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Clinical Trials: Immunotherapy

Phase I Trial of an ICAM-1-TargetedImmunotherapeutic-Coxsackievirus A21 (CVA21)as an Oncolytic Agent Against Non Muscle-Invasive Bladder CancerNicola E. Annels1, David Mansfield2, Mehreen Arif1, Carmen Ballesteros-Merino3,Guy R. Simpson1, Mick Denyer1, Sarbjinder S. Sandhu4, Alan A. Melcher2,Kevin J. Harrington2, Bronwyn Davies5, Gough Au5, Mark Grose5, Izhar Bagwan1,Bernard Fox3, Richard Vile6, Hugh Mostafid1, Darren Shafren5, and Hardev S. Pandha1

Abstract

Purpose: The CANON [CAVATAK in NON–muscle-invasive bladder cancer (NMIBC)] study evaluated a novelICAM-1–targeted immunotherapeutic-coxsackievirus A21 as anovel oncolytic agent against bladder cancer.

Patients and Methods: Fifteen patients enrolled in this"window of opportunity" phase I study, exposing primarybladder cancers to CAVATAK prior to surgery. The first 9patients received intravesical administration of monotherapyCAVATAK; in the second stage, 6 patients received CAVATAKwith a subtherapeutic dose ofmitomycinC, known to enhanceexpression of ICAM-1 on bladder cancer cells. The primaryendpoint was to determine patient safety and maximumtolerated dose (MTD). Secondary endpoints were evidence ofviral replication, induction of inflammatory cytokines, anti-tumor activity, and viral-induced changes in resected tissue.

Results: Clinical activity of CAVATAK was demonstratedby induction of tumor inflammation and hemorrhage

following either single or multiple administrations ofCAVATAK in multiple patients, and a complete resolutionof tumor in 1 patient. Whether used alone or in combinationwith mitomycin C, CAVATAK caused marked inflammatorychanges within NMIBC tissue biopsies by upregulating IFN-inducible genes, including both immune checkpoint inhibi-tory genes (PD-L1 and LAG3) andTh1-associated chemokines,aswell as the inductionof the innate activator RIG-I, comparedwith bladder cancer tissue from untreated patients. No signif-icant toxicities were reported in any patient, from either virusor combination therapy.

Conclusions: The acceptable safety profile of CAVATAK,proof of viral targeting, replication, and tumor cell deathtogether with the virus-mediated increases in "immunologicalheat" within the tumor microenvironment all indicate thatCAVATAK may be potentially considered as a novel therapeu-tic for NMIBC.

IntroductionNon–muscle-invasive bladder cancer (NMIBC) is a highly

prevalent cancer with lifelong risk of occurrence. Transurethralresection (TUR) of all visible lesions is a standard treatment forNMIBC (1), but is accompaniedwith a high tumor recurrence rateranging from50% to 70%aswell as a high tumor progression rate

between 10% and 20% over a period of 2 to 5 years (2, 3). Thus,guidelines recommend intravesical chemotherapy and immuno-therapy in the management of NMIBC to reduce these risks ofrecurrence and progression (4). Immunotherapy with BacilleCalmette–Guerin (BCG) decreases frequency and delays time tocancer recurrence and progression in patients with NMIBC (1, 5).Unfortunately, one third of NMIBC patients experience seriousside effects of local and systemic BCG infection, and one third donot respond (6, 7). Treatment options for BCG-refractory patientsare limited, and patients often undergo cystectomy. Combinedwith a potential worldwide shortage of BCG (8), there is an urgentneed to develop novel therapies for this disease.

The success of BCG treatment forNMIBC stems, uniquely, fromthe BCG-induced immune response. Antigen-presenting cells inthe urothelium can phagocytize BCG, followed by the presenta-tion of antigen to BCG-specific CD4þ T cells. Proinflammatorycytokines are released, resulting in a predominant Th1-cell–induced immunity with an enhanced recognition of cancer cellsthrough activated macrophages, CD8þ T cells, natural killer cells,and other effector cells (9, 10). Oncolytic viruses are emergingimmunotherapeutic agents for a broad range of malignancies. Aswell as their ability to preferentially replicate in and lyse cancercells directly, it is their induction of host immunity which isincreasingly recognized to be the major component of their

1Faculty of Health and Medical Sciences, University of Surrey, Guildford, UnitedKingdom. 2Targeted Therapy Group, Institute of Cancer Research, London,United Kingdom. 3Robert W. Franz Cancer Research Center, Earle A. ChilesResearch Institute, Portland, Oregon. 4Kingston Hospital NHS Foundation Trust,Kingston upon Thames, Surrey, United Kingdom. 5Viralytics, Sydney, Australia.6Mayo Clinic, Rochester, Minnesota.

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

N.E. Annels and D. Mansfield are joint first authors of this article.

CorrespondingAuthor:Hardev S. Pandha, University of Surrey, Daphne JacksonRoad, Manor Park, Guildford, Surrey GU2 7WG, United Kingdom. Phone: 44-01483-688550; Fax: 011-44-01483-688558; E-mail: [email protected]

Clin Cancer Res 2019;25:5818–31

doi: 10.1158/1078-0432.CCR-18-4022

�2019 American Association for Cancer Research.

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antitumor efficacy (11). Coxsackievirus A21 (CVA21), a naturallyoccurring common cold-producing enterovirus, is one such effec-tiveoncolytic agent against a rangeof solid tumors (12–15).CVA21specifically targets and lytically infects susceptible cells expressingthe CVA21 cellular receptors, intercellular adhesion molecule-1(ICAM-1), and decay-accelerating factor (DAF; ref. 16). Our grouprecently demonstrated the susceptibility of bladder cancer cell linesto CVA21, the ability to enhance oncolysis by modulating expres-sion of the viral receptor ICAM-1 by low doses of mitomycin Ctreatment, and the induction of immunogenic cell death inCVA21-treated cell lines capable of generating long-lasting protectiveantitumor immunity in the bladder mucosa (17). These resultsprovided the rationale for a phase I/II clinical trial (CANON) toinvestigate the therapeutic potential of CVA21 as a new immuno-therapy approach for the treatment of NMIBC.

This trial determined safety, feasibility, and biological effects ofescalating intravesical doses of a novel bioselected formulation ofCVA21 (CAVATAK) administered alone or in combination withmitomycin C in 15 first-line NMIBC patients prior to TURBTsurgery.

Patients and MethodsParticipants

Eligiblepatientswere�18years of age,with a clinical diagnosis ofNMIBC based on cystoscopic appearance, and which was suitablefor TUR. Eastern Cooperative Oncology Group performance status� 2,ANC> 1,500/mm3;Hb>9.0 g/dL; Platelet>100,000/mm3, andserum creatinine � 1.5 mg/dL were required. Patients with priorlocal or systemic treatments for NMIBC were excluded.

The study was conducted in accordance with UK-recognizedethical guidelines and received approval from National ResearchEthics Committee (Ref 14/LO/1804). The patients provided writ-ten-informed consent for treatment and analysis of their biolog-ical samples. The trial is registered on ClinicalTrials.gov (identi-fier: NCT02316171). Supplementary Table S1 outlines thepatients and treatment characteristics.

Study design and outcome measuresCANON was a phase I, two-part, open-label, dose-escalation

study designed to evaluate the safety and clinical activity ofintravesical CAVATAK (Coxsackievirus A21, CVA21) alone andin sequential combination with low-dose mitomycin C in 16first-line patients with NMIBC who were candidates for andwere planning to undergo TUR for treatment and staging oftheir disease (see Supplementary Materials and Methods forinclusion and exclusion criteria). This gave a relatively homo-geneous study population and facilitated collection of resectedtumor tissue for histologic, pharmacodynamics, and pharma-cokinetic analyses.

