Deﬁning Effective Combinations of Immune Checkpoint ... · Cancer Therapy: Preclinical Deﬁning Effective Combinations of Immune Checkpoint Blockade and Oncolytic Virotherapy Juan

Cancer Therapy: Preclinical

Defining Effective Combinations of ImmuneCheckpoint Blockade and Oncolytic VirotherapyJuan J. Rojas1, Padma Sampath1,Weizhou Hou1, and Steve H. Thorne1,2

Abstract

Purpose: Recent data from randomized clinical trials withoncolytic viral therapies and with cancer immunotherapies havefinally recapitulated the promise these platforms demonstrated inpreclinical models. Perhaps the greatest advance with oncolyticvirotherapy has been the appreciation of the importance ofactivation of the immune response in therapeutic activity. Mean-while, the understanding that blockade of immune checkpoints(with antibodies that block the binding of PD1 to PDL1 or CTLA4to B7-2) is critical for an effective antitumor immune response hasrevitalized the field of immunotherapy. The combination ofimmune activation using an oncolytic virus and blockade ofimmune checkpoints is therefore a logical next step.

Experimental Design: Here, we explore such combinationsand demonstrate their potential to produce enhanced responsesin mouse tumor models. Different combinations and regimenswere explored in immunocompetent mouse models of renal and

colorectal cancer. Bioluminescence imaging and immune assayswere used to determine the mechanisms mediating synergistic orantagonistic combinations.

Results: Interaction between immune checkpoint inhibitorsand oncolytic virotherapy was found to be complex, with correctselection of viral strain, antibody, and timing of the combinationbeing critical for synergistic effects. Indeed, some combinationsproduced antagonistic effects and loss of therapeutic activity. Aperiod of oncolytic viral replication and directed targeting of theimmune response against the tumor were required for the mostbeneficial effects, with CD8þ and NK, but not CD4þ cells medi-ating the effects.

Conclusions: These considerations will be critical in the designof the inevitable clinical translation of these combinationapproaches. Clin Cancer Res; 21(24); 5543–51. �2015 AACR.

See related commentary by Slaney and Darcy, p. 5417

IntroductionThe last 5 years have seen the emergence of antibody-mediated

blockade of immune checkpoints as a key new weapon in theanticancer arsenal (1, 2). The anti-CTLA4 inhibitor ipilimumabhas been approved for the treatment of melanoma (3, 4), while apanel of monoclonal antibodies targeting the interaction of PD1and PDL1 have also demonstrated promising responses in asuccession of clinical trials (5, 6). Together, these trials havedemonstrated the clinical need to overcome the tumor's capacityto shut down the T-cell response in the creation of an effectivecancer immunotherapy.

The field of oncolytic virotherapy has also recently demon-strated its potential to produce clinically effective cancer treat-ments,with data fromseveral recent randomized trials resulting inimpressive response rates (7, 8). One factor that has united themost successful oncolytic vectors has been the expression of animmune-activating transgene (GM-CSF), an indication that a key

determinant of the activity of oncolytic viruses is their capacity toactivate and target the immune response (9, 10). This has sincebeen confirmed in a multitude of preclinical studies (11–13),such that the oncolytic virus platformmight best be considered animmunotherapeutic.

We have previously developed several oncolytic vectors, pri-marily focusing on vectors based on vaccinia virus (14–17).These provide several advantages as immunotherapies beyondtheir long historical use as vaccines: (i) They can induce anadaptive immune response raised against tumor antigens as aresult of their selective replication within the tumor microen-vironment (18, 19). This in situ vaccination effect results inproduction of CTL targeting relevant tumor antigens withoutthe need for any prior interrogation of the tumor. (ii) Viralreplication within the tumor can at least transiently overcomelocalized immunosuppression, something that most traditionalvaccine approaches fail to achieve. However, in many cases,once the oncolytic virus is cleared by the host immune response,the immunosuppressive environment is apparently restored andthe tumor relapses. The combination of oncolytic virus and theblockade of immune checkpoint inhibitor therefore is anappealing strategy.

Although there has been much interest in this combination,including the proposed clinical combinations of the oncolyticHSV T-Vec (Amgen) and ipilimumab (Yervoy, Bristol MyersSquibb) in the treatment of melanoma (Clinical Trials.govNCT01740297), there have been very little supportive datareported to date. Here, we examine the combination of oncolyticvaccinia with several different immunotargeting monoclonalantibodies.

