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Comparative Oncology Evaluation of IntravenousRecombinant Oncolytic Vesicular StomatitisVirus Therapy in Spontaneous Canine CancerShruthi Naik1,2, Gina D. Galyon3, Nathan J. Jenks4, Michael B. Steele4,Amber C. Miller1, Sara D. Allstadt3, Lukkana Suksanpaisan5, Kah Whye Peng4,Mark J. Federspiel6, Stephen J. Russell1,2, and Amy K. LeBlanc3

Abstract

Clinical translation of intravenous therapies to treat dissem-inated or metastatic cancer is imperative. Comparative oncol-ogy, the evaluation of novel cancer therapies in animals withspontaneous cancer, can be utilized to inform and accelerateclinical translation. Preclinical murine studies demonstratethat single-shot systemic therapy with a vesicular stomatitisvirus (VSV)-IFNb-NIS, a novel recombinant oncolytic VSV,can induce curative remission in tumor-bearing mice. Clinicaltranslation of VSV-IFNb-NIS therapy is dependent on compre-hensive assessment of clinical toxicities, virus shedding, phar-macokinetics, and efficacy in clinically relevant models. Dogsspontaneously develop cancer with comparable etiology, clin-ical progression, and response to therapy as human malignan-cies. A comparative oncology study was carried out to investi-gate feasibility and tolerability of intravenous oncolytic VSV-IFNb-NIS therapy in pet dogs with spontaneous cancer. Nine

dogs with various malignancies were treated with a singleintravenous dose of VSV-IFNb-NIS. Two dogs with high-gradeperipheral T-cell lymphoma had rapid but transient remissionof disseminated disease and transient hepatotoxicity thatresolved spontaneously. There was no shedding of infectiousvirus. Correlative pharmacokinetic studies revealed elevatedlevels of VSV RNA in blood in dogs with measurable diseaseremission. This is the first evaluation of intravenous oncolyticvirus therapy for spontaneous canine cancer, demonstratingthat VSV-IFNb-NIS is well-tolerated and safe in dogs withadvanced or metastatic disease. This approach has informedclinical translation, including dose and target indication selec-tion, leading to a clinical investigation of intravenous VSV-IFNb-NIS therapy, and provided preliminary evidence of clin-ical efficacy and potential biomarkers that correlate with ther-apeutic response. Mol Cancer Ther; 17(1); 316–26. �2017 AACR.

IntroductionEffective treatment of advanced, metastatic, and disseminated

cancers requires therapeutic agents that can be administeredintravenously to reach diffuse sites of malignancy. Currentintravenous chemotherapy regimens are arduous, protracted,

and associated with significant side effects. Oncolytic viruses(OV) describe the use of viruses engineered to destroy cancer.OV therapies can be administered intravenously to selectivelyamplify in and kill malignant cells, and potently stimulate theimmune system to recognize tumor antigens that can potentiallycontrol or eradicate disseminated tumor (1–3). The oncolyticvirotherapy approach is a burgeoning field, with recent com-pletion of a landmark phase III clinical trial and FDA approval ofthe first oncolytic therapy, Talimogene Laherparepvec (an engi-neered oncolytic Herpesvirus), for the intratumoral treatment ofrelapsed melanoma (4). Clinical trials evaluating intravenouslyadministered OV therapies to date, however, have demonstratedlimited efficacy in cancer patients (5).

Vesicular stomatitis virus (VSV) is rapidly replicating RNAvirus of the Rhabdoviridae family with a natural tropism formalignant cells (6). VSV is ideally suited for development as anintravenous cancer therapy due to (i) the low occurrence of pre-existing immunity; (ii) weak pathogenicity in humans; (iii)potent immunogenicity to stimulate antitumor immunity; and(iv) the ability to manufacture high titer clinical-grade virusstocks for intravenous administration (7). VSV has been engi-neered to express IFNb and the sodium iodide symporter (NIS).IFNb enhances VSV specificity by activating antiviral innateimmune responses in normal cells, resulting in selective ampli-fication of malignant cells with defective Type I IFN responses(1, 8). The NIS gene is normally expressed in thyroid follicularcells and is responsible for iodide accumulation needed for

1Department of Molecular Medicine, Mayo Clinic College of Medicine, Rochester,Minnesota. 2Vyriad, Inc., Rochester, Minnesota. 3Small Animal Clinical Sciences,College of Veterinary Medicine, University of Tennessee, Knoxville, Tennessee.4Toxicology and Pharmacology Laboratory, Mayo Clinic, Rochester, Minnesota.5Imanis Life Sciences, Rochester, Minnesota. 6Viral Vector Production Labora-tory, Mayo Clinic, Rochester, Minnesota.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Current address for A.K. LeBlanc: DVM Diplomate ACVIM (Oncology), Director,Comparative Oncology Program, Center for Cancer Research, National CancerInstitute, Building 37, Room 2144, Bethesda, MD 20892. Phone: 240-760-7093;E-mail: [email protected]; and current address for S.D. Allstadt: DVM Dip-lomate ACVIM (Oncology), Veterinary Specialists of North Texas, 12101 Green-ville Avenue, Suite #114, Dallas, TX 75243. Phone: 972-437-9499; E-mail:[email protected]

Corresponding Author: A.K. LeBlanc, National Cancer Institute, 37 ConventDrive, Room 2144, Bethesda, MD 20982. Phone: 240-760-7093; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-17-0432

�2017 American Association for Cancer Research.

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thyroid hormone synthesis. Engineering OVs to express NISallows the use of NIS-specific radiotracers to noninvasivelyimage virus biodistribution both in preclinical and clinicalsettings. In addition, NIS-expressing OVs present the opportu-nity to utilize radiovirotherapy to enhance tumor killing bycombination with therapeutic radioisotopes (9, 10). Previousevaluation of VSV-IFNb-NIS in an immune competent, synge-neic murine myeloma model demonstrated that single-shotsystemic therapy induced rapid and durable remission of mye-loma tumors. Detailed analysis of the mechanism of actionrevealed that intravenously administered VSV-IFNb-NIS repli-cates selectively in and rapidly kill tumor cells, and stimulate aT-cell–mediated antitumor immune response to induce dura-ble tumor remission (11).

