repurposing sunitinib with oncolytic reovirus as a novel ......cancer therapy: preclinical...

Cancer Therapy: Preclinical

Repurposing Sunitinib with Oncolytic Reovirus asa Novel Immunotherapeutic Strategy for RenalCell CarcinomaKeith A. Lawson1,2, Ahmed A. Mostafa1,2, Zhong Qiao Shi1,2, Jason Spurrell1,2,WenqianChen1,2, JunKawakami3,KathyGratton1,2, Satbir Thakur1,2, andDonaldG.Morris1,2

Abstract

Purpose: In addition to their direct cytopathic effects, oncolyticviruses are capable of priming antitumor immune responses.However, strategies to enhance the immunotherapeutic potentialof these agents are lacking. Here, we investigated the ability of themulti-tyrosine kinase inhibitor and first-line metastatic renal cellcarcinoma (RCC) agent, sunitinib, to augment the antitumorimmune response generated by oncolytic reovirus.

Experimental Design: In vitro, oncolysis and chemokine pro-duction were assessed in a panel of human and murine RCC celllines after exposure to reovirus, sunitinib, or their combination.In vivo, theRENCAsyngeneicmurinemodel of RCCwas employedto determine therapeutic and tumor-specific immune responsesafter treatment with reovirus (intratumoral), sunitinib, or theircombination. Parallel investigations employing the KLN205 syn-geneic murine model of lung squamous cell carcinoma (NSCLC)were conducted for further validation.

Results: Reovirus-mediated oncolysis and chemokine produc-tion was observed following RCC infection. Reovirus monother-apy reduced tumor burden and was capable of generating asystemic adaptive antitumor immune response evidenced byincreased numbers of tumor-specific CD8þ IFNg-producing cells.Coadministration of sunitinib with reovirus further reducedtumor burden resulting in improved survival, decreased accumu-lation of immune suppressor cells, and the establishment ofprotective immunity upon tumor rechallenge. Similar resultswereobserved for KLN205 tumor–bearing mice, highlighting thepotential broad applicability of this approach.

Conclusions: The ability to repurpose sunitinib for augmen-tation of reovirus' immunotherapeutic efficacy positions thisnovel combination therapy as an attractive strategy ready forclinical testing against a range of histologies, including RCC andNSCLC. Clin Cancer Res; 22(23); 5839–50. �2016 AACR.

IntroductionDespite the improvements in progression-free (PFS) and over-

all survival (OS) associated with the use of targeted therapy,metastatic renal cell carcinoma (mRCC) remains an incurabledisease. With a five-year survival rate of less than 10%, thismalignancy remains a significant health issue (1). As such, theneed for the development of novel therapeutic strategies for thisdisease is obvious. Sunitinib, a multi-tyrosine kinase inhibitortargeting VEGFR, PDGFR, C-KIT, RET, CSF-1R, and FLT-3 iscurrently a first-line therapy for mRCC (2). While this drug hashistorically been associated with potent antiangiogenic activity,

recent clinical studies have highlighted its immune-modulatoryeffects. Indeed, after two cycles of oral sunitinib therapy at 50 mgdaily for 4weeks every 6weeks,mRCCpatients display an increasein the percentage of IFNg-producing T cells relative to treatment-na€�ve patients (3). Moreover, the immune suppressive type-2T-cell cytokine response and accumulation of T regulatory cells(Treg) and myeloid-derived suppressor cells (MDSC), which arecharacteristic of mRCC patients, are reversed after sunitinib ther-apy (3, 4). Accordingly, this evidence has generated significantinterest in targeting Tregs andMDSC with sunitinib to reverse themRCC-induced immunosuppressive microenvironment andenhance the antitumor immune response generated by immu-notherapeutics (5). The feasibility of this approach has beendemonstrated in an immunocompetent syngeneic murine modelof RCC (RENCA) inwhich the downregulation ofMDSC and Tregby sunitinib enhanced intratumoral infiltration and activation ofadoptively transferred CD8þ T cells, leading to tumor regression(6). Furthermore, sunitinib has also been demonstrated to reducetumor burden and improve OS in mouse models of melanoma,hepatocellular carcinoma, and colorectal metastasis in immuno-therapeutic models, highlighting the efficacy and widespreadutility of this approach against multiple histologies (7–9).

In the current study, we investigated the ability of sunitinib toenhance the antitumor immune response generated by thedsRNA oncolytic reovirus. To date, reovirus is one of the mostclinically advanced oncolytic viruses (OV), demonstrating mod-est efficacy in phase II clinical trials as a monotherapy and incombination with platinum- and taxane-based chemotherapy

1Department of Oncology, University of Calgary, Calgary, Alberta, Canada. 2TomBaker Cancer Centre, Calgary, Alberta, Canada. 3Southern Alberta Institute ofUrology, University of Calgary, Calgary, Alberta, Canada.

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

K.A. Lawson and A.A. Mostafa contributed equally to this article.

Current address for Keith Lawson: Division of Urology, University of Toronto,Toronto, Ontario, Canada.

Corresponding Author: Donald G. Morris, Tom Baker Cancer Centre, 1331, 29Street NW, Calgary, Alberta T2N 4N2, Canada. Phone: 403-521-3347; Fax: 403-283-1651; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-16-0143

�2016 American Association for Cancer Research.