1Department of Surgery, University of Pittsburgh Cancer Institute,University of Pittsburgh, Pittsburgh, Pennsylvania. 2Department ofImmunology, University of Pittsburgh Cancer Institute, University ofPittsburgh, Pennsylvania.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

CorrespondingAuthor:SteveH.Thorne,UniversityofPittsburghCancer Institute,1.46e Hillman Cancer Center, 5117 Centre Avenue, Pittsburgh, PA 15213. Phone:412-623-4896; Fax: 412-623-2525; E-mail: [email protected].

doi: 10.1158/1078-0432.CCR-14-2009

�2015 American Association for Cancer Research.

ClinicalCancerResearch

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Published OnlineFirst July 17, 2015; DOI: 10.1158/1078-0432.CCR-14-2009

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Materials and MethodsCell culture and viruses

Renca (murine renal adenocarcinoma) cell line was obtainedfrom ATCC. MC38 cell line (murine colon adenocarcinoma) wasa kind gift from Dr. David Bartlett (University of PittsburghCancer Institute, Pittsburrgh, PA). Cell lines were maintained inrecommended culture media containing 5%–10% FBS at 37�C,5% CO2. Cell lines have not been authenticated by the authorsbeyond their ability to form tumors in syngeneic mouse models.

All recombinant vaccinia strains used in this work are derivedfrom the Western Reserve (WR) strain (BEI Resources). Thedouble-deleted strains vvDD andWR.B18R-.TK- (B18R- in short)have been described previously (15, 20). These contain deletionsin the tk gene and in the vgf or B18R viral genes, respectively. Inaddition, both strains express the firefly luciferase gene from thesynthetic vaccinia promoter pE/L (21), which allows monitoringof luciferase expression as a surrogate indicator of viral replication(22). Viruses were titered, manufactured, and purified as previ-ously described (23).

Animal modelsAll animal studies were approved by the University of Pitts-

burgh Institutional Animal Care and Use Committee. C57/BL6and BALB/c female mice (6–8 weeks old) were purchased fromThe Jackson Laboratory. Renca or MC38 tumor cell lines wereimplanted subcutaneously at 5� 105 cells permouse into BALB/cor C57/BL6 mice, respectively. Oncolytic vaccinia viruses wereinjected intravenously (tail vein) at 2 � 108 pfu/mouse whentumors reached approximately 50 to 100 mm3.

Anti-mouse CTLA4 (9D9) and anti-mouse CD25 (PC-61.5.3)antibodies (BioXCell) were injected intraperitoneally at 100 or200 mg/mouse/dose, respectively, with treatments consisting of3 doses each 3 days apart. Mouse IgG2b k Isotype Control(BioXCell) was used as a control. For depletion experiments,anti-mouse CD8 (2.43), anti-mouse CD4 (GK1.5), and anti-mouse IFNg (XMG1.2) were purchased from BioXCell,and anti-mouse Asialo-GM1 was purchased from Wako Pure

Chemicals (Richmond, VA). Mice were injected intraperitoneallywith 500 mg at days 1 and 2 after tumor implantation, followed by250 mg injection every 5 days till the end of the experiment.

Tumor volume was monitored by caliper measurement anddefined by V(mm3)¼ p/6�W2� L, whereW and L are the widthand the length of the tumor, respectively. Data are expressed astumor size relative to the beginning of the therapy (100%). ForKaplan–Meier survival curves, end point was established at�750mm3. Animals whose tumor size never achieved the thresholdwere included as right-censored information.

Bioluminescence imagingViral gene expression was determined through biolumines-

cence imaging of luciferase expression in vivo. A dose of 4.5 mgof D-luciferin (GoldBio) was injected intraperitoneally per mousebefore imaging on an IVIS2000 (PerkinElmer; 2% isoflurane).Images were analyzed using LivingImage software (PerkinElmer).

IFNg ELISPOTsFor ELISPOT assays, splenocytes weremixedwith tumor cells or

splenocytes from na€�vemice infected with UV-inactivated vacciniavirus at 5:1 ratio. Na€�ve splenocytes were used as control. 96-wellmembrane filter plates (EMDMillipore) coated with 15 mg/mL ofmonoclonal anti-mouse IFNg antibody AN18 (Mabtech, Inc.)were used. Cells were maintained for 48 hours at 37 oC and spotswere detected using 1 mg/mL of biotinylated anti-mouse INFgantibody R4-6A2-biotin (Mabtech). Plates were developed usingan ABC kit and an AEC substrate kit for peroxidase (VectorLaboratories, Inc.). Specific spotswere counted and analyzedusingan ImmunoSpot Analyzer and ImmunoSpot software from CTL.