Clinical translation of novel cancer therapies minimallyrequires preclinical assessment of drug toxicity, safety, andpharmacologic effect in disease-bearing animals (12). FDAguidance recommends additional preclinical assessment of virusshedding and viremia for clinical development of OV therapies(13). Rodent models, although valuable to establish therapeuticutility and define mechanisms of action, limit evaluation ofnovel cancer therapies due to dissimilarity in size, physiology,and disease compared with humans (14). Comparative oncol-ogy, the study of naturally occurring cancer in animals, allowsevaluation of novel cancer treatments in pet dogs, which aresimilar in size, genetic make-up, and physiology, diagnosed withspontaneous cancers, which has highly comparable etiology,progression, immune landscape, and heterogeneity as humancancer (15–19). The comparative oncology approach allowscollection of biological specimens in sufficient quantities toserially monitor virus shedding, pharmacokinetics, and phar-macodynamics, making it a particularly powerful method tofacilitate and inform clinical development and dosing regimensof novel intravenously administered OV therapies, while facil-itating the development of novel treatments for canine cancer.Correlative studies carried out during the evaluation of novelcancer therapies in spontaneous, heterogeneous canine cancercan potentially yield insights into determinants of therapeuticresponse and facilitate identification and testing of clinicallyrelevant biomarkers of exposure and response to intravenouslyadministered OV therapies.

Previously, a preclinical rapid dose-escalation study ofintravenous VSV-hIFNb-NIS administration in purpose-bredhealthy, immune-competent dogs indicated that an intrave-nous dose of 1010 TCID50 was well-tolerated in dogs with dose-limiting toxicities (DLT) observed at a 10-fold higher doseincluded hepatotoxicity and coagulopathy (20). This articledescribes a clinical feasibility study to test the tolerability ofintravenous VSV-IFNb-NIS in client-owned dogs with sponta-neous cancer. The main objectives were to evaluate clinicalresponse to and tolerability of a single intravenous dose of VSV-IFNb-NIS, expressing either human or canine IFNb, thus allow-ing a comparison of VSV therapy with species specific, biolog-ically active IFNb versus nonspecific, but partially active IFNb.In addition, we establish the shedding profile in biologicalsamples (urine, feces, and buccal swab samples) while alsomonitoring of viremia and development of neutralizing anti-bodies against VSV. Correlative pharmacokinetic analyses werecarried out to investigate relationships between clinical diseaseresponses and quantitation of VSV RNA copies in blood andsustained expression of human IFNb. Pharmacokinetic moni-

toring was utilized to evaluate the impact of dose modificationof intravenous VSV-IFNb-NIS administration, in order toexplore whether repeated intravenous dose administrationcould extend persistence of infectious virus in blood. Thesedata were expressly collected to directly inform clinical doseselection, clinical trial design, and monitoring and initiation ofa phase I study evaluating intravenous VSV-IFNb-NIS therapy inpatients with relapsed or refractory cancer.

Materials and MethodsGeneration and characterization of recombinant oncolytic VSV

Recombinant oncolytic VSV-IFNb-NIS was generated as previ-ously described (11). For the purpose of this study, we alsogenerated VSV expressing canine IFNb andNIS. Briefly, restrictionsite–flanked cDNA coding for canine IFNbwas incorporated intoa single NotI site at the M/G junction in the pVSV-XN2 plasmidcontaining the VSV antigenome. VSV-cIFNb-NIS viruswas rescuedand amplified in BHK-21 cells as previously described (21).Growth kinetics of recombinant VSVs was tested by infection ofBHK-21 cells (MOI 3.0, 1 hour at 37�C). Viral titer was measuredin supernatant by overlay on BHK cells of serially diluted super-natant to measure Tissue culture infective dose (TCID50) deter-mined using the Spearman and Karber equation. Radio-iodideuptake was confirmed in vitro as previously described by incuba-tion of BHK-21 cells following infection with recombinant VSVs,in the presence of radiolabeled NaI (I125 at 1 � 105 cpm) � 100mmol/L potassium perchlorate (KClO4; ref. 9). Functional IFNbexpression was confirmed by ultracentrifugation of supernatant(Beckman Coulter Life Sciences) following infection of BHK-21cells with recombinant VSVs to remove virus. Diluted supernatantwas added to human melanoma Mel-624 cells or canine Madin–Darby Canine Kidney (MDCK) cells for 12 hours, followed byinfection with VSV-GFP at an Multiplicity of Infection (MOI) of1.0. Cell viability was assessed by MTT (3-(4,5-dimethylthiazlyl-2)-2,-5-diphenyltetrozolium bromide) assay (ATCC 30–1010K).

Cell cultureBHK-21 cells were purchased in 2012 from the American

Type Cell Culture, and MDCK cells were generously provided byP. Harris (Mayo Clinic) in 2013. Both lines were cultured inDMEM supplemented with 10% FBS, 100 U/mL penicillin, and100 mg/mL streptomycin in 5% CO2. Mel-624 melanoma cellswere generously provided in 2011 by R. Vile (Mayo Clinic) andwere grown in DMEM supplemented as described above. Celllines are routinely tested for mycoplasma contamination aftercells are thawed and cultured immediately before use but werenot otherwise tested or authenticated.

