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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.
CancerResearch
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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.
Diaconu et al.
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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).
Oncolytic Adenovirus Coding for CD40L
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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.
Diaconu et al.
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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.
Oncolytic Adenovirus Coding for CD40L
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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.
Diaconu et al.
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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.
Oncolytic Adenovirus Coding for CD40L
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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.
Diaconu et al.
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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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