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Cancer Therapy: Preclinical

The Efficacy of Oncolytic Adenovirus Is Mediatedby T-cell Responses against Virus and Tumor inSyrian Hamster ModelXiaozhu Li1, Pengju Wang2, Hang Li1, Xuexiang Du1,3, Mingyue Liu1,3,Qibin Huang1, Yaohe Wang2,4, and Shengdian Wang1

Abstract

Purpose: Oncolytic adenoviruses (Ad) represent an innova-tive approach to cancer therapy. Its efficacy depends on mul-tiple actions, including direct tumor lysis and stimulation ofantiviral and antitumor immune responses. In this study, weinvestigated the roles of T-cell responses in oncolytic adenoviraltherapy.

Experimental Design: An immunocompetent and viral repli-cation–permissive Syrian hamster tumor model was used. Thetherapeutic mechanisms of oncolytic Ad were investigated by T-cell deletion, immunohistochemical staining, and CTL assay.

Results: Deletion of T cells with an anti-CD3 antibodycompletely demolished the antitumor efficacy of oncolytic Ad.Intratumoral injection of Ad induced strong virus- and tumor-specific T-cell responses, as well as antiviral antibody response.

Both antiviral and antitumor T-cell responses contributed to theefficacy of oncolytic Ad. Deletion of T cells increased viral repli-cation and extended the persistence of infectious virus withintumors but almost abrogated the antitumor efficacy. Preexistingantiviral immunity promoted the clearance of injected oncolyticAd from tumors but had no effect on antitumor efficacy. Strik-ingly, the repeated treatment with oncolytic Ad has strong ther-apeutic effect on relapsed tumors or tumors insensitive to theprimary viral therapy.

Conclusions: These results demonstrate that T cell–mediatedimmune responses outweigh the direct oncolysis in mediatingantitumor efficacyof oncolytic Ad.Ourdatahave ahigh impact onredesigning the regimen of oncolytic Ad for cancer treatment. ClinCancer Res; 23(1); 239–49. �2016 AACR.

IntroductionOncolytic viruses (OV) are self-replicating, theoretically tumor

selective, and possess an ability to directly lyse cancer cells. OVsare being developed as an attractive therapeutics for cancers (1).Viral replication is quite immunogenic. An active host immuneresponse against the viruses that rapidly eliminates the virus hadbeen considered as an important barrier to the successful cancervirotherapy. Indeed, several studies showed that suppressing theimmune system enhanced the efficacy of oncolytic adenovirus(Ad; refs. 2, 3). However, there has been accumulating evidencethat the virus-induced immune activation can generate innate andadaptive immune responses that are critical to mediating theantitumor responses. Recently, preclinical and clinical data sug-gested that virotherapymight in fact act as cancer immunotherapy(4, 5). The interactions betweenoncolytic viruses, tumor cells, and

the immune system are critical to the outcome of antitumorefficacy of OVs.

Like many OVs, Ad replication is species specific and humanAds replicate poorly in cells frommost other species likemice andrats. Consequently, most published efficacy data have come fromimmunodeficient mice bearing human tumor xenografts. Thesemodels cannot adequately address the effect of the host immunesystem on the vector, tumor, as well as the toxicity and biodis-tribution of the vector in normal tissues. The Syrian hamster hasbeen characterized as a suitable immunocompetent, replication-permissive animal model for the assessment of human oncolyticAds (6, 7). Intratumoral injection of oncolytic Ad resulted in viralreplication and necrosis of tumors, suppression of primary andmetastatic lesions, viral replication in the liver and lungs, and anti-Ad antibody induction. Wold and colleagues used this model tostudy the effects of preexisting anti-Ad immunity and immuno-suppression on replication and antitumor efficacy of oncolytic Advectors. They found that immunosuppression induced by elim-ination of all white blood cells with cyclophosphamide abrogatedanti-Ad immune response, allowed sustained Ad replication, andsignificantly improved tumor suppression (2). However, thepreimmunization, which induced a persistent high-level of neu-tralizing anti-Ad antibody and disabled the detection of theinfectious virus injected in tumors, had no effect on the antitumorefficacy of oncolytic Ad in immunocompetent animals. In thepreimmunized tumor-bearing animals, cyclophosphamide treat-ment had no effect on tumor growth, although the titer ofneutralizing anti-Ad antibody stayed steady at all time points,and tumors had moderate amount of virus (8). These resultssuggest that curative viral oncolysis on its own probably only

1CAS Key Laboratory of Infection and Immunity, Institute of Biophysics, ChineseAcademyof Sciences, Beijing, China. 2National Centre for International Researchin Cell and Gene Therapy, Sino-British Research Center for Molecular Oncology,Zhengzhou University, Zhengzhou, China. 3University of the Chinese Academyof Sciences, Chinese Academy of Sciences, Beijing, China. 4Centre for MolecularOncology, Barts Cancer Institute, Queen Mary University of London, London,United Kingdom.

Corresponding Author: Shengdian Wang, Institute of Biophysics, ChineseAcademy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101, China.Phone: 8610-6488-8493; Fax: 8610-6484-6538; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-16-0477

�2016 American Association for Cancer Research.

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occurs in the instance when tumors are completely devoid ofany virus defense system. On the other hand, promotion ofimmune responses by arming oncolytic Ad with immunosti-mulatory molecules in the immunocompetent hosts signifi-cantly enhanced antitumor effects (9–11). Thus, the hostimmune responses will probably be critical to the efficacy ofoncolytic virotherapy, although they also mediate the rapidviral elimination.

