Oncolytic Virus and Anti–4-1BB Combination Therapy Elicits Strong Antitumor Immunity against...

2012;72:1651-1660. Published OnlineFirst February 7, 2012.Cancer Res Liza B. John, Linda J. Howland, Jacqueline K. Flynn, et al. Strong Antitumor Immunity against Established Cancer

4-1BB Combination Therapy Elicits−Oncolytic Virus and Anti

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Microenvironment and Immunology

Oncolytic Virus and Anti–4-1BB Combination Therapy ElicitsStrong Antitumor Immunity against Established Cancer

Liza B. John1, Linda J. Howland1, Jacqueline K. Flynn1, Alison C. West1, Christel Devaud1,Connie P. Duong1, Trina J. Stewart1, Jenny A. Westwood1, Z. Sheng Guo5, David L. Bartlett5,Mark J. Smyth1,2,3,4, Michael H. Kershaw1,2,3,4, and Phillip K. Darcy1,2,3,4

AbstractOncolytic virotherapy using vaccinia virus (Vv) has shown some encouraging antitumor responses in mouse

models and patients, but the breadth of efficacy in clinical trials has been somewhat limited. Given that antitumoreffects have correlated with increased host immune responses, we hypothesized that improved therapeuticoutcomesmay be achieved by using oncolytic virus (OV) in combinationwith a potent immune agonist reagent. Inthis study, we carried out a preclinical evaluation of a genetically engineered strain of oncolytic vaccinia virus(Vvdd) for its capacity to induce antitumor responses when combined with an agonist antibody (Ab) specific forthe costimulatory molecule 4-1BB (CD137). In immune-competent syngeneic mouse models of cancer, thiscombination therapy significantly reduced the growth of established subcutaneous tumors relative to eithertreatment alone. Importantly, the development of pulmonarymetastatic lesions was also reduced. Tumor growthinhibition was associated with increased numbers of CD11bþ and CD11cþ myeloid cells in the tumor draininglymph nodes, greater infiltration of CD8þ effector T and natural killer (NK) cells, and amore sustained presence ofneutrophils at the tumor site. Depletion of T or NK cells or neutrophils reduced efficacy, confirming theircontribution to an effective therapeutic response.We further extended this conclusion through results from IFNg-deficient mice. In summary, our findings offered a proof-of-concept for a combinatorial approach to enhance theantitumor efficacy of an OV, suggesting a strategy to improve their use as an immunotherapeutic treatment forcancer. Cancer Res; 72(7); 1651–60. �2012 AACR.

IntroductionOncolytic viruses (OV) offer an exciting new anticancer

strategy with the potential to target both localized tumorsand more advanced metastatic lesions (1–3). A distinct advan-tage of using OV is their capacity to replicate selectively withintumor cells leading to tumor cell death. Several DNA virusesgenetically engineered to enhance tumor tropism while reduc-ing pathogenicity are currently undergoing clinical investiga-tion, including modified strains of adenovirus, herpes simplex

virus, and vaccinia virus (Vv; refs. 3, 4). Vv, in particular, causesno known disease in humans, except for rare individuals withseverely impaired immune function, providing a firm founda-tion for its safe use. Nevertheless, as a further safety measure,genetic engineering of the Western Reserve (WR) strain of Vvwas undertaken to generate either the Vvddmutant, by doubledeletion of the viral thymidine kinase (TK) and viral growthfactor (VGF) genes, or the Vsp mutant by deletion of 2 viralserpin genes SP1 and SP2. Importantly, both Vvmutants retainthe potent oncolytic activity of wild-type Vv but display asignificant reduction in pathogenicity when examined in vivo(5).

Following OV administration in mice, some studies haveshown significant antitumor effects against localized tumors,with evidence suggesting that the host immune response maycontribute to viral induced antitumor responses (6–8).Although detailed mechanistic studies are lacking, resultssuggested that enhanced therapeutic efficacy might beachieved by using OV in combinationwith agents that increasethe activation and strength of host antitumor immuneresponses.

The cellular receptor 4-1BB (CD137) is an important medi-ator of T-cell activation and persistence, particularly for cyto-toxic CD8þ T cells. Administration of agonist anti–4-1BBantibody can impact on tumors in mice and in some modelseradicate small tumors when used alone or in combination

Authors' Affiliations: 1Cancer Immunology Program, Peter MacCallumCancer Centre, East Melbourne; 2Sir Peter MacCallum Department ofOncology, 3Department of Pathology, The University of Melbourne, Park-ville; 4Department of Immunology, Monash University, Clayton, Victoria,Australia; and 5The University of Pittsburgh Cancer Institute and Depart-ment of Surgery, University of Pittsburgh School of Medicine, Pittsburgh,Pennsylvania

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

L.B. John and L.J. Howland contributed equally to this work.

M.H. Kershaw and P.K. Darcy contributed equally as senior authors.

