reovirus: a targeted therapeutic progress and potential · dsrna (5). however, considering the fact...

Review

Reovirus: A Targeted Therapeutic—Progress And Potential

Radhashree Maitra1, Mohammad H. Ghalib1, and Sanjay Goel1,2

AbstractMedical therapy of patients withmalignancy requires a paradigm shift through development of new drugs with a

good safety record and novel mechanisms of activity. While there is no dearth of such molecules, one particularagent, "reovirus" is promising by its ability to target cancer cells with aberrant signaling pathways. This double-stranded RNA virus has been therapeutically formulated and has rapidly progressed from preclinical validation ofanticancer activity to a phase III registration study in platinum refractory metastatic squamous cell carcinoma of thehead and neck. During this process, reovirus has shown safety both as a single agent when administeredintratumorally and intravenously, as well as in combination therapy, with multiple chemotherapeutics such asgemcitabine, carboplatin/paclitaxel, and docetaxel; and similarly with radiation. The scientific rationale for itsdevelopment as an anticancer agent stems from the fact that it preferentially replicates in and induces lyses of cellswith an activated Kras pathway. As documented in many previous studies, the initial observation of greater tropismin Kras-compromised situation might certainly not be the sole and possibly not even the predominant reason forenhanced virulence. All the same, scientists have emphasized on Kras optimistically due to its high prevalence invarious types of cancers. Incidence of Krasmutation has been found to be highest in pancreatic cancer (85%–90%)followed by colorectal (35–45%) and lung (25–30%). Reovirus, in fact has the potential not only as a therapy butalso as a tool to unravel the aberrant cellular pathway leading to carcinogenicity.Mol Cancer Res; 10(12); 1514–25.�2012 AACR.

IntroductionExciting research in the past decade has revealed unique

viral characteristics not registered in any other microorgan-ism. Viruses harbor distinct strategies to overcome thesophisticated defense mechanisms of the infected host (1).The intricate ability to quickly gain control over the hostcellular mechanism has potentially made the virus a double-edged sword. While several viruses have been identified ascancer-causing agents, many have also been identified astherapeutically viable oncolytic elements. One importantmember of therapeutically identified viruses is RespiratoryEnteric Orphan virus commonly known as REOvirus (1). Itis naturally oncolytic with inherent propensity to replicate incells with dysfunctional cell signaling cascade including ras-activation. Preferential oncolytic properties of reovirus canbe effectively exploited as clinical therapy due to generalmildness of infection often requiring no special medicalintervention.

The search for oncolytic viruses roots from the fact thattransient cancer remission can occur following viral infec-tions (2). Supportive preclinical evidence of the novelmechanisms of anticancer activity of reovirus provided therationale for therapeutic advancement to clinical trials.Unique mode of tumor destruction encouraged oncolyticviral therapy as potential augmentation of chemotherapyand radiation thus making it a cutting edge clinical approach(3, 4).Many of the oncolytic viruses currently in clinical

testing are attenuated derivatives of prevalent humanpathogens. Typically, in recent years, they have beengenetically engineered to further reduce their pathogenic-ity and increase their oncolytic potency and enhancespecificity for cancer tissue. Virus with a double-strandedDNA genome is the most suitable candidate for suchmanipulations with greater genome stability and lesserchance of hazardous mutations. Adenoviruses and herpessimplex virus are the most suitable and thus been exten-sively engineered. Reverse genetic manipulation of RNAvirus is still a scientific challenge even with the availabilityof the present day cutting edge genetic technology. In asimilar vein, reovirus is not amenable to genetic engineer-ing, especially due to its segmented structure of thedsRNA (5). However, considering the fact that thisnaturally oncolytic, and pathologically self resolving virusseems to be tumor cytotoxic, even in the presence ofneutralizing antibodies, the lack of engineerability maynot be a constraint to its further therapeutic development.

Authors' Affiliation: 1Montefiore Medical Center, 2Albert Einstein Collegeof Medicine, Bronx, New York

R. Maitra and M.H. Ghalib contributed equally to this work.

Corresponding Author: Sanjay Goel, Department of Medical Oncology,Montefiore Medical Center, Albert Einstein College of Medicine, 1825Eastchester Road, Bronx, NY 10461. Phone: 718-904-2488; Fax: 718-904-2830; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-12-0157

�2012 American Association for Cancer Research.

MolecularCancer

Research

Mol Cancer Res; 10(12) December 20121514

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Preclinical Studies with ReovirusReovirus as a single agentIn vitro data with reovirus. Several preclinical studies

documented oncolytic characteristics of reovirus (6, 7).Initial studies with NIH-3T3 cells revealed that resistanceto reovirus infectivity can be overcome by transformationwith activated Sos/Ras oncogenes (8, 9). The findings indi-cated that usurpation of Ras signaling pathway constitutesthe basis of viral oncolysis (8). Several in vitro studies withhuman cancer cell lines documented the evident role ofreovirus in cellular cytopathy (4, 8). A systematic analysis ofthe propensity of reovirus infectivity towards 24 establishedglioma cell lines was conducted. Dramatic and widespreadcell killing after exposure to live (but not dead) reovirusoccurred in 20 (83%) cell lines. After 48 hours of infection,widespread cell death was found in U87, U251N, and A172cell lines, and almost complete cell death was seen after72 hours. In contrast, cells receiving either dead or no virusremained healthy. Furthermore, to ensure that cell lysis wasdue to viral replication, cells were reacted with rabbit anti-reovirus antibody, followed by FITC-conjugated goat anti-rabbit IgG when susceptible lines depicted the expressionof the viral antigens. Replication of reovirus in susceptiblelines was further confirmed by [35S]methionine metaboliclabeling (4).Short-term primary culture from surgically excised human

