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Therapeutics, Targets, and Chemical Biology

Immune Response Is an Important Aspect of the AntitumorEffect Produced by a CD40L-Encoding Oncolytic Adenovirus

Iulia Diaconu1, Vincenzo Cerullo1, Mari L.M. Hirvinen1, Sophie Escutenaire1, Matteo Ugolini1,Saila K. Pesonen1, Simona Bramante1, Suvi Parviainen1, Anna Kanerva1,2, Angelica S.I. Loskog3,Aristides G. Eliopoulos4, Sari Pesonen1, and Akseli Hemminki1

AbstractOncolytic adenovirus is an attractive platform for immunotherapy because virus replication is highly

immunogenic and not subject to tolerance. Although oncolysis releases tumor epitopes and provides costimu-latory danger signals, arming the virus with immunostimulatory molecules can further improve efficacy. CD40ligand (CD40L, CD154) induces apoptosis of tumor cells and triggers several immunemechanisms, including a T-helper type 1 (TH1) response, which leads to activation of cytotoxic T cells and reduction of immunosuppression.In this study, we constructed a novel oncolytic adenovirus, Ad5/3-hTERT-E1A-hCD40L, which features a chimericAd5/3 capsid for enhanced tumor transduction, a human telomerase reverse transcriptase (hTERT) promoter fortumor selectivity, and human CD40L for increased efficacy. Ad5/3-hTERT-E1A-hCD40L significantly inhibitedtumor growth in vivo via oncolytic and apoptotic effects, and (Ad5/3-hTERT-E1A-hCD40L)–mediated oncolysisresulted in enhanced calreticulin exposure and HMGB1 and ATP release, which were suggestive of immuno-genicity. In two syngeneic mouse models, murine CD40L induced recruitment and activation of antigen-presenting cells, leading to increased interleukin-12 production in splenocytes. This effect was associated withinduction of the TH1 cytokines IFN-g , RANTES, and TNF-a. Tumors treated with Ad5/3-CMV-mCD40L alsodisplayed an enhanced presence ofmacrophages and cytotoxic CD8þTcells but not B cells. Together, ourfindingsshow that adenoviruses coding for CD40L mediate multiple antitumor effects including oncolysis, apoptosis,induction of T-cell responses, and upregulation of TH1 cytokines. Cancer Res; 72(9); 2327–38. �2012 AACR.

IntroductionOncolytic adenoviruses have shown safety in clinical trials

and some efficacy has also been seen (1–4). Importantly, it hasbeen discovered that immunologic factors are critical withregard to efficacy of oncolytic viruses (5) and some investiga-tors consider thema sophisticated formof immunotherapy (6).However, clinical and preclinical results show that treatmentwith unarmed oncolytic viruses is usually not immunostimu-

latory enough to result in sustained therapeutic immuneresponse (5). In this regard, oncolytic viruses can be armedwith immunostimulatory molecules. Moreover, viral replica-tion and expression of immunomodulatory proteins within thetumor potentiates the immune system by inducing cytokineproduction and release of tumor antigens (7).

CD40L is a type II transmembrane protein expressed pre-dominately on CD4þ T cells, and it binds to the CD40 receptoron antigen-presenting cells (APC; refs. 8, 9). CD40 is expressedon macrophages and dendritic cells (DC) where its activationby CD40L leads to antigen presentation and cytokine produc-tion followed by T-cell priming and a strong innate immuneresponse (10). Interactions between CD40L and its receptorCD40 provide critical costimulatory signals that trigger T-lymphocyte expansion (8), and increase interleukin (IL)-12production that is required for the engagement of cytotoxicT lymphocytes (CTL) in the antitumor immune response (11,12). Previous reports indicate that recombinant soluble proteinCD40L (rsCD40L) has direct effects in suppression of tumorcell proliferation in vitro (13, 14) and in vivo (15, 16). Otherdirect effects of rsCD40L are stimulation of survival signalingpathways and induction of apoptosis in carcinoma cells(15, 17). Clinical trials conducted with rsCD40L have generallybeen safe, and while there are many examples of patientsbenefiting from treatment, the overall level of activity has notbeen high enough to result in successful phase III studies

Authors' Affiliations: 1Cancer Gene Therapy Group, Molecular CancerBiology Program & Transplantation Laboratory & Haartman Institute &Finnish Institute for Molecular Medicine, University of Helsinki; 2Depart-ment of Obstetrics and Gynecology, HUCH, Helsinki, Finland; 3Division ofClinical Immunology, Rudbeck Laboratory, Uppsala University, Uppsala,Sweden; and 4Laboratory of Molecular & Cellular Biology, University ofCrete Medical School and Laboratory of Cancer Biology, Institute ofMolecular Biology & Biotechnology, FORTH, Heraklion, Crete, Greece

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

S. Pesonen and A. Hemminki contributed equally to this work.

Corresponding Authors: Akseli Hemminki, Cancer Gene Therapy Group,University of Helsinki, POBox 63, BiomedicumHelsinki, Haartmaninkatu 8,Helsinki 00290, Finland. Phone: 358-9-1912-5223; Fax: 358-9-1912-5465;E-mail: [email protected]; and Sari Pesonen,[email protected]

doi: 10.1158/0008-5472.CAN-11-2975

�2012 American Association for Cancer Research.