Flow cytometryTumors were harvested from mice and mechanically disaggre-

gated and digested with triple enzyme mixture (Collagenase typeIV, DNase type IV, and Hyaluronidase type V, Sigma-Aldrich)].Cell surface and intracellular immunostaining analyses wereperformed using a Gallios Flow Cytometer (Beckman Coulter,Inc.). For intracellular staining, cells were fixed and permeabilizedusing a Foxp3 Fix/Perm Buffer Set (eBioscience). Tumor-disag-gregated cells were stained using PE-Cy7 anti-mouse CD3 (BDBiosciences), eFluor450 anti-mouse NKp46, APC anti-mouseNKg2D, FITC anti-mouse CD4, PerCP-Cy5.5 anti-mouse CD8,PE anti-mouse CD25, and APC anti-mouse Foxp3 antibodies(eBioscience).

Statistical analysisStandard Student t tests (two-tailed) were used throughout

this work, except for the comparison of survival curves, where alog-rank test was used. In all cases, significance was achieved ifP< 0.05.

ResultsAnti-CTLA4 antibody hinders vaccinia virus replication inmice

Mice harboring syngeneic subcutaneous mouse renal adeno-carcinomas (Renca cells) were injected with a single intravenousdose of oncolytic vaccinia virus and with three intraperitonealdoses of 100 mg of mouse anti-CTLA4 antibody at days 0, 3, and 6after virus injection. The schedule and doses of anti-CTLA4antibody used were determined on the basis of previously

Translational Relevance

Two of the most promising novel therapeutic platforms forthe treatment of cancer are blockade of immune checkpointsand oncolytic viral therapies. Here, we look to combine thesein preclinical mouse tumor models. The realization that inhi-bition of immune checkpoints is a critical need for successfulimmunotherapy and that the immune response activated byoncolytic viral therapies provide their most potent antitumoreffects, means that the combination of these approaches islikely to result in significant clinical benefit. The enthusiasm inthis combination is seen with the ongoing clinical combina-tion of the oncolytic T-Vec with ipilimumab. However, therehas been almost no preclinical data reported to support thiscombination to date. In this article, we not only demonstratethat clinically relevant combinations can produce significantlyenhanced responses inmouse tumormodels, but also providemechanistic insight into why some combinations are syner-gistic and others resulted in complete loss of therapeuticadvantage.

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published preclinical studies (24–26). Initially, we looked todetermine the safety of the combination and the effects of injec-tion of anti-CTLA4 antibody on the replication of vaccinia in thetumors. We monitored viral luciferase transgene expression withbioluminescence imaging as ourselves andothers have shown thisto directly correlate with viral replication (22, 27). Anti-CTLA4antibody significantly reduced viral luciferase expression fromwithin the tumors (at days 3 and5after virus injection,>5- and40-fold reduction was detected; Fig. 1A). A similar depletion in viralreplication was also observed in a second tumor model (MC38tumors implanted subcutaneously in C57/B6 mice, Supplemen-tary Fig. S1B), demonstrating that thiswas not a cell line- ormousestrain–associated effect. Reduced viral replication did, however,correlate with enhanced immune activation, as seen with theincreased numbers of CTLs recognizing vaccinia epitopes detectedin the spleens of the mice (Fig. 1B). Although increased antiviralCTL appeared as early as day 3 after treatment, it is likely thatinnate immune responses may also be enhanced with the com-bination as viral replication is reduced as early as 24 hours aftertreatment. This indicated a more robust immune response wasraised in mice when anti-CTLA4 antibody was injected togetherwith the viral therapy.

Delayed administration of anti-CTLA4 antibody improvesantitumor efficacy

A novel schedule for oncolytic vaccinia and anti-CTLA4 anti-body combination was therefore designed to permit an initialphase of viral oncolytic activity before anti-CTLA4 antibodyadministration (Fig. 1C). Anti-CTLA4 doses were thereforeinjected at days 4, 7, and 10 after virus injection, allowing aninitial phase of unhindered viral replication and spreadwithin thetumor (Supplementary Fig. S1C). Whereas simultaneous injec-tion of vaccinia virus and anti-CTLA4 antibody resulted in nosignificant antitumor benefit (compared with mice treated withsingle vaccinia therapy), when we delayed administration of theblocking antibody until after the peak of viral replication, a greaterthan 3-fold reduction (P < 0.04) in tumor volume was observed(isotype control antibody had no effect on tumor growth; Fig. 1Dand Supplementary Fig. S1A). In addition, this novel treatmentschedulewas able to significantly increase survival ofmice relativeto groups treated either with single vaccinia therapy or vacciniainjected concurrently with anti-CTLA4 (Fig. 1E).