Corresponding Author: Phillip K. Darcy, Cancer Immunology Program,Peter MacCallum Cancer Centre, Victoria, Australia. Phone: 613-965-61286; Fax: 613-965-61411; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-11-2788

�2012 American Association for Cancer Research.

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with other antibodies (9–11). Thus, combining OV adminis-tration with anti–4-1BB antibody may result in enhancedantitumor effects. Herein, we used syngeneic mouse modelsof breast and colon cancer to investigate whether and howcombination therapy using oncolytic Vvdd and anti–4-1BBagonist antibody could enhance therapeutic effects in bothlocalized and disseminated disease settings.

Materials and MethodsCell lines

The B6 mouse 24JK sarcoma and the BALB/c mouse 4T1.2breast cancer lines were kindly provided by Dr. Patrick Hwu(NIH, Bethesda, MD) and Dr. Robin Anderson (Peter MacCal-lum Cancer Centre, VIC, Australia), respectively. The humancervical cancer line HeLa-S3 and the African green monkeykidney cell line CV-1 (CCL-70 clone) were both obtained fromAmerican Type Culture Collection and maintained at 37�C in5% CO2 in RMPI-1640 media supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mmol/L glutamine, 1mmol/L sodium pyruvate, 0.1 mmol/L nonessential aminoacids, 100 U/mL penicillin, and 100 mg/mL streptomycin (Lifetechnologies). The mouse colon adenocarcinoma line MC38was obtained from Dr. Jeff Schlom (NIH, Bethesda, MD), andthe AT-3 tumor cell line was derived as previously described(12). These lines were maintained in complete Dulbecco'smodified Eagle's medium supplemented as above.

VirusesAll vaccinia viruses used in this study were derived from the

WR strain. Two low pathogenic mutants containing deletionsin the TK and VGF genes (Vvdd) or both serpin 1 and serpin 2genes (Vsp) have been previously described (5, 13). Viral stockswere prepared as previously reported (14).

MTT viability assayTumor cells were plated in a 96-well tissue culture plate at 1

� 104 cells per well (100 mL) and incubated overnight at 37�C.The virus was then titrated using 10-fold serial dilutions[multiplicity of infection (MOI) from 1 to 1� 10�5] in completemedium, before overlaying 100 mL of each dilution onto theindividual cell monolayers, in triplicate. Plates were incubatedat 37�C for 72 hours and the viability of the respective tumorcells determined using a MTT formazan viability assay (15).Optical density (OD) of the wells was read at 450 nm on a platereader (Molecular Devices VERSAmax). The mean viability ofthe virus infected tumor cells for each viral dilution wascalculated as a percentage relative to the control wells treatedwith media alone (100% survival) � SEM.

Flow cytometryTumors and the inguinal draining lymph nodes and tumors

were removed frommice at day 3, 9, and 20 posttreatment (n¼5–7 per group). Assessment of infiltrate immediately followingvirus administration was evaluated at day 3 posttreatment,whereas comparison of the monotherapeutic treated groupscompared with the combined therapy (Vvdd and anti–4-1BB)was done at days 9 and 20 posttreatment. Lymph nodes were

homogenized, passed through a 70 mm nylon sieve, and resus-pended in Fc blocking solution (anti-mouse FcR clone 2.4G2 in2% FCS/PBS) for 20 minutes at 4�C. The AT-3 tumors weredisruptedmechanically and digested further with collagenases(0.3%)/DNase (1500 U) solution (Sigma) for 40 minutes at 37�Con a shaker incubator, and a single cell suspension generatedby homogenization as described above. Cells were stained witha cocktail of antibodies for TCRb, CD4, CD8, NK1.1, CD11c,CD11b, andLy6G cell surfacemarkers. Sampleswere incubatedfor 40 minutes at 4�C and analyzed by flow cytometry (LSR-II;BD Biosciences). The data was analyzed using FlowJo softwarev8.5.3 (StanfordUniversity, Palo Alto, California), and themeanpercentage of viable cells for each cell type � SEM wasdetermined at various time points for each treatment group.