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heretofore (18). Side effects at nontarget sites limit the con-centration achievable at the target, which may restrict theefficacy of systemic rsCD40L.

The vector approach is an improvement in this regard as itcan yield higher local CD40L concentrations while reducingsystemic exposure. In this regard, a nonreplicating adenoviralvector coding for CD40L (19) has been safely tested in humans.Nevertheless, the nonreplicating platform may not be potentenough for treatment of advanced tumors and it has only beenused in local bladder tumors so far.

In this study, we hypothesized that a transcriptionallytargeted oncolytic adenovirus, which features a capsid mod-ification and is armed with CD40L, can result in potentoncolytic antitumor activity and stimulate an immuneresponse. Our previous studies in vitro (20) showed highcytotoxic effect for Ad5/3-hTERT-E1A, which is an oncolyticadenovirus featuring the human telomerase reverse transcrip-tase (hTERT) promoter for specific targeting to tumor cells.Also, the 5/3 serotype chimerism approach displays signifi-cantly enhanced gene delivery and antitumor effect whencompared with adenoviruses with a wild-type capsid (21–23).

To this end, we constructed Ad5/3-hTERT-E1A-hCD40L(CGTG-401), a new generation oncolytic virus based on Ad5/3 capsid modification for enhanced tumor transduction, tumorselectivity mediated by the hTERT promoter, and armed withCD40L. This virus was tested in vitro and in vivo for specificity,efficacy, induction of immune response, and apoptotic effect.

Materials and MethodsCell lines

Low-passage cultures of 293 and A549 from American TypeCulture Collection (ATCC; LGS standards) were used. EJ cells

were provided and authenticated by A.G. Eliopoulos (Univer-sity of Crete Medical School and Laboratory of Cancer Biology,Heraklion, Crete, Greece). MB49 cells are from Dr. K. Esuvar-anathan (National University Hospital, Singapore). Their qual-ity and identity has been monitored with regard to growthpattern in vitro and in vivo, in vitro phenotype, and Y-chromo-some positivity. B16-Ova are provided and authenticated byRichard Vile (Rochester).

AdenovirusesViruses were generated and amplified with standard ade-

novirus preparation techniques (20–22, 24–27). More in detailexplanation can be found in Supplementary Material.

The viral particle (VP) to plaque-forming units (pfu) ratiosfor Ad5/3-Luc1, Ad5/3-hTERT-E1A, Ad5/3-hTERT-E1A-hCD40L, Ad5/3-CMV-hCD40L, and Ad5/3-CMV-mCD40Lwere25, 31, 200, 138, and 86, respectively.

Cell viability assayCells were infected and 1 hour later were washed and

incubated for 4 to 8 days. Cell viability was then analyzed byMTS assay (CellTiter 96 AQueous One Solution ProliferationAssay, Promega). A competition experiment with anti-CD40Lantibody (Table 1) was carried out on EJ and A549 cell lines.MTS assay was conducted 48 hours upon infection.

Functionality of CD40LSupernatant collected 48 hours following infection was

filtered with 0.02-mm filters (Whatman 6809-1002). This wasused for 2 functionality assays, which are described more indetail in Supplementary Material.

EJ cells line was transfected with the plasmid pNiFty-Luc(InvivoGen). Supernatant was added and 1 mg/mL recombinant

Table 1. Antibodies used in the experiments

Antibody Product Method Company

FITC mouse anti-human CD40L 555699 FC BD BiosciencesFITC mouse IgG1 555909 FC BD BiosciencesAnti-human CD40 VP-C349 IHC Vector LaboratoriesRabbit anti-active caspase-3 559565 IHC BD PharmingenRabbit anti-mouse F4/80 14-4801 IHC eBioscienceRat anti-mouse CD45 550539 IHC BD PharmingenRat anti-mouse CD19 14-0193 IHC eBioscienceRat anti-mouse CD4 14-0041 IHC eBioscienceRat anti-mouse CD8 14-0083 IHC eBiosciencePE-Cy7 rat anti-mouse CD3 560591 FC BD BiosciencesFITC rat anti-mouse CD8 553030 FC BD BiosciencesPE rat anti-mouse CD19 561736 FC BD BiosciencesRat anti-mouse (NKp46)-V450 560763 FC BD BiosciencesOva-specific H-2Kb SIINFEKL (APC) F093-4A-E FC ProImmunePE-Cy7 anti-mouse CD4 25-0042 FC eBioscienceAnti-hCD40L sc-978 NAb Santa Cruz BiotechnologyAnti-calreticulin ab2907 FC AbcamAlexa-Fluor 488 IgG A21202 FC Invitrogen

Abbreviations: FC, flow cytometry; IHC, immunohistochemistry; NAb, neutralizing antibody experiment.