The study consisted of two sequential parts. Part 1 (VLA012A)was a study of the safety and tolerability of CVA21 administeredvia intravesical instillation as a single agent in subjects withNMIBC scheduled to undergo TURBT for treatment and stagingof their disease. Three cohorts consisting of 3 subjects eachreceived CVA21 on day 1 in Cohorts A1 (1 � 108 TCID50) andA2 (3� 108 TCID50)with subject in Cohorts A3 receiving to dosesof CVA21 (3� 108 TCID50) on days 1 and 2. As these doses werewell tolerated, Part 2 (VLA012B) commenced. Part 2 (VLA012B)evaluated the safety and tolerability of CVA21 administered insequential combination with low-dose mitomycin C in the samesubject population. Two dose levels and two schedules ofadministration of CVA21 were evaluated with a fixed dose ofmitomycin C (10 mg) in two cohorts of subjects. Mitomycin Cwas administered by intravesical instillation on day 1, andintravesical instillation of CVA21 (3 � 108 TCID50) occurred4 hours after instillation of mitomycin C. In subjects scheduledto receive two instillations of CVA21 (3 � 108 TCID50), theinstillations were at approximately the same time each day.Both parts of the study were open-label, with ascending dosesand increased frequency of CVA21 dosing in a standard 3þ3design. After the MTD of the combination was established inVLA012B, up to 10 additional subjects were to be enrolled atthe MTD to further explore the safety and PD of the combi-nation. This part of the study was not executed due to theobjectives of the study being met and the sponsor deciding toterminate the study. The MTD was defined in the protocol asthe highest intravesical CVA21 dose at which no more than onesubject in each 3-subject cohort experienced a dose-limitingtoxicity (DLT). The period of observation for DLTs was from thetime of first administration to 7 days later.

The highest dose of intravesical CVA21 used in this study (3 �108 TCID50) on days 1 and 2 was well tolerated and may be usedin future studies. The safety and tolerability of the treatment ofCVA21 with and without low-dose mitomycin C were assessedthrough daily physical examinations, vital signs, and review ofhematology, serum chemistry, and urinalysis data. Treatment-emergent adverse events were also reviewed (SupplementaryMaterials and Methods and Supplementary Table S2). The mito-mycin C dose (SupplementaryMaterials andMethods) was basedon in vitro studies where ICAM-1 expression was upregulatedwithout cytopathic effects (CPE) on tumor cells. Cystoscopyphotography was performed before and after treatment. Intrave-sical treatment was followed by TURBT surgery after 8 to 11 dayswhich allowed tissues to be analyzed for virus replication, apo-ptosis, evidence of viral-induced changes in immune cell infil-trates, and immune checkpoint molecules. Serum and urine werecollected on day 1 (before virus instillation), 3, 5, and 8 after virustreatment.

Translational Relevance

This report describes the first "window-of-opportunity"study of the tumor microenvironment of non–muscle-invasive bladder cancer (NMIBC) following exposure to anovel oncolytic virus, CVA21. Posttreatment tissue was direct-ly visualized and available for study after resection. No cleardifferences were seen in tumors receiving CVA21 versus priorlow-dose mitomycin-C, an agent used to increase expressionof the CVA21 entry molecule, ICAM-1. Intravesical CVA21therapy was extremely well-tolerated, was highly tumor-selec-tive, and replicated in NMIBC causing inflammatory changes(immunological "heat") in tumor but not normal bladder.Upregulation of IFN response genes and immune checkpointgenes (PD-L1 and LAG3) was evident, supporting combina-tion studies with immune checkpoint inhibitor antibodies.NMIBC is regarded as a malignancy that responds well toimmunotherapy. Oncolytic viral therapy, alone or in combi-nation with immune checkpoint blockade, may offer analternative to the 40-year-old standard-of-care, BCG therapy,but without its limiting toxicities.

Viral Targeting of Non–Muscle-Invasive Bladder Cancer

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RNA extraction and viral RNA detectionAliquots of urine were clarified by centrifugation (1 minute,

10,000 RPM) and viral RNA extracted from140 mL of supernatantusing Qiagen QIAmp Viral RNA Mini Kit (Qiagen). Controlsamples spiked with 1 � 103 and 1 � 106 copies were alsoextracted as internal controls. For detection of viral RNA, 5 mLextracted RNA was tested in triplicate with 20 mL of Qiagen'sQuantifast pathogen RT-PCR þ IC kit. Primer and Probesequences are as follows: Forward primer (KKVP3fwd): 50-GAGC-TAAACCACCAACCAATCG-'3'; Reverse primer (KKVP3rev): 50-CGGTGCAACCATGGAACAA-'3'; Probe (KKVP3): 6FAM-CACA-CACATCATCTGGGA-MGB. Samples that tested positive werefurther tested by TCID50 assay to confirm replication-competent virus.

TCID50 assayAliquots of urine samples were thawed, vortexed, and centri-

fuged (1 minute, 10,000 RPM) and supernatant serially diluted(DMEM, 2% FBS) and incubated on 70% to 90% confluentmonolayers of SKMEL-28 cells, prepared in 96-well plates,24 hours previously. Plates were incubated for 5 days beforescoring wells positive/negative for CPE and calculating the viraltiter using the Karber method.

Neutralizing anti-coxsackievirus antibody assaySK-MEL-28 cells were plated 24 hours prior to the assay. Test

serumwas serially diluted in the range 1:4 to 1:32768, and 100 mLof each dilution was mixed with 100 mL of media containing 100TCID50 CVA21 and incubated at 37�C for 1 hour. Followingincubation, the serawere added to SK-MEL-28 cells and incubatedfor 72 hours with cytopathic effect (CPE) scored visually at theendpoint and 50% neutralizing titer calculated. Positive andnegative control serum was also tested, and viral titer was con-firmed by TCID50 with each assay.

ImmunohistochemistryNote that 4 mm sections were deparaffinized before antigen

retrieval, and endogenous peroxidase blocking was performed.Slides were incubated with mouse anti-enterovirus antibody(DakoCytomation), rabbit anti-cleaved Caspase 3 (Cell SignalingTechnology), rabbit anti-HMGB1 antibody (Abcam), and mouseanti–ICAM-1 antibody (Santa Cruz Biotechnology). Subsequent-ly, slides were incubated with a horseradish peroxidase–labeledsecondary antibody (DakoCytomation), followed by detectionwith diaminobenzidine solution and counterstained with hema-toxylin (VectorLabs).

Total RNA extraction from formalin-fixed paraffin-embeddedtumor tissues

Total RNA was isolated from paraffin-embedded tumor tissuesusing a Norgen FFPE RNA Purification kit (Norgen Biotek) as perthe manufacturer's instructions. RNA concentration and puritywere measured using an Agilent 2100 BioAnalyzer.

Human Cell Death PathwayFinder RT2 Profiler PCR ArraycDNAs were reverse transcribed from RNA extracted from

CAVATAK-treated bladder tumor tissues (see above) using theRT2 First Strand Kit (SABiosciences). Comparison of the relativeexpression of 12 cell death–related genes between the sampleswas made with PCR Array: the cDNAs from those samples werecharacterized using a custom-designed Cell Death PathwayFinder

RT2 Profiler PCR Array (SABiosciences) and the RT2 SYBR Green/Rox PCR master mix (SABiosciences) on a Stratagene Mx3000PThermal Cycler. ATP5B and GAPDH housekeeping genes wereused for normalization (18), and data were analyzed with theDDCt method.

Multispectral IHC analysisTissue sections were cut at 4 mm from formalin-fixed paraffin-

embedded blocks. All sections were deparaffinized and subjectedto heat-induced epitope retrieval in citrate buffer (pH 6.0; Bio-genex). Note that 6-plex panel IHC was performed for each tissueslide using the following antibodies: anti-FoxP3 (clone 236A/E7,Abcam), anti–PD-L1 (clone E1L3N, Cell Signaling Technology),anti-CD8 (clone SP16, Spring Bioscience), anti-CD3 (clone SP7,Spring Bioscience), anti-CD163 (clone MRQ26, Ventana), andanti-Cytokeratin (clone AE1/AE3, DAKO). Antigen–antibodybinding was visualized with TSA-Cy5, TSA-Cy3, TSA-FITC, TSA-Cy5.5, TSA-Coumarin (PerkinElmer), and TSA-Alexa594 (LifeTechnologies). Microwave treatment in citrate buffer (pH 6.0)was performed between antibody detection to prevent cross-reactivity. Tissue slides were counterstained with DAPI and cover-slipped with VectaShield mountingmedia (Vector Labs). Controltissue samples were stained for each different marker. Hematox-ylin and eosin staining was performed for each sample andreviewed by a pathologist to ensure the representativity of thetissue sample. For a detailed IHC protocol for multispectralanalysis, see study by Feng and colleagues (19).