In vivo therapy experimentsWild-type B6 and IFNg�/� mice were obtained from The

Walter and Eliza Hall Institute of Medical Research (Parkville,Australia). Mice of 6 to 10 weeks of age were used for in vivoexperiments in accordance with the Peter MacCallum CancerCentre Animal Experimentation Ethics Committee guidelines.Tumors were established in mice by subcutaneous injection of5� 105 breast carcinomaAT-3 cells or 1� 106 colon carcinomaMC38 cells into the left and/or right abdominal flank of wild-type B6 or for some experiments B6.IFNg�/� mice. Oncetumors had reached 20 to 30 mm2, mice were either treatedwith Vvdd virus alone (1 � 109 plaque-forming unit (pfu) perinjection delivered intratumorally on days 0 and 2 from start oftreatment), or twice intraperitoneally with anti–4-1BB (clone3H3, IgG2a, caprylic acid/ammonium sulfate purified, endo-toxin free) antibody alone (25, 50, or 100 mg per injectiondelivered on days 4 and 6 from start of treatment), or acombination of Vvdd and anti–4-1BB antibody (25mg). Controlgroups were left untreated. Tumor growth in mice was mon-itored by twice weekly measurements using electronic calipers(length � height) and the area of the tumor determined foreach mouse. The mean tumor area � SEM was recorded foreach treatment group. To assess spontaneous metastases,mice were culled on day 38 posttreatment and the lungsimmediately perfused with 800 mL of 10% neutral bufferedformalin (NBF) via intratrachea injection. The lungs were thenexcised and fixed in 10%NBF overnight, processed, and stainedwith hematoxylin and eosin (H&E) to determine the number oflung lesions. The mean number of lung metastases � SEM foreach group was plotted. In some experiments, mice (n¼ 6 pergroup) were treated intraperitoneally with the followingdepleting antibodies: 100 mg a-MAC4 Ig control, a-CD8b(clone 53.5.8), or a-CD4 (clone GK1.5) (days�1, 0, 1 from startof treatment, plus twice weekly thereafter), a-Ly-6G (cloneNIMP-R14) (days�1, 0, plus twice weekly thereafter), or 100 mLa-asialoGM1 (WAKO) (days�1, 0, plus once a week thereafter)to determine the impact of the various cellular subsets on theantitumor response following combination therapy.

Statistical analysisThe difference in tumor growth and survival (mean area)

between the treatment groups was analyzed by 2-way ANOVAwith P < 0.05 considered significant. Difference in number of

John et al.

Cancer Res; 72(7) April 1, 2012 Cancer Research1652




lung metastases between groups was analyzed by a Mann–Whitney test. The difference in mean percentage � SEM ofeach immune cell type in the draining lymph nodes and tumorsbetween treatment groups was analyzed by 2-way ANOVA ateach time point.

ResultsSusceptibility of various tumor cell lines to vaccinia virusin vitroThe host range of vaccinia virus is known to be broad and

capable of infecting numerous species. To determine thesusceptibility of a panel of mouse cancer cell lines to killingby 2 oncolytic mutants of Vv (Vsp and Vvdd) an in vitrocytotoxicity assay was done and results quantified using aMTT viability assay. The results revealed that all 4 cell lines(breast 4T1.2 and AT-3, colon MC38, and sarcoma 24JK) weresimilarly susceptible to killing by the 2mutant viruses and thatviral induced cell death was observed at low concentrations ofvirus (MOI � 10�3; Fig. 1). Our results indicated that both Vspand Vvdd mutants of Vv are capable at producing direct

cytotoxic antitumor effects in vitro at low doses against variousmouse cancer lines of different histologic origins. Furtherinvestigation into how Vvdd modulates the immunogenicityof tumor cells in vitro found no induction of apoptosis asmeasured by Annexin Vþ or calreticulin translocation at theindicated time points (Supplementary Fig. S1A and B). Inter-estingly, we observed a trend of increased HMGB1 and ATPrelease from AT-3 cells after 16-hour treatment with Vvdd(Supplementary Fig. S1C and D). These results indicated thatVvdd OV does not cause apoptotic immunogenic cell death ofthese cell lines. Rather, we propose that Vvdd is inducing lyticcell death (necrosis) of the tumor cells, which is supported byother studies (16).

Oncolytic Vvdd combined with anti–4-1BB antibodyenhances antitumor responses against establishedtumors and metastases in vivo

Recent evidence has suggested that the host immuneresponse plays an important role in the antitumor effectsmediated by OV (6–8). Hence, we hypothesized that the useof an OV in combination with an immune stimulatory reagent(anti–4-1BB antibody) may result in enhanced antitumoreffects. Although Vsp and Vvdd mutant vaccinia virusesshowed similar cytotoxicity of tumor lines in vitro, we usedthe Vvdd virus strain initially for combination studies,although it would also be interesting in future studies tocompare combined therapeutic effects using the Vsp strain.We first examined combination therapy against the AT-3breast carcinoma line, given that it allowed us to determineantitumor efficacy in both localized and metastatic diseasesites. In the following experiment, we tested whether admin-istration of Vvdd vaccinia virus and anti–4-1BB antibody couldimpact on the growth of established subcutaneous AT-3 breastcarcinoma tumors in B6 mice. We chose intratumoral deliveryof Vvdd, given higher viral titers were observed in both AT-3and MC38 tumors compared with systemic administration(Supplementary Fig. S2). Intratumoral treatment of mice withVvdd alonewas able to significantly reduce AT-3 tumor growthcompared with nontreated control tumors, although theseeffects were modest and not significantly different to treat-ment with anti–4-1BB antibody alone. In contrast, the com-bined use of Vvdd and anti–4-1BB antibody showed enhancedgrowth inhibition of AT-3 tumors that was statistically signif-icant to either treatment alone (Fig. 2A). Furthermore, micetreated with the combined therapy resulted in an increasedpercentage survival that was significant compared with eithertreatment alone (Fig. 2B). Increasing the dosage of anti–4-1BBmonoclonal antibody (mAb) alone up to 100 mg did notsignificantly enhance the therapeutic effect as observed withthe combined therapy (Fig. 2C).We also examinedwhether ourtherapy could impact on concomitant tumors in recipientmicethat were not directly treated. In this experiment we found thatonly the combinedVvdd and anti–4-1BB antibody treatment ofestablished AT-3 tumors could significantly impact on AT-3tumors injected on the contralateral flank (Supplementary Fig.S3). This result suggested that the combination approach maynot only be effective against primary tumors but could poten-tially impact on distant metastasis at other sites.