Combination of anti-CD25 antibody with vaccinia provided notherapeutic benefit

As an alternative to immune checkpoint blockade therapy(anti-CTLA4), we looked to test whether other approaches thatalso target tumor immunosuppression [such as depletion ofregulatory T cells (Treg) with anti-CD25 therapy] also synergizedwith oncolytic vaccinia therapy. We again initially monitoredvirus replication in tumors via luciferase bioluminescence imag-ing after anti-CD25 administration. As with anti-CTLA4 combi-nation, we observed a reduction in viral kinetics when antibodytherapy began on the same day as viral treatment; however,differences were not significant (Fig. 2A). Furthermore, when theantitumor effects of vaccinia/anti-CD25 combination therapywere tested, neither regimen (injecting anti-CD25 antibody con-currently with virus or after viral replication peak) resulted inimproved efficacy relative to single oncolytic vaccinia therapy(Fig. 2B and Supplementary Fig. S2B). As a further test, anti-CD25antibody was also added before viral therapy (Supplementary

Fig. S2C), however, againno therapeutic advantagewas seen (Tregdepletion with the anti-CD25 regimen used was also confirmed;Supplementary Fig. S2A). Finally, a direct comparison of theanticancer activity of vaccinia/anti-CD25 versus vaccinia/anti-CTLA4 combination therapies confirmed the enhanced efficacyof combining oncolytic virus with blockade of CTLA4 (Fig. 2B).

Immunogenicity-enhanced oncolytic vaccinia vectors improvesynergistic effects with anti-CTLA4 antibody

As a next step, we looked to examine the importance of the viralvector used in these combination approaches. Two differentdouble-deleted oncolytic vaccinia viruses were compared in com-bination with anti-CTLA4 antibody therapy. vvDD (vgf and tkdouble-deleted vaccinia virus) has demonstrated highly tumor-restricted replication (28) that is equivalent in level and selectivityto the B18R- strain. B18R- (B18R and tk double-deleted vacciniavirus) also demonstrated highly tumor-restricted replication butthiswas coupledwith enhanced immunogenicity relative to vvDD(including increased production of cytokines and chemokineswithin the tumor; ref. 29). This is due to the loss of B18R, thatencodes a secreted type I IFN-binding protein (14). Whenboth viral strains were compared for anticancer effects in combi-nation with anti-CTLA4 antibody (Fig. 3), B18R-/anti-CTLA4treatment induced a more than 3.6-fold (P < 0.009) reductionin tumor size at sacrifice compared with PBS treatment, whilein this model vvDD/anti-CTLA4 combination only induced a1.4-fold inhibition.

B18R- oncolytic vaccinia virus exhibits potent antitumorefficacy in optimized combination with anti-CTLA4 antibodytherapy

We next looked to test in more detail the most effectivecombination of viral vector (B18R-), antibody (anti-CTLA4) andregimen (antibody treatment beginning 4 days after viral therapy)determined from the previous studies.

Mice carrying either Renca (renal adenocarcinoma) or MC38(colon adenocarcinoma) tumors were injected with a singleintravenous dose of B18R- at 2 � 108 pfu per mouse. At days4, 7, and 10 after virus injection, an intraperitoneal dose of 100 mgof mouse anti-CTLA4 antibody was administrated. PBS or singletherapy treatments were used as controls. At the time of sacrifice,combination therapy resulted in a reduction of more than 2.7-(P < 0.035) and 1.3-fold (P < 0.02) in Renca and MC38 tumormodels, respectively, relative to single B18R- therapy (Fig. 4A).

The combination induced a reduction of more than 2.8-fold(P < 0.04) in tumor volume compared with singe anti-CTLA4therapy at day 42 after treatment in Renca models. Importantly,B18R-/anti-CTLA4 combination therapy induced 3 of 12 com-plete responses in this model. For MC38 tumors, B18R-/anti-CTLA4 combination therapy did not produce as dramatic aneffect, but still reduced tumor volume 1.5-fold (P < 0.045)compared with single anti-CTLA4 therapy, a significant improve-ment by day 24 after treatment.

Vaccinia/anti-CTLA4 combination therapy resulted inenhanced systemic and tumor-specific cellular immuneresponse.

