Cancer Therapy: Preclinical

Local Delivery of OncoVEXmGM-CSF GeneratesSystemicAntitumor ImmuneResponsesEnhancedby Cytotoxic T-Lymphocyte–Associated ProteinBlockadeAchim K. Moesta1, Keegan Cooke2, Julia Piasecki3, Petia Mitchell2, James B. Rottman4,Karen Fitzgerald1, Jinghui Zhan2, Becky Yang1, Tiep Le3, Brian Belmontes2,OluwatayoF. Ikotun5, KimMerriam4,CharlesGlaus5, KennethGanley4,DavidH.Cordover4,Andrea M. Boden4, Rafael Ponce6, Courtney Beers3, and Pedro J. Beltran2

Abstract

Purpose: Talimogene laherparepvec, a new oncolytic immu-notherapy, has been recently approved for the treatment ofmelanoma. Using a murine version of the virus, we characterizedlocal and systemic antitumor immune responses driving efficacyin murine syngeneic models.

Experimental Design: The activity of talimogene laherpar-epvec was characterized against melanoma cell lines using an invitro viability assay. Efficacy of OncoVEXmGM-CSF (talimogenelaherparepvec with the mouse granulocyte-macrophage colony-stimulating factor transgene) alone or in combination withcheckpoint blockade was characterized in A20 and CT-26contralateral murine tumor models. CD8þ depletion, adoptiveT-cell transfers, and Enzyme-Linked ImmunoSpot assays wereused to study the mechanism of action (MOA) of systemicimmune responses.

Results: Treatment with OncoVEXmGM-CSF cured all injectedA20 tumors and half of contralateral tumors. Viral presence was

limited to injected tumors and was not responsible for systemicefficacy. A significant increase in T cells (CD3þ/CD8þ) wasobserved in injected and contralateral tumors at 168 hours. Exvivo analyses showed these cytotoxic T lymphocytes were tumor-specific. Increased neutrophils, monocytes, and chemokines wereobserved in injected tumors only. Importantly, depletion ofCD8þ T cells abolished all systemic efficacy and significantlydecreased local efficacy. In addition, immune cell transfer fromOncoVEXmGM-CSF-cured mice significantly protected from tumorchallenge. Finally, combination of OncoVEXmGM-CSF and check-point blockade resulted in increased tumor-specific CD8þ anti-AH1 T cells and systemic efficacy.

Conclusions: The data support a dual MOA for Onco-VEXmGM-CSF that involves direct oncolysis of injected tumors andactivation of a CD8þ-dependent systemic response that clearsinjected and contralateral tumors when combined with checkpointinhibition. Clin Cancer Res; 23(20); 6190–202. �2017 AACR.

IntroductionUnderstanding of the interactions between tumors and the

immune system has resulted in breakthrough oncology immu-notherapies, including Blincyto, a CD19-CD3 bispecific T-cellengager (BiTE), Opdivo, Tecentriq, and Keytruda, anti–pro-

grammed cell death protein (PD)-1 monoclonal antibodies, andYervoy, an anti–cytotoxic T-lymphocyte–associated protein(CTLA)-4 monoclonal antibody (1). These agents build on clin-ical and mechanistic data that demonstrate the central role oftumor-specific CD8þ T cells in mediating tumor elimination (2–4). Appropriate T-cell activation is regulated by dendritic cells(DC), which detect peripheral tumor antigen, become activated,and migrate to secondary lymphoid tissues to induce antitumorresponses (5, 6). Emergent tumors may develop strategies toevade this immune response, including expression of factors thatsuppress DC/T-cell activity (7–9).

Currently approved immunotherapies can overcome tumor-induced immune suppression by blocking negative immunoreg-ulatory signals [e.g., CTLA-4 (10) or PD-1 (11)]. Oncolytic virusesmay complement immunotherapies by selectively killing infectedtumor cells (thereby releasing tumor antigens) and promotingspecific anti-tumor immunity (by acting as viral adjuvants and byinducing proinflammatory tumor cell death); such activities arefurther enhanced by expression of transgenes designed to pro-mote adaptive immune responses (12).

The available clinical evidence supports the safety of intratu-morally administered oncolytic viruses, with modest side effects

1Oncology Research, Amgen Inc., South San Francisco, California. 2OncologyResearch, Amgen Inc., Thousand Oaks, California. 3Therapeutic Innovation Unit,Amgen Inc., Seattle, Washington. 4Pathology Department, Amgen Inc., Cam-bridgeMassachusetts. 5Research Imaging Sciences, Amgen Inc., ThousandOaks,California. 6Comparative Biology & Safety Sciences, Amgen Inc., South SanFrancisco, California.

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

C. Beers and P.J. Beltran contributed equally to this article.

Corresponding Author: Pedro J. Beltran, Amgen Inc., One Amgen Center Drive,Thousand Oaks, CA 91320. Phone: 805-447-9221; Fax: 805-375-8524; E-mail:pbeltran@amgen.com

doi: 10.1158/1078-0432.CCR-17-0681

�2017 American Association for Cancer Research.

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(13–16). Talimogene laherparepvec was designed to kill neoplas-tic cells by direct lysis and stimulation of a tumor antigen–specificadaptive immune response. Removal of the ICP34.5 gene fromherpes simplex virus type 1 (HSV-1) confers tumor-selectivereplication and markedly reduces neurovirulence (17–20). Inser-tion of human granulocytemacrophage colony-stimulating factor(GM-CSF) allows local GM-CSF expression to trigger the influxand activation of DCs, which present tumor-associated antigensfrom lysed tumor cells. These DCs are intended to prime tumor-specific CD4þ and CD8þ T cells to stimulate a systemic andantitumor adaptive immune response (21, 22). Additional mod-ifications include removal of the ICP47 gene, and immediate earlyexpression of US11 (12).

Studies using mouse syngeneic models have demonstratedantitumor activity of talimogene laherparepvec in injected andcontralateral tumors (12). These results support the developmentof adaptive antitumor immune responses and are consistent withemerging clinical evidence in treated patients (23). To gain furtherinsight into the immune-mediatedmechanisms of systemic activ-ity, we have conducted studies in syngeneicmurine tumormodelsusing a version of talimogene laherparepvec that expresses themurine GM-CSF gene (OncoVEXmGM-CSF). Here, we detail thepresence of viral particles to injected tumors only, describe lon-gitudinal changes in chemokine expression and immune-cellinfiltrates, pinpoint the key role of CD8þ cytotoxic cells insystemic efficacy, and characterize the enhanced anti-tumor activ-ity of OncoVEXmGM-CSF in combination with CTLA-4 blockade.Our data provide definitive evidence of a systemic, tumor-specificadaptive immune response triggered by OncoVEXmGM-CSF.

Materials and MethodsOncolytic viruses, cell lines, and in vitro viability assay

The engineering of talimogene laherparepvec (IMLYGIC,Amgen Inc.) is described above and by Liu and colleagues(18). OncoVEXmGM-CSF is engineered in a manner similar totalimogene laherparepvec, with the exception that the human

GM-CSF transgene is replaced with the murine GM-CSF (18). Alltumor cell lines were acquired from the ATCC, and cultured asindicated. Cells were plated in a 96-well plate at 2,000 to 10,000cell per well and incubated overnight at 37�C. Talimogene laher-parepvec was added in 1:5 dilutions starting at 10 or 100 MOI.After a 72-hour incubation, the number of cells left in each wellwas quantified using ATP-Lite (Perkin Elmer).