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across multiple solid malignancies (10–11). Of the over 1,500patients treated thus far with this agent, the maximum tolerateddose has not been reached, highlighting its tolerability andsafety. As such, reovirus is one of the most promising OVcurrently being investigated in the clinic. Despite this, however,early-phase monotherapy clinical trials have failed to demon-strate appreciable clinical effectiveness in the form of objectiveand durable complete responses, highlighting the need toaugment reovirus' therapeutic potency. Interestingly, in exper-imental murine models, reovirus administration is capable ofpriming innate and adaptive antitumor immune responses (12–14). This antitumor immunity has not only been shown tocontribute to reovirus' therapeutic efficacy, but has also beendemonstrated to result in the generation of long-term tumorimmunosurveillance (15–17). Moreover, reovirus-mediatedantitumor immunity has been demonstrated clinically in aphase I trial of a single intraprostatic injection of reovirus thatinvolved 6 patients with localized prostate cancer (18). In thisstudy, despite the robust antiviral neutralizing antibodyresponse that was seen, a significant number of tumor-infiltrat-ing CD8þ T cells were present following reovirus injection.Likewise, in a phase I clinical trial of intravenous reovirusadministration involving patients who had previously receivedcytotoxic chemotherapy and radiotherapy, increased CD8þ T-and NK-cell (CD3�/CD56þ) numbers were observed in theperipheral blood (19). Hence, in addition to its direct oncolyticeffects, reovirus is also a potent immunotherapeutic. While aconsiderable amount of literature supports reovirus' immuno-therapeutic potential, most preclinical investigations haveinstead focused on improving the systemic delivery of the viruswith immunosuppression or histology relevant cytotoxic che-motherapy, respectively (20, 21). As such, strategies to augmentreovirus-mediated antitumor immune responses for generationof long-term protective immunity capable of improving clinicalobjective responses are warranted.

Herein, the ability to augment reovirus's immunotherapeuticefficacy through combination with sunitinib was explored utili-zing clinically relevant syngeneic murine models. Collectively,this work highlights the promise of combining reovirus withsunitinib as a novel viroimmunotherapeutic approach, support-ing the investigation of this strategy in phase II clinical trials.

MethodsCell lines and virus

ACHN, A498, 786-0, L-929, KLN205, and RENCA cell lineswere obtained from the (ATCC) in July 2010. Cell lines wereauthenticated by the ATCC by short tandem repeat analysis priorto purchase. No further authentication was done by the authors.For all experiments, early passage cells were utilized. ACHN,A498, andKLN205were cultured inminimum essentialmedium;786-0 and RENCAwere cultured in RPMI; and L-929was culturedin DMEM (Invitrogen). All media were supplemented with 10%heat-inactivated FCS (Invitrogen). Cultures were free of antibio-tics and negative for mycoplasma as determined by routinetesting. Reovirus, Dearing strain serotype 3 was grown in L-929cells then purified and titered as described previously (18).Sunitinib was purchased from Selleck Chemicals.

Cell viability assayCells were seeded at a density of 3 � 103 (A498, 786-0) or 8 �

103 (ACHN, RENCA) cells/well into 96-well microtiter plates andincubated for 24 hours in 10% FCS-containing media. Reovirus,sunitinib, or their combinationwas then added to eachwell for 48hours. Subsequently, drug-containing media were replaced withmedia containing WST-1 (diluted 10:1) and absorbance wasquantified utilizing a BioTek plate reader. Percent viability wascalculated as the absorbance ratio of treated/untreated cells mul-tiplied by 100. Additionally, photomicrographs of reovirus cyto-pathic effects at 48 hours after infectionwere capturedwith a ZeissAxiovert 200M microscope at 10� zoom.

Viral progeny assayACHN, 786-0, A498, and RENCA cells were treated with

reovirus in 12-well microtiter plates in their respective media forup to 72 hours. The cultures were then freeze-thawed 3 times andto quantify progeny production, supernatants were harvested andplaque titrated on monolayers of L-929 cells in semi-solid medi-um for 72 hours as described previously (18).

Chemokine expression assaySupernatants were harvested from RENCA cells infected

with live (RV) or UV-inactivated reovirus (DV) for 12 or 24hours in 6-well plates. The multiplexing analysis was per-formed using the Luminex 100 system (Luminex) by EveTechnologies Corp. Thirty-two markers were simultaneouslymeasured in the serum using a MILLIPLEX Mouse Cytokine/Chemokine 32-plex kit (Millipore) according to the manufac-turer's protocol. Assay sensitivities for these analytes rangedfrom 0.2 to 63.6 pg/mL.

In vitro synergy assayDose–response curves for all cell lines to reovirus and sunitinib

were generated. Calcusyn software (Biosoft) was utilized to gen-erate effective dose for 50 percent cytotoxicity (ED50) values foreach cell line to reovirus and sunitinib from the dose responsedata. Cell lines were then treated with escalating doses of reovirusand sunitinib concurrently at a fixed ratio of ED50:ED50. Cellviability was quantified as per the cell viability assay and com-bination index (CI) values were generated using CalcuSyn soft-ware with a CI value < 1 denoting an synergistic response, > 1denoting an antagonistic response, and 1 denoting an additiveresponse.

Translational Relevance

In this study, we demonstrate the ability to repurposesunitinib, a multi-tyrosine kinase inhibitor and first-line met-astatic renal cell carcinoma (RCC) agent, to potentiate theimmunotherapeutic efficacy of the oncolytic virus, reovirus.This novel therapeutic strategy not only improved tumorresponses and overall survival in a murine model of RCC, butalso, resulted in the generation of a systemic protective anti-tumor immune response. We validated our findings in amurine model of lung squamous cell carcinoma (SCC),highlighting the potential broad applicability of this treatmentapproach. Taken together, these results provide clear rationaleto investigate this promising viroimmunotherapeutic strategyin early-phase clinical trials for a broad range of tumorhistologies.