Microscopy and image analysis of multiplexed IHC: phenotypecell quantification in high-resolution images

Digital images were captured with PerkinElmer Vectra 2.0platform following hot spot lymphocyte assessment: Tumor areaswith the highest immune cell (CD3þCD8þ) infiltrates werescanned at 20X and selected for analysis. Three images of 0.36mm2 each per tissue sample were analyzed with InForm Software(PerkinElmer). The total number of cells were enumerated for thefollowing phenotypes: PD-L1þ tumor cells, PD-L1þ other cells,CD3þPD-L1þ, CD3þPD-L1- FoxP3þ, CD3þCD8þPD-L1þ,CD3þCD8þPD-L1-, CD163þPD-L1þ, CD163þPD-L1- in the stro-ma and tumor compartment.

Detection of urinary HMGB1Urine samples of patients and controls were tested using a

commercially available HMGB1 ELISA kit (ST51011; IBL Inter-national GmbH). The assay was conducted according to themanufacturer's instructions. Urinary HMGB1 was expressed asHMGB1/Cr ratio (mg/mmolCr) to correct for differences indilution.

Detection of urinary cytokine levelsQuansys Biosciences was contracted to test the urinary cytokine

levels in 12 CANON patients, 15 untreated NMIBC patients, and13 healthy controls using their Q-Plex Human Cytokine multi-plexed ELISA array. Thawed samples were diluted with the appro-priate Quansys sample dilution buffer at a ratio of (sample:buffer) 1:2 (50%). Polypropylene low-binding 96-well plateswere used to prepare the samples and standards prior to loadingthe Q-Plex plate. Each dilution was measured in triplicate, a totalof 3 wells per sample. The intensity of chemiluminescence fromeach array was measured using the Q-View chemiluminescent

Annels et al.

Clin Cancer Res; 25(19) October 1, 2019 Clinical Cancer Research5820




imager (Quansys Biosciences) and quantified using the Q-Viewsoftware (Quansys Biosciences).

NanostringAll RNA samples included in the study passed quality control

requirements (as assessed by the RNA integrity number) of theplatform. Digital multiplexed NanoString nCounter analysis sys-tem (NanoString Technologies)-based gene expression profilingwas performed on 100 ng total RNA from each sample as inputmaterial according to themanufacturer's instructions. NanostringRNA analysis of 700 immune-related genes was performed usingthe nCounter GX Human PanCancer Immune profiling Kit (XT)on the nCounter Analysis System. Analysis and normalization ofthe raw Nanostring data were performed using nSolver AnalysisSoftware v1.1 (Nanostring Technologies).

Statistical analysisCorrelations were evaluated by the Pearson tests. All P values

were calculated using two-tailed test. P values < 0.05 were con-sidered statistically significant. Analyses were performed usingGraphPad Prism.

ResultsPatients, study design, and toxicity of the trial

Fifteen patients were recruited into this study. All patientspresented with cystoscopically visible bladder tumors positivefor urine cytology and were scheduled to undergo a TUR of theirbladder tumor (TURBT) as part of their standard clinical care. Thepatients' clinical characteristics are shown in SupplementaryTable S1, and the design of the clinical trial (CANON) is illus-trated in Fig. 1A and in Supplementary Materials and Methods.Intravesical administration of CAVATAK either as a single agent orin combination with mitomycin C was generally well toleratedwith no grade 2 or higher product-related adverse events observed(Supplementary Table S2). Six patients developed urinary tractinfections whichwere attributed to displacement of bacteria fromthe urethra into the bladder during catheterization. This wasprevented in further patients by a 5-day course of oral amoxicillincommencing the day of first catheter insertion. The primaryendpoint of this trial was to determine patient safety and MTD.Secondary objectives were assessment of viral replication, anti-tumor activity, and viral-induced changes in immune cellinfiltrates.

Figure 1.

A and B, Tumor response: pre- and posttreatment cystoscopy. Cystoscopic photographywas performed in all subjects to record the appearance of their tumorbefore and after intravesical CAVATAK treatment. Representative images pre- and post-CAVATAK treatment are shown for 2 patients (B001 and B008).






Tumor response: pre- and posttreatment cystoscopyThe clinical trial design of CANON (Fig. 1A), administering

CAVATAK a week before the patients underwent a TURBT fromwhich tissue was then available to study, gave a uniquewindow of opportunity to study the effect of this oncolyticvirus on bladder cancer. Response was defined as complete(disappearance of tumor, i.e., pathologic complete response)or macroscopic increase of hemorrhage compared with pre-treatment. Due to the heterogeneous size, shape, and preexist-ing hemorrhage/necrosis, only observational comparison waspossible. Cystoscopic photography was performed in all sub-jects to record the appearance of their tumor before and afterintravesical CAVATAK treatment. As shown in Fig. 1B, 8 to11 days following intravesical CAVATAK treatment surfacehemorrhage and inflammation of tumors was observed in anumber of the patients, and one complete response wasconfirmed by histology. Supplementary Fig. S1 shows the urinecytology slide from this complete responder (patient B008)confirming he did have bladder cancer prior to the intravesicalCAVATAK treatment.

Viral-induced antitumor activity in TUR tissueAll patients underwent surgery 8 to 11 days after treatment

with CAVATAK monotherapy or in combination with mitomy-cin C, which allowed us to evaluate specific tumor targeting ofthe virus in the resected tumors. IHC staining for enterovirusprotein indicated viral infection in 12 of 14 tissues available forstudy with varying amounts of positive tumor stainingobserved (Fig. 2A). No viral staining was detected in surround-ing stromal areas or in areas of tumor tissues displaying normalglandular change within urothelium (Fig. 2B) consistent withselective CAVATAK targeting to, and/or replication in, malig-nant cells. Viral protein positivity in the tumor appeared to becorrelated with those tumors that displayed at least localizedareas of ICAM-1 positivity, in keeping with our previous workshowing the necessity of ICAM-1 for CVA21 infection of blad-der cancer cells (17). One patient (B004) displayed extensivehomogeneous ICAM-1 expression by the tumor cells, n ¼ 5displayed < 30%, n ¼ 4 < 10% ICAM-1 tumor positivity, and n¼ 4 no ICAM-1 tumor positivity was detected. IHC analysis ofthe other CVA21 cellular receptor, DAF, showed strong homo-geneous DAF expression by the tumor cells in all patient cases(Supplementary Fig. S2). Hematoxylin and eosin–stained sec-tions of the viral-positive tissues showed areas of nonviabletumor and apoptotic bodies which was further confirmed bystaining for cleaved caspase 3. Tissues that demonstrated min-imal to no viral infection also displayed little to no cleavedcaspase 3 positivity and were confirmed as viable tumor fromthe hematoxylin and eosin stains. The detection of significantareas of nonviable tumor based on hematoxylin and eosinstaining as well as cleaved caspase 3 was not observed inuntreated historic bladder cancer control tissues (data notshown) providing further evidence that the increased tumorcell apoptosis observed in the viral-positive tumors was due toCAVATAK-induced cell death.