glioma have shown100% sensitivity to reoviral oncolysis (4).Studies conducted to test the ability of reovirus to infect andkill primary cultures of brain tumors freshly excised from 9patients with glioma intriguingly depicted complete growtharrest and cell death (100%), although similar level ofreovirus-mediated cellular cytopathy was not documentedwith 7 primary meningioma cultures (4). The ability ofreovirus to lyse all primary glioma cell cultures derived fromsurgical specimen suggested that a substantial proportion ofgliomas may respond to reovirus treatment. It can be arguedthat the number of specimens being relatively small, theobservations might not reflect the comprehensive tumorproperties of gliomas. The oncolytic spectrum of reovirus hasalso been studied in 6 breast cancer cell lines where a highsusceptibility was documented (10). An infectivity of 10MOI (multiplicity of infection) was used in the study.Furthermore, the controlHs578Bst normalmammary glandepithelial cell line used in the study did not show anycytopathic effect (CPE) to the virus confirming the fact thatreovirus preferentially targets the transformed cells sparingthe normal ones (9). Contemporary studies with melanomacell lines and primary cultures from fresh resected tumorsrevealed similar results. The transformed cells allowed viralreplication followed by caspase-dependent cytotoxicity (11).Normal melanocytes conversely resisted viral replication.Notably, it was also reported that all of the screened breastcancer and melanoma cell lines had activated Kras.The basis of the ability of reovirus to target and kill tumor

cells but not infect nonproliferating normal cells lies in itsability to usurp the highly activated signaling pathway foundin tumor cells (4). This ability is most clearly established forRas or elements in its downstream pathways.Ras activation is

very common in malignant gliomas, colorectal (CRC) can-cers, as well as pancreatic malignancies.Natural affinity of reovirus to kill and lyse cancer cells and

the prevalence of Kras mutation in many of the studiedmodels apparently instigated the researchers to attempt todraw a correlation between Kras mutation and viral onco-lysis. It is to be noted that the fact is yet to be substantiatedwith scientific evidence. A somewhat similar situation wasfaced by the scientific community while developing thetherapeutically viable adenovirus Onyx-01. The antitumoractivity of the virus was initially proposed to be solelydependent on the status of p53 but deeper introspectionrevealed that lytic activity was observed in both p53 negativeand WT conditions and the complex molecular mechanismof virus mediated oncolysis is yet to be clearly defined (12).Further studies to elucidate cellular events favoring reovi-

rus-mediated apoptosis confirmed that colon cancer celllines, HEK293 and HCT116 displayed elevated b-cateninexpression to promote reovirus-mediated oncolysis by down-regulation of NF-kB (3). Independent studies reported thatreovirus activates human dendritic cells to promote innateantitumor immunity (13). The exact role of different immuneeffector cells in oncolytic virus–mediated tumor regression hasnot yet been clearly defined. It is plausible that dendriticcells (DC) are likely to play a coordinating role in virus-mediated immune response as key antigen presenting cells(APC) that recognize the viral infection and regulate bothinnate and adaptive immunity (11). The fact that reovirus hasbeen found to be effective in tumor cytopathy despite increas-ing neutralizing anti reovirus antibody (NARA) in the serumlogically indicates the intricate role of the innate immunesystem in the virus-mediated oncolytic process. The obser-vation that reovirus-activatedDCs enhance the innate naturalkiller (NK) cells and cytotoxicT cells (Tc) by release of solublefactors inducing tumor cell killing via exocytosis clearlysupports the hypothesis. The role of NK cells in tumorregression has been well documented in mouse model bothby direct tumor recognition as well as via DC activation (14).Reovirus-induced DC maturation also stimulated the pro-duction of proinflammatory cytokines interferon (INF)-a,tumor necrosis factor (TNF)-a, interleukin (IL)-12p70, andIL-6. Activation of DCs by reovirus was not dependent onviral replication, whereas cytokine production was inhibitedby the blockade of PKR (protein kinase receptor) and NF-kBsignaling. These observations provide a hint that reovirus-mediated DC activation and the downstream immune sig-naling is multimodal. Although systemic delivery activatesthe adaptive immune system and triggers a robust antibodyresponse, intratumoral (ITu) injection of the virus results insuccessful tumor destruction by activation of the innateimmune effector cells within the tumor microenvironment.Hence, reovirus recognition by DCs may trigger innateeffecter mechanisms to complement the virus's direct cyto-toxicity, potentially enhancing the efficacy of reovirus as atherapeutic agent (13). Intravenous administration of reovirusin conjunction with immunosuppressants has been found tobe therapeutically more effective indicating that the neutral-izing antibodies are not completely blunted (15). All the same,