Animal care and useFemale BALB/c mice (Charles River Laboratories), 6 to 8 weeks

of age were cared for in accordance with the "Guide for the Careand Use of Laboratory Animals" (24). Animals were housed atAssociation for Assessment and Accreditation of Laboratory Ani-mal Care International–accredited facilities (at Amgen) in venti-lated micro-isolator housing on corncob bedding. All protocolswere approved by an Institutional Animal Care and Use Com-mittee. Animals had ad libitum access to sterile pelleted feed andreverse osmosis–purified water and were maintained on a 12:12-hour light:dark cycle with access to environmental enrichmentopportunities.

Tumor growth evaluation in subcutaneous murine tumormodels

Single-agent and combination efficacy: mouse B-lymphomacells (cell line A20) or mouse colon carcinoma cells (cell lineCT-26) were injected subcutaneously in the right and left flanks ofmice (2 � 106 cells). Tumor volume (mm3) was measured usingelectronic calipers twice per week. Once tumors reached anaverage of approximately 100 mm3, animals were randomizedinto groups (10 mice per group) such that the average tumorvolume (in both flanks) at the beginning of treatment adminis-tration was uniform across treatment groups. Animals were thenadministered three intratumoral injections of OncoVEXmGM-CSF

or vehicle every third day. Anti-CTLA-4 antibody was adminis-tered intraperitoneally twice per week for four doses, beginningon the same day at the same time as OncoVEXmGM-CSF. Clinicalsigns, body weight changes, and survival (tumors reached800 mm3) were measured two to three times weekly until studytermination.

CD8þ depletion and adoptive cell transferCD8þ T-cell depletion was achieved by treating animals with

antimouse CD8a antibody (clone 53-6.72; BioXCell) intraperi-toneally, 500 mg per mouse, 3 days prior to tumor cell implan-tation and then twice per week until study termination.

Donor immune cells derived from spleen and axillary lymphnodes (LNs) were processed into single-cell suspensions bymechanical dissociation. The splenocytes were then depleted ofred blood cells by ammonium chloride lysis before being mixedwith LN cells. A total of 2.5 � 107 donor immune cells weretransferred intravenously into new Balb/c recipients and thenchallenged with A20 tumor cells (2 � 106) on the left subcuta-neous flank. The appearance of tumor formation was monitored/measured for the ensuing 50 days.

ImmunohistochemistryTumors were either embedded in optimal cutting temperature

(OCT) compound or fixed in 10% neutral buffered formalin.Frozen sections were cut at 6 mmand fixed in an acetone/absoluteethanol solution (75%/25%). Paraffin sections were cut at 5 mm,baked at 56 to 60�C for at least 60 minutes, deparaffinized inxylene, and rehydrated through graded ethanol solutions. Epitope

Translational Relevance

Breakthrough oncology immunotherapies targeting check-point inhibition allow preexisting, tumor-specific cytotoxic Tcells to replicate and attack the patient's tumor. Unfortunately,the combination of an immunosuppressive tumor microen-vironment and/or lack of tumor-specific cytotoxic T cellsrenders these immunotherapies ineffective in most patientswith cancer. Oncolytic virus immunotherapy is able to fosteran inflammatory tumor microenvironment and generate newtumor-specific T-cell responses representing the ideal partnerfor checkpoint inhibitor therapy. Our results provide mecha-nistic data supporting the systemic efficacy of talimogenelaherparepvec in combination with CTLA-4 blockade. Thesignificant efficacy enhancement observed in these animalmodels and in the recent phase II clinical study combiningtalimogene laherparepvec with ipilimumab in patients withadvanced melanoma suggest that this combination is a viableoption to generate specific and efficacious antitumor adaptiveimmune responses in patients with cancer who might notbenefit from single-agent therapy with checkpoint inhibitors.

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retrieval was performed by boiling the slides in DIVA Decloakingsolution (Biocare Medical, #DV2004G1).

IHC was performed on frozen sections with the followingmarkers—HSV-1 (BiocareMedical, #APA-3027AAK), CD3 (clone#145-2C11; BD Biosciences #550275), F4/80 (clone #CI:A3-1;AbD Serotec #MCA497R), CD8 (clone #YTS 169AG 101HL;Thermo Scientific #MA1-70041), p46 (clone #29A1.4; eBios-ciences #11-3351), and CD103 (clone #2E7; AbD Serotec#MCA4705GA). IHC was performed on formalin-fixed, paraf-fin-embedded (FFPE) sections with the followingmarkers—HSV-1, F4/80, Granzyme B (clone #16G6; eBiosciences #13-8822-82),and B220 (clone #RA3-6B2; BD Biosciences #553086).

Morphometric analysis was performed by staining tissue sec-tions via IHC with an anti-B220 antibody to identify the A20tumor regions. The tumor areas, or region of interest (ROI) wereoutlined manually (nonviable regions were excluded) andapplied to serial images to evaluate staining for cell markers. Thearea occupied by CD3þ, CD8þ, CD103þ, or Granzyme Bþ cells isexpressed as percentage of the total tumor area.

Digital droplet PCR analysis for HSV-1 DNATotal DNA was extracted from pulverized tissue samples

(tumor or liver) and whole blood using Qiagen's DNeasy Kit(Qiagen). Digital droplet PCR (ddPCR) was performed usingBio-Rad QX200 System (Bio-Rad). The following sequenceswere used to amplify HSV-1 thymidine kinase gene—forwardprimer, 50-CGATATCGTCTACGTACCCG-30; reverse primer, 50-ATATCTCACCCTGGTCGAGG-30; and probe, 50-FAM-CGAT-GACTT/ZEN/ACTGGCGGGTG-IBFQ-30. Viral copy-numberwas normalized against mouse hypoxanthine guanine phos-phoribosyl transferase (HPRT) gene. The following primers andprobe sequences were used to detect murine HPRT—forwardprimer, 50-GCTCCACTTTGAAACAGCTG-30; reverse primer, 50-CTTTTTCCAAATCCTCGGCA-30; and probe 50-HEX-TGCAGAT-TAGCGATGATGAACCA- BHQ-1-30. DNA was digested withHaeIII before PCR amplification. Thermal cycling conditionsare 95�C for 10 minutes, then 40 cycles at 94�C for 30 seconds,and 60�C for 1 minute, followed by 98�C for 10 minutes.

Gene product analysisOCT-embedded or flash-frozen tissues were processed using a

TissueLyzer (Qiagen). mRNA was extracted using RNEasy col-umns (Qiagen) and converted to cDNA using the High-CapacitycDNA Reverse Transcription Kit (Applied Biosystems). qPCRassay primer sets consisting of amplification primers and labeledprobes for individual gene assays were synthesized by IntegratedDNA Technologies. Pre-amplification and qPCR reactions werecarried out using TaqMan PreAmpMaster Mix and Universal PCRMastermix (Applied Biosystems), respectively, and then analyzedon Fluidigm Biomark HD using 96.96 integrated fluidics chips(Fluidigm). Expression data for individual samples are normal-ized to the geometric mean of four housekeeping genes (HKGs)Actb, Gapdh, Ipo8, and Tbp (dCt ¼ Ctgene of interest � GeomeanCtHKGs) and displayed as normalized gene expression (NGE ¼2�DCt with an arbitrary scaling factor of 1,000). IFN response genesignature is comprisedof the geometricmeanof theNGEvalues ofIfi27l1, Ifi44, Ifit1, Mx1, and Oas1b.