Study enrollment and clinical care of client-owned dogsClient-owned dogs receive medical care at the University of

Tennessee College Of Veterinary Medicine (UTCVM). The Uni-versity of Tennessee-Knoxville (UTK) animal facilities are regis-tered with the United States Department of Agriculture and havean assurance on file with theOffice of Laboratory AnimalWelfare.TheUTK animal care and use program (IACUC) is fully accreditedby theAssociation forAssessment andAccreditationof LaboratoryAnimal Care, International (AAALAC-I). Animal care was provid-ed in adherence to the AAALAC-I guidelines with oversightprovided by theUTKOffice of Laboratory Animal Care. Followinga cancer diagnosis, pet-owners give written informed consent to

Comparative Oncology Informs Development of Oncolytic VSV

www.aacrjournals.org Mol Cancer Ther; 17(1) January 2018 317




enroll their pet dog in this study. All study procedures wereapproved by the University of Tennessee-Knoxville InstitutionalAnimal Care and Use Committee (UTK-IACUC). Consented dogswere confirmed to meet study eligibility criteria as indicated inSupplementary Table S1. Prior to entry into the study, dogs werehoused in USDA-compliant and IACUC-approved housing unitswithin the UTCVM laboratory animal facilities. Dogs were fedonce daily in the morning a commercially available dry dog food(Hill's Science Diet Canine Maintenance) and had tap water todrink ad libitum. Following treatment, studydogsweremoved intoa USDA/IACUC/UTK-Biosafety office-approved housing unitwithin the UTCVM for 7 days.

Test article and intravenous administrationPreclinical grade VSV-hIFNb-NIS or VSV-cIFNb-NIS was man-

ufactured at the Mayo Clinic Viral Vector Production labs. Viruslots were confirmed negative benzonase and endotoxin contam-inations. Viruswas shipped ondry ice and received at an approvedBSL2 laboratory at the University of TennesseeMedical Center forstorage. Virus stocks were thawed and formulated by dilution insterile saline based on calculated dose immediately prior toadministration to dogs in a final volume of 10mL. The test articlewas administered as a 2- to 5-minute intravenous bolus via asterilely placed intravenous cephalic catheter.

Clinical monitoring protocolDogs were fasted overnight prior to virus administration and

monitored continuously on the day of virus administration (day0). Rectal temperature, heart and respiratory rates, and generalattitude/demeanorwere recorded every 1 to 2hours or as the dogs'condition dictated. Appetite and presence of any gastrointestinalupset (e.g., vomiting, diarrhea) was noted. On every day of study,the dogs' vital signs (rectal temperature, heart, and respiratoryrates), overall attitude and appetite, and physical exam findingswere recorded. Clinical pathology wasmonitored by collection ofblood drawn via Vacutainer for a complete blood count (CBC),serum biochemistry and electrolyte panel (CHEM), and coagu-lation panel (COAG). All analyses were performed at the Uni-versity of Tennessee College of Veterinary Medicine ClinicalPathology and Microbiology Laboratories. Tumor burden wasmonitored as indicated in Supplementary Table S2. Diseaseburden in dogs with peripheral nodal lymphoma in dogs wasassessed and monitored using criteria previously established bythe Veterinary Cooperative Oncology Group (VCOG; ref. 22).

Viremia, pharmacokinetic, and virus shedding studiesVirus pharmacokinetics and viremia were assessed in whole

blood collected inRNAprotect animal blood tubes (Qiagen). RNAwas isolated using Qiagen RNeasy animal blood system andanalyzed qRT-PCR to detect VSV-N gene copies as previouslydescribed (19). Blood was also collected in a heparinized CPTtube to avoid clotting and centrifuged to separate peripheralblood mononuclear cells (PBMC) and plasma. PBMCs and plas-mawere assessed to detect infectious virus and VSV-N gene copiesby qRT-PCR in isolated RNA. Briefly, PBMCs were subject tofreeze-thaw and centrifuged, and supernatant was collected andtested for infectious virus by titration on BHK cells. Biologicalsamples, specifically urine, buccal swabs, and fecal samples, werecollected and tested to monitor virus shedding as previouslydescribed (20). Briefly, urine was collected by cystocentesis orurinary tract catheterization in a sterile falcon tube, centrifuged,

and urine cell pellet was collected. Buccal swabs were collectedusing a sterile Omni swab (Whatman). Urine cell pellet andbuccal swab samples were tested to detect infectious virus, usingan infectious virus recovery (IVR) assay, and virus genome, byqRT-PCR detection of VSV-N gene copies. Feces was collectedfresh and processed. RNA was isolated from fecal supernatantusing the Qiagen Viral RNA mini Kit, and qRT-PCR was utilizedto detect VSV-N gene copies.

Antibody neutralization assaySerum samples were collected using Vacutainer serum separa-

tor tubes (BD Life Sciences) and tested to detect neutralizingantibodies against VSV as previously described (20). Briefly,complement inactivation was carried out by incubation of serumat 56�C. Dilutions of serum were preincubated with 500 TCID50

VSV-GFP, and the virus/serum mix were added to previouslyplated Vero cells in triplicate. Cytotoxicity was assessed 48 hourslater by measurement of CPE-positive wells at each serum dilu-tion, and the minimum virus dilution that did protect cells fromVSV-induced CPE was recorded.

Measurement of human IFNb in plasmaPlasma samples were collected at indicated time points and

frozen at �80�C. Plasma samples were diluted and assayed fordetection of human IFNb using the Verikine high-sensitivityhuman IFNb ELISAKit (PBL assay science). The limits of detectionfor this assay were 60 to 7,500 pg/mL human IFNb.

Surgical resection and histopathology analysisUTK01, a 12-year-old spayed female mixed breed research

dog weighing at UTCVM, was diagnosed with anal adenocar-cinoma and received a single dose of VSV-hIFNb-NIS. Diseasestabilization occurred, and thus 45 days after VSV treatment,UTK01 underwent surgical resection of the adenocarcinoma.Resected tumor was assessed by standard hematoxylin andeosin staining and immunohistochemistry to detect canineCD3þ lymphocytes.