In recent years, a lot of clinical data in cancer immunotherapyclearly demonstrated that T-cell response is the dominant fighteragainst cancers. The cellular antiviral immune responsemay limitefficacy of virotherapy by eliminating tumor infection via clear-ance of infected tumor cells. Alternatively, clearance of infectedtumor cells may play a key role in tumor regression. Some studiesshowed that T-cell response triggered by oncolytic virotherapyincluded virus-specific cytotoxic activity, which might play a rolein tumor therapy (12–14). Although T cells are major compo-nents for both antivirus and antitumor immunity, they have beenrarely studied in oncolytic Ad therapy in immunocompetent,replication-permissive hosts. In this study, we evaluated theeffects of T cells in oncolytic Ad therapy by using an anti-Syrianhamster CD3 mAb that can delete T cells in hamsters. We foundthat oncolytic Ad therapy induced strong virus-specific andtumor-specific T-cell responses. Deletion of T cells extended thepersistence of infectious virus in tumors, but almost abrogated theantitumor efficacy. These results suggest that T cell–mediatedimmune responses outweigh direct oncolysis of Ad in mediatingantitumor efficacy in immunocompetent hosts.

Materials and MethodsCell lines, viruses, and reagents

The Syrian hamster pancreatic carcinoma cell line HPD-1NRwas purchased from German Collection of Microorganisms andCell Culture, and kidney tumor cell line HAK was originallypurchased from the ATCC. JH293, the human kidney epithelialcell line transformed with Ad5 DNA, was obtained from CancerResearch UK Central Cell Services. All cell lines were cultured inbasal medium (HPD-NR and HAK, RPMI1640; JH293, DMEM)supplemented with 10% FBS (Hyclone), 2 mmol/L L-glutamine(Hyclone), 100 IU/mL penicillin, and 50 mg/mL streptomycin.

Cells were tested negative for mycoplasma. Cells were not furtherauthenticated.

Wild-type adenovirus type 5 (Ad5) and E1A-deleted Ad5(dl312) were from Sino-British Research Centre, ZhengzhouUniversity (Zhengzhou, China), and grown on HEK-293 cells asdescribed previously (15).

The mouse mAb against Syrian hamster CD3e (clone 4F11,IgG1 isotype) and anti-KLH mAb were prepared as describedpreviously (16).

In vivo treatmentsFour- to 5-week-old female Syrian (golden) hamsters (Meso-

cricetus auratus) were obtained from Vital River Corp. and inoc-ulated subcutaneously with 1 � 106 HPD-1NR cells. The tumor-bearing animals were treated by intratumoral injection of Ad5 orPBS every other day for six times from day 11 after tumorinoculation. The injections were introduced through a singlecentral tumor puncture site, and 3 to 4 needles tracts were maderadially from the center while the virus was injected as the needlewas withdrawn. To delete T cells, an anti-hamster CD3 mAb(4F11) was injected intraperitoneally at doses of 500 mg perinjection every 5 days from the day before the viral therapy, andanti-KLH mAb was used as control. Tumor size was measuredtwice weekly using digital calipers, and tumor volume was cal-culated using the formula: volume¼ (length�width2�p/6). Allanimal experiments were approved by the Animal Welfare andResearch Ethics Committee of the Institute of Biophysics, ChineseAcademy of Sciences (Beijing, China) and were conducted inaccordance with institutional and national regulations.

CTL assaysCytotoxic T lymphocyte (CTL) assays were done as previously

described with a little modification (17). In briefly, splenocyteswere stimulated in vitro with 60Co-irradiated HPD-1NR tumorcells infectedwith dl312 at a ratio of 5:1 in RPMI1640medium for4 days. The lymphocytes were isolated by Lymphocyte SeparationMedia (LTS1077, TBD Corp.) as effector cells. Both HAK cellsinfected with dl312 for 24 hours and HPD-1NR cells withoutinfection were stainedwith 10 mmol/L CFSE (CFSEhigh) as specifictarget cells. HAK cells without viral infection were stained with0.5 mmol/L CFSE (CFSElow) as control target cells. A mixture ofspecific and control target cells at a 1:1 ratio was incubated witheffector cells at different E/T ratios in a round-bottom 96-wellplate for 16 hours. Then, the cells were harvested and stainedwith7-aminoactinomycin D (7-ADD). The CFSE profiles were ana-lyzed using 7-AAD�CFSEþ gate. Cytotoxicity was determined bythe following formula: cytotoxicity (%) ¼ 100% � [1�ratio(target/control)þeffector/ratio (target/control)�effector].

Assessment of virus in tumorsFresh tumor tissues were homogenized and titrated on JH-293

cells to determine the 50% tissue culture infective dose (TCID50)as described previously (18). Viral DNA levels were evaluated asdescribed previously (16). Results were expressed as genome copynumber/gram based on the weight of tumor tissues.

IHCThe harvested tumor tissues were processed and stained respec-

tively with anti-hamster CD3 mAb (4F11), anti-Ad5 E1A mAb(MA5-13643, Thermo Scientific), and anti-Ad5 Hexon mAb(ab8249, Abcam), followed by relevant peroxidase-conjugated

Translational Relevance

Oncolytic viruses are able to directly lyse cancer cells andrepresent an innovative approach to cancer therapy. Virusesadditionally represent a potent immunologic "danger signal,"which facilitates the generation of antiviral and antitumorimmune responses. The roles and the relative therapeuticimportance of the direct tumor lysis, antiviral and antitumorimmunity in antitumor efficacy of oncolytic virotherapy haveremained uncertain. We explored the effects of T cells inoncolytic adenoviral therapy by using a deleting anti-Syrianhamster CD3 mAb. We identified the key roles of T cells inoncolytic adenovirus (Ad) therapy, in which viral-specific T-cell response may act as a bridge linking Ad infection andoncolysis to the antitumor immune responses. These resultsshed light on developing new therapeutic regime of oncolyticAd by enhancing antigen-specific T-cell responses.