To evaluate the mechanisms driving the most effective combi-nation of oncolytic vaccinia and anti-CTLA4 antibody, we exam-ined the immune response raised against and within the tumor.Mice bearing Renca tumors were treated as before. Controls

Oncolytic Virus Combination with Checkpoint Inhibitors

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Figure 1.Combining oncolytic vaccinia virus and anti-CTLA4antibody therapies. A, anti-CTLA4 antibody injection reduces vaccinia virus replication in the tumor in vivo. Balb/cmice with subcutaneous Renca tumors (renal adenocarcinoma) were randomized and injected with a single intravenous dose of 2� 108 plaque-forming units (pfu)per mouse of oncolytic B18R- vaccinia virus (VV). In the combination group, 100 mg of mouse anti-CTLA4 antibody was injected intraperitoneally on days0, 3, and 6 after virus administration. Bioluminescence imaging was used to follow viral luciferase transgene expression from within the tumor. Mean values of9 to 10 animals þ SD are plotted. Representative luciferase signals at day 3 after injection are also depicted (tumors are circled). B, viral/anti-CTLA4 combinationresults in increased levels of vaccinia-specific cytotoxic T cells (CTL). Mice were treated as in A, adding PBS and single therapy with anti-CTLA4 antibody asadditional controls. At days 3 and 8 after virus injection, spleens were harvested and quantified by the IFNg ELISpot assay for vaccinia-reacting T cells. Values ofindividual mice and means � SEM of the different treatments are plotted. C, alternative schedule for vaccinia virus and anti-CTLA4 antibody combination.Anti-CTLA4 antibody doses were administrated at days 4, 7, and 10 after virus injection, in an approach designed to permit an initial period of viral replication.D, injection of anti-CTLA4 antibody after vaccinia virus replication improves therapeutic activity of combination therapy. Mice (Balb/c bearing Renca tumors)were treated as before or in combination with anti-CTLA4 antibody as depicted in C. Relative tumor growth and Kaplan–Meier survival curves (E) are plotted. Forsurvival curves, the end point was established at a tumor volume �750 mm3. Mean values of 7 to 8 mice/group þ SE are plotted. � , P < 0.05, comparedwith the VV group; f, P < 0.05, compared with the PBS group; c, P < 0.05, compared with the anti-CTLA4 group; #, P < 0.05, compared with the VVþanti-CTLA4day 0 group.

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included PBS, single B18R- therapy, or single anti-CTLA4 therapy(injected at days 0, 3, and 6). Mice were sacrificed at day 11 aftervirus administration and evaluated for specific CTLs in the spleenby the ELISpot assay and for immune cell populations in tumorsby flow cytometry. Combination therapy was able to significantlyincrease the numbers of CTLs recognizing tumor cell antigenscompared with any of the controls (Fig 4B). When CD3þCD4þ

populations in tumors were quantified, a significant percentageincrease was observed after treatment with B18R-/anti-CTLA4combination therapy relative to any other treatment (Fig. 5A andC). An increase in the percentage of CD3þCD8þ cells infiltratingthe tumor was also observed, but appeared to be more closelyassociated with replication of the virus in the tumor (Fig. 5B andC), with both virus-treated groups displaying high levels of thesecells. Finally, to ensure that the increased CD3þCD4þ populationinfiltrating the tumors did not represent Tregs, additional stainingfor CD25 and FoxP3 was used (Fig. 5D). We observed that in thecontrol group, about 40% of the CD3þCD4þ cells present a Tregphenotype (CD25þFoxp3þ). Anti-CTLA4 treatment barelyreduced this percentage, but treatment with B18R- virus droppedamounts to 17%, and this improved further to only 13% when

anti-CTLA4was combinedwithoncolytic virus. Although very fewNKorNK-T cells were detected in the tumor (<0.01%of cells), thisnumber was also significantly increased only when the combi-nation of B18R- virus and anti-CTLA4 antibody was used incombination (Fig 5E and Supplementary Fig. S4).

Vaccinia/anti-CTLA4 combination therapy synergistic effectsrequire CD8þ T cells, NK cells, and IFNg, but not CD4þ T cells

To define the host factors critical for the therapeutic advantageseen with the B18R-/anti-CTLA4 combination, viral replicationand antitumor effect experiments were repeated in Renca tumor-bearing mice depleted for CD4þ T cells, CD8þ T cells, or NK cells(Fig. 6). It was seen that both CD8þ T cells and NK cells wererequired for the therapeutic advantage (while antitumor effectswere maintained after depletion of CD4þ T cells; Fig. 6B). Deple-tion of CD8þ T cells but not NK cells or CD4þ T cells alsosignificantly enhanced viral replication, indicating this cell lineagewas responsible for both reduced viral replication and enhancedantitumor effects during B18R-/anti-CTLA4 combination (Fig.6A). Interestingly CD8þ T cells appeared responsible for reducedviral replication in the tumor, even at times as soon as 1 day after