ResultsTest article characterization

Recombinant VSV-expressing IFNb and NIS, VSV-IFNb-NIS,was previously generated and characterized demonstratingrapid cytotoxicity against myeloma and lymphoma cell linesin vitro, and potent therapeutic efficacy following single-shotintravenous treatment in an immune-competent syngeneicmurine myeloma model (11, 21). For the purpose of this study,we generated a VSV-expressing canine IFNb and NIS, VSV-cIFNb-NIS. Preclinical virus stocks of VSV-hIFNb-NIS andVSV-cIFNb-NIS were prepared and tested to ensure test articleintegrity, sterility, and absence of endotoxins. Virus viability,activity, and functional gene expression of purified stocks ofVSV-hIFNb-NIS and VSV-cIFNb-NIS were confirmed in vitrofollowing infection of susceptible BHK cells, indicating bothtest viruses replicated rapidly and comparably, albeit with aslightly reduced maximal titer compared with rVSV-GFP in BHKcells (Fig. 1A). Functional NIS expression was confirmed byperchlorate-sensitive radio-iodine uptake in infected BHK cells(Fig. 1B). Functional human and canine IFNb expression wastested in human Mel-624 (melanoma) cells and canine MDCK

Naik et al.

Mol Cancer Ther; 17(1) January 2018 Molecular Cancer Therapeutics318




cells, respectively. Virally expressed IFNb derived from super-natant of infected BHK-21 cells induced protective antiviralinnate immune responses. Human IFNb from VSV-hIFNb-NIS–infected cells was protective in human Mel-624 cells (Fig. 1C),whereas canine and human IFNb (from VSV-cIFNb-NIS andVSV-hIFNb-NIS–infected cells) were protective in MDCK cells,indicating human IFNb is partially cross-reactive in caninetissues. These assays confirm the replicative capacity and func-tional gene expression of the recombinant oncolytic VSV testarticles under investigation in this study.

Study design and enrollmentA fixed-dose feasibility study investigating tolerability, as a

primary endpoint, and efficacy, as a secondary endpoint, ofintravenously administered recombinant oncolytic VSV-hIFNb-NIS or VSV-cIFNb-NIS was implemented in client-owned dogswith spontaneous cancer at a starting intravenous dose of 1 �1010 TCID50/0.5 m2, having previously demonstrated the toler-ability of this dose in purpose bred laboratory Beagle dogs (20).Owners of dogs diagnosed with cancer at the University ofTennessee Veterinary Medical Center provided written informed

consent and dogs to assess compliance with study eligibilitycriteria (Supplementary Table S1). One research dog and eightclient-owned dogs were enrolled initially into the study, eachreceiving a single intravenous dose of VSV-cIFNb-NIS orVSV-hIFNb-NIS. A tenth dog with metastatic osteosarcoma wasenrolled and received two intravenous doses of VSV-hIFNb-NIS,with the second dose administered 48 hours following the first.All procedures were carried out with approval of the Universityof Tennessee IACUC and Institutional Biosafety Committee.

Treatment and monitoringOne research dog and 9 client-owned dogs diagnosed with

various spontaneous malignancies were enrolled including 4dogs with B-cell lymphoma and 2 dogs with T-cell lymphoma(Supplementary Table S2). Enrolled dogs underwent a preas-sessment to confirm diagnosis of malignancy, establish tumorburden and clinical pathology, and collect biological samplesat baseline (Fig. 1D). Dogs were housed in ABSL-2 housing andfasted overnight prior to virus administration. Dose was cal-culated based on surface area conversion, where each dogreceived 1 � 1010 TCID50/0.5 m2 (Table 1). Virus was diluted

Figure 1.

Characterization of recombinant oncolytic vesicular stomatitis viruses expressing human or canine IFNb and NIS. VSV-expressing canine or human IFNband the NIS were tested in vitro to evaluate (A) virus replication in BHK-21 cells by infection with VSV-hIFNb-NIS, VSV-cIFNb-NIS, or VSV-GFP at anMOI of 3.0 and measurement of virus titer in TCID50. B, Functional NIS expression by measurement of radio-iodide (125I) uptake 24 hours afterinfection of BHK-21 cells (MOI 1.0) in the presence (þ) or absence (�) of NIS-specific inhibitor KCLO4. Expression of (C) functional human or canineIFNb expression by protection of human Mel-624 (top panel) or canine MDCK (bottom panel) cells, respectively, following exposure to ultracentrifuged virusinfection supernatant (using VSV-GFP cell supernatant as a control), followed by infection with VSV-GFP (MOI 0.1). Cell viability was assessed 48 hours laterby MTT assay and shown as a percentage of control mock–infected cells. D, Veterinary clinical study flow diagram outlining procedures for (i) enrollment, (ii)preassessment, (iii) treatment, (iv) acute monitoring, and (v) long-term monitoring.






in sterile saline to a 10 mL injection volume and administeredto each dog via a sterilely-placed intravenous cephalic catheterin a 2- to 5-minute intravenous bolus. Dogs were monitoredcontinuously on the day(s) of virus administration to recordtemperature, vitals, and general demeanor, and blood sampleswere collected for pharmacokinetic monitoring. Continuedmonitoring included physical examinations, and collection ofblood, urine, buccal swab, and fecal samples, to monitorclinical pathology, viremia, shedding, and antibody responseas indicated (Fig. 1D). Disease burden was monitored bymethods specific to indication type as listed in SupplementaryTable S2. Peripheral lymphoma burden and response weremeasured using response criteria established by the VCOG. Asummary of dogs treated, signalment, indication, dosereceived, clinical toxicities, virus shedding, and treatment out-come are provided in Table 1.