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Clin Cancer Res; 23(1) January 1, 2017 Clinical Cancer Research240




secondary antibodies. The peroxidase activity was detected withDAB substrates (ZLI-9019, Zhongshan Golden Bridge Biotech-nology). Apoptosiswas detected in situusing theRoche TUNELKit(11684817910, In Situ Cell Death Detection Kit, POD, Roche)according to the manufacturer's instructions. For assessment ofthe positive staining proportion of tumors, each samplewas cut inat least three sections at different, equally spaced depths, and fivehigh-power fields (HPF, 200�) were randomly selected for eachsection. A total of more than 15 high-powered fields for eachsample were counted, and the exact number of positive cells wasdetermined per HPF by an image analysis system (Image-Pro Plus6.0). The values for the different fields were then averaged toobtain the final values of each sample for statistical analysis.

Detection of anti-Ad5 antibodiesAnti-Ad5 antibodies were detected as described previously

(16). The relative levels of total antibodies in all samples wereshown as the value of OD450nm.

Statistical analysisStatistical analysis was done with GraphPad Prism 5 software.

The results are expressed as means with the SDs (�SD) whenappropriate. Differences between groups were analyzed using thetwo-tailed Student t test or two-way ANOVA. P < 0.05 (�) wasconsidered significant, and P < 0.01 (��) was considered highlysignificant.

ResultsAntitumor efficacyof oncolytic AddependsonT cells inhamstertumor model

To investigate the role of T cells in antitumor efficacy ofoncolytic Ad therapy, Syrian hamsters were inoculated subcuta-neously with HPD-1NR pancreatic carcinoma cells and treatedwith 6 intratumoral injections of oncolytic adenovirus (Ad5) orPBS at 1-day interval from day 11 after tumor inoculation whenthe tumors were 6 to 7mm in diameter. Meanwhile, anti-hamsterCD3 mAb (4F11) or control Ig was injected intraperitoneally at a3-day interval from the day before the start of viral therapy.Consistent with the previous studies (6), Ad therapy markedlyinhibited tumor growth compared with intratumoral injection ofPBS. Moreover, the antitumor efficacy of Ad was dose dependent(Fig. 1A). The Ad treatment with a low dosage of 5� 108 plaque-forming unit (PFU) per injection inhibited tumor growth andcaused transient tumor regression, leading to significant delay oftumor growth in most animals (80%). One of 5 animals in thegroup was cured and remained tumor free for more than 60 days(data not shown). The regressed tumors in other animals startedgrowing rapidly about 2 weeks after viral treatment. Ten timeshigher dosage (5�109PFU/injection) ofAdmarkedly suppressedtumor growth and caused tumor regression in all animals, inwhich tumors were completely disappeared in 3 of 5 animals. Therecurrent tumors also started to grow about 2 weeks after Ad5treatment. When T cells were deleted, Ad therapy still markedlyinhibited tumor growth as well during the vector injections.However, it could not cause tumor regression even with a highdosage of Ad, and all tumors started to grow about one week afterAd treatment, which was one week earlier than the recurrenttumors in immunocompetent animals. Importantly, the tumorgrowth after Ad treatment in T cell–deficient animals was fasterthan that in immunocompetent animals. These data showed that

oncolytic Ad vector effectively inhibited tumor growth duringviral treatment but failed to cause tumor regression in the T cell–deficient animals. Deletion of T cells caused rapid regrowth of thesuppressed tumors after viral therapies. To further assess thecontributions of CD4þ and CD8þ T cells to the antitumor effect,we compared the effect of T-cell deletion with that of CD4þ T-celldeletion in the therapeutic efficacy of oncolytic Ad, by using ananti-mouse CD4 antibody that cross-reacted with T cells ofhamsters (19). Compared with deletion of total T cells, deletionof CD4þ T cells marginally attenuated the antitumor effect ofoncolytic Ad, suggesting that the antitumor T-cell response ismajorly mediated by CD8þ T cells (Fig. 1B). These results dem-onstrate that T-cell responses, especially CD8þ T cell–mediatedresponse, play a critical role in the therapeutic effect of oncolyticAd, especially in mediating tumor regression and preventingtumor recurrence after viral treatment.

To determine whether the T-cell responses initiated by Advector result in immunologic memory, the hallmark of adaptiveimmunity, we evaluated the cured hamsters for long-term pro-tection by tumor rechallenge with or without T-cell deletion. Thehamsters that underwent complete tumor regression followingAdtreatment were rechallenged with 2 � 106 HPD-1NR cells (twotimes of the primary tumor inoculation) after primary tumors hadnot been detected for one month. All animals rejected the rechal-lenged tumors. However, once the cured animals were intraper-itoneally injected with anti-CD3 mAb at the day before therechallenge, all the rechallenged tumors grew progressively (Fig.1C). This result strongly supports that oncolytic Ad therapygenerates antitumor memory T cells capable of protecting thehost from rechallenge, and presumably against tumor relapse.