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Figure 1. Susceptibility of variousmouse cancer lines to oncolytic VspandVvdd vaccinia virus. Monolayers of mouse cancer cell lines (breast celllines 4T1.2 and AT-3; colon cell line MC38 and sarcoma cell line 24JK)were grown to 80% confluency in 96-well tissue culture plates, beforeinfection with serial dilutions of vaccinia virus (Vsp or Vvdd) usingmultiplicities of infection (MOI) ranging from1 to10�5, in triplicate for eachdilution. At 72-hour postinfection, MTT formazan was added to each welland incubated for a further 4 hours at 37�C. The mean percentage ofviable cells � SEM was determined relative to nontreated control wellsgrown under the same conditions. Representative data from 2experiments conducted are shown.

Combined Immunotherapy Utilizing Oncolytic Virus and Anti–4-1BB Antibody

www.aacrjournals.org Cancer Res; 72(7) April 1, 2012 1653




Given that combined Vvdd and anti–4-1BB therapyresulted in strong antitumor effects against localized AT-3tumors, we next wanted to determine whether this therapywould impact on breast cancer metastases in vivo. In thisexperiment, mice were injected subcutaneously with AT-3tumor cells and lungs were obtained from both nontreatedgroups and mice treated with combined Vvdd and anti–4-1BB therapy at day 38 posttreatment and subjected to H&Estaining to evaluate the number of metastatic lung lesions.Strikingly, this data revealed a significant reduction in thenumber of metastatic lesions present in the lungs of micetreated with the combination therapy compared with thenontreated control group (Fig. 3A–E). To show the broadutility of this therapy, Vvdd and anti–4-1BB antibody com-bined treatment also significantly reduced the growth ofMC38 colon carcinoma cells in mice compared with eithertreatment alone (Supplementary Fig. S4). Collectively, thesedata showed for the first time that the combination therapyof Vvdd and anti–4-1BB antibody could significantlyenhance antitumor effects against established cancer andmetastases in syngeneic immune-competent mice.

Antitumor effects correlate with increased immune cellinfiltrate in the draining lymph nodes and tumorsfollowing combination therapy

To gain insight into the mechanism responsible for theenhanced antitumor effects observed in mice treated with acombination of Vvdd and anti–4-1BBmAb, the draining lymphnodes and tumors of mice from each treatment group weretaken at various time points posttreatment and analyzed byflow cytometry to quantify the different immune cell subtypespresent. With the exception of a decrease in CD8þ T cells, wefound no significant difference in other innate or adaptive celltypes infiltrating the draining lymph nodes following virusadministration at day 3 posttreatment (before anti–4-1BBantibody injection) compared with nontreated mice (Fig. 4Aand B). In contrast, there was a notable increase in thepercentage of both CD11cþ and CD11bþ cells at day 9 post-treatment in the lymphnodes ofmice treatedwithVvdd, whichwas significantly increased following combined Vvdd andanti–4-1BB antibody treatment (Fig. 4C). Interestingly, thenumber of CD8þ T cells decreased further at day 9 followingVvdd treatment alone or combined therapy suggesting thatthese cells may have migrated toward the tumor site (Fig. 4D).There was no difference in percentage of CD4þ T cells at eithertime point following Vvdd treatment or combined therapycompared with nontreated mice. As expected, there werelimited numbers of NK (NK1.1þ) or NKT cells (NK1.1þ TCRbþ)within the draining lymph node, which was not altered withany of these therapies.