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hCD40L protein (Abcam) was used as a positive control. Cellswere lysed and luciferase activity was measured (LuciferaseAssay System, Promega). Ramos-Blue cells were stimulatedwiththe supernatant and their activity was measured with theQUANTI-Blue assay reagent (InvivoGen).

Immunogenicity of cell deathCalreticulin exposure. Cells were infected with 100 VP/

cell. Twelve hours later cells were stained with anticalreticulinantibody and Alexa-Fluor 488 IgG was used as secondaryantibody for flow cytometric analysis.Extracellular ATP. Cells were infected for 2 hours with

100 VP/cell. Supernatant was collected after 18 hours andanalyzed with ATP Determination Kit (A22066, MolecularProbes; Invitrogen).HMGB1 release. Cells were infected with 100 VP/cell.

Twenty-four hours later, supernatant was collected andHMGB1 was measured with ELISA kit (ST51011; IBLInternational).

ApoptosisCells infectedwith viruses and uninfected cells asmockwere

analyzed with TACS Annexin-V kit according to manufacturerinstructions (4830–250-K, Immuno Diagnostic). Tumors col-lected from nude and C57Bl/6 mice were homogenized andstained for Annexin-V with the same kit and results arepresented as percentage over the unstained cells.

Quantitative PCRMB49 cell line was infected for 2 hours with 100 VP/cell.

Infection media was removed and cells were collected atdifferent time points. Tumors from nude mice bearing EJ andA549 tumors were collected at the end of the experiment andDNAwas extracted by theQIAampDNAMini Kit (Qiagen). PCRamplification was based on primers and probe targeting the E4gene (28).

Replication assay in vitroMB49 cells were infected with Ad5/3-CMV-mCD40L and

Ad5/3-Luc1. Cell killing was assessed byMTS assay at differenttime points after infection.

Flow cytometry and FACS arrayCells and tissues were stained according to manufacturer

instructions with respective antibodies (Table 1) and analyzedon BDLSR (BD Biosciences). Results were plotted with FlowJosoftware (Tree Star, Inc.).Cytokines were analyzed from supernatant of cultured

splenocytes according to the manufacturer's protocol (BDCytometric Bead Array Mouse Flex Sets; BD Biosciences).Cells infected and fixed with 70% ethanol were stained with

propidium iodide (P4864; Sigma Aldrich) and analyzed byfluorescence-activated cell-sorting (FACS) array for cell-cycleanalysis.

ImmunohistochemistryTissue sections were incubated with primary antibody

according to manufacturer instructions (Table 1) followed by

detection kits either for rabbit using LSAB2þ Dako System(K0673; DakoCytomation) or IHC Select Kit (DAB150-RT;Millipore) for the antibodies raised in rats. Sections werecounterstained with hematoxylin. Photographs were takenwith an Axioplan2 microscope (Carl Zeiss) equipped withAxiocam (Zeiss).

Animal experimentsAll animal protocols were reviewed and approved by the

experimental animal committee of the University of Helsinki(Helsinki, Finland) and the Provincial Government of SouthernFinland. Mice were obtained from Taconic at 4 to 5 weeks ofage.

Immunodeficient models (nudemice) 106 A549 or EJ cells (n¼ 5 mice/group) and 5 � 105 MB49 cells (n ¼ 6 mice/group)were injected subcutaneously. When tumors reached the sizeof approximately 5� 5 mm2, virus was injected intratumorallyat 108 VP/tumor (A549 and EJ tumors) and 3 � 108 VP/tumor(MB49 tumors) on days 0, 2, and 4.

Immunocompetent models (C57Bl/6 mice) 5 � 105 MB49cells (n ¼ 7mice/group) and 2.5 � 105 B16-Ova cells (n ¼8 mice/group) were injected subcutaneously. Viruses wereinjected intratumorally at 3 � 108 VP/tumor on days 0, 2,and 4. Spleens from C57Bl/6 mice with MB49 tumors wereminced and cultured for cytokine analysis. Tumors andorgans from C57Bl/6 mice with B16-Ova tumors weresmashed, filtered through a 70-mm filter, and cultured for24 hours. Apoptosis for tumors and flow cytometric analyseswere conducted by flow cytometry according to manufac-turer instructions (Table 1).