T cells are associated with elimination of infectious Ad andcytolysis of tumors

T-cell deficiency has a contrasting effect on antitumor efficacy ofoncolytic Ad in Syrian hamster with the reported cyclophospha-mide-induced immunosuppression, in which all immune cellswere deleted (2). Thus, it is interesting to see the effects of T cellson viral replication in tumor and their contributions to antitumorefficacy. To this end, tumor-bearing hamsters were treated with 6intratumoral injections of low dosage of Ad5 with or withoutdeletion of T cells, and the infectious viral yields, viral proteinexpression, and viral DNA level in tumor tissues were determinedat the indicated timepoints. The infectious virions of Ad5detectedby viral replication assay remained elevated at the same level(�106 PFU/g or so) in both tumors of immunocompetent and Tcell–depleted animals within 3 days after the first viral injection.Then, in immunocompetent animals, virus levels were decreasedapproximately 10-fold (105 PFU/g) by day 7. At day 11, just oneday after the last viral injection, only 1 of 3 animals showed adetectable virus in tumors, which is only slightly (about 2-fold)higher than the detection limit (1 � 103 PFU/g). In contrast, theamount of infectious virus within tumors of T cell–deficientanimals still remained elevated (�107 PFU/g or so) at day 7 afterthe first viral injection, and then started to gradually decrease andreached to approximately 104 PFU/g by day 17. All animals inboth groups had no detectable infectious virus in tumors 21 daysafter the first viral injection (Fig. 2A). Consistent with the yields ofinfectious virus in tumors, viral protein E1A and hexon detectedby IHC were highly expressed in tumors of T cell–deficientanimals at day 7 after the first viral injection and dramaticallydecreased at day 17, while the expression of viral proteins was

Oncolytic Ad Therapy Is Mediated by T-cell Responses

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significantly lower in tumors from the immunocompetent ani-mals. The numbers of tumor cells expressing E1A and hexon were3- to 4-fold higher in tumor sections of T cell–deficient animals atboth day 7 and 17 as compared with the tumor sections from theimmunocompetent animals. At day 21, the viral proteins wererarely detected in tumors of both animals (Fig. 2B). Thus, viralreplication was initially similar in tumors injected with oncolyticAd, regardless of immune status of animals. About oneweek later,the infectious virus and virus-infected tumor cells began to rapidly

decline in immunocompetent animals compared with that in Tcell–deficient hamsters. This timing suggested that the infectiousvirus and virus-infected tumor cells were eliminated by adaptiveantiviral immunity developed in immunocompetent animals.Although T-cell deficiency allowed intratumoral virus levels toremain elevated for prolonged periods, the antitumor effects ofoncolytic Ad were severely impaired in T cell–deficient hamsters(Fig. 1). These results further support the critical role of T cells inoncolytic Ad therapy.

Figure 1.

Deletion of T cells by antibody severely impairs antitumor effects of oncolytic Ad. Syrian hamsterswere inoculated subcutaneouslywith 1� 106 HPD-1NR cells, 5� 108

PFU (Adlow), or 5� 109 PFU (Adhigh) of Ad5, or PBS were injected intratumorally on day 11, 13, 15, 17, 19, and 21 (dashed arrow) after tumor inoculation. Anti-hamsterCD3 (4F11) or control antibodies (ConIg) were injected intraperitoneally at doses of 500 mg/injection every 4th day from the day before the viral therapy(day 10 after tumor inoculation) to the end of the experiment (solid arrow, mIg: mouse Ig). A, Tumor growth curves of individual animals were shown. One of threeexperiments was shown; n¼ 5 animals per group. B,Anti-mouse CD4 (GK1.5), anti-hamster CD3 (4F11), or control antibodies (ConIg) were injected intraperitoneallyat doses of 500 mg/injection every 4th day from the day before the viral therapy to the end of the experiment. n ¼ 7 animals per group. Mean tumor size and SEMare displayed. Statistical analysis was conducted using two-way ANOVA test on time points after viral therapy. �� , P < 0.001. C, Ad5-treated, tumor-free hamsters(n ¼ 5 animals/group pooled from Adlow and Adhigh-treated animals) were rechallenged with 2 � 106 HPD-1NR cells on a different site from primary tumorat least 1 month after complete rejection of primary tumors. Anti-CD3e (4F11) or control antibodies were injected intraperitoneally every 4th day from theday before the tumor rechallenge to the end of the experiment. Tumor sizes were measured twice weekly. Mean tumor size and SEM are displayed. Data arerepresentative of three independent experiments.

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Figure 2.

T-cell depletion extends the existence of infectious virus and decreases apoptosis in tumors treated with Ad5. A–C, Tumor-bearing hamsters were treated with 5�108 PFU Ad5 (arrow) and 4F11 or control antibody as they were treated in Fig. 1. The hamsters were sacrificed at 1.5 hours, and 1, 3, 7, 11, 17, and 21 days afterthe first virus injection (n¼ 3/time point), and tumorswere harvested.A,Viral loads in tumor tissue homogenateswere determinedby TICD50on JH-293 cells.B, Theexpression of viral protein E1A and hexon was determined by IHC. ConIg, control antibodies. Left, representative anti-E1A and hexon staining of sectionsof tumors harvested at day 7, 17, and 21 (original magnification, 400�; bars, 36 mm); right, statistical analysis of positive staining cell number within tumors. C, Viralgenome was determined by a quantitative PCR assay (copy number/g). Each point represents the yield from one animal. Dotted line, sensitivity of theassay. D, Tumor-bearing hamsters were injected intratumorally with 5 � 108 PFU of Ad5 or PBS on day 11, 13, 15, and 17 after tumor inoculation. The virus-treatedgroups were injected intraperitoneally with anti-CD3e (4F11) or control antibodies at doses of 500 mg per injection at the day before the first viral injectionand 4 days later. At the day after the last viral injection, Syrian hamsterswere sacrificed and tumorswere harvested for immunohistochemical analysis. n¼ 3 animalsper group. Representative anti-CD3e, anti-E1A, and TUNEL staining of sections of tumors derived from three different groups of Syrian hamsters (originalmagnification, 200�; bars, 73 mm; top). Bottom, statistical analysis of positive staining cell number within tumors. � , P < 0.05. �� , P < 0.01. �� , P < 0.001. ND, notdetected. Data are representative of four independent experiments.