Flow cytometryThe tumor-draining LNs (TDLNs) and spleens were isolated,

and single-cell suspensionswere generated byusing protocols and

reagents for dissociation of mouse spleen provided by Miltenyiand the GentleMACS Octo instrument (Miltenyi). Lymphocyteswere stained with anti-CD3 (clone 145-2C11 or 17A2), anti-CD69 (H1.2F3), anti-CD49b (DX5), anti-CD19 (6D5), anti-Ly6C(HK1.4), and anti-Ly6G (1A8), and AH16-14 loaded H2-Ld dex-tramer (Immudex). Cells were run on either the Miltenyi MACS-Quant VYB or a BD LSR Fortessa and analyzed using FlowJosoftware (FlowJo LLC).

Enzyme-linked ImmunoSpot assaysFor enzyme-linked ImmunoSpot (ELISPOT) assays, 96-well

plates with a nitrocellulose filter base (Millititer HA; Millipore)were coatedwithpurified anti-IFNg (2mg/mL) antibody. EnrichedT cells (mouse T-cell kit, Miltenyi) from OncoVEXmGM-CSF- orvehicle-treated A20 tumor-bearing mice on days 4, 7, 11, and 14after treatment were added to the plates in a 10:1 ratio with eitherA20 or CT-26 tumor cells.

Alternatively, splenocytes fromdifferent treatment groupswereused in peptide restimulation assays. Splenocytes (8 � 105) wereincubated with control peptides (GFPs) or the AH1 peptide(SPSYVYHQF) at a final concentration of 1 mmol/L for 20 hoursat 37�. The AH1 peptide is an immunodominant Ag derived fromthe envelope protein (gp70) of the endogenous murine leukemiavirus presented by the MHC class I Ld molecule (25). Spots wereenumerated using a CTLS6 Fluorospot analyzer (CTL).

Positron emission tomography imaging of OncoVEXmGM-CSF

activityMice bearing 150 mm3 A20 tumors in both flanks received an

intratumoral injection of 5 � 106 PFU OncoVEXmGM-CSF (n ¼ 4)or saline (n ¼ 4) into the left flank tumor. Animals received anintravenous injection of 100 mCi [18F]FHBG (26) into the lateraltail vein 24 hours after OncoVEXmGM-CSF treatment. Two hoursafter [18F]FHBG injection, animals were anesthetized (2–3%isoflurane in 100% oxygen) for anatomical x-ray computedtomography (CT) and 15-minute static PET imaging (InveonPET/CT; Siemens Medical Solutions). Attenuation-correctedimages were reconstructed using the OSEM OP-MAP algorithm,and images were analyzed for tumor [18F]FHBG uptake usingInveon Research Workplace (Siemens Medical Solutions).

Statistical analysisIn vivo efficacy data were analyzed by Kaplan–Meier anal-

ysis of median survival of mice treated with single agents orsingle agent versus combination. Unpaired Mann–Whitneynonparametric test was used to analyze morphometric IHCanalysis, and P < 0.05 was considered statistically significant.One-way ANOVA with Dunnett's correction was applied foranalysis of gene expression differences. Multiple comparisonunpaired T-test with Holm–Sidak correction was appliedto test the significance of ex vivo T-cell reactivities andflow cytometric analysis of individual populations in a timecourse manner.

ResultsTalimogene Laherparepvec has broad oncolytic activity againsthuman melanoma and mouse tumor cell lines

To characterize the oncolytic activity of talimogene laherpar-epvec against human and murine cell lines, we treated nine celllines with virus in a 72-hour viability assay. All cell lines tested

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showed sensitivity to talimogene laherparepvec. IC50 ranged from0.05 (SK-MEL-5 melanoma) to 12.7 (A20 murine B-cell lympho-ma) MOI (Fig. 1). All human melanoma cell lines displayed IC50

below 1 MOI. Syngeneic model cell lines A20 and CT-26 wereefficiently lysed by talimogene taherparepvec, although IC50 forCT-26 was 100-fold lower.

OncoVEXmGM-CSF detection, replication, and oncolysis arerestricted solely to the injected tumor

Previous studies have demonstrated that local delivery ofOncoVEXmGM-CSF results in oncolysis but the mechanism ofantitumor efficacy in noninjected tumors remains unclear (12).We established a syngeneic A20 contralateral tumor model tostudy systemic efficacy. In OncoVEXmGM-CSF-treated animals, amajority of injected tumors showed complete regression at alldoses. In contrast, contralateral tumors at the lower dose showedno response, whereas the medium and high doses showed com-plete responses in half the tumors and growth delay in the otherhalf (Fig. 2A–D). Efficacy comparison with backbone OncoVEX(noGM-CSF activity) in thismodel at the highest dose showed nodifference in efficacy in the injected tumors but a significantdecrease in cured mice in the contralateral side (SupplementaryTable S1). Median survival was significantly increased in themedium- and high-dose groups compared with vehicle (38 daysvs. 28 days, respectively; Fig. 2E), and no changes in body weight

were observed (Supplementary Fig. S1A). Oncolysis was onlyobserved in the injected tumors (Supplementary Fig. S1B). Wemonitored the presence of viral DNA and mRNA in injected andcontralateral tumors, as well as in the blood, liver, and therespective draining LNs. Viral DNA was detected dose-propor-tionally only in injected tumors between 2 and 168 hours afterinjection and did not increase significantly with time. Viral DNA,at a level consistent with a 5� 103 PFU dose, was detected in 1/16contralateral tumors (5 � 105 PFU dose group) and probablyrepresents tissue contamination (Fig. 2F). The expression of threeHSV-1 genes, ICP27 (transcriptional regulator, immediate-earlygene), VP16 (virion maturation), and thymidine kinase (markerof HSV-1 viral replication), were detected as early as 4 hours in theOncoVEXmGM-CSF-injected tumor, with a gradual reduction of thesignal over time. Conversely, neither the OncoVEXmGM-CSF con-tralateral tumor nor the LNs draining any tumor had significantlevels of any of the three viral gene products (Fig. 2G andSupplementary Fig. S2A).

We next analyzed tumors for viral antigen and HSV-1 thymi-dine kinase activity. Histologic analysis of OncoVEXmGM-CSF-injected tumors revealed a large, well-demarcated central nonvi-able region that was not present in vehicle-injected tumors.Evaluation of IHC staining in OncoVEXmGM-CSF-injected tumortissue sections at higher magnification highlighted a central zoneof HSV-1þ cell debris, surrounded by a thin band of HSV-1-

IC50

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Figure 1.

Oncolytic activity of talimogene laherparepvec against multiple human melanoma and murine tumor cell lines. Relative luminescence units (RLUs) represent thenumber of live cells following treatment with talimogene laherparepvec for 72 hours in vitro. Tumor cells were plated in 96-well culture plates and exposed toa range of talimogene laherparepvec MOIs (1:5 dilutions). After 72 hours, cell viability was measured with ATPlite assay. ATPlite signal is plotted againstincreasing MOI. Each point represents the average � SD. IC50 is calculated using GraphPad 6.1. Data shown are representative of at least two experiments.