ELISAhCD40L and mCD40L concentration in the serum of mice

were determined with Human CD40 Ligand ELISA Kit (ELH-CD40L-001; RayBiotech Inc) and Mouse sCD40L ELISA Kit(BMS6010; Bender Medsystems) according to the manufac-turer's protocol.

Statistical analysisTwo tailed Student t test was used and a P value of less than

0.05 was considered as significant.

ResultsIn vitro and in vivo characterization of constructedadenoviruses

Replication competent Ad5/3-hTERT-E1A-hCD40L wasconstructed by inserting the hTERT promoter to controlthe E1 gene for tumor selectivity whereas hCD40L wasplaced in E3 for potentiating the immune response (Fig.1A). Our arming strategy associates transgene expressionwith viral replication to ensure high expression of thetransgene at the tumor site, starting from 8 hours afterinfection (25).

Both viruses coding hCD40L showed expression ofthe transgene in vitro on 293 cells (Fig. 1B). Expression ofhCD40L and mCD40L was confirmed also in vivo (Fig. 1C).Following intratumoral injection and subsequent secretion

Oncolytic Adenovirus Coding for CD40L

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of CD40L into blood, infection with Ad5/3-CMV-hCD40Lresulted in higher serum levels than Ad5/3-hTERT-E1A-hCD40L. The cells transduced with Ad5/3-CMV-hCD40Lcontinue to produce CD40L ad infinitum, whereas Ad5/3-

hTERT-E1A-hCD40L causes oncolysis, which limits the timeof CD40L production. Oncolysis of the CD40L-producingcell might be advantageous from a safety perspective,as CD40L can cause side effects when present at high

Figure 1. Functionality and expression of constructed adenoviruses: in vitro and in vivo. A, schematic representation of virus construction. B, flow cytometricanalysis for hCD40L expression in 293 cells at 24 hours postinfection with 10 VP/cell. IC, isotype control. C in vivo expression of CD40L in serum of mice.Results are presented as mean from all mice per group þ SEM. pg/ml, picogram/milliliter. D, functionality of virus produced hCD40L in vitro. Filteredsupernatantwas addedonEJ-transfected cells andNF-kBactivity is expressed in fold increase of luciferase expression (RLU, relative light units).Mock valueswere subtracted. Functionality of virus-produced CD40L was also confirmed by studying NF-kB/AP-1 activation in Ramos-Blue cells (right). The assay wasconducted 3 times and each time was assessed in triplicates. Data are presented as mean � SEM. ��, P < 0.001.

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concentrations (18). The human maximum-tolerated doseof rhCD40L was reported to correspond with a 2,900 pg/mLserum concentration, which is 100-fold higher than what wesaw.Ad5/3-CMV-mCD40L resulted in lower serum CD40L

levels than Ad5/3-CMV-hCD40L, presumably becausemCD40L is metabolized by murine tissues and cells, whereashCD40L is inactive in mice (29). Alternatively, cells trans-duced with adenovirus coding for an immunostimulatorymolecule could be rapidly cleared in immunocompetentmice. Accordingly, no mCD40L could be detected on day8 in the serum of mice with B16-Ova tumors infected withthe same virus (not shown).Two approaches were used to assess the functionality of

hCD40L. A549 cells were infected, supernatant was collectedand filtered through a 0.02-mm filter to obtain the hCD40Lprotein expressed by the virus. We observed a 2.3-foldincrease in NF-kB activation by Ad5/3-hTERT-E1A-hCD40Ladenovirus and a 4.5-fold increase in NF-kB/AP-1 in Ramos-Blue cells (Fig. 1D) compared with Ad5/3-hTERT-E1A–infected cells. Taken together with ELISA and FACS data(Fig. 1B–C), these results suggest that the constructed virusesexpress fully functional CD40L both in vitro and in vivo atlevels predicted to be safe in humans based on use ofrecombinant hCD40L (18).

Presence of CD40 increases virus potencyComplete cell killing induced by Ad5/3-hTERT-E1A-

hCD40L, was seen with 1,000 viral particles per cell (VP/cell)in EJ cells (Fig. 2A). In A549 cells oncolysis by Ad5/3-hTERT-E1A-hCD40L was slower than the control virus. Ad5/3-hTERT-E1A-hCD40L was more potent in CD40þ EJ cells (Supplemen-tary Fig. S1A) whereas Ad5/3-hTERT-E1A killed CD40� A549cells more efficiently (Supplementary Fig. S1B). A competitionexperiment using an anti-CD40L antibody showed that the cellkilling capacity of Ad5/3-hTERT-E1A-hCD40L was abrogatedwhereas Ad5/3-hTERT-E1A was not influenced (Supplemen-tary Fig. S1C). As reported by Gomes and colleagues (30),somewhat higher S-phase content was seen in cell-cycle anal-ysis following infection with CD40L viruses (SupplementaryFig. S1D).Moreover, EJ (CD40þ) cells showed significant apoptosis