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Clin Cancer Res; 22(23) December 1, 2016 Clinical Cancer Research5840




In vivo studies in immunocompetent syngeneic murine mousemodels

All mice in these studies were housed in pathogen-free condi-tionswith food andwater ad libitum and treatedwithin proceduralguidelines that were approved by the University of Calgary Ani-mal Care Committee. A total of 2.5 � 106 RENCA or 5 � 105

KLN205 cells were implanted into the right hind-flank of Balb/CorDBA/2mice, respectively (Charles River Laboratories) onday0.Once tumors were palpable (approximately day 5), therapywas initiated. Mice were grouped into cohorts and treated withintraperitoneal (i.p) PBS, intratumoral UV-irradiated reovirus(5�108PFU), intratumoral live reovirus (5�108PFU), sunitinib(40 mg/kg, i.p.), or a combination of these agents as indicated inthe figure legends. Biweekly caliper measurements were taken tomonitor tumor burden. Tumor volume was calculated using theformula, volume ¼ 0.52 � (width)2 � length.

For the adoptive transfer studies, spleens were extracted frommice treated as above 19 days after RENCA tumor implantation (3mice/group). After pooled single-cell suspensions were generatedby passing mechanically separated spleens through 100-mm cellstrainers (Becton Dickinson), mononuclear splenocytes wereisolated by centrifugation over a Ficoll–Hypaque gradient (GEHealthcare). A total of 1 � 107 splenocytes in 100-mL PBS werethen administered intravenously via tail-vein into each of 6recipient Balb/C mice for each group. Seven days later, all recip-ient mice were challenged with an implantation of 2.5 � 106

RENCA cells into the right hindflank and thenmeasured biweeklyfor tumor burden.

For immune depletion studies, CD8þ or CD4þ lymphocytedepletion was accomplished by intraperitoneal injection of 0.25mg depleting anti-CD8a mAb (BioXcell; CD8 Clone: 2.43; CD4Clone GK1.5) on day 5, followed by 0.1 mg on day 8, 12, and 19.

For tumor rechallenge studies, Balb/C RENCA tumor–bearingmice successfully treated with reovirus-sunitinib combinationtherapy were rechallenged with 1 � 106 RENCA cells in thecontralateral hind flank and tumor burdenwas assessed by calipermeasurement. Treatment-na€�ve Balb/C mice were challenged inthe same manner to serve as a control.

CD8þ enrichmentSpleens were processed as described above from Balb/C or

DBA/2mice bearing RENCA or KLN205 tumors, respectively, andenriched for CD8þ T cells using an EasySep Mouse CD8þ Selec-tion Kit via an EasySep magnet as per manufacturer's protocol(Stem Cell Technologies).

Flow cytometryFlow cytometry was performed as described previously (22).

Briefly, cell staining was done using FITC, Alexa Flour 488, PE,APC, PE-Cy7, or PerCP Cy5.5 conjugated rat mAbs against CD4,CD8, Foxp3, CCR7, CD62L, CD11b, and GR1 (Lyc6Gþ/Lyc6C;BD Biosciences). APC-conjugated hamster IgG against CD3e(eBioscience). Appropriate mouse IgG isotypes were used ascontrols (BD Biosciences). Intracellular Foxp3 staining was doneusing Mouse Foxp3 Buffer Set (BD Biosciences) according to themanufacturer's protocol. Intratumoral lymphocytes were stainedand analyzed by flow cytometry following collagenase type I(Gibco, cat #17100-017) treatment according to the manufac-turer's protocol. Briefly, tumor tissue was washed with PBS, cutinto a small piece (>1 � 1 mm), and incubated with collagenasetype I (100U/mL) for 4 hours at 37�C. Tumor fragments were then

disaggregated through 100-mm cell strainers, and single cells werecollected and enumerated. All Samples were run using BD AccuriFlow Cytometry (BD Biosciences) and analyzed by FlowJo soft-ware (FlowJo Enterprise).

ELISAA total of 1 � 106 CD8þ splenocytes were isolated as above

and coincubated in 1mL of complete RPMI in 24-well plates with1 � 105 RENCA cells either untreated or infected with 1 MOI(multiplicity of infection) of reovirus for the previous 12 hours.At 48hours, supernatantswere harvested for IFNg ELISA as per themanufacturer's protocol (R&D Systems) at room temperature.Briefly, capture antibody at 8 mg/mL; biotinylated detectionantibody at 0.8 mg/mL; and avidin peroxidise at 1.5 mg/mLwere utilized with ABTS substrate. The sensitivity of the assaywas 31.25 pg/mL.

ELISPOT assayCD8þ splenocytes from RENCA tumor–bearing Balb/C mice

were isolated as above from pooled cell suspension and 2 � 105

cells were subsequently cocultured with RENCA cells (typically1 � 104 cells) for 48 hours in 96-well nitrocellulose membraneplates precoated with anti-mouse IFNg mAb (BD Biosciences, cat# 552569).Detection antibody solution at 100mL/well was addedfor 2 hours followed by Streptavidin-HRP solution at 100 mL/wellfor a 1-hour incubation period. AEC substratewas then added andspots were scanned and counted using the CTL-ImmunoSpotS6 Macro Analyzer and BioSpot Software, respectively (C.T.L).The frequency of RENCA-specific CD8þ splenocytes was calcu-lated on the basis of the percentage of CD8þ T cells present in theresponding population.