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A

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OncoVEXmGM-CSF-induced lysis and HSV-1 virus are only detected in OncoVEXmGM-CSF-injected tumors. A–D, Growth of OncoVEXmGM-CSF (dose response 3� 104 to3 � 106 PFU) and vehicle-treated A20 tumors on the injected and contralateral side. Each line represents the growth pattern of a single tumor (n ¼ 10/group).Data are representative of at least three independent experiments. E, Kaplan–Meier analysis of median survival of mice treated with OncoVEXmGM-CSF vs. vehicle.Eventswere recordedwhen tumor volume exceeded 800mm3. F, ddPCR quantification analysis of viral HSV-1 DNA. Bars or points represent themean copy-numbervariation (CNV) and SD for each dose or time point (n ¼ 4). Dose–response analysis was performed at 24 hours. Time response analysis was performed at5 � 106 PFU. G, Relative expression of viral gene products (ICP27 and thymidine kinase) is shown for injected and contralateral tumors as well as inguinal TDLNsdraining each. Time points as indicated. Viral gene product expression is representative of two independent experiments with n ¼ 5/group (� , P < 0.001). H,Sections fromvehicle- andOncoVEXmGM-CSF-injected and contralateral A20 tumors at 96hours postinjectionwere stainedbyH&Eor an antibody-specific for anHSV-1 antigen. IHCdata are representative of three independent experiments, n¼ 5/group. I, In vivodetection ofOncoVEXmGM-CSF HSV-1 thymidine kinase activity by PET/CT imaging was performed using the radiolabeled penciclovir analog [18F]FHBG. [18F]FHBG accumulation in OncoVEXmGM-CSF-injected tumors compared tocontralateral or vehicle-injected tumors.

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Treatmentwith OncoVEXmGM-CSF induces a localized inflammatory response.A,Bilateral A20 tumor sections fromOncoVEXmGM-CSF (single dose of 5� 106 PFU) andvehicle control mice collected at time points indicated were processed to extract RNA. Indicated type I IFN gene signature was measured as the geometricmean of five different IFN inducible genes using Fluidigm qPCR. Statistics shown are for OncoVEXmGM-CSF-injected tumors vs. vehicle-injected using a one-wayANOVA (Dunnett's multiple comparison test). B, Expression of the GM-CSF, IFNg , CXCL2, and CXCL10 was measured in vehicle and OncoVEXmGM-CSF-treatedA20 tumor between 1 and 96 hours. Data are representative of two independent experiments.C,Viable cell counts obtained from LN disaggregates. Viable cell countby trypan exclusion harvested 96 hours after treatment and flow cytometric analysis of CD69þ CD3þ lymphocytes from vehicle- and OncoVEXmGM-CSF-treatedmiceat 2, 5, and 10 days after treatment. Data are representative of two independent experiments n ¼ 10/group; statistics shown are for one-way ANOVAwith Sidak correction for multiple testing, with comparators preselected by matched time point (OncoVEXmGM-CSF vs. vehicle) (�, P < 0.0001; �� , P < 0.01;��� , P < 0.05).

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OncoVEXmGM-CSF increases immune cells in both injected and contralateral tumor. A, Relative expression of CD3E, CD8A, and CD103 (ITGAE) was analyzed fromvehicle-injected and OncoVEXmGM-CSF-injected and corresponding contralateral tumors. Gene expression analysis detects increased infiltration into bothOncoVEXmGM-CSF-injected and contralateral tumors at 168-hour time points, compared with vehicle-injected tumors. Gene expression analysis is representative oftwo independent experiments with n ¼ 5/group. B and C, Morphometric analysis of A20 tumor serial sections by IHC. A20 tumor sections from OncoVEXmGM-CSF

(single dose of 5� 106 PFU) and vehicle control mice 168 hours (or as indicated in Fig. 4C) after injection were subjected to morphometric analysis, and the percentarea of ROI was calculated for CD3, CD8, and CD103. Statistics were generated using unpaired Mann–Whitney nonparametric test. D and E, FACS analysisof A20 tumors following OncoVEXmGM-CSF (single dose of 5 � 106 PFU) and vehicle treatment at the indicated time point (D) and at 96 hours (E). Data arerepresentative of three independent experiments (� , P < 0.0001; �� , P < 0.01; ��� , P < 0.05).

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Figure 5.

T cells fromOncoVEXmGM-CSF-treated tumor-bearingmice specifically and functionally recognize tumor antigens.A,Ex vivo restimulation reveals systemic antitumorreactivity. Splenic T cells were isolated from A20 tumor-bearing animals on days 4, 7, 11, and 14 following OncoVEXmGM-CSF (5� 106 PFU) intratumoral injection andrechallenged ex vivo for 24 hours with either CT-26 or A20 tumor cells. Data are depicted as number of IFNg-positive spots per 100,000 plated T cells. B–E,EfficacyofOncoVEXmGM-CSF followingdepletion ofCD8þT cells. Each line represents thegrowthpatternof a singleA20 tumor (n¼ 10/group).Micewere treatedwithanti-CD8 antibody or control isotype. Following confirmation of CD8þ T-cell depletion, the mice where inoculated (contralaterally) with A20 cells, randomized intofour groups when tumors reached 160 mm3, and treated with vehicle or OncoVEXmGM-CSF at 5 � 106 PFU every 3 days for the first week. Tumor volumeand bodyweightsweremeasuredwith calipers or an analytical scale, respectively. F,Kaplan–Meier analysis ofmedian survival ofmice treatedwith OncoVEXmGM-CSF

vs. vehicle, with or without anti-CD8. Eventswere recordedwhen tumor volume exceeded 800mm3.G, Schematic of adoptive cell transfermodel.H–J, Immune cellsfrom na€�ve BALB/C mice, BALB/C mice bearing A20 tumors or BALB/C mice cured of their A20 tumors with OncoVEXmGM-CSF treatment were collected andtransplanted into recipient BALB/C mice. Mice in all groups were then challenged with A20 cells. Each line represents an individual A20 tumor followedusing calipers for 50 days. All data are representative of two independent experiments (� , P < 0.01; �� , P < 0.0001).

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vs.vs.

vehicle

vehicle

vs.vs.

n.s (P = 0.11)

A

B

F

G

K L M N

H I J

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infected tumor cells undergoing oncolysis (SupplementaryFig. S2B). Ninety-six hours after intratumoral injection treatment,HSV-1 antigen was observed only in the OncoVEXmGM-CSF-injected tumors (Fig. 2H and Supplementary Table S2). Viralantigen staining was most robust at 72 hours and markedlydecreased by 168 hours. Finally, we used [18F]FHBG PETto detectHSV-1 thymidine kinase activity in live animals injected withvehicle or OncoVEXmGM-CSF. A very pronounced and signi-ficant 18FHBG tumor accumulation was only measured in theOncoVEXmGM-CSF-injected tumors (Fig. 2I and SupplementaryFig. S2C). Delivery of OncoVEXmGM-CSF systemically by intrave-nous administration did not produce detectable viral DNA intumor tissue which correlated with lack of efficacy in the A20syngeneic model (Supplementary Fig. S3).

OncoVEXmGM-CSF therapy induces a local inflammatoryresponse

Type I IFN signaling has previously been shown to be critical inrestricting the pathogenesis of HSV-1 in vivo (27). The IFN genesignature (composed offive type I IFN inducible genes) is elevatedin OncoVEXmGM-CSF-injected tumor relative to vehicle 4 hoursafter injection (Fig. 3A).

GM-CSF can activate antigen-specific immune responses (21)and may play a similar role for OncoVEXmGM-CSF. We detectedhigh levels of mGM-CSF between 4 and 96 hours, consistentwith viral presence in the injected tumor. In addition, signi-ficant increase in IFNg was detected at 4 hours postinjection inOncoVEXmGM-CSF-injected tumors. Consistent with an inflamma-tory response, the chemoattractant chemokine (C-X-C motif)ligand (CXCL) 2 and CXCL10 were both highly upregulated inthe OncoVEXmGM-CSF-injected tumor compared with vehiclebetween 4 and 96 hours (Fig. 3B). Expression of CXCL2, knownfor its chemoattractant attributes for monocytes and neutrophils,increased in time andmatched the increased number of these celltypes detected in injected tumors at 24 and 48 hours (Supple-mentary Fig. S4A).