Clinical toxicity and adverse eventsDogs were monitored by physical evaluations and clinical

pathology testing of blood samples, including CBC, bloodchemistry (CHEM), and coagulation (COAG) tests, to monitorclinical toxicities (Fig. 2). Clinical toxicities were evaluatedaccording to the Veterinary cooperative oncology group Com-

mon terminology criteria for adverse events (VCOG-CTCAE)following biological antineoplastic therapy in dogs (v1.1, July2011; ref. 22). DLTs of intravenous VSV-IFNb-NIS administra-tion include hepatotoxicity and coagulopathy (20). There wereno observable symptoms of discomfort or toxicity duringintravenous VSV-IFNb-NIS infusion or following treatment.All treated dogs developed a mild fever starting approximately3 hours after infusion that normalized by 24 hours. Only 1 dog,UTK08, a 10-year-old spayed female Boxer diagnosed withhigh-grade peripheral T-cell lymphoma who received 2.2 �1010 TCID50 VSV-hIFNb-NIS, had a notable adverse event,specifically transient hepatotoxicity indicated by an alanineaminotransferase (ALT) elevation of approximately 10-fold ofupper limit of normal (ULN) that resolved spontaneously(Fig. 2A). The ALT elevation was considered grade 3 toxicityaccording to VCOG criteria but was not considered to be doselimiting as it was both transient and self-limiting. UTK04,another Boxer diagnosed with high-grade T-cell lymphoma,had a transient ALT elevation of approximately 2-fold ULNfollowing VSV-hIFNb-NIS treatment. Notably, both dogswith transient ALT elevations also had peripheral disease remis-sion and partial responses to IV VSV therapy as describedbelow. All dogs developed transient reduction in white blood

Table 1. Summary of toxicity, biosafety, and therapeutic response

Study ID Breed, sex, and age Disease type TX (and TX time) Toxicity Safety (IVR) Outcome

1 UTK01 Mix, female (spayed)10 years

Analadenocarcinoma

1.6 � 1010 TCID50 No notableadverse events

Urine (�) STABLE DISEASEVSV-hIFN-NIS Buccal (�) 30 days; followed by

tumor resection(at diagnosis)

2 UTK02 Mix, male (Cast.)10 years

Multiple myeloma(IgA)

1 � 1010 TCID50 Mild/transientALT elev.

Urine (�) STABLE DISEASE

VSV-cIFN-NIS Buccal (�)(at diagnosis)

3 UTK03 German Shepherd,female, (spayed);8 years

B-cell lymphoma(peripheral LN)

2.4 � 1010 TCID50 Mild/transientALT increase


VSV-cIFN-NIS Buccal (�)(at diagnosis)

4 UTK04 Boxer, male (Cast.)8 years

T-cell lymphoma 2.2 � 1010 TCID50 2X ULN ALTelevation,transient,self-limiting

Urine (�) PARTIAL RESPONSEVSV-hIFN-NIS Buccal (�) Rapid disease

remission, relapsePR: �21 days

(at diagnosis)

5 UTK05 Mix, male (Cast.)6 years


1.7 � 1010 TCID50 Mild/transientALT increase

Urine (�) PROGRESSIVEDISEASEVSV-hIFN-NIS Buccal (�)

(at diagnosis)

6 UTK06 Schnauzer female(spayed) 10 years

Multifocalcutaneousmelanoma

1.02 � 1010 TCID50 Mild/transientALP increase


VSV-cIFN-NIS Buccal (�)(At diagnosis)

7 UTK07 Hound mix, female(spayed) 12 years


1.7 � 1010 TCID50 No notableadverse events

Urine (�) PROGRESSIVEDISEASEVSV-cIFN-NIS Buccal (�)

(refractory disease) (Rapid progression)

8 UTK08 Boxer, female (spayed)10 years

T-cell lymphoma 2.2 � 1010 TCID50 10X ULN ALTelevation,transient,self-limiting

Urine (�) PARTIAL RESPONSE

VSV-hIFN-NIS Buccal (�) Rapid diseaseremission, relapsePR: �36 days

(at diagnosis)

9 UTK09 Pembroke Welsh Corgifemale (spayed)11 years

B-cell lymphoma 1.2 � 1010 TCID50 No notableadverse events

Urine (�) PROGRESSIVEDISEASEVSV-cIFN-NIS

(refractorydisease)

Buccal (�)Brief, transientresponse, anddisease progression

10 UTK10 Metastatic OS,maxillary lesion,and lung mets

2 doses of 2.2 �1010 TCID50

No notableadverse events


VSV-hIFN-NISBuccal (�) SD 6 months;

maxillary lesionprogression @ 6 m@ 0 and 48 h (at

diagnosis)

NOTE: Dogs diagnosed with various types of cancer were enrolled and treated with the indicated dose of VSV-hIFNb-NIS or VSV-cIFNb-NIS. Hepatotoxicity, theprimaryDLT, is described. Safety data indicate samples tested for IVR are negative (�). Summary of therapeutic response during the 28-day study period is indicated(PR: partial response).

Naik et al.





cells (WBC) in blood, indicative of mild, transient lymphope-nia following intravenous VSV-IFNb-NIS treatment (Fig. 2B).There were no notable coagulopathies except as expected in onedog (UTK02) diagnosed with spontaneous myeloma-inducedhyperglobulinemia, wherein prolongations of prothrombintime (PT) and activated partial thromboplastin time (PTT)were documented at baseline (Fig. 2C). Finally, there were noadverse events related to repeated intravenous administrationas noted following treatment of one dog, UTK10, who receivedtwo doses of VSV-hIFNb-NIS, providing preliminary evidenceof the feasibility of repeat dosing.