We next assessed immune infiltrate into AT-3 tumorsfollowing treatment. We found a significant increase in thepercentage of both myeloid and lymphoid cell types postther-apy. This included an increase in the percentage of CD11bþ

cells and CD11bþLy6Gþ (neutrophils) at day 3 following Vvddtherapy (Fig. 5A). Both of these cell types remained elevatedafter Vvdd treatment alone or Vvdd combinedwith anti–4-1BBantibody at day 9 posttreatment (Fig. 5C). Interestingly,

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Figure 2. Oncolytic Vvdd vaccinia virus and anti–4-1BB combinationtherapy enhances inhibition of AT-3 tumor growth and survival of mice.Groups of B6 mice (n ¼ 8–21 mice per group) were injectedsubcutaneously with 5 � 105 syngeneic AT-3 tumor cells and grown topalpable size (20–30 mm2) for 10 to 14 days. The mice were then treatedeither with direct intratumor injection of Vvdd at 1� 109 pfu per injection(�2), anti–4-1BB mAb (anti-CD137) at 25, 50, or 100 mg per injection(�2 injections), or a combination of both virus and mAb at 25 mg. Resultsare represented as the mean tumor size (mm2) � SEM. Data from 3combined experiments is shown. A, combination therapy significantlyenhanced inhibition of AT-3 tumor growth comparedwith Vvdd treatmentalone (��, P < 0.001) and anti–4-1BB antibody alone (��, P < 0.001).�, P < 0.01, Vvdd-treated mice versus nontreated control mice. B,percentage survival of mice treated with combination of Vvdd andanti–4-1BB antibody was significantly increased compared with Vvdd oranti–4-1BB treatment alone and nontreated control mice (��,P < 0.0001,Vvdd and anti-4-1BB vs. Vvdd alone). C, combined Vvdd and anti–4-1BBantibody therapy significantly enhanced inhibition of AT-3 tumor growthcompared with increasing doses of anti–4-1BB antibody alone.��, P < 0.001.

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systemic delivery of 1 � 106 enriched CD11bþ cells could nottransfer immunogenicity to naive recipients bearing estab-lished AT-3 tumors (data not shown). An important observa-tionwas that the percentage of neutrophils persisted in tumorsat day 20 following combined therapy compared with thesingle-agent groups and nontreated controls (Fig. 5E). Inaddition to these myeloid cell types, we observed an increasein the percentage of CD8þT cells and natural killer (NK) cells atday 9 following either Vvdd treatment alone or combinationtherapy (Fig. 5D), and the percentage of CD8þ T cells wassustained at day 20, particularly following combined therapy(Fig. 5F). In contrast, we found that there was no significantdifferences in the percentage of lymphoid cell types in AT-3tumors at day 3 in either Vvdd treated or nontreated mice (Fig.5B), and there was no significant change in the percentage ofCD4þ T cells or NKT cells following combined Vvdd and anti–4-1BB antibody treatment (Fig. 5B, D, and F). Our data sug-

gested that oncolytic vaccinia virus present in the local tumorregion stimulates an increased number of antigen-presentingcells (APC)migrating to the draining lymphnodes fromas earlyas day 3 postadministration of virus but also induces the rapiddecline of CD8þ T lymphocytes residing in the lymph nodes. Inparallel to this, there was an elevated infiltration of CD8þ Tcells, NK cells, and myeloid cells at the tumor site followingVvdd treatment, with the addition of systemically deliveredanti–4-1BB mAb permitting the increased percentage of neu-trophils and CD8þ T cells, which were sustained out to day 20posttreatment.

Important role for both innate and adaptive immunecells in the antitumor effects mediated by oncolytic Vvddand anti–4-1BB therapy

Althoughourflowcytometry results had shownan increasedimmune infiltrate correlating with therapeutic effects

Figure 3. Combination oncolyticVvdd vaccinia virus and anti–4-1BBtherapy reduces breast cancermetastases in vivo. B6 mice (n ¼ 8mice per group) were injectedsubcutaneously with 5 � 105 AT-3cells and grown to 20 to 30 mm2.Mice were then treated by directintratumor injection of Vvdd at 1 �109 pfu per injection (�2) and anti–4-1BB mAb at 25 mg per injection (�2)or left untreated. At day 38posttreatment, lungs were harvestedfrom both groups of mice andsubjected to H&E staining forquantitation of the number ofmetastatic lesions. A–D,representative H&E sections of lungsfrom nontreated versus treated miceof 8 sections examined. E, the meannumber of lung metastases � SEMwas significantly reduced followingVvdd and anti–4-1BB combinationtherapy compared with nontreatedmice. �, P < 0.01.

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following combined Vvdd and anti–4-1BB treatment, we nextinvestigated whether these cell types were functionally impor-tant in the antitumor response by depleting these subsetsbefore therapy. For these experiments, AT-3 tumors wereestablished in mice as previously described and then groupsofmicewere treatedwith depleting antibodies for either CD8þ,CD4þ T cells, NK cells, or neutrophils before treatment withVvdd and anti–4-1BB mAb. The results showed that depletionof CD8þ T cells (a-CD8), NK cells (a-asialoGM1), and neu-trophils (a-Ly-6G) significantly reduced treatment-inducedgrowth inhibition of AT-3 tumors compared with control Igantibody–treated mice (Fig. 6). Mice depleted of CD4þ T cellsbefore therapy showed comparable antitumor effects to Igcontrol–treated mice indicating that these cells do not play animportant role in the therapeutic effect. Importantly, tumor-bearing mice injected with depleting antibodies alone showedsimilar growth kinetics to nontreated mice (SupplementaryFig. S5). Collectively, this data showed for the first time animportant role for both innate and adaptive immune cells inthe therapeutic responses mediated by combined Vvdd andanti–4-1BB antibody therapy.