Previous preclinical work from our group showed that themode of CAVATAK-induced cell death in bladder cancer cell lineswas predominantly by apoptosis (17). To ascertain the cell deathroute taken by bladder cancer cells in response to CAVATAKinfection in this human clinical setting, we analyzed the totalRNA extracted from the CAVATAK-treated tumors using a custom

Human Cell Death PathwayFinder RT2 Profiler PCR Array. Usingthis array, the expression of key genes important for the centralmechanisms of cellular death, apoptosis, autophagy, and necro-sis, was profiled, and their expression levels were compared witharchival untreated bladder cancers. As shown in Table 1, genesencoding the intrinsic apoptotic cell death pathway (BCL2L1,BAK1, and caspase-9)were predominantly upregulated comparedwith untreated bladder cancer in the majority of patient tumorsstudied. Three of the tumors also displayed upregulation of one ofthe main players in programmed necrotic cell death, the serine/threonine kinase receptor-interacting protein 3 (RIPK3). In addi-tion, seven of the tumors displayed a significant increase in theimmunogenic cell death marker, Calreticulin.

Increases in infectious virus in patient urine followingintravesical CVA21 administration

To measure viral replication within the patients' tumors, levelof viral shedding into the urine was assessed by analysis of viralcopy number by RT-PCR, and retrieval of replication-competentviruswas determined by adding urine toCVA21-sensitive SKMEL-28 cells in a tissue culture infectious dose (TCID50) assay. Betweendays 2 and 5 following the initial instillation of CAVATAK, allpatients from both the monotherapy and combination cohortsshowed an increase in viral levels with several patients (4, 5, 6, 7,8, 11, and 15), demonstrating a second peak at later time pointssuggesting more than one cycle of viral replication within thebladder of these patients (Fig. 3).

Urinary cytokine profile in CAVATAK-treated patientsWe investigated the ability of CAVATAK to modulate the

immune response in the NMIBC microenvironment as reflectedby shed urinary cytokines in 12 CANON patients, and comparedwith 15 untreated NMIBC patients and 13 age-matched healthycontrols. A 17-plex quantitative ELISA-based chemiluminescentassaywas used to screen the urines of CANON trial patients beforetreatment at day 1 and then after treatment on days 3, 5, and 8(with the exception of patient B005where nod8urine samplewasobtained). Figure 4A shows the kinetics of the CAVATAK-inducedcytokine response to 8 of the 17 cytokines studied in 6 represen-tative patients. Although the cytokine levels did not vary signif-icantly over the treatment period in the majority of patients,patient B004 was the exception displaying a peak in the level ofvirally induced cytokines (IL6, IL1a, IL1b, andTNFa) at day 3 afterinfection which then declined with a second increase from day 5.Notably, 4 of the virus-treated patients (B001, B005, B008, andB010), including the patient (B008) that demonstrated a com-plete response to CAVATAK, displayed increased levels of theproinflammatory cytokine IL23 following treatment which wasconsistently undetectable in the untreated NMIBC patients andhealthy controls (data not shown).

Increases in urinary HMGB1 levels in CAVATAK-treatedpatients

Having previously shown the release of HMGB1 by CVA21-treated bladder cancer cells in vitro and the importance of thispotent cytokine for directing an immune response (17, 20),HMGB1 levels were assessed in the urine of the virus-treatedpatients. Figure 4B shows the increases in urinary levels ofHMGB1 in selected patients (6 of 11 assessed) after CAVATAKtreatment using creatinine-normalized values of HMGB1 (pgHMGB1/mg creatinine).

Annels et al.





Interestingly, IHC analysis of this cytokine showed increasedexpression of cytoplasmic HMGB1 in CAVATAK-treated tumortissues when compared with untreated bladder cancer controls(Fig. 4C). The untreated bladder cancer control tissues displayed apredominantly nuclear localization of HMGB1 consistent withthat seen in the positive control tissue, normal kidney. This resultsuggests that CAVATAK infection may trigger the translocation ofHMGB1 to the cell cytoplasm whence, upon cell death, it can bepassively released to the extracellular environment, in keepingwith the increased levels of urinary HMGB1 in virus-treatedpatients.

CAVATAK induces upregulation of PD-L1 in the NMIBCmicroenvironment

A multispectral IHC method which allowed the simultaneousdetection of 7markers was employed to investigate changes in theimmune microenvironment of CAVATAK-treated NMIBCs com-paredwith control-untreated archival cases ofNMIBCandnormalbladder tissue. Quantitation of the CD8þ T-cell infiltrationrevealed no significant differences between the virus-treatedtumors and untreated bladder cancer or normal bladder tissuein either the stromal regions or intraepithelial regions (Supple-mentary Fig. S3). In addition, despite having shown in a previous

Figure 2.

Intravesical CAVATAK selectively targets tumor cells within the bladder. A, IHC images showing representative cases displaying ICAM-1 positivity, enterovirusprotein (VP-1), and cleaved caspase 3 (positive staining shown in brown). The hematoxylin and eosin images of the same cases show the extent of tumornecrosis/apoptotic bodies outlined by the broken black line. Magnification, x20. B, Left (Patient B006): Anti-enterovirus positivity by tumor cells compared withabsence of anti-enterovirus staining on normal urothelium (indicated by broken line). Right (Patient B004): Anti-enterovirus staining (brown) only present ontumor cells and not on stromal cells and lymphocytic infiltrate.






preclinical study an enhanced uptake of the virus in mitomycinC–treated bladder cancer cell lines, this treatment did not result inan increased CD8þ T-cell infiltration in the patient tumors com-pared with control tumor tissues. However, notably, the patient(B008) who experienced a complete response to CAVATAK treat-ment showed a considerable immune infiltrate within the biopsytaken from the bladder area where the tumor had previously beenidentified (Supplementary Fig. S4). The 7-marker multispectralanalysis also revealed an increase in PD-L1 within the stromalareas of the CAVATAK-treated tumors compared with the controlbladder tissues, although this did not reach statistical significance(Supplementary Fig. S3).

CAVATAK induces upregulation of immune response genesTo further explore the immune response to CAVATAK, Nano-

string Pan-Cancer Immune profiling was performed on RNAderived from 12 CAVATAK-treated and 7 untreated NMIBCs.CAVATAK led to elevated expression of the IFN-inducible genesIFIT1, IFIH1, OAS3, and MX1, compared with levels seen inuntreated NMIBC control tissues. Furthermore, CAVATAK led tohigher expression of the IFNg-induced chemokine genes encod-ingCXCL9, CXCL10, andCXCL11 (Fig. 5A) associatedwith a Th1-mediated immune response.

Due to the knownbarriers that hamper oncolytic virus-inducedantitumor immune responses, we were interested to explore theexpression levels of immune checkpoint molecules and immu-nosuppressive enzymes within the CAVATAK-treated tumorscompared with untreated archival NMIBCs. As shown by thehistograms in Fig. 5A, in CAVATAK-treated tumors, there was anincrease in the expression level of the immune checkpoint mole-cules PD-L1 and LAG3 as well as the amino acid–depletingenzyme, indoleamine 2,3-dioxygenase (IDO). These immuno-suppressive mechanisms could be dampening T-cell responsesand thus limiting the efficacy of CAVATAK, thus providing arationale for future combination therapies using immune check-point inhibition or IDO-1 inhibition.

Another notable result from the Nanostring immune profilingwas the significant upregulation in CAVATAK-treated tumors ofthe gene DDX58 which encodes the RIG-I–like receptor dsRNA

helicase enzyme (Fig. 5B). This specific upregulation of DDX58further confirms the infection by and sensing of CAVATAKwithinthese tumors and is in keeping with the induction of a type I IFNresponse.

ICAM-1 expression is essential for productive CAVATAKinfection

Previous work from our group has clearly shown the impor-tance of ICAM-1 expression levels on bladder cancer cell lines inorder to obtain a sufficient level of CAVATAK infection andsubsequent oncolytic effect (17). This finding is further exempli-fied in this clinical study by one particular patient (B004) who,despite receiving the lowest dose of virus (single CAVATAK doseof 1 � 108), demonstrated significant viral infection and viral-induced changes in their bladder tumor. Although all the patients'tumors expressedDAF,many of the patients' tumors only showedfocal regions of ICAM-1 tumor positivity. Notably, patient B004'sresected CAVATAK-treated tumor displayed a high level of ICAM-1 expression throughout the tumor, enabling a high level of virusinfection and apoptotic tumor cell death (Supplementary Fig.S5B). Furthermore, the virus-treated tumor displayed cytoplasmicHMGB1 expression, a high level of urinary cytokines (Fig. 4A),immune infiltration, increased levels of stromal PD-L1, and ahighlevel of perforin expression indicating a clear immune response tothis productive infection (Supplementary Fig. S5B). Perforin-positive cells were also detected in four other CANON patients(B006, B007, B008, and B010), but not at the same high prev-alence (Supplementary Fig. S5A). Such perforin-positive cellswere not detected in untreated bladder cancer (n ¼ 10 tissuesstudied) and normal bladder tissues (n ¼ 10 tissues studied).