While evaluating the TDLN for HSV-1 antigen, we observ-ed a marked enlargement of the TDLN proximal to theOncoVEXmGM-CSF-injected tumor at 48 hours (SupplementaryFig. S4B) but not the contralateral or vehicle-treated TDLN.Enlargement was characterized by an increased number of viablecells. We next investigated the lymphocyte populations by flowcytometry at 2, 5, and 10 days after OncoVEXmGM-CSF treatment.An increase in the percentage of activated T cells (CD69þCD3þ) inthe OncoVEXmGM-CSF-injected TDLN was observed at days 2 and5, but not at day 10 (Fig. 3C).

Systemic modulation of lymphocytes after OncoVEXmGM-CSF

intratumoral treatmentNext, we performed gene expression analysis in tumors har-

vested from OncoVEXmGM-CSF-treated and vehicle-treated ani-mals for CD3, CD8, and CD103 at 48 and 168 hours aftertreatment (Fig. 4A). At 168 hours posttreatment, mRNA expres-sion levels of all three markers were increased in both theOncoVEXmGM-CSF-injected and OncoVEXmGM-CSF contralateraltumors compared with the corresponding tumors from vehicle-treated animals. IHC and FACS analysis were performed tosupport the findings from gene expression analysis. Morphomet-ric analysis of serial tissue sections stained using IHC showed that5.5% of the ROI area was occupied by CD3þ cells in both thevehicle-injected (SD ¼ 3.2%) and contralateral tumors (SD ¼1.2%) at 168 hours after injection (Fig. 4B). There was a statis-tically significant increase in the percent tumor area occu-pied by CD3þ cells in both the OncoVEXmGM-CSF-injected(14.24% � 2.29% of ROI, P < 0.01 by Mann–Whitney test) andOncoVEXmGM-CSF contralateral tumors (11.97% � 2.89% ofROI, P < 0.05) compared with the respective tumors from vehi-cle-treated animals. Longitudinal changes in CD3þ T cells incontralateral tumors were confirmed by IHC and FACS analysis.Significant increases in CD3þ T cells were measured at 168 hoursby both techniques (Fig. 4C and D). Similarly, the percent areaoccupied by CD8þ cells was significantly increased in both theOncoVEXmGM-CSF-injected and contralateral tumors at 168 hourscompared with relevant controls (Fig. 4B). The percentage oftumor area occupied by CD103þ cells, a marker of tissue residentT cells and a pro-inflammatory subset of DCs (28), was signifi-cantly elevated in the OncoVEXmGM-CSF-injected versus vehicle-injected tumor (P < 0.01) but not in contralateral tumors(Fig. 4B). T-regulatory cells (Tregs) known for their immuno-suppressive role in the tumor microenvironment were also sig-nificantly decreased in contralateral tumors following treatmentwith OncoVEXmGM-CSF (Fig. 4E).

T-cell-specific antitumor responses activated by OncoVEXmGM-CSF

We set out to investigate the ability of the systemic T cells foundin OncoVEXmGM-CSF-treated animals to react to tumor-specificantigens. Pan-T cells were harvested and purified from tumor-bearing animals at days 4, 7, 11, and 14 post-intratumoralOncoVEXmGM-CSF or vehicle administration and used in an ELI-SPOT assay. At all time points tested, the number of IFNgþ spotswas significantly increased in the OncoVEXmGM-CSF-treated T cellscultured with A20 tumor cells compared with vehicle–treated Tcells (Fig. 5A). In addition, tumor responses were largely specific

Figure 6.Enhanced efficacy of OncoVEXmGM-CSF in combination with anti–CTLA-4 antibodies in A20 and CT-26 tumor models. A, Relative expression of CD80 and CTLA-4 intumors andTDLNs. Statistical significancewasdeterminedusing aone-wayANOVAusingDunnett's correction,with significance indicated vs. vehicle–injected tumor(� , P < 0.05; �� , P < 0.01; ��� , P < 0.001). Representative of two independent experiments with n¼ 5/group. B–E, Enhanced efficacy of OncoVEXmGM-CSF with CTLA-4antibodies in the A20 tumormodel. Growth of vehicle, OncoVEXmGM-CSF (5� 106 PFU), anti–CTLA-4, and combination tumors on the injected and contralateral side.Each line represents the growth pattern of a single tumor (n ¼ 10/group). F, Kaplan–Meier analysis of median survival of mice treated with single agents vs.combination. Eventswere recordedwith tumor volume exceeding 800mm3.G–J, Enhanced efficacy of OncoVEXmGM-CSF with CTLA-4 antibodies in the CT-26 tumormodel. Growth of vehicle, OncoVEXmGM-CSF (5 � 106 PFU), anti–CTLA-4, and combination tumors on the injected and contralateral side. Each line represents thegrowth pattern of a single tumor (n ¼ 10/group). K, Kaplan–Meier analysis of median survival of mice treated with single agents vs. combination. Events wererecordedwith tumor volume exceeding 800mm3. L andM, Pharmacodynamic assessment of systemic anti-AH1 CD8þ T cells. Percent of splenic CD8þ T cells reactingwith the AH1 antigen (L:EliSpot & M:FACS) were quantified from vehicle, OncoVEXmGM-CSF (5 � 106 PFU), anti–CTLA-4, and combination treated mice. N,Pharmacodynamic assessment of tumor anti-AH1 CD8þ T cells (black bars) and tumor Tregs (white bars) from vehicle, OncoVEXmGM-CSF (5� 106 PFU), anti–CTLA-4,and combination treated tumors. Spleens were collected 7 days after the last treatment dose (n ¼ 5). Data representative of two independent experiments.� , P < 0.05; �� , P < 0.01; ��� , P < 0.001.

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to A20 and not to CT-26. Reactivity peaked at day 11. HSV-1–derived viral antigens did not appear to play a significant role inthe systemic immune response visualized here, as there was nodifference in T-cell reactivity when A20 stimulator cell lines werepre-exposed to virus prior to ex vivo restimulation with splenic Tcells (Supplementary Fig. S5A).

To further characterize the activity of these systemic T cells invivo, weperformedCD8þdepletion experiments and adoptive celltransfer experiments. Depletion of CD8þ cells (>95%) using anti-CD8 antibodies (Supplementary Fig. S5B) had profound negativeeffects on the efficacy of OncoVEXmGM-CSF (Fig. 5B–F). CD8þ celldepletion affected the growth of both injected and contralateralA20 tumors by removing tumor volume variability from thevehicle-treated group (Fig. 5B and C). Most importantly, deple-tion of CD8þ cells fromOncoVEXmGM-CSF-treatedmice abrogatedall the efficacy driven by OncoVEXmGM-CSF on contralateraltumors (0/10 vs. 5/10 cures) and also had a profound negativeeffect on efficacy in the injected tumors (1/10 vs. 9/10 cures) (Fig.5D and E). The survival benefit produced by OncoVEXmGM-CSF inthismodel was not observedwhen CD8þ cells were depleted (Fig.5F vs. Fig. 1E).