Development of Reovirus for Cancer Therapy

www.aacrjournals.org Mol Cancer Res; 10(12) December 2012 1515




the fact that antitumor activity is observed even in thepresence of virus-neutralizing antibodies can also indicate aplausible role of the host cellular machinery in camouflagingthe virus and thus preventing its recognition by the specificantibodies. In a very elegantly conducted translationalresearch study, it has been shown that replicating virus isdetectable in cellular compartment of the blood, namely inmononuclear, granulocyte and platelets, and this may be amechanism of protection from NARA (16). Furthermore, inthis same clinical trial, when patients' tumors were harvested,replicating virus was evident in tumor tissue but not fromnormal liver, showing some evidence of selective tropism formalignant cells.PKR plays crucial inhibitory role in efficient viral

replication essential for infective virion production andoncolysis (refs. 7, 17; Fig. 1). Expression of PKR isupregulated in response to INF released by infected cells(7). Binding to viral RNA/initial transcripts results in PKRdimerization, autophosphorylation, and activation (7).The viral S1 segment mRNA has been shown to be apotent activator of PKR (18). Once activated, PKR blocksthe primary and secondary reovirus protein translation.In Ras-transformed cells, PKR is not activated, allowingunabated viral replication and effective assembly of viralproteins for production of infection efficient virions(7, 17). Specific chemical inhibitors of PKR phosphory-lation restore reovirus translation in untransformed cellsproviding evidence for a direct role of PKR in definingresistance to reovirus replication (9). Ras activation issuggested to release the translational blocks in the trans-formed cells. However, the exact molecular mechanism ofcoordination between Ras activation and inhibition ofPKR-mediated viral translational remains elusive (Fig. 1).The recently discovered novel oncogene CUG2 (cancer

upregulatory gene-2), inhibits the expression of PKR andactivatesRas and p38MAPK (19). Studies further confirmedthat inhibition of p38 MAPK or Ras blocks reoviral pro-liferation even in the presence of CUG2 indicating thepossibility of multimodal crosstalk between Ras activationand inhibition of PKR phosphorylation (19). It is pertinenttomention that PKR dimerization and autophosphorylationis critical for effective viral propagation but the definitivecontribution of Kras mutation in the process is still elusive.Aberrant cell signaling cascade generates many anomalousevents within the cell and exactly how it facilitates theinhibition of PKR dimerization is yet to be defined clearly.Investigation of the mechanism of apoptosis in reovirus-

infected HEK293 cell line concluded a significant role ofTRAIL (TNF-related apoptosis-inducing ligand). The apo-ptotic event was successfully inhibited by anti-TRAIL anti-bodies or death receptor (DR)4 and DR5 (20). Similarinvolvement of TRAIL was confirmed in cell lines derivedfrom 2 different human lung (A157/H549) and breastcancers (MDA231/ZR75-1; refs. 20, 21). Reovirus infectionsynergistically sensitizes these cancer cell lines to killing byexogenous TRAIL. The observed sensitization was associ-ated with an increase in the activity of the death receptor-associated initiator caspase-8, and was inhibited by the

peptide IETD-fmk, suggesting that reovirus sensitizescancer cells to TRAIL-induced apoptosis in a caspase-8–dependent manner. Enhanced sensitization was also foundto be associated with increased cleavage of PARP, a substrateof the effector caspases 3 and 7 (22).In vivo experience using animal models of cancer.

Tumor regression studies in animals with reovirus as singleagent showed dramatic results. Pronounced effects oftenwith complete tumor regression was documented in vivo in 2subcutaneous (P ¼ 0.0002 for both U251N and U87) andin 2 intracerebral (P ¼ 0.0004 for U251N and P ¼ 0.0009for U87) human malignant glioma mouse models (4).Immunocompetent C3 mice implanted with Ras-trans-formed C3H10t1/2 fibroblast showed complete regressionof tumor after ITu injection of reovirus (23). Similar strategyof ITu injection resulted in significant tumor regression inSCID mice with subcutaneously implanted v-erbB–trans-formed NIH-3T3 cells (23), and in SCID-NOD mice withsubcutaneously implanted human malignant glioma (4, 8).Subcutaneous tumor allograft studies in immune competentC3H mice showed strong inhibition of tumor growth withintravenous administration of reovirus (15). A 74% cure ratewas observed in comparatively less immunocompromisednude mice harboring human malignant glioma with singleintralesion injection of reovirus. SCID-NOD mice bearingsubcutaneous U251N xenografts (4) or multiple myelomaRPMI8226GFPþ cells introduced via tail vein for tumorestablishment, when treated with single injection of live ordead reovirus showed a striking regression of live virus–treated tumors (24). Immunofluorescence analysis showedthat reovirus replication was restricted to the tumor masswithout spreading to the underlying normal tissue.

Reovirus as combination therapyAssociation with radiotherapy. Combination therapy

involving radiation and reovirus was evaluated both in vitroand in vivo (21). The CRC cell line HCT116 was treatedwith reovirus and radiation individually, and in combina-tion, showed a marked synergy in the combinational subset.In vivomurine studies depicted similar synergy in nude micewith tumor induced by HCT116/SW480 cell implantationas well as in C57BL6 mice by B16 melanoma cells mediatedtumor induction (21).The relative tumor volume (RTV) whenmeasured in nude

mice xenografted with RH30 and SK-ESI human sarcomaand treated with similar doses revealed lowest mean tumorvolume and longest event-free survival in the group receivingthe combination treatment. TUNEL assay conducted withtumor biopsies showed enhanced apoptotic activity in com-bination therapy as compared with single agent (25).Association with chemotherapy. In vitro studies to

determine augmentation of viral therapy with chemotherapywas done using gemcitabine, fluorouracil, cisplatin, anddoxorubicin in HCT116 cells. Reovirus was synergisticwith all 4 drugs across a wide range of concentration (26).The synergistic consequences observed with gemcitabineadvanced further onto in vivo evaluation of xenograftednude mice. A remarkable synergy was confirmed with no

Maitra et al.