Lactate dehydrogenase cytotoxicity assayCD8þ splenocytes from KLN205 tumor–bearing DBA/2 mice

were isolated as above from pooled cell suspension and subse-quently cocultured with 1 � 104 KLN205 cells in a 96-well platefor 24 hours. Supernatant was collected and cytotoxicity wasmeasured by CytoTox Non-Radioactive Cytotoxicity Assay kitaccording to themanufacturer's protocol (Promega). Cytotoxicitywas calculated using the formula: effector (experimental) – effec-tor (spontaneous) – target (spontaneous)/target (maximum) –

target (spontaneous) � 100.

Statistical analysisStatistical analysiswaspreformedutilizingGraphPadversion6.

Unpaired two-tailed t tests and two-way ANOVA followed byBonferroni post hoc tests were used to determine significancebetween experimental groups. Kaplan–Meier analysis togetherwith log-rank sum test was utilized to determine significancefor in vivo survival benefits. Statistical significance was defined asP values being < 0.05 unless otherwise stated.

ResultsReovirus replicates in human and murine RCC cell linesresulting in oncolysis, chemokine production, and synergisticcytotoxicity when combined with sunitinib

Reovirus is a nonenveloped dsRNAvirus that has demonstratedoncolytic activity against a wide variety of malignancies in bothpreclinical and clinical studies. As this virus has not been inves-tigated to date against RCC, the first objective was to determine its

Viroimmunotherapy for Renal Cell Carcinoma

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Lawson et al.





oncolytic activity in a panel of humanRCC cell lineswith differingvon Hippel Lindau (VHL) gene status (786-O VHL�/�, A498VHL�/�, ACHN VHLþ/þ). Treatment of 786-O, ACHN, andA498 cell lines with reovirus resulted in a dose-dependentdecrease in cell viability (Fig. 1A). Light microscopy of all celllines exposed to virus confirmed cytopathic effect, highlighted byplasmamembrane blebbing, cell surface detachment, and round-ing up of the RCC cell lines (Fig. 1B). A rise in viral titer was alsoobserved after reovirus treatment, confirming a productive lyticinfection of the RCC cells (Fig. 1C).

Reovirus has been demonstrated to initiate innate immuneresponses characterized by the production of proinflammatorychemokines including RANTES, MIP-1-a, MCP-1, KC, IP-10, andMIG across a variety ofmelanoma and prostate cancer cell lines inaddition to its direct oncolytic effects (14, 17). On the basis ofthesefindings, we characterized the ability of reovirus to stimulateproduction of these chemokines during infection of the murineRCC RENCA cell line. This cell line was studied to provide proof-of-principle of chemokine generation during reovirus oncolysis ofRCC in vitro to support subsequent in vivo experiments utilizingthis cell line. Similar to the human RCC cell lines, infection ofRENCA cells with reovirus resulted in viral replication and cyto-toxicity (Fig. 1A–C). Furthermore, after 24 hours of reovirusinfection at a concentration of both 0.007 MOI (ED50) and 1MOI increased chemokine expression was observed, highlightingthe potential of utilizing this agent to generate an inflammatoryoncolytic response against RCC (Fig. 1D). Importantly, chemo-kine production was observed in viable RENCA cells 12 hoursafter infection suggesting chemokine production was a product ofviral replication rather than cell lysis (Supplementary Fig. S1).

To assess in vitro synergy between reovirus and sunitinib, CIvalues as per the Chou and Talalaymethodwere determined (23).Dose response data for reovirus and sunitinib on RCC cells wereused to calculate ED50 values (Fig. 1E and Supplementary Fig.S2A), with median effect plots demonstrating acceptable linearcorrelation coefficients (r>0.9). Treatment of cellswith increasingdoses of reovirus and sunitinib at fixed ratios (ED50:ED50)revealed a synergistic or additive cytotoxic response (CI � 1) forboth the ACHN and A498 cell lines across all doses studied(Fig. 1F). Reovirus–sunitinib synergywas also observed inRENCAand 786-O cells; however, the magnitudes of effect were highlydependent on concentration (Fig. 1F).

Reovirus demonstrates in vivo therapeutic efficacy as amonotherapy and in combination with sunitinib

The RENCA murine model of RCC is an established immuno-competent syngeneic model for studying novel immunothe-rapeutics against this disease preclinically (24). This model wasemployed to investigate whether or not reovirus has therapeuticefficacy against RCC in vivo and determine the ability of sunitinibto augment this activity. Sunitinib dose–response studies were

conducted in vivo to determine the optimal dose to administer incombination with reovirus. A dose range of 20 to 60 mg/kg waschosen as it is clinically relevant and has previously been utilizedin the RENCA model (6). A dose of 40 mg/kg of sunitinibsignificantly decreased tumor burden while not eradicatingtumors (Supplementary Fig. S2B) and as such was chosen forsubsequent experiments. Reovirus was administered at a dose of 5� 108 plaque forming units (PFU) based on previous reports inimmunocompetent murine models (13).

In combination experiments, sunitinib was administered priorto reovirus and on a continuous basis as this approach has beendemonstrated to improve immunotherapeutic efficacy by allow-ing sunitinib to precondition the tumor microenvironmentthrough downregulation of immune suppressor cells, includingMDSCs (25). RENCA tumor–bearing balb/C mice were treatedwithmonotherapy sunitinib (i.p), reovirus (i.t), or a combinationof these agents.UV-irradiatednonreplicating reovirus (DV; i.t)wasadministered as a control. Sunitinib treatment was initiated oncepalpable tumors were present (approximately day 5) and contin-ued for 14 days while reovirus was administered on day 8, 11, and14. Although, relative to DV and PBS controls, reovirus given as amonotherapy significantly reduced tumor burden, the use of thisagent in combination with sunitinib resulted in a significantlyincreased reduction in tumor volume (P < 0.001 by two-wayANOVA; Fig. 2A). Tumor burden was not plotted beyond 18 daysas considerable central necrosis in large tumors occurred limitingaccurate measurement. Instead, overall survival was followed toestablish long-term therapeutic efficacy. Kaplan–Meier analysis oftwo independent experimentswith pooled results (8mice overall)revealedanoverall survival benefit (P<0.05, log-rank sumtest) forthosemice receiving reovirus in combinationwith sunitinib versuseither agent used as a monotherapy (Fig. 2B). Death in all caseswas a result of tumor progression as determined by tumor sizegreater than 2 centimeters in a single dimension or greater than20% loss of body weight as per Animal Care institutional guide-lines. Notably, animal body weights remained reasonably stableduring treatment and no therapy-specific toxicities were observed(Supplementary Fig. S3).