DiscussionNMIBC remains a highly prevalent and significant health

problem worldwide, requiring intrusive surveillance and highhealth economic costs. It is the prototypical example of successfulimmunotherapy with high primary response rates to BCG likelyinvolving a combination of innate and adaptive immuneresponses initially to BCG, followed by tumor-specific T-cell

Table 1. CAVATAK induces the intrinsic apoptotic cell death pathway in bladder tumors

Canon Fold increase in cell death pathway genes compared with untreated bladder cancerCohort patient Caspase8 BCL2L1 Caspase9 BAX BAK1 Caspase3 Caspase7 RIPK3 HMGB1 Calreticulin

A1 1 � 108 CVA21 B001 20.1 24547.43 20217.04 6.9 23062.87 23959.07 65231.64 63009.61 11.6 7848.95B003 1.05 10 3198.73 0.81 1811.9 ND ND ND 2.21 NDB004 2.4 ND ND 2.4 6453.13 ND ND ND 11.26 ND

A2 3 � 108 CVA21 B005 2.38 7260.15 2462.29 0.85 1433.96 ND ND ND 0.83 4198.87B006 1.21 3630.08 ND 1.12 1765.41 ND ND ND 0.49 4707.63B007 1.38 2648.18 10 0.69 2938.34 ND ND ND 1.11 2027.92

A3 (3 � 108)x2 B010 1.45 ND ND 0.72 1630.14 ND ND ND 0.69 NDCVA21 B011 .187 ND ND 1.24 ND ND ND ND ND ND

B1 (1 � 10�)x2 B012 1.56 ND ND 0.63 1424.05 ND ND ND 0.59 NDCVA21þMitC B013 6.18 4531.54 3294.36 3.07 2979.35 ND 12466.63 25723.23 2.33 3470.15

B2 (3 � 108) x2 B015 3.95 6375.33 5001.98 2.27 4294.53 12188.93 4523.69 ND 3.3 5036.77CVA21þMitC B016 137.75 43036.83 56590.97 22.49 180705.9 29191.98 106706.2 476885.8 31.04 3870.45

LOWEST HIGHEST

NOTE: Total RNA was isolated from the CANON trial patient paraffin-embedded tumor tissues and from archival untreated non–muscle-invasive bladder tumorblocks (n ¼ 6). The cDNAs reverse transcribed from the RNA were characterized using a custom-designed Cell Death PathwayFinder RT2 Profiler PCR Array(SABiosciences). The results are displayed as the fold increase in each gene compared with the average expression for that gene from n ¼ 6 untreated bladdercancers.Abbreviation: ND, not determined.

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Figure 3.

Increased levels of CAVATAK in patient urine indicate secondary viral replication within the bladder. Viral shedding was assessed in the urines of patients beforeand between days 2 and 5 after initial intravesical treatment with CAVATAK. The blue line represents the analysis of viral copy number by RT-PCR, and the redline depicts the levels of replication-competent virus as determined by a TCID50 assay. Indications of secondary viral replication were evident in patients 4, 5, 6, 7,8, 11, and 15.






responses (9). The unrestricted BCG-induced vigorous inflam-matory response in the entire bladder mucosa is highly problem-atic and often results in incomplete treatment programs.

Oncolytic viruses are emerging as highly potent cancer immu-notherapeutics, which in many ways have similar properties toBCG (21). They target cancers through direct cytotoxicity and alsoinduce cancer-specific adaptive immunity. Their ability to convert

"cold" tumors into "hot" tumors through inflammation makesthem potential partners for combination with immune check-point inhibitors (22), and there are currently a large number ofclinical trials ongoing with this combination. This includesdelivery of CVA21 into metastatic melanoma deposits alone(CALM, ClinicalTrials.gov Identifier: NCT01227551) or com-bined with anti–CTLA-4 antibody (MITCI ClinicalTrials.gov

Figure 4.

A, Urinary cytokine levels in patients followingCAVATAK treatment. The Q-Plex HumanCytokine multiplexed ELISA array (QuansysBiosciences) was used to assess the level andkinetics of the CAVATAK-induced cytokineresponse in the urine of CAVATAK-treatedpatients on days 1, 3, 5, and 8 after viraltreatment. Representative results are shownfor 6 patients. B and C, CAVATAK-specificinduction of HMGB1. B, Urinary HMGB1levels were measured in the trial patients onday 1 (before CAVATAK treatment) and thenon days 3, 5, and 8/9 after viral treatmentusing an HMGB1 ELISA kit (ST51011; IBLInternational GmbH). Urinary HMGB1 wasexpressed as HMGB1/Cr ratio (mg/mmolCr) tocorrect for differences in dilution. Six of the 11patients tested showed amarked increase inHMGB1 levels on day 3, 48 hours after viraltreatment. C, Screening of HMGB1 expression(brown staining) in untreated non–muscle-invasive bladder cancer (3 representativecases shown: control 1–3) and CAVATAK-treated bladder cancer (3 representative casesshown: B004, B003, and B007) by IHC. Theuntreated bladder cancer cases showednuclear localization of HMGB1 consistentwith that seen in the control tissue, normalhuman kidney. In contrast, the CAVATAK-treated bladder cancers expressed HMGB1within the cytoplasm (magnification, x20;inset images, x40).

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Identifier: NCT02307149) and anti–PD-1 (CAPRA, Clinical-Trials.gov Identifier: NCT02565992). These studies confirmedthat CVA21 exposure causes a marked immune inflammatoryand very durable local and abscopal antitumor response.

NMIBC is an ideal model for oncolytic immunotherapy witheasy and direct access to bladder cancer, their visualization,delivery of high doses of virus, no risk of neutralizing antibodylimitation, complete control of length of exposure through

Figure 5.

CAVATAK treatment upregulates IFN-induced genes and immune checkpoint molecules within the microenvironment of NMIBC tissue. Nanostring Pan-CancerImmune profiling was performed on RNA derived from 12 CAVATAK-treated and 7 untreated NMIBCs. A, CAVATAK treatment induced elevated expression ofthe viral RNA response genes IFIT1, IFIH1, OAS3, andMX1, and IFNg-induced chemokine genes encoding CXCL9, CXCL10, and CXCL11 compared with levels seen inuntreated NMIBC control tissues. Of the immune checkpoint inhibitory and immunosuppressive genes, PD-L1, PD-L2, and LAG3 were upregulated in theCAVATAK-treated tumors compared with untreated NMIBC controls. B, CAVATAK treatment induced elevated expression of the RIG-I–like receptor dsRNAhelicase enzyme that is encoded by the DDX58 gene. The levels of significance as determined by an unpaired t test which refer to the average expression ofupregulated genes in the CAVATAK-treated patients compared with the average expression of untreated NMIBC control tissues are indicated: � , P < 0.05;�� , P < 0.01; �� , P < 0.001.






catheterization, and sequencing of repeat doses and/or com-bination treatments based on existing schedules used for BCGtherapy.