Mol Cancer Res; 10(12) December 2012 Molecular Cancer Research1516




residual tumor tissues remaining at the end point of the studyin more than 95% of the mice (27).Another independent study of reovirus in combination

with cisplatin, mytomycin, vinblastine, or gemcitabine inNSCLC cells lines documented synergy in cell lines sensitiveto the chemotherapeutic drugs and antagonism in cell linesthat are drug resistant. The taxanes showed reasonablesynergy when administered in combination even in drug-resistant cell lines (28). Similarly C57BL/6 rodent with B16melanoma–induced tumor when treated by combinationtherapy of reovirus and cisplatin showed a synergistic reduc-

tion in RTV as compared with those receiving monotherapy(29).In context of animal models of human tumors, athymic

mice bearing xenografts of osteosarcoma, Ewings, and syno-vial sarcoma when treated in similar fashion also confirmed asynergistic antitumor activity in comparison with mono-therapy (30). Docetaxel administered in combination in celllines and PC-3 prostate cancer mouse model confirmedsynergy, which diminished with increasing docetaxel con-centration (31). The confinement of viral proteins within thetumor mass was confirmed by several studies including

Reovirus

Kra

s W

T

Kras m

utant

GAP RAS

RAS

GDP

GTP

GEF

GAP

RAS

??

RAS

GDP

GTP

GEF

Phagocytosis

Endosome

Viraluncoating

CUG2

Translation blocked

Translation

Cell lysis

release of infective virions

Protein

Virus assembly

Transcripts

P-PKR

P-elF2α elF2α

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ds-RNA

Figure 1. A schematic illustration of the current hypothesis of the fate of reovirus upon entering the host cell by phagocytosis. The status of the host cellsignaling cascadedetermines the downstreamconsequences.Kras (Kirsten rat sarcoma viral Oncogene) is amembrane-boundmolecular switchwhich bindsto and catalyses the hydrolysis of GTP (guanosine triphosphate) to initiate a cell signal. The hydrolysis is facilitated by GTPase activating protein(GAP) and guanine nucleotide exchange factor (GEF) with subsequent conversion to inactive Kras GDP (guanosine diphosphate) complex. On the left isrepresented the k-raswild-type (WT) environment, which prevents the translation of viral transcripts and abrogates reovirus assembly. The double-strandedRNA (dsRNA)-dependant protein kinase R (PKR) plays a crucial role in the viral translation block by dimerization and autophosphorylation to phospho-PKR(P-PKR), which in turn promotes the phosphorylation of translation initiation factor 2 subunit alpha (eIF2a) to P-eIF2a. The dashed arrow depicts thetranslational inhibition by P-eIF2a in Kras WT cells. In Kras-compromised situation (shown on right), the autophosphorylation of P-PKR is inhibited withunabated viral translation and subsequent virion assembly and cell lysis. The factors contributing to the crosstalk between defective Kras and compromisedPKR phosphorylation are unknown and thus represented by (?). Cancer upregulatory gene 2 (CUG2) frequently elevated in variety of tumor tissues hasbeen recently characterized to promote reoviral proliferation by activation of Kras and inhibition of PKR phosphorylation. CUG2 has been proposed as abiomarker to identify the subset of cancers that might benefit from reoviral therapy.






immunohistochemical evaluation of U87 xenograftedrodent (8). The broad oncolytic spectrum of reovirus assingle agent and in combination along with remarkablesuccess of the animal study platform provided the scientificconfidence to proceed onto clinical trials. Although muchpreclinical work has been carried out establishing thatreovirus could serve as a cancer therapy, the mechanismsgoverning the permissiveness of transformed cells to reovirusinfection remain to be fully characterized. Furthermore, asreovirus replicates in cancers of very diverse origin, it is likelythat the virus exploits cellular signals that occur often intransformation and tumorigenesis. A deeper understandingof the molecules involved in antiviral defense and thepatterns they recognize will allow harnessing them for bettertherapeutic strategies. Thus, delineating this usurpationshould in turn shed light on signaling that is commonamong various cancers, and may reveal novel therapeutictargets.Although the first hint of reovirus susceptibility (32) was

noted in 1977, it was not until 1990s that any scientific cluewas obtained regarding the preferential replication of reo-virus in transformed cells. Since then, systematic researchhas provided some clues on the molecular synergy of effi-cient reovirus oncolysis in ras-compromised cancer cells.Although the preliminary findingmight not be substantial indefining the viral tropism, it has nevertheless logicallypermitted clinicians to attempt the use of reovirus as treat-ment towards ras-mutated carcinogenicity especially in sit-uation where no other well established treatment regime isavailable.Incidentally, reovirus is intrinsically oncolytic without

need for any genetic manipulation. It is a naturally occurringvirus that possesses the right level of potency that renders itrelatively harmless to normal cells while having strong lyticeffect on ras-compromised cancer cells. Investigations on themechanisms of selective viral oncolysis in ras-mutated tumorcells will help unravel the complex cellular pathways involvedin cellular transformation which in turn will not only help inidentifying novel targets for cancer therapy but also shedlight on which cancer backgrounds are compatible with viraloncolysis strategies.