Sunitinib augments reovirus-mediated antitumor immuneresponse through reversal of tumor-inducedimmunosuppression

As reovirus infection results in the production of proinflam-matory chemokines and consequent priming of innate and adap-tive immunity (14–17), the antitumor immune response gener-ated by reovirus treatment of RCC in vivo was determined. Micewere treated as in Fig. 2A with sunitinib, reovirus, or combinationtherapy and CD8þ splenocytes were harvested and coculturedwith RENCA cells or reovirus -infected RENCA cells for antigenicstimulation prior to IFNg ELISA. In mice receiving reovirusmonotherapy, a significant IFNg responsewasdemonstrated from

Figure 1.Reovirus has direct oncolytic effects against human and murine RCC. A, Cell viability of human (786-O, A498, ACHN) and murine (RENCA) RCC cell linestreated with escalating doses of reovirus (RV) determined by WST-1 assay. B, Pictures of RCC cells infected with 40 (786-O, A498, ACHN) or 0.007 (RENCA) MOIof UV-irradiated virus (DV) or RV for 48 hours, taken with a Zeiss Axiovert 200M microscope at 10� zoom. C, RV titer over 72 hours after infection of RCCcell lines (40 MOI) determined by plaque titration assay. D, Chemokine levels in supernatants from RENCA cells infected with RV at ED50 and 1 MOI for 24 hoursdetermined by luminex analysis. E, Sunitinib and reovirus ED50 doses after RCC cell line treatment for 48 hours calculated by CalcuSyn software. F, CI values(�SEM) forRCCcell lines treated for 48hourswith sunitinib and reovirus at afixed ratio of ED50:ED50 calculated byCalcuSyn software. Synergistic values in bold. In allpanels error bars ¼ SEM for at least three independent experiments. P < 0.001 (��) by two-tailed Student t test.






harvested CD8þ splenocytes after stimulation with reovirus-infected RENCA cells (Fig. 3A). Interestingly, sunitinib therapysignificantly augmented this reovirus-generated antitumorimmunity, as a robust increase in IFNg production (fourfold)in those mice that received reovirus in combination with suni-tinib was observed (Fig. 3A). In mice receiving PBS, DV, orsunitinib,minimal or no IFNg response was seen. Notably, whileCD8þ-reactive splenocytes were not observed in reovirus orcombination therapy–treated mice by ELISA when RENCA cellswere used alone as an antigenic stimulus, their presence wasconfirmed by the higher sensitivity ELISPOT assay (Fig. 3B–C).To further establish an immunotherapeutic mechanism of reo-virus and combination therapy, CD8 depletion studies wereconducted (>98% depletion confirmed; SupplementaryFig. S4) confirming OS benefit observed with reovirus or com-bination therapy was CD8 mediated (Fig. 3D). Interestingly, weobserved that CD4 depletion (>98% depletion confirmed; Sup-plementary Fig. S4) as a monotherapy resulted in a therapeuticbenefit, consistent with recent reports, highlighting the ability to

enhance tumor immunogenicity with this approach throughTreg depletion (Fig. 3E; ref. 26). However, as expected, CD4depletion did not abrogate the therapeutic effects of reovirus orcombination therapy. Taken together, these results suggest thatreovirus generates a systemic adaptive antitumor immuneresponse against RCC that is augmented by sunitinib.

As sunitinib is known to reverse tumor-induced immunosup-pression (4), we next immunophenotyped splenocytes andtumor-infiltrating immune populations in mice receiving reovi-rus, sunitinib, or combination therapy. Consistent with a viroim-munotherapeutic effect, reovirus monotherapy resulted in anincreased accumulation of CD8þ splenocytes, as well as, CD8þ

tumor-infiltrating lymphocytes (Fig. 4A–C). This was associatedwith a concomitant accumulation of splenic and intratumoralMDSCs as well as intratumoral Tregs (Fig. 4D–F). As hypothe-sized, sunitinib reversed this reovirus-induced immunosuppres-sive effect preventing the accumulation of splenic MDSCs andintratumoral MDSCs and Tregs (Fig. 4D–F), highlighting this as apotential mechanism explaining sunitinib's ability to augmentreovirus-mediated adaptive immune responses (Fig. 3A–C).

To determine whether reovirus or combination therapyresulted in the establishment of a protective immune response,adoptive transfer experiments were conducted. Here, splenocytesfrom mice treated with reovirus, sunitinib, or combination ther-apy were isolated and intravenously transferred into treatment-na€�ve mice (Fig. 5A). These mice were then challenged with asubcutaneous injection of RENCA cells and followed for tumorinitiation and burden. Interestingly, only those mice receivingsplenocytes from mice treated with combination therapy dem-onstrated reduced tumor growth rate relative to controls,highlighting an established protective immune response in thisgroup (P < 0.05 between all groups by two-way ANOVA; Fig. 5B).To further support these findings, tumor rechallenge experi-ments were conducted with mice that had achieved a completeresponse to combination therapy versus mice that were treat-ment na€�ve. Indeed, this demonstrated that pretreated curedanimals were protected against tumor rechallenge, whereasna€�ve mice formed rapidly growing tumors (Fig. 5C). Overall,these results confirmed establishment of protective immunityfollowing combination therapy.