Figure 2.Combination of vaccinia viruswith anti-CD25 antibody did not provide any therapeutic advantage. A, anti-CD25 antibody therapy effect on vaccinia virus replication.Balb/c mice bearing Renca tumors were injected intravenously with 2 � 108 pfu/mouse of oncolytic vaccinia virus (VV, strain B18R-). For the combinationgroup, a dose of 200 mg of mouse anti-CD25 antibody was also injected intraperitoneally at days 0, 3, and 6 after virus administration. Viral luciferase expressionfrom within the tumor was quantified at indicated time points by bioluminescence imaging. Mean values of 7 to 8 animals þ SD are plotted. Bioluminescencesignals from one representative animal of each group at day 3 after administration are also shown (tumors are circled). B, mice (Balb/c with subcutaneousRenca tumors) were treated intravenously with 2 � 108 pfu of VV (n ¼ 10–12 per group). For combination groups, anti-CTLA4 or anti-CD25 antibodies wereinjected with 100 or 200 mg/mouse, respectively, at days 4, 7, and 10 after virus injection. PBS was injected intraperitoneally as a control. Tumor growth wasfollowedby calipermeasurements.MeansþSEare plotted. � ,P<0.05, comparedwith the PBSgroup; #,P<0.05, comparedwith theVVgroup; f,P<0.05, comparedwith the VVþ anti-CD25 day 4 group.






viral treatment. The importance of CD8þ T cells was supportedthrough depletion of IFNg , which also resulted in loss of thera-peutic advantage and enhanced viral tumor-specific replication(Supplementary Fig. S5). NK cells appear to be required for theantitumor effect, but do not limit viral replication.

DiscussionIt is evident that the blockade of immune checkpoint alone is

rarely curative, but has the capacity to synergize with othertherapies that selectively activate the immune response. As such,the realization that the immune response raised by oncolytic viraltherapies is a critical mechanism mediating their therapeuticactivitymeans that the combination of these twoplatformswouldbe logical and appealing (30). However, despite the fact thatclinical trials have been proposed combining the oncolyticHSV T-Vec with ipilimumab, little preclinical data have been reported onsuch combinations (31).

Here, we examine approaches to combine oncolytic vacciniaviruses with different monoclonal antibodies that target cancer-mediated immunosuppression. Significantly improved antitu-mor responses were demonstrated in several mouse tumor mod-els, providing strong support for the clinical translation of thisapproach.However, it was initially seen that careful considerationwas needed to identify the correct combination of antibody, viralstrain and especially in the timing of application of the differenttreatments. Incorrect combination resulted in a loss of benefit andpotentially antagonistic effects.

In initial studies combining anti-CTLA4 blocking antibodywith vaccinia virus, no therapeutic benefit was seen (Fig. 1D). Inthese studies the antibody treatment was begun at the same timeas viral inoculation, and imaging of viral luciferase transgeneexpression demonstrated that viral gene expression was reducedby more than 40-fold relative to virus used alone (Fig. 1A). Thisindicated that a robust antiviral immune response was beingraised leading to premature clearance of the virus. Indeed, this

Figure 4.Optimized combination therapyresults in synergistic anticanceractivity. A, Renca (left) orMC38 (right)tumors were implanted into Balb/c orC57/Bl6 mice, respectively. Mice wereinjected with PBS or 2 � 108 pfu ofB18R- oncolytic vaccinia virus (VV)through the tail vein. For theanti-CTLA4 group, 100 mg ofanti-CTLA4 antibody was injectedintraperitoneally at days 0, 3, and 6.For the combination group, anti-CTLA4 antibody doses wereadministrated at days 4, 7, and 10 aftervirus injection. Tumor volumes weremeasured, and relative tumor volumeþ SE of the 12–15 mice/group isplotted. B, combination therapyincreases cytotoxic T cells recognizingtumor antigens. Cellular immuneresponses to tumor cells wasevaluated by the IFNg ELISpot assay.At day 11 after virus administration,spleens were harvested from Balb/cmice bearing Renca tumors andtreated as in A. Splenocytes wereevaluated for CTLs recognizing Rencacells. Values of individual mice andmeans�SEMare depicted. � ,P <0.05,compared with the PBS group; f,P<0.05, comparedwith theVVgroup;#, P < 0.05, compared with theanti-CTLA4 group.