Clinical Experience with ReovirusReovirus has been therapeutically tested in 325 (as

reported) patients administered ITu or intravenously eitheras a monotherapy or in combination with radiotherapy orchemotherapy. The first human trial with reovirus began inJuly 2001, and since then a total of 27 clinical trials havebeen completed/initiated/planned as phase 1, 2 and 3.Thirteen trials have been completed, whereas 12 are ongoingand 2 have been announced. Of these 27 trials, 6 will besponsored by the National Cancer institute. Of the 16 trialswith reported clinical data, clinically formulated reovirus wasinjected ITu in 6 trials enrolling 90 patients, whereas theremaining 10 clinical trials involved the intravenous routeenrolling 235 patients (Table 1). The current dosing regi-men is intravenous of 3� 1010 TCID50 on days 1 to 5 over60 minutes repeated every 3 or 4 weeks in both mono- and

combination therapy. As with the preclinical observations,the clinical experience with reovirus has been interesting,evolving, and has shown promise. Summaries of the resultsare provided in Table 1.

Reovirus as intratumoral injectionsThe first human phase 1 trial was conducted in 18 patients

with metastatic or recurrent solid tumors with easy access tothe tumor to allow for direct ITu injection, and measure-ment by direct observation or palpation. The treatment waswell tolerated without any observation of dose-limitingtoxicity (DLT) and/or maximum tolerated dose (MTD;ref. 33). The second trial was based on the preclinicalexperience of reovirus on prostate-derived cell lines (34).Six patients with prostate cancer (stage T2/T3) received asingle transrectal ultrasonography (TRUS)-guided reovirusinjection followed by radical prostatectomy after 3 weeks.Histologic analysis after prostatectomy of the injected lesionsand other synchronous lesions showed CD8 T-cell infiltra-tion and evidence of caspase-3 activity within the reovirus-injected areas. The third clinical trial was based on thepreclinical data showing reovirus activity in malignant gli-omas (4). While oncolytic viruses have been used in clinicaltrials in malignant gliomas (35–38), this study was the firstto use reovirus. A total of 12 patients (10 glioblastomamultiforme, 1 anaplastic astrocytoma, and 1 anaplasticoligoastrocytoma) were treated at 3 dose-escalating levels(ongoing trial). Antibody studies showed a seroconversionrate of 83% consistent with other clinical studies. Subse-quently, a combined phase 1/2 study (currently ongoing)was started in recurrent malignant gliomas with a singleITu infusion (using transcranial catheters) of reovirusdirectly into the intracerebral tumor over 72 hours.Accrual to the phase I portion has been completed, butthe data are pending.In another 2-stage phase I trial, dose-escalating reovirus

was given ITu along with palliative radiotherapy to 23patients with advanced cancers (39). There was no evidenceof exacerbation of skin toxicity induced by radiation. Therewas marked efficacy with 7 of 14 evaluable patients havingexperienced a partial response (PR). In another phase 2,multicenter, clinical trial, ITu reovirus was evaluated whencombined with low-dose radiotherapy (20 Gy) in patientswith advanced cancer (40). This study showed safety andefficacy of the combination with 4 patients with PR and 2with minimal response.

Reovirus as systemic administrationThere have been 2 single agent, phase 1 trials of reovirus

as a systemic intravenous infusion. One clinical trial wasconducted at our center, MontefioreMedical Center, AlbertEinstein College of Medicine (Bronx, NY; ref. 41). This wasa single center dose-escalation trial and enrolled 18 patientswith refractory solid tumors. Overall, toxicities were minor,with only 2 patients experiencing grade 2 events, withoutan observation of a single DLT. One patient with breastcancer who received 7 cycles developed grade 2 fever andgrade 1 chills that progressively worsened with repeated

Maitra et al.





Tab

le1.

Sum

maryof

clinical

trialswith

reporteddata.

Thistable

summarizes

thecu

rren

tdatafrom

completedan

don

goingclinical

trialswith

Reo

virus

Study#

Indication

Other

therap

yNum

ber

of

patients

Pha

se

Lowes

tdose

(TCID

50)

Highe

stdose

(TCID

50)