Sunitinib augments reovirus immunotherapeutic effect in amurine model of lung SCC

To confirm a broader utility of reovirus–sunitinib viroimmu-notherapy as a novel therapeutic strategy, we conducted parallelinvestigations in a syngeneic murine lung squamous cell carci-nomamodel employing the KLN205 cell line. Consistent with theresults in the RENCA model, KLN205 cells demonstrated sensi-tivity to reovirus oncolysis in vitro and displayed a dose-variablesynergistic response when treated with combination therapy(Supplementary Fig. S5A–C). In vivo, combination therapy alsoresulted in decreased KLN205 tumor burden as well as improvedOS relative to monotherapy (Fig. 6A and B). Moreover, this effectwas associated with increased splenic CD8þ lymphocytes with amemory phenotype (CD8þ/CCR7þ/CD62L�; Fig. 6C and D) anda concomitant prevention of reovirus-induced MDSC accumula-tion (Fig. 6E). Furthermore, isolated CD8þ splenocytes frommicetreated with combination therapy demonstrated increased effec-tor function in coculture cytotoxicity assays with KLN205 cells,confirming enhanced adaptive immunity (Fig. 6F). Taken togeth-er, these results establish a viroimmunotherapeutic benefit for

Figure 2.

Reovirus combined with sunitinib results in decreased tumor burden andimproved overall survival in the RENCA murine model. Balb/C mice wereimplanted with RENCA (2.5 � 106 cells) subcutaneous tumors and treated withPBS, sunitinib (40 mg/kg i.p), UV-irradiated reovirus (DV; 5 � 108 pfu i.t),live reovirus (RV; 5 � 108 pfu i.t), or a combination of these agents.Sunitinib was given daily for 14 consecutive days starting on day 5 after RENCAimplantation. Reoviruswas administered three times (day8, 11, 14).A,Tumor sizewas followed with caliper measurements. N ¼ 6 mice per group. Errorbars¼ SEMof tumorswithin each group. �� , P <0.001;�� , P < 0.01; � , P < 0.05 bytwo-way ANOVA. Data representative of two independent experiments. B,Kaplan–Meier plot of mice treated as per A. N ¼ 8 mice per group. Analysisrepresents data pooled from two independent experiments involving 3 and5 mice, respectively. �� , P < 0.001 by log-rank between PBS and RV orSunitinib þ RV. � , P < 0.05 by log-rank between RV and Sunitinib þ RV.

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Figure 3.

Reovirus induced antitumor immune response is augmented by sunitinib. A, CD8þ cells were separated from pooled spleens of RENCA tumor-bearingmice within each treatment group from Fig. 2A and stimulated with reovirus-infected RENCA cells. IFNg production was quantified by IFNg ELISA. N ¼ 3 mice.B and C, CD8þ cells were separated from pooled spleens of RENCA tumor-bearing mice treated as per Fig. 2A and stimulated with RENCA cells.Percentage of RENCA-specific IFNgþ cells determined by ELISPOT assay. N ¼ 5 mice. Representative wells (B) and their quantitation (C) is shown. In bothpanels, error bars ¼ SEM of experimental triplicate. �� , P < 0.001, by two-tailed Student t test. D and E, Kaplan–Meier plot demonstrating OS for micepretreated with depleting anti-CD8a (D) or anti-CD4a (E) antibodies (i.p) followed by treatment as in Fig. 2A. ��, P < 0.01, by log-rank test betweensunitinib þ RV versus sunitinib þ RV þ CD8 Ab. N ¼ 5 mice. Data from panel D and E Represent a single experiment and are presented separately for clarity.






Figure 4.

Reovirus–sunitinib therapy increases tumor and splenic immune stimulatory cells while preventing accumulation of immune suppressor cells. Pooled splenocytes(A, B, D) and tumor single-cell suspensions (C, E, F) from RENCA tumor–bearing mice treated as per Fig. 2A were immunophenotyped by flow cytometry.N ¼ 5 mice/group. In all panels, error bars ¼ SEM of experimental triplicate. �� , P < 0.001, �� , P < 0.01, by two-tailed Student t test. Source of cells indicated inparentheses.

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reovirus–sunitinib combination therapy against murine lungSCC, highlighting the broad potential of this novel treatmentparadigm.

DiscussionMetastatic renal cell carcinoma remains an incurable disease

with an urgent need for novel therapeutics to be developed thatimpact overall survival. Immunotherapeutic strategies againstRCC are of particular interest given that durable completeresponses have only been demonstrated in patients receivingcytokine (IL-2) immunotherapy, as well as recent evidencehighlighting the efficacy of immune checkpoint inhibition ofPD-1 (27–29). The current study represents the first preclinicalevidence of reovirus efficacy as both a direct oncolytic andimmunotherapeutic agent for use against RCC. Beyond this, theability to repurpose sunitinib, a first-line approved mRCCagent, for augmentation of reovirus' immunotherapeutic effi-cacy was confirmed. This not only positions reovirus-sunitinibcombination therapy as an attractive novel treatment strategy,but further supports the growing body of literature highlightingthe benefit of harnessing the immune system to improveoncolytic virotherapy.