Figure 3.Therapeutic activity of oncolytic vaccinia in combination with anti-CTLA4antibody is viral strain dependent. A total of 2� 108 pfu of oncolytic vacciniavirus (B18R- or vvDD) were administrated intravenously to Balb/c micebearing subcutaneous Renca tumors. At days 4, 7, and 10 after virus injection,a dose of 100 mg of anti-CTLA4 antibody was injected intraperitoneally.B18R- displayed greater inhibition of tumor growth relative to vvDD. Relativetumor volume after virus administration is plotted (n ¼ 12–15 mice/group þSE). � , P < 0.05, compared with the PBS group; f P < 0.05, compared withthe vvDDþanti-CTLA4 day 4 group.

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combination was also shown to result in a significant increase inthe level of antiviral CTL (Fig. 1B) and viral replication wasrestored after depletion of either CD8þ T cells or IFNg (Fig. 6Aand Supplementary Fig. S4A). This is potentially important asseveral groups are looking to express antibodies blocking immunecheckpoints directly from oncolytic vectors (32). We have previ-ously used exogenous regulation of cytokine transgene functionto down regulate cytokine function for a period of around 4 daysafter initial treatment (22, 33). This allowed an initial phase ofviral oncolytic activity and unhindered replication within thetumor, prior to a secondary phase of immunotherapeutic activitythat could be enhanced through subsequent stabilization of thecytokine function.Using a similar tactical approach, itwas felt thataddition of anti-CTLA4 antibody at later times after viral therapycould result in improved therapeutic activity.

Thiswasindeedconfirmed(Fig.1D),withinitiationofanti-CTLA4therapy 4 days after viral delivery found to result in significantlyimproved antitumor effects in mouse syngeneic tumor models.

Several different monoclonal antibody therapies target cancer-mediated immunosuppression, including both blockade ofimmune checkpoints as well as direct depletion of suppressiveimmune cell types that are known to accumulatewithin the tumormicroenvironment (34). One example of the latter approach usesanti-CD25 antibody to deplete Tregs within the tumor. It wasdetermined, however, that independent of whether anti-CD25was added prior to, at the same time as, or after viral therapy therewas no therapeutic advantage seen with anti-CD25 antibodycombinedwithoncolytic vaccinia (relative to vaccinia used alone)(Fig. 2B and Supplementary Fig. S2). This somewhat surprisingresult may be due to the fact that oncolytic vaccinia alone wasactually found to be an effective means to reduce the levels ofTregs in the tumor microenvironment (Supplementary Fig. S3A).This also highlights the fact that the important contribution ofthe anti-CTLA4 antibody to the synergistic combination withoncolytic vaccinia appears to be dependent on its activation ofCD8þ T cells rather than on depletion of Tregs.

Figure 5.Altered T-cell repertoire in the tumorafter vaccinia/anti-CTLA4 combinationtherapy. Balb/c mice with subcutaneousRenca tumors were treated as before(Fig. 4), and tumors were harvested atday 11 after virus injection and evaluatedfor lymphocyte populations by flowcytometry. Numbers of CD3þCD4þ (A)and CD3þCD8þ (B) cells per 200,000total cells are plotted. C, representativedistributions of CD4þ and CD8þ

populations within CD3þ populationwithin the tumor. D, percentage of Tregs(CD25þFoxp3þ) within the CD3þCD4þ

population of the tumor. Values forindividual tumors and means þ SEM areplotted. E, numbers of NK cells(NKp46þNKg2DþCD3�) and NK-T cells(NKp46þNKg2DþCD3þ) per 200,000events within the tumor. � , P < 0.05,compared with the PBS group;#, P < 0.05, compared with the anti-CTLA4 group; f, P < 0.05, comparedwith the B18R- group.






We have also previously demonstrated that some viral muta-tions result in production of viral vectors with enhanced immu-noactivation properties (29). For example, although vacciniastrains carrying the thymidine kinase deletion typically used tomediate tumor selectivity in oncolytic vaccinia vectors such as JX-594 (Pexa-Vec, Jennerex, now part of Sillajen; ref. 10), vvDD (28)or GLV-1h68 (GL-ONC1, GeneLux; ref. 35) did result in immuneactivation, this could be enhanced if the viral B18R gene (asecreted type I IFN-binding protein; ref. 36) was also deleted.When the oncolytic vaccinia strain vvDD was compared head tohead with a comparable strain carrying the B18R gene deletion(WR.TK-.B18R-) in combinations with anti-CTLA4 antibody, itwas found that the "immune activation enhanced" virus (B18R-)was significantly more potent (Fig. 3). Therefore, viral strain andbackbone are also important considerations when designingcombination therapies with blockade of immune checkpoints.