when infected with Ad5/3-hTERT-E1A-hCD40L (Supplemen-tary Fig. S2A). In contrast, no apoptosis was seen on A549 cellssuggesting that CD40 is needed for CD40L to cause apoptoticcell death whereas oncolysis is chiefly a nonapoptotic phe-nomenon at least in this cell line (Supplementary Fig. S2A).These experiments indicated that the innate oncolytic potencyof Ad5/3-hTERT-E1A-hCD40L is comparable with a highlypotent control virus and that CD40þ cells are killed moreefficiently than CD40� cells.

Immunogenicity of CD40L-coding viruses in vitroCalreticulin exposure and ATP and HMGB1 release have

been proposed as in vitro measurable indicators of immu-nogenic cell death (31). We found significant enhancementof each of these features after infection of CD40þ EJ cellswith Ad5/3-hTERT-E1A-hCD40L (Fig. 2B–D). In contrast,

only HMGB1 release was enhanced in CD40� cell lineA549 (Fig. 2D).

In vivo efficacy of adenoviruses expressing hCD40L inimmunodeficient animals xenografted with EJ CD40þ

and A549 CD40� tumorsLack of productive replication of human adenovirus in

mouse cells and inactivity of hCD40L in mouse tissues com-plicate preclinical evaluation of Ad5/3-hTERT-E1A-hCD40L.We elected to isolate the antitumor mechanisms into differentmouse models.

In immunodeficient mice the replication-deficient virusAd5/3-CMV-hCD40L had no effect on A549 CD40� tumorswhereas in EJ CD40þ tumors induced a significant decreaseof tumor growth (Fig. 3A and B; Supplementary Fig. S2B).The oncolytic Ad5/3-hTERT-E1A-hCD40L was found aspotent as the positive control virus in both tumor models(Fig. 3C and D), and signs of virus replication was also seenin both sets of mice (Supplementary Fig. S2C). Thus, theoncolytic effect of Ad5/3-hTERT-E1A-hCD40L is not abol-ished by transgene expression or CD40L/CD40-mediatedbiologic effects (Fig. 3D). Nevertheless, it was interestingthat less virus genomes were seen in CD40Lþ tumors,perhaps suggesting less viable tumor cells capable of virusproduction (Supplementary Fig. S2C). Alternatively, apopto-sis induction could affect virus titers.

CD40L promotes apoptosis in CD40þ EJ tumors in nudemice

It has been suggested that interaction of CD40L with CD40can cause apoptosis of tumor cells (15, 32) and thus this wasstudied by staining for caspase-3. Some apoptosis was inducedby control oncolytic adenovirus Ad5/3-hTERT-E1A as reportedfor oncolytic adenoviruses (33). Also, replication-deficientadenovirus Ad5/3-CMV-hCD40L induced apoptosis in tumorsdue to hCD40L expression and its apoptotic effect (15). Nev-ertheless, much more apoptosis was seen in the tumorsinjected with Ad5/3-hTERT-E1A-hCD40L (Fig. 4). The cas-pase-3 data were confirmed by analyzing Annexin-V (Supple-mentary Fig. S2A).

Antitumor activity of CD40L in syngeneicimmunocompetent animal models

In immunocompetent mice with subcutaneous MB49CD40þ bladder carcinoma tumors (34) there was a significantincrease in antitumor activity in the group treated with Ad5/3-CMV-mCD40L (Fig. 5A). Ad5/3-CMV-mCD40L inducedapoptosis suggesting that antitumor activity was partiallydue to apoptosis induced by either binding of mCD40L toCD40 or immunologic effects triggered by mCD40L (Fig. 5B).As apoptosis was not seen in the same experiment carriedout in vitro (Supplementary Fig. S3A), the latter may be morelikely. Interestingly, T cells seemed necessary for the thera-peutic effect, as when the same experiment was carried out inT-cell–deficient mice, no efficacy was seen (Fig. 5C). How-ever, the presence of a therapeutic effect in the absence ofT cells could depend on the cell line to some degree (Fig. 3;ref. 30).






In an immunocompetent but poorly immunogenic model(B16-Ova), Ad5/3-CMV-mCD40L was just as effective as thecontrol virus, the efficacy likely mediated by immune recog-nition of adenovirus per se. Also in this model apoptosis wasseen suggesting that it may be involved in immunologicclearance of infected tumor cells (Fig. 5D).