The conducted in vitro studies demonstrate the ability of reo-virus to induce oncolysis against RCC, as has been described formultiple tumor histologies (Fig. 1A–C).Oncolysis of humanRCCcell lines was seen within 48 hours of infection with all ED50

values being less than 40MOI,which is comparablewith that seenwith other solid malignancies (18, 30–32). Notably, the RENCAcell line demonstrated significantly greater in vitro sensitivityrelative to human RCC cells, consistent with previous reportshighlighting increasedmurine cell line sensitivity to reovirus (33).While the precisemechanism for the enhanced sensitivity remainsunknown, this observation suggested a direct oncolytic in vivoresponse could be achieved. As such, the established sensitivity ofthe RENCA cell line coupled with the observed production ofproinflammatory chemokines known to be involved in thepriming of reovirus-mediated innate and adaptive immunity(RANTES, MIP-1a, MCP-1, KC, IP-10 and MIG) supported itsuse for in vivo investigation of reovirus immunotherapeuticefficacy against RCC as a model to establish preclinical proof-of-principle (14, 17) (Fig. 1D). Moreover, these results alsohighlight that similar to infection of melanoma and prostatecancer cells, reovirus replication induces an inflammatory celldeath response against RCC (14, 18).

The therapeutic utility of the in vitro studies was confirmed invivo utilizing the RENCA immunocompetent murine model ofRCC. Treatment of mice with reovirus and sunitinib resulted in areduction in tumor burden relative to both PBS and DV controls(Fig. 2A). Furthermore, in those mice receiving reovirus andsunitinib, tumor-specific CD8þ splenocytes could be isolated(Fig. 3A-B), highlighting the ability to generate a systemic adap-tive antitumor immune response.

The combination of OV with relevant standard-of care treat-ments will likely be essential for their successful translationinto clinical practice. The observation that the combination ofsunitinib, a first-line mRCC therapeutic, with reovirus results ina significant regression in tumor burden and improved OSrelative to either of these agents given as monotherapies pro-vides proof-of-principle to investigate these agents in combi-nation for superior antitumor activity (Fig. 2A and B). Thepotential to broaden this treatment paradigm to other histol-ogies was confirmed by demonstrating combination therapyefficacy in the KLN205 murine lung SCC model (Fig. 6A and B).While the observed benefit in this model was significant, it wasnot as robust as seen with the RENCA model possibly due to

Figure 5.

Combination therapy induces a protective immune response against tumorrechallenge. A, Adoptive transfer experimental scheme adapted from Gujar andcolleagues (17). Briefly, mice (donor) were implanted with RENCA tumors andtreated as shown in Fig. 2A. On day 19, splenocytes were harvested andtransferred to treatment-na€�vemice (recipient) by tail vein injection, followedbyRENCA (2.5 � 106 cells s.c.) tumor challenge. Arrow indicates treatmentstart date, X indicates treatment end date. B, Tumor size in recipient micechallenged with RENCA cells s.c. N¼ 6mice/group. Error bars¼ SEM of tumorswithin each group. �� , P < 0.01, by two-way ANOVA. Data representative of twoindependent experiments. C, Pretreated (reovirus þ sunitinib) micedemonstrating curative response (N¼ 3) and a cohort of treatment-na€�ve mice(N ¼ 5) were challenged with RENCA tumors (1 � 106 cells, s.c). Tumor burdenover time is displayed. �� , P < 0.001, by two-tailed Student t test.






disparities in sensitivity to reovirus oncolysis or differences inimmunogenicity.

To better understand the direct cytotoxic effects of reovirus–sunitinib combination therapy on RCC, the Chou and Talalaymethod was utilized (23). This line of experimentation demon-strated the potential for reovirus and sunitinib to synergisticallyinduce cell death, highlighting enhanced direct cytotoxicity as a

plausible mechanism explaining the superior therapeutic efficacyseen with these agents in combination in vivo (Fig. 1F). Interest-ingly, a recent report has demonstrated the ability of sunitinib toenhance viral replication through targeting innate immune path-ways such as double stranded RNA protein Kinase R (PKR) andRNaseL, shedding light on a possiblemechanism for the observedin vitro synergy (34). Moreover, as reovirus-mediated apoptosis

Figure 6.

Sunitinib augments reovirusimmunotherapeutic efficacy in asyngeneic immunocompetent murinemodel of lung squamous cellcarcinoma. DBA/2 mice wereimplanted with KLN205 (5� 105 cells)subcutaneous tumors and treated asdescribed in Fig. 2A. Sunitinib wasgiven daily for 14 consecutive daysstarting on day 6 after KLN205implantation. Reovirus wasadministered four times (day 9, 12, 15,18). A, Tumor size was followed withcaliper measurements. N¼ 8 mice pergroup. Error bars ¼ SEM of tumorswithin each group. �� , P < 0.001;��, P < 0.01; � , P < 0.05 by two-wayANOVA. B, Kaplan–Meier plot of micetreated as in A. N ¼ 4 mice per group.� , P < 0.05 by log-rank between PBSor RV or sunitinib and sunitinib þ RV.C–E, Splenocytes from KLN205tumor–bearing mice treated as perA (sacrificed day 23) wereimmunophenotyped by flowcytometry. N¼ 5mice per group. In allpanels, error bars ¼ SEM ofexperimental triplicate. �� , P < 0.001;��, P < 0.01; � , P < 0.05 by two-tailedStudent t test. F, CD8þ splenocyteswere isolated from KLN205 tumor-bearing mice treated as per A andcocultured with KLN205 cells.%KLN205 cytotoxicity wasdetermined by LDH assay. Errorbars ¼ SEM of experimental triplicate.� ,P <0.05 by two-tailed Student t test.N ¼ 5 mice per group.