Oncolytic Vvdd and anti–4-1BB combination therapyrequires IFNg to elicit optimal antitumor effects

Given that one of the mechanisms used by CD8þ T cells andNK cells for eliciting effective antitumor responses is throughtheir secretion of IFNg , we wanted to determine whether thiscytokine was playing an important role in the therapeuticeffects observed. In this experiment we compared therapeuticresponses against established AT-3 tumors following Vvdd andanti–4-1BB mAb treatment in wild-type B6 and B6.IFNg�/�

mice. Growth inhibition of AT-3 tumors was significantlyreduced in IFNg�/� mice compared with wild-type miceindicating an important role for IFNg (Fig. 7). Importantly,growth of untreated AT-3 tumors was comparable in bothnontreated B6 wild-type and IFNg�/� mice (Fig. 7). Takentogether, our data showed that oncolytic Vvdd vaccinia virusand anti–4-1BB antibody combined therapy can potentlystimulate host immune responses to deliver a strong thera-peutic effect against established cancer.

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Figure 4. Combined Vvdd and anti–4-1BB antibody therapy increasespercentage of APCs but reduces CD8þ effector T cells in the tumordraining lymph nodes. Tumor draining lymph nodes were taken at day 3frommice treated with Vvdd alone or nontreatedmice before anti–4-1BBantibody treatment and at day 9 from mice treated with Vvdd or anti–4-1BB antibody alone or combined Vvdd and anti–4-1BB antibody, andexamined by flow cytometry (n ¼ 5–7 mice per group). A and C, meanpercentage � SEM of CD11cþ cells, CD11bþ cells, and neutrophils(CD11bþ Ly6Gþ) at days 3 and 9. B and D, mean percentage � SEM ofnatural killer cells (NK1.1þ), natural killer T cells (NK1.1þ TCRbþ), CD4þ

and CD8þ T cells at days 3 and 9. ��, P < 0.01; ��, P < 0.001, betweentreatment groups.

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Figure 5. Combined Vvdd and anti–4-1BB antibody therapy increasespercentage of innate and adaptive immune cells at the tumor site. Tumorswere taken from mice in each treatment group at days 3, 9, and 20posttreatment (n ¼ 5–7 mice per group). The tumors were analyzed byflow cytometry to quantify the mean percentage � SEM of immune cellsubtypes present in the tumor at each time point. A, C, and E, meanpercentage � SEM of CD11cþ cells, CD11bþ cells, and neutrophils(CD11bþ Ly6Gþ) at days 3, 9, and 20. B, D, and F, mean percentage �SEM of natural killer cells (NK1.1þ), natural killer T cells (NK1.1þ TCRbþ),CD4þ and CD8þ T cells at days 3, 9, and 20. �, P < 0.05; ��, P < 0.01;��, P < 0.001, between treatment groups.

John et al.





DiscussionOncolytic virotherapy for the treatment of cancer is

gaining momentum, with several new viral agents currentlyundergoing phase II/III clinical evaluation including natu-rally occurring reovirus (Reolysin), and 2 engineered virusesincorporating the gene encoding granulocyte macrophagecolony–stimulating factor (GM-CSF) into herpes simplexvirus (OncoVEX) and vaccinia virus (JX-594; ref. 2, 17).Despite the exciting clinical translation of these biologicagents, the exact mechanisms by which they induce tumorregression presently remains unclear. A large number of invivo studies using OV have been evaluated in xenograftmouse models which have indicated an inherent capacityof OV to be able to infect, replicate, and propagate within thetumor resulting in tumor cell lysis (13, 18–25). However,more recently it has become apparent that a complexinterplay between the virus, tumor, and the host immunesystem exists that determines the outcome of virotherapy(6–8, 25, 26). Given that current evidence suggests an impor-tant role of the host immune system in the antitumor effectmediated by OV, we hypothesized that enhanced therapeuticresponses might be achieved using a combined approach

involving administration of vaccinia virus with an immuneagonist anti–4-1BB antibody. Other studies have reportedstrong antitumor effects combining systemic anti–4-1BBadministration with other reagents delivered intratumorallysuch as CpG or interleukin-12 and GM-CSF (27, 28). Ourresults showed that administration of Vvdd in combinationwith systemic delivery of anti–4-1BB antibody could signif-icantly inhibit the growth of established subcutaneous syn-geneic cancer cell lines AT-3 and MC38, compared witheither treatment alone, and reduce spontaneous AT-3 met-astatic lesions in the lung. It is presently unclear whetherthis is entirely because of direct effects on primary tumorgrowth or because of emerging metastasis being destroyedby the elicited immune response. We showed that theantitumor effects following combined therapy correlatedwith increased numbers of dendritic cells in tumor draininglymph nodes and the increased presence of CD8þ T cells, NKcells, and neutrophils at the tumor site. Indeed, enhancedantitumor responses following combination therapy wasdependent on these cell types as depletion of each of thesesubsets reduced the overall therapeutic effect. Furthermorewe showed a key role for IFN-g , an important cytolyticcytokine secreted by both CD8þ T and NK effector cells.Thus this study describes for the first time that using animmune agonist antibody can significantly increase antitu-mor responses mediated by OVs in a combined attackagainst established cancer.