Therapeutic responseTherapeutic response was monitored by measuring disease

burden as indicated (Supplementary Table S2) for 28 daysfollowing VSV-IFNb-NIS therapy depending on the cancer typeand was observed to be variable (Table 1). Disease burden indogs with peripheral lymphoma was measured using estab-lished guidelines (23). Two dogs with advanced peripheral T-cell lymphoma (UTK04 and UTK08), each treated with a singleintravenous dose of 2.2 � 1010 TCID50 VSV-hIFNb-NIS, hadrapid disease remission of disseminated peripheral lymphoma

indicated by reduction in diameters of all peripheral lesions(Fig. 3A and B) resulting in measurable partial responses of 40%and 60%, respectively (Fig. 3C). In both cases, remission wastransient, lasting approximately 21 and 36 days before diseaserelapsed. UTK08, who had rapid remission of peripherallesions, received an additional intratumoral injection of 3 �1010 TCID50 VSV-hIFNb-NIS on day 19 in the right mandibularlymph node lesion that was relapsing (Fig. 3B, depicted byarrow), with no measurable impact on lesion size. Two dogswith spontaneous anal adenocarcinoma and advanced periph-eral B-cell lymphoma had stable and progressive disease, respec-tively. Three of five dogs treated with VSV-cIFNb-NIS, includingdogs with myeloma, peripheral B-cell lymphoma, and multi-focal cutaneous melanoma, had stable disease, whereas twodogs with advanced peripheral B-cell lymphoma had diseaseprogression. UTK01, a dog with anal adenocarcinoma, hadsurgical resection of the tumor 45 days after receiving intrave-nous VSV-hIFNb-NIS treatment. Histopathologic analysis of theresected tumor indicated a high infiltration of T cells comparedwith historical controls of the same tumor type (SupplementaryFig. S1) indicative of tumor-specific immune infiltration fol-lowing intravenous VSV treatment.

Figure 2.

Clinical pathology testing of canine blood samples. Blood was collected from enrolled dogs at baseline and following systemic VSV therapy. Selected analysesare shown indicating (A) liver function tests (ALT and AST), (B) CBC including WBC and LYMPH, and (C) coagulation testing (PT and PTT). Normalrange is delineated by the shaded region. AST, aspartate aminotransferase; ALP, alkaline phosphatase; LYMPH, percentage lymphocytes.






Virus sheddingShedding studies were carried out to monitor release of infec-

tious materials following intravenous administration of replica-tion-competent oncolytic VSV. Buccal swabs, urine (separatedinto urine cell pellet and supernatant), and fecal samples werecollected. All samples were assessed using highly sensitive qRT-PCR to detect VSV-N gene copies. Quantitative RT-PCR analysesindicated some buccal swab, urine, or fecal samples had detect-able VSV-N gene copies, though all sampleswere close to or belowlimit of detection (LOD; Supplementary Fig. S2). Detection ofviral RNA by qRT-PCR is not indicative of infectious virus, and inaccordance with FDA recommendations (13), buccal swabs andurine samples were also assayed to detect presence of infectiousvirus using an IVR assay by overlay on susceptible BHK-21 cells,indicating no detectable infectious virus. Some buccal swabsamples were toxic to cells in culture due to the predictablepresence of nonviral contaminants, and absence of infectiousvirus was confirmed by filtration and repeat of overlay of cellsupernatant and confirming absence of VSV RNA by qRT-PCR.

These data indicate the absence of virus shedding in biologicalexcreta following intravenous VSV-IFNb-NIS administration intumor-bearing dogs.

Virus pharmacokinetics in bloodThe comparative oncology approach is amenable to pharma-

cokinetic monitoring. Serial blood sample collection was carriedout starting at 10 minutes and up to 6 hours following intrave-nous VSV-IFNb-NIS administration to detect VSV RNA in wholeblood, and both VSV RNA and infectious virus in isolated PBMCsand plasma. Pharmacokinetic studies indicate an immediatetransient decline (in the first 60 minutes) and then stabilizationof VSV RNA over 6 hours (Fig. 4A). Infectious virus was detectedonly in PBMC samples (Fig. 4B) and not in plasma (Fig. 4D).Measurement of VSV RNA biodistribution in blood indicates thatintravenously administered VSV localizes to PBMCs and does notremain in plasma (Fig. 4C and D). These data also indicate thatVSV RNA is detectable at higher quantities in PBMCs comparedwithwhole blood. Notably, the two dogswithmeasurable diseaseremission, UTK04 and UTK08, had an approximately 10-foldhigher VSV-N gene copy number in blood compared with dogswith no measurable disease remission, peaking at approximately2 hours after IV VSV-IFNb-NIS administration. The highest quan-tity of infectious virus was recovered in PBMCs isolated fromUTK08, the dog with the best response to intravenous VSV-IFNb-NIS therapy. These data indicate that assessment of isolatedPBMCs is the most sensitive method to measure both VSV RNAand infectious virus. Furthermore, these data provide preliminaryindication that virus pharmacokinetics may correlate with ther-apeutic response to intravenously administered oncolytic VSVtherapy.

Viremia and neutralizing antibodiesBlood samples were analyzed to monitor viremia and pres-

ence of neutralizing antibodies following intravenous VSV-IFNb-NIS therapy. Virus genomes were detectable in wholeblood at 24 hours after infusion and decayed rapidly betweendays 3 to 7, with genomes undetectable (or below LOD) by day14 (Fig. 4E). Anti–VSV-neutralizing antibodies were detectablein serum by day 7 in most treated dogs (Fig. 4F), coinciding withclearance of virus from blood. Analysis of antibody response indogs indicated there was a delay in generation of neutralizingantibodies against VSV in a myeloma-bearing dog, UTK02, andlower antibody titers in dogs with B-cell malignancies (e.g.,UTK07, UTK09). The immune-suppressed state of patients withB-cell malignancies may affect generation of antiviral antibo-dies, but this observation would need to be confirmed in a largercohort of tumor-bearing dogs.

Monitoring virus transgene expressionVirus activity can be monitored by measurement of expres-

sion or biological activity of encoded transgenes (10). Here, wemeasured the human IFNb levels in plasma from 3 dogs treatedwith VSV-hIFNb-NIS, using this as a surrogate soluble marker ofthe total burden virus-infected cells. One dog treated with VSV-cIFNb-NIS and a non–tumor-bearing (NTB) dog administeredwith a single dose of VSV-hIFNb-NIS were included as controls.These data indicate that human IFNb is measurable in plasmaand rapidly increases following VSV-hIFNb-NIS administrationin tumor-bearing or non–tumor-bearing dogs. Human IFNb

Figure 3.