Our preclinical study had indicated ICAM-1–targeted cytotox-icity after CVA21 exposure and the inductionof immunogenic celldeath in bladder cancer cell lines, enhanced by exposure to low,noncytotoxic doses of mitomycin-C through induction of ICAM-1 expression (17). DAF expression was not dysregulated acrossnumerous cell lines andwas not predictive of CAVATAK infection.In the current neoadjuvant clinical study, 15 patients receivedintravesical CVA21 � low-dose mitomycin C prior to tumorresection. The purpose of the study was to determine safety andtolerability for this approach, but also biological endpoints as a"window of opportunity" approach including tumor response,changes in the immune environment, and evidence of virusreplication. Importantly, the study was not attempting to com-pare efficacy or immunogenicity with BCG, but aimed to providea clear picture of immunomodulatory effects of CAVATAK intreatment-na€�ve tissue. All patients had detailed prior imaging toexclude locally advanced and metastatic disease. All patients hadpositive urine cytology, but evidence of muscle invasion wouldhave only been possible after TURBT: this was not evident in anypatient post-TURBT, and despite the cohort all being urine cytol-ogy positive, not all were high-grade tumors. The treatment wasextremely well-tolerated with minor toxicities reported. Objec-tively, clinical photography indicated virus-induced hemorrhagicand inflammatory changes in 3 patients, but notably there was noevidence of a general inflammation of the normal bladder, incontrast towhatwould be expectedwith BCG therapy. The patientwith complete response showed clear evidence of acute inflam-mation within the preexisting area of tumor within the bladderproviding further evidence for the role of the immune response inthe therapeutic outcome of this viral treatment.

We found evidence of secondary viral replication in mostpatients in urine samples between 2 and 5 days after CVA21treatment by both viral copy number by RT-PCR and TCID50,representing replication-competent virus production. IHC stain-ing for enteroviral protein was shown to be specifically associatedwith cancer cells and not normal urothelium in all but 2 patients(in whom the tissue was nonevaluable due to damage atresection).

After intravesical delivery of CVA21, no virus was detectedin serum in any patient, nor was there evidence of inductionof a systemic neutralizing antibody response in either cohort(Supplementary Table S3). After systemic delivery of CVA21 inan ongoing study (STORM, https://clinicaltrials.gov/ct2/show/NCT02043665), approximately 80% of patients demonstrated aneutralizing antibody response at day 8 after treatment (personalcommunication with Viralytics Ltd.).

Wepreviously demonstrated the importance of ICAM-1 expres-sion for CVA21 uptake in bladder cancer cell lines (17). In thisstudy, ICAM-1 expression was mostly focal, in 8 of the 14 patienttumors studied. This proportion was lower than expected andmaybe explained by the paucity and quality of tissue after TURBT,and heat artifacts to adjacent tissue. Indeed, the importance ofICAM-1 expression for a productive CVA21 infection was clearlydemonstrated by one particular patient (B004) whose tumordisplayed high, widespread ICAM-1 expression which was asso-ciated with high levels of virus protein expression, apoptosis,HMGB-1 in urine, urinary cytokines, and immune infiltrationwith PD-L1 induction.

Virus infection was associated with apoptosis, determined bycleaved caspase 3 expression in patient tumor tissues. In preclin-ical studies, we found the mode of cell death in bladder cancerlines was through the intrinsic apoptosis pathway; in patienttissue, we found this was also the case in the majority of caseswith additional evidence of necroptosis in 3 patients. Usingbladder cancer cell lines, we had previously determined thatCVA21 induces a type of cancer cell death that is immunogen-ic (17, 23). In this clinical study, 6 of 11 patients, despite wideintra-and interpatient variations in urine pH, specific gravity, andmetabolite content, showed increased levels of one of the hall-marks of immunogenic cell death, high mobility group box 1(HMGB1) in the urine post-CVA21 treatment. Furthermore, IHCanalysis of HMGB1 expression in the tumor tissues of the studypatients revealed a greater degree of cytoplasmic expression.Unfortunately, we were unable to correlate the cytoplasmicHMGB1 expression revealed in tissue with peaks in urinaryHMGB1 as we excluded any patient urine samples that displayedhematuria to prevent false-positive results. HMGB-1 is one of themost abundantly secreted DAMPs following oncolytic virusinfection (24–28) and is thought to aid the therapeutic efficacyof oncolytic viral therapy by acting as a potent immunostimula-tory molecule and chemoattractant for monocytic cellular infil-tration during virus infection (29).

In addition to levels of HMGB1 in the urine, the levels ofinflammatory cytokines were also evaluated to see whether theyreflected local immunomodulation caused by CVA21 infection.Such an analysis of urinary cytokine profiles has previouslybeen used to determine responders and nonresponders to BCGtherapy (30). Looking at the kinetics of the cytokine responsesin individual patients, there were increases in classical virallyinduced cytokines (31) on day 3 after infection, often with asecond peak at day 8. Interestingly, one cytokine that did differbetween bladder cancer controls and CVA21-treated patientswas the levels of IL23 which was consistently undetectablein the untreated NMIBC patients, but was elevated in four ofthe virus-treated patients, including the patient that demon-strated a complete response to CAVATAK. IL23 has been shownto have significant antitumor effects in various models of cancerand is associated with the promotion of cell-mediated immuneresponses and activation of cytotoxic T lymphocytes (CTL) ornatural killer (NK) cells (32–35).

One of the key aims of the study was to induce immunological"heat" into the NMIBC tumor microenvironment we have pre-viously observed as relatively "cold." Multispectral IHC analysisdid not show significant differences in the quantitation of CD8þ Tcells in the stromal or intraepithelial regions between the CAVA-TAK-treated and untreated bladder tumors. Although CAVATAKtreatment did not appear to result in increased TILs, furtheranalysis revealed that the CD8þ TILs that were present in thevirus-treated tumors displayed amore activated phenotype basedon perforin expression, and several cases revealed higher PD-L1expression in the stroma. Interestingly, although NK cell activa-tion has been shown to be a prominent feature of the immuneresponse to BCG (36, 37), CD56 IHC staining of these virus-treated tumors did not show any evidence for significant NK cellinvolvement in the immune response to CAVATAK in the bladdertumor microenvironment.

An even more comprehensive analysis of the expression ofimmune response genes in patient tissue compared with tissuefrom untreated, surgically resected NMIBC of similar tumor stage

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https://clinicaltrials.gov/ct2/show/NCT02043665https://clinicaltrials.gov/ct2/show/NCT02043665https://clinicaltrials.gov/ct2/show/NCT02043665http://clincancerres.aacrjournals.org/

and grade was performed using Nanostring analysis. UntreatedNMIBC had significant expression of genes associated with localimmunosuppression such as IDO and ARG-2. Data have alreadyindicated that such IDO gene expression is a feature of moreaggressive NMIBC, emphasizing the potential immunosuppres-sive role of IDO (36). As expected, as a result of virus infection/replication, the induction of IFN-inducible genes (IFIT1, IFIH1,MX1) and IFNg-induced chemokine genes was observed. Inkeeping with CVA21-associated induction of IFN, we also foundmarked increases in expression of immune checkpoint moleculesPD-L1 and LAG-3 as well as the amino acid–depleting enzyme,IDO presumably triggered to dampen down the immuneresponse to CVA21. The pattern of response is very similar tochanges observed after intratumoral injection ofmetastaticmalig-nant melanoma in the studies mentioned earlier (CALM andCAPRA. We demonstrated activation of innate immunity byCVA21-infected tumors by the induction of RIG-I gene (retinoicacid–inducible gene I) expression. RIG- is a RIG-I–like receptordsRNAhelicase enzyme that is encoded by theDDX58 gene. RIG-Iis part of the RIG-I–like receptor family, which also includesMDA5 and LGP2, and functions as a pattern recognition receptorwhich is a sensor for a number of viruses (38).

Although this study has obvious limitations in terms of size,some heterogeneity of patient tumor size, and distribution, itdoes provide valuable insights into the potential efficacy andutility of oncolytic immunotherapy for this condition. Thestudy ended at the point of TURBT resection so although nosafety concerns arose from any patient at any time after thestudy, formal follow-up tumor evaluations were not possiblewithin the scope of the study. The scheduling of mitomycin-C/CAVATAK was based on in vitro demonstration of ICAM-1upregulation following mitomycin C (within 24 hours), butmay have been suboptimal in the trial context. The circa 11 dayswas judged by the ethics committee as the limit of an accept-able temporary delay of the TURBT procedure. The translation-al aspects were limited by tissue quality following surgery, andmeasuring urinary markers such as cytokines and HMBG-1 ischallenging. We were unable to take distant random bladderbiopsies for comparison at surgery due to the risk of tumorseeding. Although Nanostring allowed evaluation of all of theavailable tumors, multispectral analysis is limited by its selec-tivity and focus on very small areas within the whole tumorsample. The role and value of mitomycin C for ICAM-1 mod-ulation were still unclear.