One advantage of using OVs such as vaccinia is theircapacity for expressing various transgenes, including costi-mulatory molecules or cytokines, to induce stronger anti-tumor responses. The incorporation of B7.1 or lymphocyte

Tum

or

area

(m

m2 )

140

120

100

80

60

40

20

0

Days posttreatment

Nontreated

MAC4 Igα-CD8α-CD4α-Ly-6G

α-asialoGM1

0 5 8 10 12 15 19

******

Figure 6. Depletion of various innate and adaptive immune cells reducesthe therapeutic effect observed following Vvdd and anti–4-1BB antibodytherapy. B6 mice (n ¼ 6 mice per group) were injected with 5� 105 AT-3cells andallowed to establish to 20 to30mm2. Themicewere then treatedwith either 100 mg a-MAC4 Ig control, a-CD8b (clone 53.5.8), or a-CD4(clone GK1.5) depletion antibodies (days�1, 0, 1 from start of treatment,plus twiceweekly thereafter),a-Ly-6G (cloneNIMP-R14; days�1, 0, plustwice weekly thereafter), 100 mL a-asialoGM1 (days �1, 0, plus once aweek thereafter), or left untreated. Antibody treated groups were thentreated with Vvdd at 1 � 109 pfu (days 0, 2 from start of treatment) andanti–4-1BB mAb at 25 mg (days 4 and 6). The area of the subcutaneoustumor wasmonitored 2 to 3 times a week with electronic calipers and themean tumor size � SEM recorded. Significant reduction of AT-3 tumorgrowth inhibition was observed for a-CD8b (��, P < 0.01), a-Ly-6G(�,P <0.05), anda-asialoGM1 (��,P<0.001) depletedgroups, comparedwith control MAC4 Ig or a-CD4 antibody–treated mice. Representativedata from 2 experiments conducted are shown.

Tum

or

area

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m2 )

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100

50

0

Days posttreatment

*

0 4 7 9 11

B6.IFNy–/– Nontreated

B6.IFNy–/– treated

B6.WT Nontreated

B6.WT treated

Figure 7. Important role for IFNg in the therapeutic effect followingcombined Vvdd and anti–4-1BB antibody therapy. B6wild-typemice andB6.IFNg�/� mice (n ¼ 6 mice per group) were injected with 5 � 105 AT-3cells and grown to 20 to 30 mm2. Mice were then treated by directintratumor injection of Vvdd at 1 � 109 pfu (days 0, 2 from start oftreatment), followed by anti-4-1BB mAb at 25 mg (days 4 and 6) or leftuntreated. The area of the subcutaneous tumor was monitored 2 to 3times a week with electronic calipers and the mean tumor size (mm2) �SEM recorded. Therapeutic effects were significantly reduced in B6IFNg�/� mice compared with B6 wild-type mice following combinedtherapy (�, P < 0.01).






function–associated antigen-3 into vaccinia virus has beenshown to induce tumor-specific T-cell responses in meta-static melanoma patients (29–31). Similarly, an oncolyticadenovirus armed with TNF-related apoptosis-inducingligand was shown to induce efficient melanoma cell killingand reduced tumor growth in mice (32). Another studyshowed that oncolytic vaccinia virus expressing 4-1BBLcould mediate effective antitumor responses against B16melanoma cells in mice compared with nonengineered virus,although these effects were observed only when combinedwith host lymphodepletion (29). This is less than ideal asimmunotherapies involving host lymphodepletion is oftenassociated with toxicity and may result in depletion ofimmune cells important for the therapeutic effect. In con-trast, we observed potent antitumor effects in vivo in 2different cancer models following combined vaccinia virusand anti–4-1BB therapy that did not require prior condi-tioning of the host.

Previous studies have reported that engagement of 4-1BB ondendritic cells using anti–4-1BB mAb enhances their produc-tion of inflammatory cytokines and enhances their ability toactivate T cells (33). Our study is consistent with this astreatment of mice with Vvdd alone increased both numbersof CD11bþ and CD11cþ cells in the tumor draining lymph nodewhichwere further increased following administration of anti–4-1BB antibody. Interestingly we observed a concomitantdecrease in numbers of CD8þ T cells from day 9 followingtreatment, suggesting that these cells have become activatedfollowing engagement of APCs in the lymphnode andmigratedto the tumor site.