Grade3/4

toxicities

Effica

cy

Viral

detec

tion

serum/C

SF/

stool/urine/

fece

sAntibody

resp

ons

eReferen

ceCommen

ts

Intratum

oral

1Solid

tumors

181

107(PFU

)10

10(PFU

)Non

e1CR,1

PR,

8SD

Serum

Yes

33

2Prostate

6Pilo

t10

7(PFU

)Non

e1PR,4

SD

Urin

eYes

343

Maligna

ntgliomas

121

1�

107

1�

109

Tran

sien

tGGT

elev

ation

1SD

Saliva,

Fece

sYes

6Solid

tumors

XRT

231

1�

108

1�

1010

Non

e7PR,7

SD

Non

eYes

3920

–36

Gy

7Maligna

ntgliomas

151/2

1�

108

1�

1010

NR

NR

NR

NR

8Solid

tumors

XRT-20

Gy

162

1�

1010

Non

e4PR,2

MR,

7SD

NR

Yes

40

Intrav

enou

s4

Solid

tumors

181

1�

108

3�

1010

Non

e1PR,7

SD

Urin

e,Serum

,Saliva,

Fece

sYes

41

5Solid

tumors

331

1�

108

3�

1010

Flu-likesy

ndrome

3MR,7

SD

Urin

e,Serum

,Saliva,

Fece

sYes

44

CPK-M

B/TropI

elev

ation

Lympho

pen

iaNeu

trop

enia

9Solid

tumors

Gem

citabine

1000

mg/m2

121

1�

109

3�

1010

Flu-like

synd

rome

48

Neu

trop

enia

AST/ALT

/GGT

elev

ation

Trop

Ieleva

ton/

STch

ange

s10

Solid

tumors

Doc

etax

el75

mg/m2

241

1�

109

3�

1010

Febrile

neutropen

ia4PR,3

MR,

7SD

Urin

e,Serum

,Saliva,

Fece

sYes

54

Thromboc

ytop

enia

GIs

ymptoms

Hyp

okalem

iaHyp

oten

sion

NR

NR

11Solid

tumors

Carbop

latin

AUC

531

1/2

3�

109

3�

1010

Mye

losu

ppress

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8PR,6

SD

56Effica

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Pac

litax

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5mg/m

2

Hyp

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patientswith

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(Con

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don

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Tab

le1.

Sum

maryof

clinical

trialswith

reporteddata.

Thistable

summarizes

thecu

rren

tdatafrom

completedan

don

goingclinical

trials

with

Reo

virus

(Con

t'd)

Study#

Indication

Other

therap

yNum

ber

of

patients

Pha

se

Lowes

tdose

(TCID

50)

Highe

stdose

(TCID

50)

Grade3/4

toxicities

Effica

cy

Viral

detec

tion

serum/C

SF/

stool/urine/

fece

sAntibody

resp

ons

eReferen

ceCommen

ts

13Colorec

tal

canc

erwith

liver

metas

tase

s

102

1�

1010

Yes

16Viru

sse

lectively

detec

ted

inca

ncerou

sliver

tissu

e14

Sarco

mas

532

3�

1010

Neu

trop

enia

19SD

NR

NR

Optic

neuritis

15Hea

dan

dne

ckca

ncer

Carbop

latin

AUC

514

23�

1010

Neu

trop

enia

4PR,2SD

NR

NR

57

Pac

litax

el17

5mg/m

2

Hyp

okalem

iaFa

tigue

Ane

mia

Nau

sea

Hyp

oten

sion

16NSCLC

Carbop

latin

AUC

622

23�

1010

Mye

losu

p-

pression

6PR,13

SD

NR

NR

58

Pac

litax

el20

0mg/m

2

Hyp

oten

sion

Fatig

ueGIs

ymptoms

17Pan

crea

ticca

ncer

Gem

citabine

800mg/m

2

182

3�

1010

Neu

trop

enia

1un

confi

rmed

PR

NR

NR

52

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administration. She had a PR and her tumor had a mutationin ras codon 12 (42, 43). In the second, 33 patients enrolled,of which 10 patients showed stable disease (SD; ref. 44).Overall, 28 patients showed an increase in NARA titers to amaximum at 4 weeks, which remained constant duringsubsequent cycles (45). Despite the NARA response, whichcould have blunted the viral delivery to tumor site (via rapidneutralization), viable virus was shown in post treatmentbiopsies after 2 cycles (45).More recently, in a phase 2 study that enrolled patients

with advanced CRCwith liver metastasis, reovirus was givenintravenously for 5 consecutive weeks before the plannedliver metastesectomy. Among the 10 patients treated so far,there is evidence that reovirus selectively targets tumor cellsversus normal liver cells (60% of patients have shown noevidence of reovirus in their normal liver cells). In 2 patients,only necrotic tissue was seen, whereas reovirus was detectedin the immune cells of the tumor of 1 patient. This newexciting information shows that reovirus can be deliveredspecifically and selectively as a monotherapy while sparingthe normal liver cells (ongoing trial). The first completedphase II trial of single agent reovirus targeted 53 patients withsoft tissue and bone sarcomas withmetastasis to lung. A totalof 19 patients showed SD. One patient with synovialsarcoma had SD for more than 80 weeks.There is strong preclinical rationale to combine intrave-

nous infusion of reovirus with cytotoxic chemotherapy likegemcitabine, carboplatin, or docetaxel (15, 27, 28, 46, 47).The phase 2 dose of reovirus was based on two-phase 1monotherapy trials (41, 44). It was postulated that chemo-therapy might blunt the immune-mediated viral clearancewithout significantly increasing the toxicity profile. In thefirst combination phase I study, intravenous reovirus (on day1–5 of each cycle with the starting dose at 3� 109 TCID50)was administeredwith gemcitabine at 1,000mg/m2 on day 1and day 8 (21-day cycle) in dose-escalating cohorts (48).However, with the observation of 2 DLTs [grade 3 ALT(alanine aminotransferase) rise and grade Troponin I] thedose of reovirus was amended to a 1-day treatment. Onepatient with nasopharyngeal carcinoma [(NPC) which iscaused by an Epstein Barr virus (EBV) infection] showed aPR, possibly due to the expression of EGFR (EBV-inducedexpression). The combination showed clinical activitydespite the potential NARA response, and ongoing workis unraveling the intricate details of mechanisms involved inreovirus-medicated oncolysis and resistance (49, 50). On thebasis of this clinical (48) and prior preclinical findings (51), aphase 2 clinical trial in patients with metastatic pancreaticcancer is ongoing, and preliminary data suggests a clinicalbenefit rate of 58%with prolonged SD (52). Preclinical datasuggestive of hepatic toxicity associated with reovirus (53)and the observation of the ALT elevation when combinedwith gemcitabine (48) should be factored in dealing withsituations where patients have compromised hepatic func-tion or are taking potentially hepatotoxic medications, suchas acetaminophen.Therapeutically formulated reovirus has been combined