There was no significant difference in virus DNA levels titratedby real-time PCR between the tumors of T cell–deficient andimmunocompetent hamsters. Viral DNA level remained elevatedin tumor until day 17 and could not be detected at day 21 after thefirst viral injection (Fig. 2C). These data suggest that T cells play amajor role in elimination of infectious virus, but have less effecton clearance of viral genome. The elimination of viral genomeshould be mediated by innate immune system.

To directly assess T-cell responses and virus replication intumors, as well as their contributions to tumor cytolysis, wehistologically assessed T-cell infiltration, virus replication, andcell apoptosis in tumors onday7 after thefirst viral injectionwhenthe level of intratumoral virus started to become different inimmunocompetent and T cell–deficient animals. Compared withPBS treatment, oncolytic Ad therapy increased tumor infiltrationof CD3þ T cells and resulted in expression of viral protein E1A andmarked apoptosis of tumor cells. Deletion of T cells dramaticallyeliminated tumor-infiltrating CD3þ T cells and increased expres-sion of E1Aprotein but significantly decreased apoptosis of tumorcells (Fig. 2D). These data showed that the viral replication wasnot correlated with cytolysis in tumors even at day 7 after viralinjection, while T-cell response was associated with suppressionof virus replication and increased apoptosis in tumors.

Overall, these results demonstrate that oncolytic Ad therapyrapidly induces T-cell responses, which eliminate the virus-infected tumor cells and mediate antitumor efficacy althoughthey also decrease the yield of infectious virus in tumors.

Oncolytic Ad therapy resulted in both tumor- and virus-specificCTL activities

CD8þ T cells are major effector cells for immune responsesagainst tumors and virus infection. Next, we assessed the tumor-and virus-specific CTL activities induced by oncolytic Ad therapy.To exclude the influence of tumor size on the antitumor T-cellresponses, splenocytes were harvested at day 4 and 7 after thefirst viral injection and stimulated in vitro for 4 days with HPD-1NR tumor cells infected with replication-deficient dl312, anE1A-deleted Ad5. CTL activities were detected by an in vitrokilling assay (17) using an irrelevant hamster renal cancer cellHAK as an internal reference. Tumor-specific cytotoxicity wasdetected against HPD-1NR tumor cells without virus infection,and virus-specific cytotoxicity was detected against dl312-infected control HAK tumor cells. Relative to cytolysis of HAK,the splenocytes from PBS-treated animals had no detectablecytolytic effects on any target cells. The splenocytes from Ad-treated animals displayed clear cytotoxicity against dl312-infected HAK tumor cells 4 days after the first viral injection,but no cytotoxicity against HPD-1NR tumor cells, suggesting thatonly anti-virus CTL response was induced at this moment. Onday 7, Ad treatment-induced CTLs not only had increasedcytolytic activity against dl312-infected HAK tumor cells but alsodisplayed strong cytotoxicity against HPD-1NR cells, indicatingthat a strong tumor-specific CTL activity also appeared at thistime point (Fig. 3A). These results demonstrate that oncolytic Adtherapy induces both strong tumor- and virus-specific CTLs, andvirus-specific CTL responses are first developed. The virus-specificCTLs have strong cytolytic effect on virus-infected tumor cells,suggesting that antiviral CTL response also contributes to onco-lytic efficacy.

To further study the impact of anti-viral immunity on thetherapeutic efficacy of oncolytic Ad5, 1 � 106 HPD-1NR cells

were inoculated, respectively, in right and left flanks of hamsters.The right tumors were treated with six intratumoral injections oflow dosage of Ad5 (5 � 108 PFU/injection) or PBS. The viraltreatment caused transient regression of the virus-injected tumorsafter the third vial injection (about 6 days after the first viralinjection) and also markedly inhibited the growth of the untreat-ed left tumors after completion of Ad treatment (about 14 daysafter the first viral injection; Fig. 3B). Because the viral treatmentjust transiently inhibited tumor growth in T cell–deficient animals(Fig. 1A), the regression of the virus-injected right tumors sug-gested the therapeutic effect of antiviral immunity, while the

Figure 3.

Ad5 therapy successively induced antiviral and antitumor CTL responses.A,CTLassay. The tumor-bearing hamsters were injected intratumorally (i.t.)with 5� 108 PFUofAd5or PBS every other day for 4 times fromday 11 posttumorinoculation and sacrificed 4 and 7 days after the first injection of virus,respectively. Splenocytes from 3 animals per groupwere pooled and stimulatedwith irradiated HPD-1NR tumor cells infected with replication-deficient dl312 (anE1A-deleted Ad5) for 24 hours in vitro for 4 days. HPD-1NR and dl312-infectedHAK cells were respectively stained with 10 mmol/L CFSE (CFSEhigh) as specifictarget cells. HAK cells without virus infection were stained with 0.5 mmol/LCFSE (CFSElow) as control target cells. CTL activities were examined againstHPD-1NR cells and dl312-infected HAK cells with different effector-to-targetratios (E:T) by an in vitro killing assay using amixture of CFSEhigh-labeled specifictarget cell (HPD-1NR or dl312-infected HAK) and CFSElow-labeled control cell(HAK). Cytotoxicity was expressed as cytotoxicity (%) ¼ (100% � [1�ratio(target/control)þeffector/ratio (target/control)�effector]). ND, not detected. Dataare representative of two independent experiments. B, Syrian hamsters wereinoculated subcutaneously with 1 � 106 HPD-1NR cells in right and left flanks. Atotal of 5� 108 PFU Ad5 or PBS were injected intratumorally in right tumors onday 10, 12, 14, 16, 18, and 20 (dashed arrow) after inoculation. n ¼ 7 animals pergroup. Mean tumor size and SEM are displayed. Statistical analysis wasconducted using two-way ANOVA test on time points after viral therapy.�� , P < 0.001; ns, not significant.