To evaluate the direct role ofOncoVEXmGM-CSF in the specificityof a memory response, we injected mice with immune cells fromna€�ve mice, mice bearing A20 tumors, and mice cured fromcontralateral tumors through OncoVEXmGM-CSF treatment. Re-challenged animals that received na€�ve cells or cells from A20tumor-bearing mice showed no protection (Fig. 5G–I). Bothgroups showed established tumors at day 10 in 9/10 mice.However, mice receiving immune cells from OncoVEXmGM-CSF-cured mice demonstrated resistance to rechallenge with A20 cellswith only two out of 10mice growing tumors at day 32 (Fig. 5J). Asimilar level of protectionwas also observedwhenOncoVEXmGM-

CSF cured mice were directly challenged with A20 cells (Supple-mentary Fig. S6).

Significant enhancement in systemic efficacy in combinationwith CTLA-4 blockade

We sought to determine if changes in immune regulatorymolecules CD80 and CTLA-4 could be detected after OncoV-EXmGM-CSF treatment. By FACS analysis, CTLA-4 and CD80 wereupregulated in the OncoVEXmGM-CSF-injected TDLN at the early48-hour timepoint, whereas significant tumor upregulation wasobserved in injected tumors at 168 hours (Fig. 6A).

Given the observed upregulation of CD80 and CTLA-4, weset to determine whether a combination of OncoVEXmGM-CSF

with anti–CTLA-4 antibodies could result in enhanced effi-cacy in contralateral tumors. Treatment of A20 tumors withOncoVEXmGM-CSF in combination with anti–CTLA-4 antibodiesresulted in a significant increase in median survival and completeregressions compared with either single agent alone (Fig. 6B–F).The combination produced cures in 90% of the contralateraltumors (Fig. 6E). To extend our observations to other models,we performed a combination study with anti–CTLA-4 antibodiesin the CT-26 model (Fig. 6G–K). This model demonstratedsome resistance to OncoVEXmGM-CSF in contralateral tumors,where tumor growth delays may be seen, but tumor regressionswere rarely observed in monotherapy (Fig. 6I). The combinationof OncoVEXmGM-CSF with anti–CTLA-4 resulted in completecures of all injected tumors and regressions in 80%of contralateraltumors (with 6/10 cures) (Fig. 6J). More importantly and similarto what was observed in the A20 model, the median survival

in the combination group was more than double that in thevehicle-treated group and was significantly better than eithersingle agent alone (Fig. 6K). Interestingly, treatment in the combi-nation group seemed to significantly delay the dynamics of theOncoVEXmGM-CSF monotherapy response resulting in large tu-mors (>500mm3) suddenly regressing and disappearing (Fig. 6J).

Finally, we used the previously described AH1 antigenin CT-26 cells as a surrogate to assess the ability ofOncoVEXmGM-CSF, CTLA-4, and the combination treatment torelease tumor antigens and stimulate antitumor-specific T-cellresponses. Quantification of systemic (splenic) anti-AH1 CD8þ Tcells by ELISpot or by dextramer staining using FACS demon-strated a significant increase in AH1 reactive T cells inmice treatedwith OncoVEXmGM-CSF, CTLA4 blockade, or the combination(Fig.6L and M). Quantification of local (tumor) anti-AH1 CD8þ

T cells showed a significant increase only in the combinationgroup. Consistent with the effect of OncoVEXmGM-CSF in the A20model, a significant decrease in Tregs was also observed in theCT-26model. This effect was greater in combination with CTLA-4blockade (Fig. 6N).

DiscussionIn OPTiM, a randomized phase III clinical trial of intralesional

talimogene laherparepvec versus subcutaneously GM-CSF inunresectable, metastatic melanoma, the primary endpoint ofdurable response rates was met, with a significant increase in thetreatment arm versus control (HR 8.9; 16.3% vs. 2.1%; P < 0.001;ref. 29). At the lesion level, a �50% reduction in tumor area wasseen in 64% of injected lesions, 32% of noninjected subcutane-ously and nodal lesions, and 16% of distant visceral lesions (30).These findings can be explained by direct oncolysis or byenhanced systemic antitumor immunity. Clinical trials to eluci-date the mechanism of action (MOA) and the potential use oftalimogene laherparepvec in combinationwith checkpoint block-ade (CTLA-4 and PD-1) are underway (NCT01740297 andNCT01740297). However, given the potential benefits forpatients, preclinical studies to further elucidate itsMOAare criticalfor optimizing future clinical use in combination with noveltherapies that perturb the cancer immunity cycle.

Although evidence indicates systemic efficacy is provided by atalimogene laherparepvec-triggered tumor-specific immuneresponse, data demonstrating that virus does not directly lysedistant tumor cells has been lacking. Using OncoVEXmGM-CSF, wedemonstrate that oncolysis alone cannot account for systemicefficacy. Despite robust tumor growth inhibition in the contra-lateral tumor, no signals of oncolysis (viral DNA,mRNA, antigen,or thymidine kinase activity) were detected in contralateraltumors, blood, and liver with highly sensitive (31, 32) assays(ddPCR andqPCR). These data support the tumor selective natureof OncoVEXmGM-CSF replication described previously (12) andpoint to an immune-mediated MOA as its principal mode ofsystemic efficacy.

The A20 B-cell lymphoma model used in this study models animmunogenic tumor as evidenced by the diffuse staining of CD3.Despite this infiltration, detectionof systemic antitumor reactivityin the absence of treatment is minimal. The localized innateimmune response to the injected oncolytic virus and subsequentlysed tumor cells creates type I and type II IFN responses. Thenotion that such localized inflammatory response is capable oftransforming an immunosuppressive microenvironment into an

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inflammatory one is an attractive therapeutic hypothesis forseveral reasons. First, altering the tumor immunosuppressiveniche may be sufficient to unleash tumor-reactive T cells that areotherwise limited in their function (9). Second, this type ofimmunogenic cell death is thought to be a key factor in generatingnovel systemic antitumor responses and enabling efficacyenhancement through checkpoint blockade (23, 33, 34). There-fore, unlike tumor vaccinations, talimogene laherparepvec offersa novel strategy with no requirement for elucidating patient-specific tumor antigens.

Despite the potency of the inflammatory response elicited byOncoVEXmGM-CSF, these effects appear to be transient and local-ized to the injected tumor and adjoining LN. Systemic efficacywould theoretically still be governed by the same principles ofinteraction between the immune system and the tumor. Evidenceof this can be seen in the upregulation of T-cell checkpointmolecules concomitant with infiltration of T-cells into theinjected and contralateral tumors. In effect, intratumoral admin-istration of OncoVEXmGM-CSF appears to increase the reactivity ofsplenic T cells even at early time points. Subsequently, an expan-sion of tumor-reactive T cells evaluated by secretion of IFNg andby CTL killing is detected at time points that would be consistentwith amplification and/or de novo priming. Without determiningantigen specificity of the T cells, it is impossible to distinguishtumor-reactive from virus-specific T cells. However, a preponder-ance of evidence suggests that the systemic adaptive immuneresponse contains a significant portion of tumor-specific T cellsthat are not present (or present at lower levels) in untreatedtumor-bearing animals: (i) the absence of viral antigens in thecontralateral tumor; (ii) ex vivo tumor cell line reactivity; (iii)transplantability of tumor rejecting immune cells; (iv) the loss ofefficacy upon CD8þ cell depletion; (v) enhanced systemic efficacyin combination with CTLA-4 blockade. The increase in tumor-reactive T cells found in the splenic compartment as early as day 4posttreatment is consistent with a very rapid mobilization andexpansion of tumor-reactive T cells. At later time points, theadaptive response is expanded both systemically, as evidencedby the expansion of splenic tumor-reactive T cells and by theinfiltration of lymphocytes into the contralateral tumor. Activa-tion of tumor-reactive T cells may be accomplished by expansionand proliferation of the preexisting tumor-infiltrating lympho-cytes (TIL) or by uncovering of novel tumor-derived antigenspresented in secondary lymphoid organs. Even thoughwe did notstudy the uncovering of novel tumor-derived antigens in ourcurrent experiments, our assessment of anti-AH1 CD8þ T cellsin the spleen and tumor following OncoVEXmGM-CSF, CTLA-4blockade, or the combination is consistent with the expansionand proliferation of preexisting TILs. The models described heredo not allow us to pinpoint if efficacy is driven by a preexistingantigen like AH1, by novel tumor antigens or by the combination.However, previous approaches have shown that release of tumor-associated antigens by localized tumor destruction can lead toenhanced systemic responses and epitope spreading (35–37).