with docetaxel in 24 patients with advanced cancer (54). Of

all patients treated, 46% experienced grade 3 or greaterneutropenia, consistent with that observed with docetaxelmonotherapy (65%) at the same dose and schedule. MTDwas not technically reached as only one DLT, of grade 4neutropenia, was encountered. Four PR were observed inbreast, stomach, gastroesophageal, and ocular melanomaalong with 3 minor responses in mesothelioma, prostatecancer, and squamous cell cancer of the head and neck. Only2 patients showed evidence of viral shedding. Pharmacoki-netic studies revealed no change in docetaxel clearance by theaddition of reovirus. Docetaxel showed no effect on NARA,a finding that was inconsistent with preclinical data (55).In a separate phase I trial, reovirus was added to the

combination of carboplatin and paclitaxel (56). With pre-liminary signs of clinical activity, the phase 2 expansioncohort was enriched with patients with squamous cellcarcinomas of head and neck (HNSCC), and remarkably,8 of 19 (42%) evaluable patients showed PR. The toxicitieswere consistent with earlier results. This study showed thesafety and efficacy of this combination, leading to a targetedphase II study in patients with HNSCC with a RR of 31%(57), and finally to a randomized phase III study of carbo-platin and paclitaxel with orwithout reovirus in patientswithplatinum refractory HNSCC (ongoing trial). The samecombination has also been tested in patients with advancednon–small cell lung cancer (58). As reported at the 14thWorld Lung Cancer Congress, the combination was welltolerated and of the 23 patients entered, 6 patients had a PRand 13 had SD.

Neutralizing anti-reovirus antibodiesAn important and critical issue when considering the

clinical use of reovirus is the presence of preexisting andthe development of a rapid massive rise of titer of "ontherapy" NARA. This phenomenon of NARA developmenthas been consistently observed across the single agent and inthe chemotherapy combination trials. It is clearly evidentthat despite the presence of preexisting NARA, there is adetectable 100-fold increase in the viral titer, which has beensuggested to be both desirable and unwarranted. On onehand, it allows for limiting the toxic effects of the virus andprotects patients from the unwanted side effects of the virusinfection, whereas, on the other, it may compromise itsbeneficial potent anticancer effects. Prior preclinical in vivostudies in murine models using C3H mice have shown thatblunting the immune response using cyclosporine A or antiCD4/CD8 antibodies leads to improved efficacy (15).Similarly, cyclophosphamide, when used as a modulatingagent, was effective in ensuring tumor seeding of the intra-venously delivered reovirus in tumors, an entity which waspreviously not attainable (59). By modifying the dose of thecyclophosphamide, the authors also showed that it modu-lates, but does not ablate, the NARA response.The concomitant delivery of reovirus with chemotherapy

has been purported to be of benefit, partly by the attenua-tion of the NARA response. However, in combinationchemotherapy trials, specifically with docetaxel, and carbo-platin–paclitaxel, there was a significant rise in NARA titer






and no discernable increase in toxicity (46, 56). Anotherinteresting phenomenon observed in these trials was that incomparison with the single-agent studies, the rate of rise ofthe NARA titer was lower, thereby leading to a delay in theachievement of the peak titer. Slower development ofNARA may have beneficial effects by enhancing tumorseeding of the virus leading to greater efficacy withoutcompromising safety. Interestingly, the only agent that hadsome suggestion of the ability to blunt the NA response wasgemcitabine (48). This is not surprising as gemcitabine hasbeen shown in murine models to specifically affect thegeneration of antibodies by B cells (49) and to suppressmyeloid suppressor cells (60). Furthermore, independent ofthe attenuation of theNARA response, gemcitabine has beenreported to tip cellular immunity favoring reovirus-initiatedantitumor immune response (51, 60). The attenuatedNARA response allowed for greater virus replication leadingto greater toxicity of the combination as compared with thesingle-agent profile, an important but not entirely unex-pected clinical observation. Therefore, careful monitoring ofimmune function should remain an important considerationin future combination trials.Moreover, it has been suggestedthat because of the interference of the adaptive immunity ofthe host, the most effective systemic delivery of reovirus willbe achieved through rapid, repeated high doses of viruswithin the first week of treatment before theNARA responsehas been boosted (45). The current dosing schema of 5continuous daily intravenous injections of the virus every 3to 4 weeks, therefore, has some clinical and scientificrationale.

Summary, Conclusions, and the FutureIn summary, reovirus has shown promise with far reaching

implications for future drug development. While initiallydeveloped as an anticancer agent based on the scientificpremise that viral replication is supported in ras-drivencancer cells; the paradox lies in that the clinical developmenthas not been driven by this fact. Moreover, clinical benefithas also been observed in patients with tumors where theincidence of ras mutations has historically been very low.This clearly suggests that a second important clinical phe-nomenon is underway in promoting virus efficacy. As dis-cussed extensively earlier in this review, evidence suggeststhat reovirus, similar to other viruses, manipulates theimmune system tomount an anticancer response. As intrigu-ing as this fact is, it only further affirms that the translation ofscience from "bench" to "bedside" is challenging, to say theleast.Further characterization of the activity of the virus is

clearly required and it is imperative that it be in the form ofa concerted effort of laboratory scientists with an interestin tumor biology, virologists, physician scientists, andacademic clinicians, with a common sense of purpose. Itis also critical that the patient be entirely integrated intothis effort. The availability of tumor tissue to study both,the effect of this therapy and the biology behind the drivingforce of the cancer is absolutely essential and cannot beoverstated.

