis of complementarity of its mode of action withthat of the vaccine format it will be combined with (30). Inthat regard, the efficacy of mRNA administered into lymphnodes depends on its uptake and its ability to create a CTLinducing milieu. We conclude that these prerequisites aremet through the codelivery of TriMix mRNA, as it allowsantigen mRNA uptake, confers a high T-cell stimulatorycapacity to DCs, and as such enhances their ability tostimulate antigen-specific immunity.

Disclosure of Potential Conflicts of InterestThe use of DCs electroporated with TAA mRNA and TriMix is the topic of a

patent application (WO2009/034172) onwhichA. Bonehill andK. Thielemans arefiled as inventors. None of the authors receive any support or remunerationrelated to this platform. No potential conflicts of interest were disclosed.

AcknowledgmentsThe authors thank Petra Roman, Elsy Vaeremans, and Xavier Debaere for

their technical assistance and Prof.M.Moser andO. Leo for critical reading of themanuscript.

Grant SupportThis work was supported by grants from the Interuniversity Attraction Poles

Program-Belgian State-Belgian Science Policy, the National Cancer Plan of theFederal Ministry of Health, the Stichting tegen Kanker, the Vlaamse Kanker Liga,an Integrated Project and anEUFP6-fundedNetwork of Excellence, an IWT-TBMprogram, the FWO-Vlaanderen, and the Scientific Fund Willy Gepts of theUniversity hospital Brussels. C. Goyvaerts, L. Goethals are doctoral fellows andA. Bonehill, K. Breckpot are postdoctoral fellows of the FWO-Vlaanderen. J. Penis a doctoral fellow of the IWT. S. Maenhout is funded by an Emanuel Van DerSchueren grant from the Vlaamse Kanker Liga.

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

Received September 8, 2011; revised January 6, 2012; accepted January 13, 2012;published OnlineFirst February 15, 2012.

Van Lint et al.





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2012;72:1661-1671. Published OnlineFirst February 15, 2012.Cancer Res Sandra Van Lint, Cleo Goyvaerts, Sarah Maenhout, et al. Antitumor TherapyPreclinical Evaluation of TriMix and Antigen mRNA-Based

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