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retarded growthof theuntreated left tumors suggested the effect ofantitumor immunity, which appeared later than that of antiviralimmunity. This result is consistent with the virus- and tumor-specific CTL activities.

Preexisting anti-Ad immunity promotes viral clearance oftumors through T cells but has no effect on therapeutic efficacy

Preexistent anti-Ad antibodies are highly prevalent in thehuman population. The neutralizing anti-Ad antibody is likelyto be the major factor of limiting the therapeutic efficacy of Ad byblocking viral infection and spreading (20, 21). So we nextmonitored anti-Ad antibody responses in immunocompetentand T cell–deficient hamsters treated with oncolytic Ad. Obviousanti-Ad antibody responses were detected in immunocompetenthamsters one week after the first viral injection and continuouslyincreased, but, anti-Ad antibody was hardly detected in T cell–deleted animals at all time points (Fig. 4).

To investigate the relative contributions of anti-viral antibodyand T cells to viral clearance and their effects on antitumorefficacy, hamsters were preimmunized with one dose of Ad5 (1�1010 PFU) by intramuscular injection, and subcutaneously inoc-ulated with HPD-1NR tumor cells one month later. Then, onco-lytic Ad therapy was performed in the preimmunized and na€�vetumor-bearing hamsters with or without T-cell deletion. Thepreimmunization induced persistent high level of anti-Ad anti-body in plasma and tumor tissues, which was not influenced byAd therapy and T-cell deletion (Fig. 5A). The infectious Ad couldnot be detected in tumors of preimmunized immunocompetentanimals even at the day after the first viral injection. However, adramatic viral amplification was detected in tumors of preimmu-nized animals with T-cell deletion and showed a similar dynamicprocess with that in tumors of immunocompetent hamsterswithout preimmunization (Fig. 5B, left). These results demon-strated that preexisting anti-Ad immunity rapidly eliminated theinfectious Ad from tumors once Ad was injected. Furthermore,infectious Ad was majorly eliminated by T cells, and the preexist-ing anti-Ad antibodies could not effectively prevent viral infectionof tumor cells. DNA detection showed the same kinetics of virusgenome clearance from tumors in the preimmunized hamsters

with orwithout T-cell deletion as that in nonimmunized hamsters(Fig. 5B, right). These data suggest that the injected infectious viruscould effectively infect tumor cells before it was cleared by thepreexisting antiviral immunity. Meanwhile, it further supportsthat Ad genome is cleared by innate rather than adaptive immu-nity. Consistent with the previous study (8), the preexisting anti-Ad immunity did not affect the antitumor efficacy of oncolytic Ad.However, T-cell deletion also abrogated the antitumor effect ofoncolytic Ad in preimmunized animals (Fig. 5C). These resultsfurther prove that T-cell responses play major roles in antitumorefficacy of oncolytic Ad, even under the condition of preexistinganti-Ad immunity that can rapidly eliminate the infectious virusfrom tumors.

Repeated oncolytic Ad therapy eradicates the primary therapy–resistant tumors

Next, we wonder whether repeated oncolytic Ad therapy hastherapeutic effects on the tumors that grow up from the primaryoncolytic Ad therapy. To this end, tumor-bearing hamsters weretreated with six intratumoral injections of low dosage of Ad5 (5�108 PFU/injection). Ad treatment caused complete regression oftumors in 5 of 20 hamsters. The other tumors just transientlyshrank. Twelve days after the last viral injection when the shrunktumors started to grow, the tumor-bearing hamsters were ran-domly divided into two groups that were respectively treated withanother six injections of high-dose Ad (2� 109 PFU/injection) orPBS as control. All tumors kept growing in the PBS-treatedanimals. Ad treatment caused complete regression of tumors in5 of 8 animals. The growth of tumors was also delayed in other 3hamsters (Fig. 6). This result demonstrates that repeated oncolyticAd therapy has strong therapeutic effect on relapsed tumors ortumors insensitive to the primary viral therapy.Most interestingly,the same treatment with high dosage of Ad eliminated therelapsed tumors in more than 60% hamsters (Fig. 6), versus theprimary tumors in 40% hamsters (Fig. 1A). Moreover, therelapsed tumors were at least two times bigger than the primarytumors when they were treated with Ad. These data suggest thatthe antiviral recall immune response triggered by the repeated Adtherapy might have greater therapeutic effect on relapsed tumors.