CD40L induces antitumor immune responses byrecruiting cytotoxic T cells at the tumor site andmodulating the cytokine profile toward T-helper cellresponses

An important part of the putative antitumor activity ofCD40L coding viruses is their effect on APCs. Splenocyte

Figure 2. A, oncolytic potency of Ad5/3-hTERT-E1A-hCD40L on EJ (CD40þ) and A549 (CD40�). Infected cell lines were analyzed by MTS assay. Cell viabilitywas assessed relative to mock-uninfected cells. B, calreticulin exposure on EJ (CD40þ, left) and A549 (CD40�, right). C, extracellular ATP release on EJ(CD40þ, left) andA549 (CD40�, right). The experiments (B andC)were carried out 3 times. D,HMGB1 release onEJ (CD40þ, left) andA549 (CD40�, right). Dataare presented as mean from triplicates plus SEM for B, C, and D. �, P < 0.05; ��, P < 0.001.

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analysis showed increased cytokine levels in the grouptreated with Ad5/3-CMV-mCD40L (Fig. 6A). IL-12 inductionsuggests activation of APC including macrophages and DCs.IFN-g , TNF-a, and RANTES are indicators of T-helper cell(TH1) type immunity and suggest induction of a cytotoxicT-cell response.To correlate this to the cellular level, we analyzed histologic

sections of tumors. Enhanced recruitment of macrophages(F4/80) and leukocytes (CD45) was seen, but only a smallincrease in B lymphocytes (CD19), suggesting that the infiltratewas mostly T cells (Fig. 6B). Analysis of T-cell subsets showedthatmost of these cells were CD8þ cytotoxic T cells, although asmaller increase was seen also in CD4þTH cells (Fig. 6C). Thesefindings indicate that production of mCD40L in syngeneicMB49 tumors prompted a strong antitumor immune responsemediated through TH1 responsive elements and cytotoxic T-cell infiltration (Fig. 6A–C). The effect was due tomCD40L as itwas not seen with the control virus. Also, the effect was notimpacted by oncolysis as Ad5/3-CMV-mCD40L is an E1-delet-ed virus not capable of replication in MB49 cells (Supplemen-tary Fig. S3B and S3C).

DiscussionAdenoviruses have many appealing characteristics as

replicating oncolytic agents, including their unparalleledcapacity for infection of a wide range of tumors, stabilityin vivo, and a good efficacy/safety profile in humans (2, 4, 35,36). Importantly, they can be armed with transgenes toimprove their efficacy. One perceived limitation of adeno-viruses is their immunogenicity. However, as the immunesystem of patients with cancer has failed to eliminate thetumor because of the immunosuppressive nature of thetumor environment, immunogenicity becomes an advan-tage. This effect can be potentiated by retaining replicationcompetence and arming with immunostimulatory moleculessuch as CD40L.

We constructed Ad5/3-hTERT-E1A-hCD40L, which features6 important aspects. (i) Tumor transduction is improved byAd3 serotype chimerism. (ii) Tumor selectivity is achieved byinserting the hTERTpromoter in front of E1A. (iii) Recruitmentand stimulation of APCs for induction of a TH1-type andcytotoxic T-cell response by CD40L. (iv) Apoptosis of CD40þ

tumors through CD40–CD40L interaction. (v) The gp19k/6.7K

Figure 3. Antitumor efficacy in immunodeficient mice. A and B, efficacy of replication-deficient adenovirus Ad5/3-CMV-hCD40L in A549 (CD40�; A) or EJ(CD40þ; B) tumors. C andD, efficacy of replication-competent adenoviruses in CD40� (C) andCD40þ (D) tumors. Data are presented asmean�SEM. Arrowsindicate virus injection. Tumor growth is expressed as percentage increase from first day of virus injection. �, P < 0.05; ��, P < 0.01; ��, P < 0.001.






deletion in E3A to increase tumor selectivity (25) and (vi)antitumor immune response (25). Adenoviruses were foundeffective in inducing high level CD40L expression in CD40þ andCD40� cells. The levels of CD40 andCD40L expression could becrucial in regulating 2 important processes with oppositeconsequences: proliferation or retardation of tumor cell

growth. For example, in lymphomas low levels constitutiveengagement of CD40 can result in neoplastic cell growth (32)whereas high concentrations of CD40L induce inhibition oftumor growth (13, 14). Thus, in the worst case, using recom-binant CD40L or a nonreplicating virus as a gene transfervector might enhance growth of some tumors. Therefore, it ismore attractive to use an oncolytic platform, which ensuresthat transduced tumor cells are ultimately killed by oncolysis.In this approach, CD40L secretion or release from lysing cellscan nevertheless cause an apoptotic bystander effect on tumorcells nearby. However, the main use may be the immunosti-mulatory effect, which was the focus of this study.