To support this contention, analysis of the tumor cellinfiltrate by flow cytometry revealed a significant increase inthe number of CD8þ T cells in mice following treatment withVvdd and anti–4-1BB mAb therapy from day 9 onwardscompared with either treatment alone. We also observed asignificant increase in number of NK cells at the tumor sitefollowing combined therapy. An important role for both ofthese effector cells was shown in subsequent depletion experi-ments. Although the exact role of how these cells were medi-ating antitumor effects is presently unclear, our studiesrevealed that this was possibly through secretion of the cyto-kine IFN-g , given the therapeutic effect was reduced in IFN-ggene–targeted mice. Future studies will determine whetherIFN-g is exerting antitumor effects through mechanisms suchas enhancing antigen presentation on tumor cells, increasingtumor susceptibility to apoptosis and/or inducing cell-cyclearrest (34–38), or through the recruitment and activation ofendogenous immune effector cells, regulation of leukocyte–endothelium interactions, and/or induction of antiangiogen-esis (34, 39–41).

Interestingly, our data revealed that both Vvdd alone andin combination with anti–4-1BB antibody increased num-bers of neutrophils (CD11bþ Ly6Gþ) at the tumor site thatwere sustained up to day 20 in the combined treatmentgroup. The importance of these cells was further supportedby depletion experiments using a-Ly-6G mAb, whichresulted in reducing the therapeutic effect (42). Althoughmany studies have linked neutrophils with having a protu-

morigenic role (43), our results are consistent with anotherstudy showing that neutrophils could mediate antitumoreffects by blocking vital blood supply to the tumor followingtheir activation (8). Furthermore, neutrophils have thecapacity to release reactive oxygen species, secrete pro-teases, and impair microvascular perfusion and may beexerting antitumor effects in our model through a numberof these different mechanisms (8, 43–46). Another recentstudy suggested that neutrophils can be polarized into N1and N2 phenotypes that possess either protumorigenic andantitumorigenic roles similar to macrophages (43, 47).Whether this is the case in our model remains to beclarified. Neutrophils have been reported to express 4-1BB,and ligation of this receptor has been shown to enhancetheir survival (48, 49). Our data is consistent with this as weobserved sustained numbers of neutrophils at the tumorsite following therapy. Nevertheless, both the presence ofviral induced danger signals mediated by Vvdd and 4-1BBcostimulation in combination was required to mediate thiseffect.

In conclusion, our studies have shown for the first time thatadministration of an immune agonist antibody can enhancethe antitumor properties of an OV against localized anddisseminated cancer. However, it remains unclearwhat impactanti–4-1BB also had on the anti-vaccinia immune responseand potentially decreasing this could further potentiate thistherapy. Our data highlighted a critical role for both adaptiveand innate immune host cells in mediating the therapeuticeffects, suggesting that future therapies combining immuneagonist antibodies with OVs that can stimulate the endoge-nous host immune system could lead to improving therapeuticoutcomes in patients.

Disclosure of Potential Conflicts of InterestD.L. Bartlett has ownership interest (including patents) in Jennerex; he is a

consultant and is on the advisory board of Jennerex. No potential conflicts ofinterest were disclosed by the other authors.

Authors' ContributionsConception and design: L.B. John, L.J. Howland, M.J. Smyth, M.H. Kershaw,P.K. Darcy.Development of methodology: L.B. John, L.J. Howland, Z.S. Guo, P.K. Darcy.Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): L.B. John, L.J. Howland, J.K. Flynn, A.C. West, C.Devaud, C.P. Duong, T.J. Stewart, J.A. Westwood, Z.S. Guo, P.K. Darcy.Analysis and interpretation of data (e.g., statistical analysis, biostatistics,and computational analysis): L.B. John, L.J. Howland, J.K. Flynn, A.C.West, T.J.Stewart, M.J. Smyth, M.H. Kershaw, P.K. Darcy.Writing, review, and/or revision of themanuscript: L.B. John, L.J. Howland,T.J. Stewart, D.L. Bartlett, M.J. Smyth, P.K. Darcy.Administrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): L.J. Howland, D.L. Bartlett, P.K. Darcy.Study supervision: M.H. Kershaw, P.K. Darcy.

AcknowledgmentsThe authors thank the technicians of the Peter MacCallum Cancer Centre

Experimental Animal Facility for animal care and the Histology Department fortheir assistance in processing of H&E sections of lung tissue following therapyand Nicole McLaughlin for preparation of the depleting antibodies.

Grant SupportThis work was funded by Project Grants from the National Health and

Medical Research Council (NHMRC), Cancer Council of Victoria, and the Susan

John et al.





Komen Breast Cancer Foundation. T.J. Stewart was supported by a NationalBreast Cancer Foundation Fellowship and M.J. Smyth by an NHMRC AustralianResearch Fellowship. M.H. Kershaw and P.K. Darcy were supported by a NHMRCSenior Research Fellowship and Career Development Award, respectively.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact.

Received August 22, 2011; revised December 29, 2011; accepted January 13,2012; published OnlineFirst February 7, 2012.

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