Remission of peripheral T-cell lymphoma following intravenous VSV-hIFNb-NIS treatment. Disease burden was monitored in two responding dogs, (A)UTK04 and (B) UTK08, both with peripheral T-cell lymphoma, who receiveda single intravenous dose of 2.2 � 1010 TCID50. Disease remission wasmonitored by serial caliper measurements and shown as percentage changein individual lesions compared with baseline measurement, includingmandibular, prescapular, popliteal, and inguinal nodal lesions. C, Therapeuticresponse in responding dogs was assessed based on response criterionestablished by the VCOG with disease burden shown as a percentage ofbaseline based on mean sum LD (longest diameter) of five target lesions.

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expression peaked at approximately 6 hours after intravenousVSV-hIFNb-NIS administration (Supplementary Fig. S3). Plas-ma levels of human IFNb in responding dogs (UTK04 andUTK08) were only slightly higher at 90 minutes after treatmentbut were detectable for a longer time (up to 7 days aftertreatment) compared with nonresponsive (UTK05) or NTBcontrol. These data demonstrate facile monitoring of bothquantity and duration of transgene expression that may provideconvenient biomarkers of therapeutic response following treat-ment of intravenously administered recombinant oncolytictherapies.

Feasibility of repeated intravenous VSV-IFNb-NISadministration

Pharmacokinetic studies provide a preliminary indicationthat higher levels of viral RNA in blood may correlate withtherapeutic response to intravenous VSV-IFNb-NIS therapy.

Modification of dose regimen, including repeated dosing, maybe utilized to improve therapeutic response by optimizingpharmacokinetics and improving bioavailability of cancertherapies (24, 25), particularly following intravenous admin-istration. To test the feasibility of repeated intravenousVSV-IFNb-NIS dosing, we enrolled one dog, UTK10, withmaxillary osteosarcoma (OSA) and metastatic lung noduleswho received 2 doses 2.2 � 1010 TCID50. Treatment was welltolerated with no notable adverse events associated with repeat-ed VSV administration (Fig. 2) and no detectable sheddingof infectious virus (Supplementary Fig. S2). As previouslyobserved, VSV genomes were detected primarily in PBMCs(Fig. 5A). Infectious virus (denoted by asterisks) was detectablein PBMCs following the first and second doses of VSV-hIFNb-NIS, indicating that repeated dosing can extend the persistenceof infectious virus in blood. Correlative monitoring of humanIFNb levels in plasma indicated that human IFNb was not

Figure 4.

Assessment of virus pharmacokinetics, biodistribution, viremia, and antibody neutralization following intravenous VSV-IFNb-NIS treatment. Blood sampleswere collected immediately following intravenous VSV-IFNb-NIS treatment in tumor-bearing dogs for pharmacokinetic studies. RNA was isolated from(A) whole blood to detect VSV-N gene copies by qRT-PCR. B, PBMCs were isolated to detect infectious virus by overlay on susceptible BHK cells tomeasure TCID50. Virus biodistribution was assessed in (C) PBMCs or (D) plasma, indicating that VSV-N genes were detectable primarily in PBMCs andbelow LOD in plasma. Whole blood and serum were collected to monitor (E) viremia and (F) anti–VSV-neutralizing antibodies at indicated time pointsfollowing intravenous VSV-IFNb-NIS treatment.






extended or increased due to repeated VSV-hIFNb-NIS admin-istration (Fig. 5B). Dogs with metastatic OSA generally have amedian life expectancy of 2 to 3 months, but this is highlyvariable depending on site of metastasis (bone vs. soft tissues)and type of treatment sought by pet owners (26). UTK10 hadstable disease for 6 months before progression of the maxillarylesion led to humane euthanasia. Chest radiographs taken atthe time of euthanasia indicated no significant progression ofthe pulmonary metastatic nodules.

DiscussionThis study provides the first evaluation of intravenous OV

therapy in dogs with spontaneous cancer. OV therapy usesengineered viruses to induce durable tumor remissionthrough a combination of virus replication, lytic destructionof tumor cells, and activation of immune-mediated tumorrecognition and elimination (3). OV products are generallyattenuated and have reduced virulence and pathogenicitycompared with the wild-type strains (1). Clinical develop-ment of novel cancer therapies requires comprehensive pre-clinical assessment of clinical toxicities and therapeutic effi-cacy (12). In addition, clinical translation of replication-com-petent OV therapies requires assessment of virus shedding andbiosafety (13). Although useful to perform a preliminaryassessment of toxicity, efficacy, and mechanisms of action ofnovel anticancer agents (27), preclinical rodent models limit

clinically relevant translational assessment due to disparitiesin body size, physiology, genetic, and immunologic attributesbetween rodents and humans. Comparative oncology allowsclinically relevant evaluation of novel OV therapies, as natu-rally occurring canine cancers recapitulate the genetic andmolecular heterogeneity and tumor immune microenviron-ment of human cancer (15). The ability to collect seriallybiological samples from the same animal, including bloodand biological excreta, enables clinical and correlative mon-itoring. Clinical monitoring of clinical toxicities and biosafetyincluding viremia and virus shedding is critical to clinicaltranslation. Correlative monitoring including virus pharma-cokinetics and pharmacodynamics, transgene expression, andimmune responses allow us to investigate determinants andidentify biomarkers that are predictive of or correlate withclinical toxicity or efficacy.