Expression of CTLA-4 (38), which is known to increase upon T-cell activation, is also consistent with an ongoing adaptiveimmune response. It appears that the inflammatory local micro-environment induced by the administration ofOncoVEXmGM-CSF,evidenced by the increased expression of CD80 and local pro-duction of IFN,may provide a critical boost to antigen-presentingcells and thereby lead to the generation of a productive immuneresponse. Therefore, it is likely that combining intratumoral

talimogene laherparepvec treatment with blockade of CTLA-4would further enhance anti-tumor efficacy beyond what can beachieved by monotherapy alone. The first evidence of this type ofsynergistic effect was described for another oncolytic virus byAllison and colleagues (37).Whenwe combineOncoVEXmGM-CSF

with CTLA-4 blockade the complete regression of many large(>400 mm3) CT-26 tumors confirms this synergistic effect. Thislevel of efficacy is similar to that achieved by the combination offour different therapeutic approaches in a recently publishedreport (39). Talimogene laherparepvec is currently being inves-tigated in combination with the anti–CTLA-4 antibody ipilimu-mab for the treatment of unresected melanoma, where 56% ofpatients experienced objective responses (by Immune-RelatedResponse Criteria) of both injected and distant tumors (40).

In summary, here we expand our understanding of theMOA oftalimogene laherparepvec in order to optimize its future clinicalbenefit for patients. Our data clearly demonstrate that intratu-moral administration of OncoVEXmGM-CSF drives the develop-ment of systemic antitumor immunity that can be enhanced incombination with checkpoint blockade.

Disclosure of Potential Conflicts of InterestA.K. Moesta and J.B. Rottman hold ownership interest (including patents) in

Amgen. R. Ponce reports receiving other commercial research support from JunoTherapeutics. C. Beers is an employee of Tizona Therapeutics and is a consul-tant/advisory board member for Oncorus. P.J. Beltran holds ownership interest(including patents) in Amgen. No potential conflicts of interest were disclosedby the other authors.

Authors' ContributionsConception and design: A.K. Moesta, J.B. Rottman, T. Le, C. Glaus, R. Ponce,C. Beers, P.J. BeltranDevelopment ofmethodology:A.K.Moesta, K. Cooke, J. Piasecki, J.B. Rottman,K. Fitzgerald, J. Zhan, T. Le, K. Merriam, C. Glaus, K. Ganley, C. Beers,P.J. BeltranAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A.K. Moesta, J. Piasecki, P. Mitchell, J.B. Rottman,K. Fitzgerald, J. Zhan, B. Yang, T. Le, B. Belmontes, O.F. Ikotun, K. Merriam,C. Glaus, D.H. Cordover, A.M. BodenAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A.K. Moesta, K. Cooke, J. Piasecki, P. Mitchell,J.B. Rottman, K. Fitzgerald, T. Le, B. Belmontes, O.F. Ikotun, C. Glaus,D.H. Cordover, A.M. Boden, R. Ponce, C. Beers, P.J. BeltranWriting, review, and/or revision of themanuscript:A.K. Moesta, J.B. Rottman,K. Merriam, C. Glaus, R. Ponce, C. Beers, P.J. BeltranAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): P. Mitchell, T. Le, K. Merriam, D.H. Cordover,A.M. BodenStudy supervision: A.K. Moesta, K. Cooke, P. Mitchell, C. Glaus, C. Beers,P.J. Beltran

AcknowledgmentsWe thank Angela Chong for technical assistance with gene expression

profiling and Annie Mirsoian for assistance with flow cytometry panel design.We thank Emily Plummer who provided medical writing services on behalf ofAmgen Inc. Assistance with formatting the manuscript to meet journal speci-fications was provided by Gurpreet Kaur, CACTUS Communications, on behalfof Amgen Inc.

Grant SupportThe study was funded by Amgen, Inc.The costs of publication of this articlewere defrayed inpart by the payment of

page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received March 8, 2017; revised May 27, 2017; accepted July 10, 2017;published OnlineFirst July 13, 2017.

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References1. Mellman I, Coukos G, Dranoff G. Cancer immunotherapy comes of age.

Nature 2011;480:480–9.2. FridmanWH, Pages F, Sautes-Fridman C, Galon J. The immune contexture

in human tumours: impact on clinical outcome. Nat Rev Cancer 2012;12:298–306.

3. Galon J, Costes A, Sanchez-Cabo F, Kirilovsky A, Mlecnik B, Lagorce-PagesC, et al. Type, density, and location of immune cells within humancolorectal tumors predict clinical outcome. Science 2006;313:1960–4.

4. Vesely MD, Kershaw MH, Schreiber RD, Smyth MJ. Natural innate andadaptive immunity to cancer. Annu Rev Immunol 2011;29:235–71.

5. BarryM,BleackleyRC.Cytotoxic T lymphocytes: all roads lead todeath.NatRev Immunol 2002;2:401–9.

6. Weigelin B, Krause M, Friedl P. Cytotoxic T lymphocyte migration andeffector function in the tumor microenvironment. Immunol Lett 2011;138:19–21.

7. Pardoll DM. The blockade of immune checkpoints in cancer immuno-therapy. Nat Rev Cancer 2012;12:252–64.

8. DunnGP, Bruce AT, IkedaH,Old LJ, Schreiber RD.Cancer immunoediting:from immunosurveillance to tumor escape. Nat Immunol 2002;3:991–8.

9. Chen DS, Mellman I. Oncology meets immunology: the cancer-immunitycycle. Immunity 2013;39:1–10.

10. Ribas A. Tumor immunotherapy directed at PD-1. N Engl J Med 2012;366:2517–9.

11. Hamid O, Robert C, Daud A, Hodi FS, HwuWJ, Kefford R, et al. Safety andtumor responses with lambrolizumab (anti-PD-1) in melanoma. N Engl JMed 2013;369:134–44.

12. Liu BL, Robinson M, Han ZQ, Branston RH, English C, Reay P, et al.ICP34.5 deleted herpes simplex virus with enhanced oncolytic, immunestimulating, and anti-tumour properties. Gene Ther 2003;10:292–303.

13. Wollmann G, Ozduman K, van den Pol AN. Oncolytic virus therapy forglioblastoma multiforme: concepts and candidates. Cancer J 2012;18:69–81.