Theoptimal combinationofmonoclonalantibody(anti-CTLA4),viral strain (B18R-) and treatment regimen (antibody therapybegun4 days after viral delivery) resulted in significantly enhanced ther-apeutic responses in different mouse cancer models (Fig. 4A),including renal cancer and colorectal cancer models in differentmouse genetic backgrounds (BALB/corC57/BL6). The combinationwas most effective against the Renca tumor model and less effectiveagainst MC38 (but still significantly better than either therapy usedalone). It is possible the MC38 model may be less immunogenic,and soenhanced inductionof antitumorCTLmaynotbepossibleormay remain ineffective at enhancing therapeutic responses. Theenhanced therapeutic activity of this combination is primarilyimmune mediated as (i) the combination resulted in significantlygreater numbers of CTLs in the spleen that target tumor antigens(relative to either therapy used alone; Fig. 4B); (ii) the combinationalso resulted in significant increases in the number of CD3þCD4þ Tcells in the tumor relative to either therapy alone (coupled to adecrease in the relative amounts of Tregs), and an increase in CD3þ

CD8þT cells (although thiswasonly significant relative to control oranti-CTLA4 used alone; Fig. 5); and (iii) depletion of either CD8þ Tcells, IFNg or NK cells (but not CD4þ T cells) resulted in loss of thetherapeutic advantage seen with the combination (Fig. 6 andSupplementary Fig. S4). It therefore appears that immune enhancedoncolytic vaccinia strains (such as WR.TK-.B18R-) can activate anadaptive immune response targeting tumor-associated antigens that

is dependent on CD8þ T cells and requires NK cell involvementduring early immune activation (Supplementary Fig. S3B; althoughNKcells return to close tobaseline levels in the tumorbyday 10 afterviral treatment). Under single viral therapy treatment, this CD8þ T-cell immune response is blunted by premature shut down of T-cellproliferation, an effect that can be overcome by adding anti-CTLA4antibody. If the antibody is added too early, then the virus cannotinduce the correct immune response before its immunomediatedremoval and the benefits are lost.

Together, these data demonstrate how the correct combinationof oncolytic virus and anti-CTLA4 antibody results in robustinduction of antitumor CTL coupled to targeting of localizedimmune suppression within the tumor. This combination there-fore more efficiently activates the immune response to target thetumor as well as blocking the capacity of the local tumor micro-environment to suppress the resultant immune response, leadingto significantly improved therapeutic effects. The clinical exam-ination of these combinations is therefore an exciting prospect.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: J.J. Rojas, S.H. ThorneDevelopment of methodology: J.J. Rojas, S.H. ThorneAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J.J. Rojas, P. Sampath, W. Hou, S.H. ThorneAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J.J. Rojas, W. Hou, S.H. ThorneWriting, review, and/or revision of the manuscript: J.J. Rojas, S.H. ThorneAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): W. Hou

Grant SupportThis work was supported by R01CA140215 and R01CA178766, while core

facilities funded under the CCSG (P30CA047904) were used, including in vivoimaging, small animal, and flow cytometry facilities.

The costs of publication of this articlewere defrayed inpart 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 August 11, 2014; revised July 2, 2015; accepted July 8, 2015;published OnlineFirst July 17, 2015.

Figure 6.Depletion of different immune cell subsets alters vaccinia virus replication and anti-tumor activity of combination therapy. A, replication of vaccinia virus (VVstrain B18R-) is increased by depletion of CD4þ, CD8þ, and NK cells. Balb/C mice injected with CD4�, CD8�, and NK-depleting antibodies were challenged withRenca tumors and treated as before (Fig. 4). Viral luciferase expression from within the tumor was quantified at indicated time points by bioluminescenceimaging. Mean values of 12 to 13 animals þ SD are depicted. B, CD8þ and NK cells are essential for the antitumor activity of vaccinia virus/anti-CTLA4 combinationtherapy. Mice were treated as in A, and tumor growth was monitored (þSE, 12–15 mice/group; � , P < 0.05, compared with the PBS group; #, P < 0.05compared with the no-depletion group).


Rojas et al.




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2015;21:5543-5551. Published OnlineFirst July 17, 2015.Clin Cancer Res Juan J. Rojas, Padma Sampath, Weizhou Hou, et al. and Oncolytic VirotherapyDefining Effective Combinations of Immune Checkpoint Blockade

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