Here, we demonstrate the utility of the comparative onco-logy approach and establish the safety, both in terms oftoxicity and shedding, of intravenous oncolytic VSV-IFNb-NIS administration in dogs with spontaneous cancer. Clini-cal monitoring indicates that hepatotoxicity is the primaryDLT following intravenous VSV-IFNb-NIS therapy, notablyobserved only in dogs that had disease remission, suggest-ing this DLT may be associated with virus bioavailabilityand/or tumor lysis. Virus shedding studies confirm theabsence of infectious virus shedding in biological excreta.These data support clinical translation and inform the designof adverse event monitoring for human clinical trials. Thisapproach also allowed testing of novel oncolytic therapies indogs with various, spontaneously occurring, and heteroge-neous malignancies. Results demonstrate that single-shotintravenous VSV therapy can induce remission of dissemi-nated cancer. The rapid speed of remissions of peripherallesions observed in dogs with advanced T-cell lymphomasuggests it is due to virus-mediated tumor destruction.Although the results of this study are limited due to thesmall sample size, the observed responses led to inclusionof T-cell lymphoma as a target indication in a phase I humanclinical trial evaluating intravenous VSV-hIFNb-NIS therapy(NCT03017820, clinicaltrials.gov).

Correlative monitoring including pharmacokinetics studiesand measurement of transgene expression in blood provideswith preliminary data indicating approximately 10X higherVSV RNA detectable and sustained IFNb expression in bloodfrom dogs with measurable disease remission (compared withdogs with stable or progressive disease). These studies suggestmethods to enhance virus pharmacokinetics that may improvevirus bioavailability and therapeutic response. Soluble serummarker measurement, however, does not indicate location ofvirus infection, and early IFNb detection may be due toinfection of PBMCs rather than tumor cells. Single-PhotonEmission Computed Tomography or Positron EmissionTomography/Computed Tomography imaging to monitorviral NIS expression would be a more effective method toserially and noninvasively monitor virus biodistribution andconfirm tumor-specific virus replication (10).

Findings from this study have contributed to clinical trans-lation of oncolytic VSV-IFNb-NIS therapy including selectionof a safe clinical starting dose to avoid protracted dose esca-lation, and design of clinical monitoring protocols and cor-relative assays to assess toxicities and virus shedding. These

Figure 5.

Assessment of virus pharmacokinetics and transgene expression followingtwo-dose intravenous VSV-IFNb-NIS treatment in a dog with spontaneousosteosarcoma. A, RNA was isolated from whole blood, PBMCs, andplasma collected immediately following VSV-IFNb-NIS treatment todetect VSV-N gene copies by qRT-PCR. Infectious virus was detected inPBMCs, and samples positive for recovery of infectious VSV are denotedusing an asterisk (�). B, Human IFNb was monitored in plasma collectedat indicated time points following administration of two IV doses ofVSV-IFNb-NIS (denoted by ~).

Naik et al.





findings supported approval and initiation of a phase I clini-cal trial to evaluate intravenous VSV-IFNb-NIS therapy inpatients with relapsed or refractory multiple myeloma andT-cell lymphoma, the latter indication included due toobserved disease remission in T-cell lymphoma–bearing dogs.These studies provide preliminary indication of factors thatcorrelate with, and may be predictive biomarkers of therapeu-tic response including virus pharmacokinetics in blood andsustained transgene expression, and support the developmentof oncolytic VSV as canine cancer therapy to benefit pets andpet-owners.

Traditional pathways of cancer drug development are longand expensive. Although introduction of accelerated pathwaysof drug approval has expedited cancer drug development, costsare high and accessibility is limited. The utilization of com-parative oncology will inform new product development andrefine selection of products to advance to clinical develop-ment, potentially reducing the time, cost, and risk of drugdevelopment.

Disclosure of Potential Conflicts of InterestS. Naik, Kah Whye Peng and S.J. Russell are co-founders and officers of

Vyriad, Inc., a biotechnology company developing engineered viruses for cancertherapy including the Vesicular stomatitis virus oncolytic virus platformdescribed in this study. No potential conflicts of interest were disclosed by theother authors.

Authors' ContributionsConception and design: S. Naik, S.J. Russell, A.K. LeBlancDevelopment of methodology: S. Naik, G.D. Galyon, K.W. Peng, S.J. Russell,A.K. LeBlanc

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S. Naik, G.D. Galyon, N.J. Jenks, M.B. Steele,A.C. Miller, S.D. Allstadt, L. Suksanpaisan, M.J. Federspiel, A.K. LeBlancAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Naik, M.B. Steele, A.C. Miller, L. Suksanpaisan,K.W. Peng, M.J. Federspiel, A.K. LeBlancWriting, review, and/or revision of the manuscript: S. Naik, S.D. Allstadt,K.W. Peng, M.J. Federspiel, S.J. Russell, A.K. LeBlancAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S. Naik, G.D. Galyon, S.D. Allstadt, A.K. LeBlancStudy supervision: S. Naik, S.D. Allstadt, A.K. LeBlanc

AcknowledgmentsWe wish to acknowledge Christina Mazcko for assistance in article

preparation. Financial support for this clinical trial was provided by grantsfrom the Morris Animal Foundation (#D14CA-002, A.K. LeBlanc); a Dis-covery Research Program and Career Enhancement Program award, sup-ported in part by the Public Health Service grant number P50 CA097274from the University of Iowa/Mayo Clinic Lymphoma Specialized Programof Research Excellence (UI/MC Lymphoma SPORE) and the NationalCancer Institute (S. Naik); Mayo Clinic Discovery Translational Programgrant (DTP2012, S.J. Russell and S. Naik); and Intramural ResearchProgram of the National Institutes of Health (NIH), NCI, Center for CancerResearch. The results and interpretations do not reflect the views of theU.S. Government.

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 May 11, 2017; revised July 13, 2017; accepted November 3, 2017;published OnlineFirst November 20, 2017.

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2018;17:316-326. Published OnlineFirst November 20, 2017.Mol Cancer Ther Shruthi Naik, Gina D. Galyon, Nathan J. Jenks, et al. Canine CancerOncolytic Vesicular Stomatitis Virus Therapy in Spontaneous Comparative Oncology Evaluation of Intravenous Recombinant

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