DiscussionAn intriguing aspect of oncolytic virotherapy is that by their

very nature they potently stimulate multiple arms of the immunesystem. In return, the immune system exerts multiple effects onthe outcome of tumor therapy: some positive, some negative. Themost important thing should be to determine the roles and therelative importance of each key components of immune system inantitumor efficacy of oncolytic virotherapy. Tomore fully explorethe roles of T cells in oncolytic Ad therapy in the hamster model,we developed amAbagainst Syrian hamster CD3 (mAB4F11) thatcan efficiently delete T cells in vivo (16). Here, we show thatdeletion of T cells remarkably impairs the antitumor efficacy ofoncolytic Ad, especially the long-term control of tumor recur-rence. On the other hand, the deletion of T cells also abrogatesproduction of anti-Ad antibodies, delays the clearance of infec-tious Ad in tumors, andmaintains elevated levels of intratumoralinfectious virus for prolonged periods. The elevated infectiousvirus levels in tumors and the impaired antitumor efficacy ofoncolytic Ad in T cell–deficient hosts fully demonstrate theimportance of T cell–mediated immune responses in antitumor

Figure 4.

The effect of T-cell deletion on anti-Ad antibody response induced by Ad5therapy. Tumor-bearing hamsters were treatedwith 5� 108 PFU of Ad5 (arrow)and 4F11 or control antibody as they were treated in Fig. 1. Sera wereharvested on indicated days, and the levels of anti-Ad5 antibodyweremeasuredby ELISA assay. Each point represents the yield from one animal. Data arerepresentativeof three independent experiments. ConIg, control antibodies; OD,optical density.






effects of oncolytic Ad therapy. Cyclophosphamide treatmentwasshown to cause deletionof totalwhite blood cells and aprolongedhigh infectious Ad level in tumors in Syrian hamsters, whichsignificantly enhanced the antitumor efficacy of oncolytic Ad (2,3). The opposite effects of these two kinds of immunodeficiencyon the antitumor efficacy of oncolytic Ad further demonstrate thatT cells are key components of immune system mediating antitu-mor effects of oncolytic Ad, whose antitumor effect far outweighsits negative effects on the direct oncolytic efficacy of Ad.Moreover,

this opposite effect suggests that the responses of other non-T cell–immune components have negative effects on antitumor efficacyby limiting the oncolysis of Ad in T cell–deficient condition.

T cell–mediated immunity in oncolytic virotherapy includesvirus-specific and tumor-specific responses. Their relationshipand the contribution of virus-specific immune response to thetherapeutic efficacy have been the hot topics of discussion inoncolytic virotherpy (22, 23). The adaptive immune response hasbeen of serious concern for the delivery and spread of oncolytic

Figure 5.

The contributions of anti-Ad antibody and T-cell responses to viral clearance in tumor and antitumor efficacy of Ad5 therapy. Hamsters were immunizedintramuscularly with 1 � 1010 PFU of Ad and inoculated with HPD-1NR cells one month later. Meanwhile, another group of na€�ve animals were also inoculatedwith HPD-1NR cells. The tumor-bearing hamsters were treated with 5 � 108 PFU of Ad5 (arrow) and 4F11 or control antibody as they were treated in Fig. 1. A, Seraand tumor tissue were harvested on the indicated days, and the levels of anti-Ad5 antibody were measured by ELISA. ConIg, control antibodies. B, Tumortissueswere harvested at the indicated times, and viral loads (left) and genome (right) in tumorwere determined by TICD50on JH-293 cells and the quantitative PCRassay, respectively. C, Tumor sizes were measured twice weekly. Error bars, mean � SDs from 7 to 8 animals per group. Statistical analysis was conductedusing two-way ANOVA test on time points after viral therapy. �� , P < 0.001; ns, not significant. Data are representative of two independent experiments.

Li et al.





virus in tumors. T-cell response plays a dominant role in immuneresponse against viral infection by clearance of virus-infected cells.Our in vitro CTL assay shows that oncolytic Ad therapy inducesstrong antiviral and antitumor CTL activities. Deletion of T cellsincreases viral replication and markedly delays the clearance ofvirus, whichwas indicated by increased expression of viral proteinE1A and elevated infectious virus levels in tumor tissues. Theseresults demonstrate that antiviral T-cell responses play importantroles in eliminating the infectious virus from tumors,whichmightlimit the direct oncolytic effect of Ad. On the other hand, antiviralT-cell response itself should have a function in antitumor efficacyat the same time. The central problem in cancer immunotherapy isthat most tumor-associated antigens are nonmutated self-anti-gens that have triggered both central and peripheral tolerance(24). Viral proteins expressed in the infected tumor cells are strongnon-self-antigens that can trigger robust virus-specific T-cellresponse as being logically immunodominant over self-derivedtumor epitopes on the tumor cells. The CTL assay shows thatoncolytic Ad therapy induces strong virus-specific CTL responseagainst the virus-infected tumor cells, which was produced earlierthan the tumor-specific CTL response. Thus, the killing of virus-infected tumor cells by virus-specific CTLs should also contributeto oncolytic efficacy, especially at the early stage of the oncolytictherapy. Importantly, the oncolytic virus infect and debulktumors, which leaves any residual tumors for the immune systemand leads to the release of "danger signals" and tumor antigensthat could allow for antigen spreading and reduce local immu-nosuppression. So, theAd-specific T-cell response appears tobe animportant component of Ad-mediated antitumor responses.