Our previous data showed that Ad5/3-hTERT-E1A has sig-nificantly higher oncolytic potency compared with wild-typeAd5 (24). Moreover, an oncolytic adenovirus driven by hTERTpromoter has shown good safety data in humans (37). In fact,Ad5/3-hTERT-E1A is the fastest oncolytic adenovirus we havedeveloped and thus it is an ambitious control virus (38). Wecompared Ad5/3-hTERT-E1A-hCD40L with Ad5/3-hTERT-E1A and found that both viruses were equally effective withregard to oncolytic potency in vivo (Fig. 3). This was animportant finding as expression of transgenes can sometimesinhibit the potency of viruses (39) and Ad5/3-hTERT-E1A-hCD40L was slower than Ad5/3-hTERT-E1A on A549 cells invitro, which might have been due to a 6-fold difference (200 vs.31 VP/pfu) in total to functional particles and viruses weredosed according to the former. It is therefore striking that Ad5/3-hTERT-E1A-hCD40L was as potent as Ad5/3-hTERT-E1A onCD40þ cells (Figs. 2 and 3). In vitro, Ad5/3-hTERT-E1A-hCD40Lhad more antitumor activity on CD40þ cells than on CD40�

cells, whereas opposite was true for Ad5/3-hTERT-E1A(Supplementary Fig. S1A and S1B). Because tumor size mea-surements may not be the optimal approach for studyingtherapeutics with immunologic modes of action, further stud-ies—including survival experiments-–would be useful. Ulti-mately, human data are needed to evaluate the actual benefitof arming with CD40L.

Although the biggest use of Ad5/3-hTERT-E1A-hCD40Lmight be in the context of CD40þ tumors, where all 3 antitumoractivities (oncolysis, apoptosis, and immune stimulation)would contribute, there are reports showing that CD40Lactivates APCs even when the tumor is CD40� (40, 41). Thus,the potential use of the virus is not restricted to CD40þ tumors.In particular, the relative contribution of apoptosis versusimmune response to the overall efficacy needs to be studiedfurther. Also, it is not yet fully clear which immune cells aremost relevant for antitumor effects and our data suggest aputative role for many classes of cells. As even unarmedoncolytic adenoviruses have shown use in humans (2, 4, 42),Ad5/3-hTERT-E1A-hCD40L might represent an improvementregardless of CD40 status of the tumor.

Clinical and preclinical work in the field of tumor immu-nology and vaccine development has shown that induction ofan antitumor immune response can be achieved with severalapproaches (43). However, this has only rarely correlated withtumor control in patients. Instead, the first successful immu-notherapeutic feature either trained/stimulated T cells toovercome tumor-mediated immunosuppression or antibodies

Figure 4. Caspase-3 expression in EJ (CD40þ) tumors. Nudemice bearingEJ tumors were injected thrice with viruses. Tumors were collected 26days after first virus injection and analyzed by immunohistochemistry forinduction of apoptosis (active caspase). Staining was carried out on alltumors and representative photographs are shown. Positive staining isshown in brown. Photographs were taken at � 20 magnification.

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capable of downregulation of immunosuppression (44–46).Many investigators also use preconditioning to "make room"for activated T cells and reduce immunosuppressive cells (47).Thus, a critical lesson is that breaking the immunologictolerance acquired by tumors may be required for successfulimmunotherapy.With regard to oncolytic adenoviruses coding for immu-

nologically active transgenes, there are no suitable animalmodels in which adenoviruses can exert their replicationeffect together with evaluation of the immunologic effect.

Syrian hamsters are semipermissive for human adenovirus(5, 28), but no rodent models are known to be sensitive tohuman CD40L. Therefore, we used different models toisolate the effect of oncolysis, apoptosis, and induction ofantitumor immunity. Of particular interest are the immu-nologic aspects of the approach, which were studied withAd5/3-CMV-mCD40L. Even in the absence of oncolysis,Ad5/3-CMV-mCD40L significantly inhibited tumor growthin the syngeneic MB49 model and some animals wereeventually cured, which is well in accordance with previous

Figure 5. Ad5/3-CMV-mCD40Linhibits MB49 (CD40þ) tumor growthin an immunocompetent animalmodel but has no effect in theabsence of T cells. A, efficacy of Ad5/3-CMV-mCD40L in C57Bl/6 micebearing subcutaneous MB49tumors. Tumor growth is expressedas percentage increase from first dayof virus injection. Data are presentedas mean � SEM. Arrows indicatevirus injection. ��, P < 0.001. B,immunohistochemistry analysis ofapoptosis (active caspase-3) inMB49 tumors. Active caspase-3expression is shown in brown.Photographs were taken at � 10magnification.C, left, efficacyofAd5/3-CMV-mCD40L nude mice bearingMB49 tumors. Tumor size wasfollowed and plotted relative to thesize on first day of injection. Data arepresented as mean þ SEM. Right,flow cytometry for Annexin-V inMB49 tumors from nude mice.Arrows indicate virus injection. ��, P< 0.001. D left, efficacy of Ad5/3-CMV-mCD40L in C57Bl/6 micebearing B16-Ova tumors. Right,apoptosis (Annexin-V) in the sametumors. Arrows indicate virusinjection. Tumor growth is expressedas percentage increase from first dayof virus injection. ��, P < 0.01.






data obtained with a noncapsid-modified virus Ad5-mCD40L (26).