Lawson et al.




and autophagy have previously been well characterized in othercancer cell lines and ex vivo human tumor specimens (35–39), wehypothesize the observed synergymay reside in sunitinib's abilityto sensitize RCC cells to these alternate modes of cell death.

In addition to direct synergistic effects on cell viability theseresults also support augmentation of antitumor immunity as acontributing mechanism mediating the observed in vivo synergybetween reovirus and sunitinib. This is evidenced by a significantincrease in the production of IFNg from tumor specific CD8þ

splenocytes isolated from mice treated with reovirus-sunitinibcombination therapy (Fig. 3A and B). Augmentation of a thera-peutic immune response by sunitinib is further establishedthrough our CD8 depletion (Fig. 3D and E), adoptive transfer(Fig. 5B), and tumor rechallenge experiments (Fig. 5C), whichconfirm the ability to generate a systemic protective immuneresponse with combination therapy. This finding has significanttranslational potential given the recent results published byZamarin and colleagues who established that intratumoral treat-ment of primary tumors with immunogenic oncolytic viruses hasthe ability to clear metastatic disease once protective immunityhas been established; known as the abscopal effect (40). As such,our work supports the potential of circumventing longstandingissues with intravenous OV administration and viral immuneclearance by application of intratumoral approaches to generatesystemic immune responses for treatment of metastatic disease.Notably, destruction of tumor vasculature has also been observedthrough intratumoral endothelial cell sensitization to oncolyticvirotherapy after resolution of sunitinib therapy (41). As such, it islikely that sunitinib and reovirus work through multiple inde-pendent mechanisms to achieve the observed in vivo synergyincluding both immune and nonimmune mediated.

Sunitinib also has been reported to enhance therapeuticimmune responses through reversing tumor-induced immunesuppression in patientswithmRCC (3). This activity resides partlyin its ability to downregulate the levels of circulating and intra-tumoral MDSC, which have been demonstrated to orchestratemRCC immune suppression through direct T-cell inhibition aswell as stimulating the upregulation of Tregs (3–4). Given this,mechanistic investigations were focused on correlating MDSCand Treg levels with tumor specific CD8þ splenocyte IFNg pro-duction between mono- and combination therapy groups in vivo.Consistent with an inflammatory cell death, reovirus monother-apy administration induced a marked rise in both immunestimulatory (CD8þ and CD4þ lymphocytes) and suppressor(MDSC and Treg) populations within the spleen and tumor,similar to recent reports in the IP8 ovarian peritoneal carcinoma-tosis model (ref. 33; Fig. 4A–F). In keeping with our hypothesis,the accumulation of immune suppressor cells could be preventedby combination of reovirus with sunitinib, correlating with theobservation that mice treated with combination therapy produce

more tumor-specific CD8þ splenocytes. These results were con-sistent with our findings in the KLN205 murine lung squamouscell carcinomamodel, wheremice treatedwith reovirus–sunitinibcombination therapy had increased numbers of tumor-reactiveCD8þ cells and increased memory T cells, while splenic MDSCaccumulation was prevented (Fig. 6C–F). Given the previouslydocumented ability of these immune suppressor cell populationsin mediating resistance to reovirus-mediated immunity (42), ourresults suggest sunitinib prevention of MDSC and Tregs accumu-lation as a potential mechanism for enhancing reovirus-mediatedadaptive immunity. Our laboratory is currently focused on elu-cidating the precise immunemechanism underlying the observedimmunotherapeutic efficacy of reovirus–sunitinib combinationtherapy.

Taken together, we believe these findings provide proof-of-principle for the study of this novel viroimmunotherapeuticparadigm against a broad range of malignancies, including RCC.Beyond this, as sunitinib is currently a first line mRCC approvedtherapeutic and reovirus is in advanced phase III clinical trials, webelieve the rapid translation of these findings in the setting of aclinical trial for patients with this incurable cancer is warranted.

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

Authors' ContributionsConception and design: K.A. Lawson, A.A. Mostafa, J. Spurrell, D.G. MorrisDevelopment ofmethodology: K.A. Lawson, A.A. Mostafa, Z.Q. Shi, J. Spurrell,K. Gratton, D.G. MorrisAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K.A. Lawson, A.A. Mostafa, Z.Q. Shi, J. Spurrell,W. Chen, J. Kawakami, K. Gratton, S. ThakurAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): K.A. Lawson, A.A. Mostafa, Z.Q. Shi, W. Chen,K. Gratton, D.G. MorrisWriting, review, and/or revision of themanuscript:K.A. Lawson, A.A.Mostafa,Z.Q. Shi, J. Spurrell, J. Kawakami, S. Thakur, D.G. MorrisAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): K.A. Lawson, A.A. Mostafa, Z.Q. Shi, J. Spurrell,K. Gratton, D.G. MorrisStudy supervision:K.A. Lawson, A.A.Mostafa, J. Spurrell, J. Kawakami,D.G.Morris

Grant SupportThis work was supported by a graduate studentship award from the Alberta

Cancer Foundation (to K.A. Lawson) and by operating grants from the AlbertaCancer Foundation and Alberta Innovates Health Solutions (awarded to D.GMorris).

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 January 18, 2016; revised April 11, 2016; accepted April 18, 2016;published OnlineFirst May 24, 2016.

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Lawson et al.




2016;22:5839-5850. Published OnlineFirst May 24, 2016.Clin Cancer Res Keith A. Lawson, Ahmed A. Mostafa, Zhong Qiao Shi, et al. Immunotherapeutic Strategy for Renal Cell CarcinomaRepurposing Sunitinib with Oncolytic Reovirus as a Novel

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