A majority of the human population is seropositive for Ad5,which is acquired as a childhood infection. Elimination of thevector by preexisting immunity to Ad poses a possible concernwith respect to achieving significant antitumor efficacy (25–27).The adaptive immune responses and, in particular, neutralizingantibodies were shown to be a common and powerful inhibitoryend response to infection involving a variety of oncolytic viruses,including Ad (28–30). To study the effect of the preexisting anti-Ad immunity on the viral clearance and therapeutic efficacy ofoncolytic virotherapy, we immunized the hamsters with one doseof intramuscular injection of Ad. Consistent with the previousstudy (8), the preimmunization induced persistent high level ofanti-Ad antibody in plasma and tumor tissues. Correspondingly,the infectious Ad could not be detected in tumors of the pre-immunized animals even at a very early time after the viral

injection. However, once T cells were deleted in the preimmu-nized hamsters, the infectious Adwas easily detected in the tumortissues and showed a similar dynamic process with that in tumorsof immunocompetent hamsters without preimmunization,although T-cell deletion had no effect on the titer of anti-Adantibody in the preimmunized hamsters. These results demon-strate that infectious Ad is majorly cleared from tumors by theantiviral T cells rather than antibodies, and the preexisting anti-Adantibody could not effectively block viral infection of tumor cells.Anti-Ad antibodies might have an important role in preventingthe systemic toxicity of oncolytic Ad by inhibiting Ad spilloverfrom the tumor and replication in normal tissues (8). The iden-tical kinetics of viral DNA clearance from the tumor in T cell–deleted or immunocompetent hamsters with or without preim-munizationdemonstrate that the adaptive immunity has no effecton the clearance of oncolytic Ad genome, and the innate immu-nity may be responsible for clearance of virus DNA from tumorsfollowing intratumoral injection of oncolytic Ad. Most impor-tantly, even though the preimmunization induced a robust sys-temic immune response against Ad, the preexisting antiviralimmunity had no effect on the therapeutic efficacy of oncolyticAd therapy, which is consistent with the previous study (8). Thedetection of viral DNA in tumors demonstrates that the injectedinfectious virus could effectively infect tumor cells before it wascleared by the preexisting antiviral immunity. This finding is notsurprising, as T-cell response eliminates the virus by destruction ofthe virus-infected tumor cells only after the virus infected thetumor cells and expressed viral antigen during their replication.This offers us an opportunity to improve oncolytic virotherapy byengineering the oncolytic viruses to increase their rapid spreadingand infection, and delay their replication within cells.

Because of the viral immunogenicity, it was generally acceptedthat the therapeutic efficacy of multiple injections of the samevirusmight be limited by the antiviral immune responses.Numer-ous strategies havebeendeveloped to circumvent this hurdle (31).For example, two or more antigenically distinct viruses wereapplied so that the specific immunity that arises subsequently tothe first virus does not inhibit the therapeutic effects of the secondtherapeutic virus (16). Alternatively, the therapeutic viruses weremasked from antibody neutralization with chemical conjugates(32, 33).However, the studies in the immunocompetent and viralreplication–permissive hamster model, including our results,demonstrated that preexisting antiviral immunity did not affectthe therapeutic efficacy of vector after intratumoral injection of

Figure 6.

The efficacy of repeated Ad5 therapy on progressive tumors. Tumor-bearing hamsters were injected intratumorally with 5� 108 PFU of Ad5 on day 12, 14, 16, 18, 20,and 22 (dashed arrow) after tumor inoculation. After 11 days of last injection, the hamsters with growing tumor were treated with six intratumoral injections(2� 109PFUofAd5or PBS; solid arrow). Tumor sizesweremeasured twiceweekly. Tumorgrowth curves of individual animalswere shown.Data are representativeoftwo independent experiments.






Ad (8). Most importantly, we, for the first time, have shown thatinstead of attenuating the efficacy of oncolytic Ad, the repeatedoncolytic Ad therapy significantly enhanced therapeutic effect onrelapsed tumors compared with the first Ad treatments for theprimary tumors. The repeated Ad treatment would trigger robustrecall immune responses against the virus, which cause violentdisruption to the virus-infected cells. The sudden death of largeamounts of tumor cells will dramatically ameliorate local immu-nosuppression and release tumor antigens that trigger robustrecall immune response against tumors, eventually leading tothe elimination of tumors. We can infer from our study that thevirus-specific T-cell response ought to play a critical role in efficacyof oncolytic Ad therapy by enhancing tumor oncolysis andtriggering a long-term antitumor immune response.

In conclusion, we have identified the key roles of T cells inoncolytic Ad therapy in an immunocompetent, replication-per-missive hamster tumor model. Our results suggest that viral-specific T-cell response may play important role in oncolytictherapy as a bridge linking Ad infection and oncolysis to theantitumor immune responses. These results shed light on devel-oping new therapeutic regime of oncolytic Ad by enhancingantigen-specific T-cell responses.

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

Authors' ContributionsConception and design: X. Li, Y. Wang, S. WangDevelopment of methodology: X. Li, P. Wang, S. WangAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): X. Li, H. Li, X. Du, S. WangAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): X. Li, P. Wang, Y. Wang, S. WangWriting, review, and/or revision of the manuscript: X. Li, Y. Wang, S. WangAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): X. Li, X. Du, M. Liu, Q. Huang, Y.Wang, S.WangStudy supervision: Y. Wang, S. Wang

AcknowledgmentsWewould like to thankXudong Zhao andXiaofei Guo for offering the facility

equipment support.

Grant SupportThis work was supported by grants from the National Key Basic Research

Program of China (2012CB917101) and the Natural Science Foundation ofChina (91231204; to S. Wang).

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 February 29, 2016; revised June 23, 2016; accepted June 25, 2016;published OnlineFirst July 19, 2016.

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2017;23:239-249. Published OnlineFirst July 19, 2016.Clin Cancer Res Xiaozhu Li, Pengju Wang, Hang Li, et al. Responses against Virus and Tumor in Syrian Hamster ModelThe Efficacy of Oncolytic Adenovirus Is Mediated by T-cell

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