Initial reports suggested that CD40–CD40L interactionsplay a role in potentiating B cells accompanied by B-cellproliferation and differentiation for consequent inductionof humoral responses (8, 48). The effect of CD40L on B cells(Supplementary Fig. S4) and T cells (Supplementary Figs. S5and S6) might depend on context; the expression of CD40Lin tumors might skew the response in the direction of acytotoxic response while expression in lymph nodes couldhave a different effect. In our case, when MB49 tumors wereanalyzed for CD19, we did not notice a significant increaseof these cells in the tumor. The same was seen in organsfrom syngeneic B16-Ova–bearing mice (Supplementary Fig.S6). Intriguingly, in T-cell–deficient mice with MB49tumors, we noticed a significant increase of B cells in thespleens but not at the tumor site (Supplementary Fig. S4).However, even looking at tumors may not accurately reflectthe entire story as T cell may spend themselves by theirantitumor actions.

This is supported by our findings in the syngeneic MB49model where macrophages and T cells, instead of B cells,

were recruited to tumors (Fig. 6). Macrophages are potentAPCs and known for their capacity to induce IL-12. In turn,IL-12 production stimulates release of TH1-type cytokinesincluding RANTES, IFN-g , and TNF-a for induction of acytotoxic T-cell response. All of these effects were seen inour studies (Fig. 6).

In conclusion, we report significant antitumor effects forCD40L expressing adenoviruses including Ad5/3-hTERT-E1A-hCD40L. An important part of the effect is induction of a TH1-type immune response, which results in accumulation ofcytotoxic T cells at the tumor site. Taken together, these dataset the stage for clinical studies with Ad5/3-hTERT-E1A-hCD40L, which are currently ongoing.

Disclosure of Potential Conflicts of InterestA. Hemminki is shareholder and consultant/advisory board member for

Oncos Therapeutics, Ltd. I. Diaconu has ownership interest (including patents)for Oncos Therapeutics. A. Kanerva has ownership interest (including patents)and is a consultant/advisory board member for Oncos Therapeutics. A.S.I.Loskog has ownership interest (including patents) as an inventor of patentowned by Alligator Bioscience AB, and is the consultant/advisory boardmemberfor NEXTTOBE AB. S. Pesonen has ownership interest (including patents) instock options from Oncos Therapeutics Inc. No potential conflicts of interestwere disclosed by the other authors.

Figure 6. Host immune responsesin C57Bl/6 syngeneic murinemodels. A, cytokine analysisin supernatant from culturedsplenocytes of C57Bl/6 micebearing MB49 tumors.��, P < 0.01. B and C,immunohistochemical analysis ofMB49 tumors from C57Bl/6 mice:macrophage-F4/80, leukocytes-CD45, and B-lymphocytes-CD19(B) and helper CD4 and cytotoxicCD8 T cells (C). Positive stainingfor all these markers is shownin brown. Photographs weretaken at �10 magnification.

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AcknowledgmentsThe authors thank Aila Karioja-Kallio, Kikka Holm for expert assistance and

Nina Peitsaro for flow cytometric analysis.

Grant SupportThis study was supported by Helsinki Biomedical Graduate School, Orion

Farmos Research Foundation, K. Albin Johansson Foundation, the EuropeanResearch Council, ASCO Foundation, Finnish Cancer Foundation, HUCHResearch Funds (EVO), Sigrid Juselius Foundation, Academy of Finland, Biocen-

trum Helsinki, Biocenter Finland, University of Helsinki, and the EuropeanCommission FP6 program Apotherapy (contract number 037344). Akseli Hem-minki is K. Albin Johansson Research Professor of the Foundation for the FinnishCancer Institute.

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 7, 2011; revised January 17, 2012; accepted February 23,2012; published OnlineFirst March 6, 2012.

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2012;72:2327-2338. Published OnlineFirst March 6, 2012.Cancer Res Iulia Diaconu, Vincenzo Cerullo, Mari L.M. Hirvinen, et al. Produced by a CD40L-Encoding Oncolytic AdenovirusImmune Response Is an Important Aspect of the Antitumor Effect

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