It is important to note here that using viruses to targetcancer is not a new phenomenon. In fact, viral approaches tocancer have been attempted for over half a century with littlesuccess (61, 62). One of the most extensively studiedproducts is ONYX-015, an attenuated E1B-55K chimerichuman group C adenovirus, which preferentially replicateswithin and lyses tumor cells that are p53 negative (12).Almost a decade later, another novel adenovirus mutant,ONYX-053, was created that showed that loss of E1B-55K–mediated late viral RNA export, rather than p53 degrada-tion, restricts ONYX-015 replication in primary cells. It wasexperimentally proven that in contrast to the initial hypoth-esis, tumor cells that supportONYX-015 replication providethe RNA export function of E1B-55K (63). There was greatenthusiasm for this approach when a similar product, thegenetically modified adenovirus H101, made by ShanghaiSunway Biotech obtained commercial approval in China inthe treatment of advanced head and neck cancer (64). Onceagain, despite the promises of early in vivo lab work suggest-ing tumor specificity, these viruses do not specifically infectcancer cells; however, they still retain some preferential cellkill for cancer cells. As of the last report, response rates wereapproximately doubled for H101 plus chemotherapy ascompared with chemotherapy alone; however, survivalinformation is unknown. Another limitation of thisapproach is that it seems to render maximum benefit whengiven as a direct ITu injection and the patient experiences afebrile response (65). Another virus in late-stage clinicaldevelopment is Oncovex, a second generation oncolyticherpes simplex virus that expresses GM-CSF (granulocytemacrophage-colony stimulating factor) and in which dele-tion of infected cell protein (ICP) 34.5 provides tumorselectivity. This has been tested as a single-agent ITu therapyand in combination with radiotherapy (66, 67). It is cur-rently in phase III clinical development to test efficacy inmalignant melanoma (ongoing trial).Other approaches using virus-mediated oncolysis have

included the use of the oncolytic adenovirus ICOVIR-5 asa treatment for malignant gliomas, PV 701, an attenuatedform of the Newcastle virus (68, 69) and the vesicularstomatitis virus (70–73). Even more intriguing, the mea-sles virus is also being evaluated as an oncolytic virus, withencouraging data in breast and ovarian cancer (74–76).The reader is referred to more detailed reviews on onco-lytic viruses (77–79). These examples of viral oncolytictherapy elucidate the multiple challenges that we face indeveloping new viral therapies for cancer, includingreovirus.Reolysin is the trade name of the therapeutic version of

human reovirus formulated and developed by OncolyticsBiotech Inc.. It is a translucent light blue liquid containing apurified isolate of 1� 1011 TCID50 (tissue culture infectivedose) of replication competent reovirus serotype 3 Dearingstrain per milliliter in a phosphate-buffered solution and isused in many of the clinical trials. The virus in its clinicalformulation as Reolysin has rapidly progressed throughclinical development to a phase III trial in platinum-refrac-tory HNSCC (ongoing trial). An interesting pathway of the

Maitra et al.





clinical development of reovirus lies in metastatic CRC;where patients, whose tumors harbor a mutation in the Krasoncogene, are ineligible to receive the anti-EGFR monoclo-nal antibodies, cetuximab and panitumumab (80, 81). Onthe basis of preclinical in vivo data that the combination ofreovirus and irinotecan is particularly synergistic in the ras-mutant cancer cells (82), the phase I study of the combi-nation of FOLFIRI (folinic acid, 5-fluorouracil, and irino-tecan) with reovirus is currently targeting patients with aKras mutation, with the potential to fulfill an unmet medi-cal need. As mentioned earlier, preclinical observations ofpreferential viral tropism under specific mutational status isnot always replicable in human subjects. The targeting ofreoviral therapy on Kras-mutated mCRC subjects is notselected on the basis of the preclinical promises of enhancedvirulence under Kras conditions but rather due to lack ofany U.S. Food and Drug Administration (FDA)-approvedtherapy for platinum-refractory mCRC subset of patients.This clearly exemplifies the future of drug development:

the potential of a safe and effective drug whose developmentis biomarker driven. The current climate of research and

health care in the United States is at a crossroads with majorchanges expected in the manner that health care is deliveredby caregivers, received by patients, and paid for by third partypayers, including the governments and the private insuranceplans. The success or failure of a drug being tested in clinicwill be highly dependent on its absolute effectiveness in anappropriate patient profile based on a validated biomarker.This is clearly a "win win" situation for all the partiesinvolved: the patient only receives the medication that ishighly likely to benefit him/her, thereby also avoidingunnecessary toxicities, and will also bring down the cost ofhealthcare by paying only for effective therapies. A word ofcaution is appropriate here: despite the encouraging devel-opment of reovirus so far, the "proof of the pudding is in theeating", and unless it can show improvement and patientbenefit over the current standard of care in a well-conductedphase III trial, it will be relegated to the confines of history, ashave many of its predecessors.

Received March 15, 2012; revised July 23, 2012; accepted September 20, 2012;published OnlineFirst October 4, 2012.

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2012;10:1514-1525. Published OnlineFirst October 4, 2012.Mol Cancer Res Radhashree Maitra, Mohammad H. Ghalib and Sanjay Goel

Progress And Potential−−Reovirus: A Targeted Therapeutic

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