Previously, both replicating and nonreplicating viruses havebeen evaluated in preclinical models of bladder cancer (39–43).In human trials, intravesical vaccinia (Dryvax) resulted inimmune infiltration of both malignant and normal tissue (44),and an oncolytic adenovirus CG0070 expressing granulocyte-macrophage colony-stimulating factor is being evaluated ina randomized study phase II/III study after demonstratingobjectives responses of 48% to 63% in different dosing regi-mens (45, and www.clinicaltrials.gov, NCT01438112). Thesestudies all indicate the approach is feasible but have focusedmainly on recurrent disease after BCG failure, where the naturalhistory of the cancer has been altered. None of the studies hasprovided detailed mechanistic evaluation of virus proteinexpression, viral replication kinetics, and the effect on theimmune microenvironment.

The lack of significant toxicity in any patient gives us greatscope to design more prolonged dosing schedules for futurestudies. The study suggested no useful role for low-dose mito-mycin C in the context of potential ICAM-1 upregulation. Inkeeping with evolving data from clinical trials of CVA21 inmalignant melanoma, the virus-induced immune checkpointmolecules, presumably as a result of IFN induction, wouldsuggest obvious combination therapy with a checkpoint inhib-itor (46). These agents are already under evaluation, as singletreatment, in patients with NMIBC failing BCG therapy who areat high risk of relapse. Therefore, the next stage of clinicalevaluation of CVA21 in NMIBC would logically move tocombination therapy, sequencing the checkpoint inhibitorafter CVA21 therapy to provide a potentially alternative effec-tive treatment for this disease to BCG.

Disclosure of Potential Conflicts of InterestK.J. Harrington reports receiving commercial research grants from Astra-

Zeneca, Boehringer-Ingelheim, MSD, and Replimune; reports receivingspeakers bureau honoraria from AstraZeneca, Bristol-Myers Squibb, Boeh-ringer-Ingelheim, Merck-Serono, MSD, Pfizer, and Replimune; and is aconsultant/advisory board member for AstraZeneca, Bristol-Myers Squibb,Boehringer-Ingelheim, Merck-Serono, MSD, Pfizer, and Replimune. B.Davies holds ownership interest (including patents) in Viralytics. M. Groseis an employee of Viralytics. B. Fox is an employee of UbiVac, Argos, Bayer,Bristol-Myers Squibb, CellDex Therapeutics, Definiens, Janssen/Johnson &Johnson, Macrogenics, AstraZeneca, Akoya/PerkinElmer, and PrimeVax;reports receiving commercial research grants from Viralytics/Merck, Bris-tol-Myers Squibb, Bayer, Definiens, Janssen/Johnson & Johnson, Macro-genics, NanString, OncoSec, Akoya/PerkinElmer, and Shimadzu; and holdsownership interest (including patents) in UbiVac and PrimeVax. D. Shafrenreports receiving commercial research grants from, holds ownership interest(including patents) in, and is a consultant/advisory board member forViralytics. No potential conflicts of interest were disclosed by the otherauthors.

Authors' ContributionsConception and design: G. Au, B. Fox, R. Vile, H.S. PandhaDevelopment of methodology: N.E. Annels, B. Davies, B. Fox, R. VileAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): D. Mansfield, M. Arif, S.S. Sandhu, K.J. Harrington,B. Davies, M. Grose, B. Fox, R. Vile, H. Mostafid, H.S. PandhaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): N.E. Annels, D. Mansfield, C. Ballesteros-Merino,G.R. Simpson, M. Denyer, A.A. Melcher, K.J. Harrington, I. Bagwan, B. Fox,R. Vile, H.S. PandhaWriting, review, and/or revision of themanuscript:N.E. Annels, D.Mansfield,C. Ballesteros-Merino, G.R. Simpson, A.A. Melcher, K.J. Harrington, G. Au,M. Grose, I. Bagwan, B. Fox, R. Vile, H. Mostafid, D. Shafren, H.S. PandhaAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): N.E. Annels, M. Denyer, G. Au, M. GroseStudy supervision: N.E. Annels, M. Grose, H.S. PandhaOthers (perform, analysis, and report the IHC part): C. Ballesteros-Merino

AcknowledgmentsViralytics Ltd. funded this study.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received December 17, 2018; revisedMarch 6, 2019; accepted June 27, 2019;published first July 4, 2019.





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References1. Babjuk M, Burger M, Zigeuner R, Shariat SF, van Rhijn BW, Comperat E,

et al. EAU guidelines on non-muscle-invasive urothelial carcinoma of thebladder: update 2013. Eur Assoc U Eur Urol 2013;64:639–53.

2. Chen SY, Du LD, Zhang YH. Pilot study of intravesical instillationof two new generation anthracycline antibiotics in prevention ofsuperficial bladder cancer recurrence. Chin Med J (Engl) 2010;123:3422–6.

3. Addeo R, Caraglia M, Bellini S, Abbruzzese A, Vincenzi B, Montella L, et al.Randomized phase III trial on gemcitabine versus mytomicin in recurrentsuperficial bladder cancer: evaluation of efficacy and tolerance. J ClinOncol 2010;28:543–8.

4. BabjukM,OosterlinckW, Sylvester R, Kaasinen E, B€ohle A, Palou-Redorta J,et al. European Association of Urology (EAU). EAU guidelines on non-muscle-invasive urothelial carcinoma of the bladder, the 2011 update.Eur Urol 2011;59:997–1008.

5. Braasch MR, Bohle A, O'Donnell MA. Risk-adapted use of intravesicalimmunotherapy. BJU Int 2008;102:1254–64.

6. Bassi P. BCG (Bacillus of Calmette Guerin) therapy of high-risk superficialbladder cancer. Surg Oncol 2002;11:77–83.

7. Fuge O, Vasdev N, Allchorne P, Green JSA. Immunotherapy for bladdercancer. Res Rep Urol 2015;7:65–79.

8. Mostafid AH, Palou Redorta J, Sylvester R, Witjes JA.Therapeutic optionsin high-risk non-muscle-invasive bladder cancer during the currentworldwide shortage of bacille Calmette-Gu�erin. Eur Urol 2015;67:359–60.

9. Kitamura H, Tsukamoto T. Immunotherapy for urothelial carcinoma.Current status and perspectives. Cancers 2011;3:3055–71.

10. Abebe F. Is interferon-gamma the rightmarker for BacilleCalmette-Gu�erin-induced immune protection? The missing link in our understanding oftuberculosis immunology. Clin Exp Immunol 2012;169:213–9.

11. Melcher A, Parato K, Rooney CM, Bell JC. Thunder and lightning:immunotherapy and oncolytic viruses collide. Mol Ther 2011;19:1008–16.

12. Shafren DR, Au GG, Nguyen T, Newcombe NG, Haley ES, Beagley L, et al.Systemic therapy of malignant human melanoma tumors by a commoncold-producing enterovirus, Coxsackievirus A21. Clin Cancer Res 2004;10:53–60.

13. Au GG, Lindberg AM, Barry RD, Shafren DR. Oncolysis of vascularmalignant human melanoma tumors by Coxsackievirus A21. Int J Oncol2005;26:1471–6.

14. Berry LJ, AuGG, Barry RD. Potent oncolytic activity of human enterovirusesagainst human prostate cancer. Prostate 2008;68:577–87.

15. SkeldingKA,BarryRD, ShafrenDR. Systemic targetingofmetastatic humanbreast tumor xenografts by Coxsackievirus A21. Breast Cancer Res Treat2009;113:21–30.