14. Harrington KJ, Hingorani M, Tanay MA, Hickey J, Bhide SA, Clarke PM,et al. Phase I/II study of oncolytic HSV GM-CSF in combination withradiotherapy and cisplatin in untreated stage III/IV squamous cell cancer ofthe head and neck. Clin Cancer Res 2010;16:4005–15.

15. Senzer NN, Kaufman HL, Amatruda T, Nemunaitis M, Reid T, Daniels G,et al. Phase II clinical trial of a granulocyte-macrophage colony-stimulatingfactor-encoding, second-generation oncolytic herpesvirus in patients withunresectable metastatic melanoma. J Clin Oncol 2009;27:5763–71.

16. Goldufsky J, Sivendran S, Harcharik S, Pan M, Bernardo S, Stern RH, et al.Oncolytic virus therapy for cancer. Oncolytic Virother 2013;2:31–46.

17. Chou J, KernER,Whitley RJ, RoizmanB.Mapping of herpes simplex virus-1neurovirulence to gamma 134.5, a gene nonessential for growth in culture.Science 1990;250:1262–6.

18. MacLean AR, ul-Fareed M, Robertson L, Harland J, Brown SM. Herpessimplex virus type 1 deletion variants 1714 and 1716 pinpoint neuroviru-lence-related sequences in Glasgow strain 17þ between immediate earlygene 1 and the `a' sequence. J Gen Virol 1991;72(Pt 3):631–9.

19. Markert JM, Medlock MD, Rabkin SD, Gillespie GY, Todo T, Hunter WD,et al. Conditionally replicating herpes simplex virus mutant, G207 for thetreatment of malignant glioma: results of a phase I trial. Gene Ther 2000;7:867–74.

20. RamplingR, CruickshankG, Papanastassiou V,Nicoll J,HadleyD, BrennanD, et al. Toxicity evaluation of replication-competent herpes simplex virus(ICP 34.5 null mutant 1716) in patients with recurrent malignant glioma.Gene Ther 2000;7:859–66.

21. Dranoff G, Jaffee E, Lazenby A, Golumbek P, Levitsky H, Brose K, et al.Vaccination with irradiated tumor cells engineered to secrete murinegranulocyte-macrophage colony-stimulating factor stimulates potent, spe-cific, and long-lasting anti-tumor immunity. Proc Natl Acad Sci U S A1993;90:3539–43.

22. Huang AY, Golumbek P, Ahmadzadeh M, Jaffee E, Pardoll D, Levitsky H.Role of bone marrow-derived cells in presenting MHC class I-restrictedtumor antigens. Science 1994;264:961–5.

23. Kaufman HL, Kim DW, DeRaffele G, Mitcham J, Coffin RS, Kim-Schulze S.Local and distant immunity induced by intralesional vaccination with anoncolytic herpes virus encoding GM-CSF in patients with stage IIIc and IVmelanoma. Ann Surg Oncol 2010;17:718–30.

24. National Research Council (US) Committee for the Update of the Guidefor the Care and Use of Laboratory Animals. Guide for the care and useof laboratory animals. Washington (DC): National Academies Press;2011.

25. Huang AY, Gulden PH, Woods AS, Thomas MC, Tong CD, Wang W, et al.The immunodominantmajor histocompatibility complex class I-restrictedantigen of a murine colon tumor derives from an endogenous retroviralgene product. Proc Natl Acad Sci U S A 1996;93:9730–5.

26. Alauddin MM, Conti PS. Synthesis and preliminary evaluation of 9-(4-[18F]-fluoro-3-hydroxymethylbutyl)guanine ([18F]FHBG): a new poten-tial imaging agent for viral infection and gene therapy using PET. NuclMedBiol 1998;25:175–80.

27. Leib DA, Harrison TE, Laslo KM, Machalek MA, Moorman NJ, Virgin HW.Interferons regulate the phenotype ofwild-type andmutant herpes simplexviruses in vivo. J Exp Med 1999;189:663–72.

28. Broz ML, Binnewies M, Boldajipour B, Nelson AE, Pollack JL, Erle DJ,et al. Dissecting the tumor myeloid compartment reveals rare activatingantigen-presenting cells critical for T cell immunity. Cancer Cell 2014;26:638–52.

29. Andtbacka RH, KaufmanHL,Collichio F, Amatruda T, SenzerN, Chesney J,et al. Talimogene laherparepvec improves durable response rate in patientswith advanced Melanoma. J Clin Oncol 2015;33:2780–8.

30. Andtbacka RH, Ross MI, Delman K, Noyes D, Zager JS, Hsueh E, et al.Responses of injected and uninjected lesions to intralesional talimogenelaherparepvec (T-VEC) in theOPTiM study and the contribution of surgeryto response. 2014 March 12–15, 2014; Phoenix, AZ. p Abstract 52.

31. Espy MJ, Uhl JR, Mitchell PS, Thorvilson JN, Svien KA, Wold AD, et al.Diagnosis of herpes simplex virus infections in the clinical laboratory byLightCycler PCR. J Clin Microbiol 2000;38:795–9.

32. Pandori MW, Lei J, Wong EH, Klausner J, Liska S. Real-Time PCR fordetection of herpes simplex virus without nucleic acid extraction. BMCInfect Dis 2006;6:104.

33. Biron CA, Brossay L. NK cells and NKT cells in innate defense against viralinfections. Curr Opin Immunol 2001;13:458–64.

34. Kaufman HL, Ruby CE, Hughes T, Slingluff CL Jr. Current status ofgranulocyte-macrophage colony-stimulating factor in the immunotherapyof melanoma. J Immunother Cancer 2014;2:1–13.

35. Vatner RE, Cooper BT, Vanpouille-Box C, Demaria S, Formenti SC. Com-binations of immunotherapy and radiation in cancer therapy. Front Oncol2014;4:1–15.

36. Woller N, Gurlevik E, Fleischmann-Mundt B, Schumacher A, Knocke S,Kloos AM, et al. Viral infection of tumors overcomes resistance to PD-1-immunotherapy by broadening neoantigenome-directed T-cell responses.Mol Ther 2015;23:1630–40.

37. ZamarinD,HolmgaardRB, Subudhi SK, Park JS,MansourM, Palese P, et al.Localized oncolytic virotherapy overcomes systemic tumor resistance toimmune checkpoint blockade immunotherapy. Sci Transl Med 2014;6:226ra32.

38. Zhan Y, Funda DP, Every AL, Fundova P, Purton JF, Liddicoat DR, et al.TCR-mediated activation promotes GITR upregulation in T cells andresistance to glucocorticoid-induced death. Int Immunol 2004;16:1315–21.

39. Moynihan KD, Opel CF, Szeto GL, Tzeng A, Zhu EF, Engreitz JM, et al.Eradication of large established tumors in mice by combination immu-notherapy that engages innate and adaptive immune responses. NatureMed 2016doi 10.1038/nm.4200.

40. Puzanov I, Milhem MM, Andtbacka RHI, Minor DR, Hamid O, Li A, et al.Survival, safety, and response patterns in a phase 1b multicenter trial oftalimogene laherparepvec (T-VEC) and ipilimumab (ipi) in previouslyuntreated, unresected stage IIIB-IV melanoma. J Clin Oncol 2015;33(suppl): Abstract 9063.

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2017;23:6190-6202. Published OnlineFirst July 13, 2017.Clin Cancer Res   Achim K. Moesta, Keegan Cooke, Julia Piasecki, et al.  

Associated Protein Blockade−T-Lymphocyte Antitumor Immune Responses Enhanced by Cytotoxic

Generates Systemic mGM-CSFLocal Delivery of OncoVEX

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