16. Shafren DR, Dorahy DJ, Ingham RA, Burns GF, Barry RD. CoxsackievirusA21 binds to decay-accelerating factor but requires intercellular adhesionmolecule 1 for cell entry. J Virol 1997;71:4736–43.

17. Annels NE, Arif M, Simpson GR, Denyer M, Moller-Levet C, Mansfield D,et al. Oncolytic immunotherapy for bladder cancer using Coxsackie A21virus. Mol Ther Oncolytics 2018;9:1–12.

18. Andersen CL, Jensen JL, �rntoft TF. Normalization of real-time quantita-tive reverse transcription-PCR data: a model-based variance estimationapproach to identify genes suited for normalization, applied to bladderand colon cancer data sets. Cancer Res 2004;64:5245–50.

19. Feng Z, Puri S, Moudgil T, Wood W, Hoyt CC, Wang C, et al. Multispectralimaging of formalin-fixed tissue predicts ability to generate tumor-infiltrating lymphocytes from melanoma. J ImmunoTher Cancer 2015;3:47.

20. Bianchi ME, Crippa MP, Manfredi AA, Mezzapelle R, Rovere Querini P,Venereau E. High mobility group box 1 protein orchestrates responses totissue damage via inflammation, innate and adaptive immunity, and tissuerepair. Immunol Rev 2017;280:74–82.

21. Grigg C, Blake Z, Gartrell R, Sacher A, Taback B, Saenger Y. Talimogenelaherparepvec (T-Vec) for the treatment of melanoma and other cancers.Semin Oncol 2016;43:638–46.

22. Meyers DE, Wang AA, Thirukkumaran CM, Morris DG. Current immuno-therapeutic strategies to enhance oncolytic virotherapy. Front Oncol 2017;7:114.

23. Kroemer G, Galluzzi L, Kepp O, Zitvogel L. Immunogenic cell death incancer therapy. Annu Rev Immunol 2013;31:51–72.

24. Wang H, Ward MF, Fan XG, Sama AE, Li W. Potential role of highmobility group box 1 in viral infectious diseases. Viral Immunol 2006;19:3–9.

25. DonnellyOG, Errington-Mais F, Steele L,Hadac E, Jennings V, Scott K, et al.Measles virus causes immunogenic cell death in human melanoma.Gene Ther 2013;20:71–5.

26. Borde C, Barnay-Verdier S, Gaillard C, Hocini H, Marechal V, Gozlan J.Stepwise release of biologically active HMGB1 during Hsv-2 infection.PLoS ONE 2011;6:e16145.

27. Huang B, Sikorski R, Kirn DH, Thorne SH. Synergistic anti-tumor effectsbetween oncolytic vaccinia virus and paclitaxel are mediated by the IFNresponse and HMGB1. Gene Ther 2011;18:164–72.

28. Guo ZS, Naik A, O'Malley ME, Popovic P, Demarco R, Hu Y, et al. Theenhanced tumor selectivity of an oncolytic vaccinia lacking the hostrange and antiapoptosis genes SPI-1 and SPI-2. Cancer Res 2005;65:9991–8.

29. Hosakote YM, Brasier AR, Casola A, Garofalo RP, Kurosky A. Respiratorysyncytial virus infection triggers epithelial HMGB1 release as a damage-associated molecular pattern promoting a monocytic inflammatoryresponse. J Virol 2016;90:9618–31.

30. Saint F, Patard JJ,Maille P, SoyeuxP,HoznekA, SalomonL, et al. T helper 1/2 lymphocyte urinary cytokine profiles in responding and nonrespondingpatients after 1 and 2 courses of Bacillus Calmette-Guerin for superficialbladder cancer. J Urol 2001;166:2142–7.

31. Morgensen TH, Paludan SR.Molecular pathways in virus-induced cytokineproduction. Microbiol Mol Biol Rev 2001;65:131–50.

32. Kaiga T, Sato M, Kaneda H, Iwakura Y, Takayama T, Tahara H.Systemic administration of IL-23 induces potent antitumorimmunity primarily mediated through Th1-type response in associ-ation with the endogenously expressed IL-12. J Immunol 2007;178:7571–80.

33. Reay J, Kim SH, Lockhart E, Kolls J, Robbins PD. Adenoviral-mediated,intratumor gene transfer of interleukin 23 induces a therapeutic antitumorresponse. Cancer Gene Ther 2009;16:776–85.

34. Kuramoto T, Fujii R, Nagai H, BelladonnaML, Yoshimoto T, Kohjimoto Y,et al. IL-23 gene therapy formouse bladder tumour cell lines. BJU Int 2011;108:914–21.

35. Choi IK, Li Y, Oh E, Kim J, Yun CO. Oncolytic adenovirus expressing IL-23and p35 elicits IFN-g- and TNF-a-co-producing T cell-mediated antitumorimmunity. Plos One 2013;8:e67512.

36. García-Cuesta EM, Esteso G, Ashiru O, L�opez-Cobo S, �Alvarez-Maestro M,Linares A, et al. Characterization of a human anti-tumoral NK cell pop-ulation expanded after BCG treatment of leukocytes. Oncoimmunology2017;6:e1293212.

37. Brandau S, Riemensberger J, Jacobsen M, Kemp D, Zhao W, Zhao X, et al.NK cells are essential for effective BCG immunotherapy. Int J Cancer 2001;92:697–702.

38. Patel JR,García-Sastre A. Activation and regulation of pathogen sensor RIG-I. Cytokine Growth Factor Rev 2014;25:513–23.

39. Bochner BH. Gene therapy in bladder cancer. Curr Opin Urol 2008;18:519–23.

40. Ramesh N, Ge Y, Ennist DL, Zhu M, Mina M, Ganesh S, et al. CG0070, aconditionally replicating granulocyte macrophage colony-stimulating fac-tor—armed oncolytic adenovirus for the treatment of bladder cancer.Clin Cancer Res 2006;12:305–13.

41. Cozzi PJ, Malhotra S, McAuliffe P, Kooby DA, Federoff HJ, Huryk B, et al.Intravesical oncolytic viral therapy using attenuated, replication-competent herpes simplex viruses G207 and Nv1020 is effective in thetreatment of bladder cancer in an orthotopic syngeneic model. FASEB J2001;15:1306–8.

42. Simpson GR, Horvath A, Annels NE, Pencavel T, Metcalf S, Seth R, et al.Combination of a fusogenic glycoprotein, pro-drug activation and onco-lytic HSV as an intravesical therapy for superficial bladder cancer. Br JCancer 2012;106:496–507.

43. Hanel EG, Xiao Z, Wong KK, Lee PWK, Britten RA, Moore RB. A novelintravesical therapy for superficial bladder cancer in an orthotopic model:oncolytic reovirus therapy. J Urol 2004;172:2018–22.


Annels et al.




44. Gomella LG, Mastrangelo MJ, McCue PA, Maguire HC, MulhollandSG, Lattime EC. Phase 1 study of intravesical vaccinia virus asa vector for gene therapy of bladder cancer. J Urol 2001;166:1291–5.

45. Burke JM, LammDL,MengMV,Nemunaitis JJ, Stephenson JJ, Arseneau JC,et al. A first in human phase 1 study of CG0070, a GM-CSF expressing

oncolytic adenovirus, for the treatment of nonmuscle invasive bladdercancer. J Urol 2012;188:2391–7.

46. Ribas A, Dummer R, Puzanov I, VanderWalde A, Andtbacka RHI,Michielin O, et al. Oncolytic virotherapy promotes intratumoral T cellinfiltration and improves anti-PD-1 immunotherapy. Cell 2017;170:1109–19.






2019;25:5818-5831. Published OnlineFirst July 4, 2019.Clin Cancer Res Nicola E. Annels, David Mansfield, Mehreen Arif, et al. Agent Against Non Muscle-Invasive Bladder CancerImmunotherapeutic-Coxsackievirus A21 (CVA21) as an Oncolytic Phase I Trial of an ICAM-1-Targeted

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