However, the consequences of Treg depletion on long-term antitumoral T cell responses that can be induced by oncolytic virotherapy are not clear. Observations in classical infection models have shown that migratory activity of Tregs plays a central role in eliciting a protective immunity to viral infection (108). Consistent with a positive function of Tregs in shaping antigenspecific immune responses, we have observed that Treg depletion abrogated the effective antitumoral T cell induction by an oncolysis-assisted, antitumoral DC-vaccine (109). We could also show that immunosuppressive MDSC expand in Treg-depleted tumors, which may explain the failure of antitumoral T cell priming. Supporting an important role of Tregs in the priming of antigen-specific T cells, it has been described that Tregs can undergo a conversion under acute inflammatory conditions to adopt a T helper phenotype (110). Converted Tregs express proinflammatory cytokines and activate additional functions to provide effective help for triggering T cell responses against new antigens. These findings described above indicate that Tregs can essentially modulate the course of tumor therapy with oncolytic viruses. A supportive role of Treg depletion on virus spread and therapeutic efficacy of oncolysis is still unclear and possible consequences on induction of sustainable tumor-directed T cell responses require further investigations.

#### **HARNESSING ONCOLYTIC VIROTHERAPY AS IMMUNOTHERAPY**

Observations in immunocompromised xenografts have tempted to overestimate the cytolytic effects that are achievable in human patients. The situation in the immunocompetent host is completely different with positive and negative consequences for the therapeutic efficacy of virotherapy. Since it is known that T cell responses against cross-presented cellular antigens upon viral infections trigger innate immune receptor pathways such as TLRs and MyD88 (11, 12), investigations on corresponding antitumoral immunity have been intensively pursued in oncolytic virus applications in immunocompetent models. The use of oncolytic VSV in the B16-Ova model strikingly demonstrated that antitumoral effects completely depended on Type I IFN responses, which mediate both antiviral protection and antitumor therapy, whereas VSV-mediated therapy was abolished in MyD88−/− mice (111). The relevance of both innate immune activation and subsequent triggering of adaptive responses was shown in experimental models with T cell depletion studies (10). Interesting observation have been reported using herpes simplex virus variants with different replicative properties. oHSV vectors that were more rapidly cleared from the tumor but induced higher levels of DAMPs resulted in best survival. This strongly indicates that replicative potency is not the dominating factor as believed before but emphasizes the impact of the initial immune induction (112), which needs to be considered in the rational designs of novel approaches aiming at increased antitumor immunity. DC are known to play a crucial role in the generation of tumor-directed T cell responses (113). First strategies on utilizing oncolytic virotherapy to engage intrinsic activity of DC were performed with an ICP34.5 deleted herpes simplex virus coding for GM-CSF (114). Tumor infection with this oncolytic virus led to regression and protected the mice against rechallenge with tumor cells. GM-CSF-expressing HSV then entered clinical development as OncoVexGM-CSF or T-Vec (14, 115). Furthermore, virus-encoded GM-CSF not only affected DCs, but also neutrophils which were shown to contribute to antitumor effects by a GM-CSF-expressing oncolytic measles virus in CD46 transgenic mice (70). The therapeutic benefit of engaging dendritic cell activity in virotherapeutic applications was confirmed using different cytokine setups. In a preclinical breast cancer model, systemic, and intratumoral delivery of a TRAIL- /E1A-expressing oncolytic adenovirus increased plasma levels of TNFα, IFNγ, and MCP-1, proinflammatory cytokines acting as maturation signals for DCs. Inclusion of FLT3L or GM-CSFexpressing adenovirus for expansion of DCs established systemic antitumor immunity and resulted in tumor elimination (116). We obtained consistent results in a mouse model of lung cancer using intratumoral delivery of an oncolytic Ad combined with vectors encoding FLT3L and MIP-1α. Tumor-directed T cells were significantly increased and improved tumor responses were obtained. However, adaptive immune responses against the viral vector were also strongly enhanced suggesting that the balance between tumor- and virus-directed immunity remains unaltered instead of generating a favorable tumor-directed response (117). Oncolytic viruses expressing cytokines for enhanced antigen cross presentation illustrate that virotherapy can be used as a tool for a generic *in situ* vaccination without the need for detailed information about specific tumor-specific antigens. However,the approach has limitations in shifting the predominant antiviral responses in favor of antitumoral responses.

#### **ONCOLYTIC VIROTHERAPY IN DC-VACCINATIONS AND HETEROLOGOUS PRIME-BOOST SETTINGS**

For focusing the immune system during virotherapy on the tumor requires the incorporation of tumor-specific antigen targeting approaches into the therapeutic scheme. We have investigated this aspect by combining viral oncolysis and a tumor-directed DC-vaccine (117). In another study, it has been shown that a CCL5 (RANTES) expressing oncolytic vaccinia virus significantly improved the therapeutic efficacy of a tumor-directed DC-vaccine (118). In a further study, it was demonstrated that the application of a replicating adenovirus allowed for highly effective DC-vaccination, when the vaccine is administered exactly at the time of apparent virus-induced tumor inflammation (109). This approach induced potent cytotoxic T cell responses leading to significant tumor regression and complete eradication of lung colonies in an aggressive tumor model that was otherwise resistant to the DC-vaccine. A further promising direction is the development of oncolytic virus-based prime-boost strategies that express the tumor-antigen. In a heterologous treatment sequence with an adenoviral TAA-endoding vaccine and an oncolytic VSV tumor expressing the same antigen significantly enhanced tumor-directed CD8 T cell immune responses compared to single treatments. Heterologous priming worked in both directions (119,120). This approach shifted the immune responses from viral antigens to tumor-antigens and reduced viral replication in healthy tissues thereby improving efficacy and safety. Interestingly, the magnitude of tumor-specific responses after combination therapy was even higher in tumor-bearing hosts compared to tumor-free mice indicating the need of infected tumor tissue for priming antitumoral T cell reponses (120). The same group could also demonstrate that heterologous boosting not only resulted in higher numbers but also in functionally superior T cells (121). A further interesting variation of prime-boost vaccinations comes from Brinkhoff and colleagues who elicited highest antitumoral responses when the boost step by an antigen-expressing infectious agent was preceded by a non-pathogenic prime using antigen-loaded PLGA-microspheres (122).

#### **TARGETING THE TUMOR ANTIGENOME AND MUTANOME BY ONCOLYTIC VIROTHERAPY**

The use of complete antigen libraries encoded by an oncolytic virus offers a promising approach to circumvent the limitations of antigen-specific vaccinations. In a preclinical study in prostate cancer, VSV-based cDNA libraries from xenogeneic healthy prostrate tissue were used for treatment of TC2 prostrate tumors. Application of VSVs coding for such a cDNA library [Altered Selfantigen and Epitope Library (ASEL)] cured established tumors after repetitive intravenous injections. The use of ASEL conferred significantly better protection against TC2 cells than a self-antigen library from normal mouse prostate tissue. Upon application of ASEL, a TH17 response was detectable and TC2 rejection was dependent on CD4 cells, but not on CD8 T cells or NK cells (123). A subsequent study from this group demonstrated that an approach of virus-encoded melanoma cDNA libraries can be used to identify tumor-associated antigens that have the ability to cure melanoma (124). Virus-expressed cDNA libraries were effective against melanoma thereby inducing only mild signs of autoimmunity. The xenogenic, altered self-source is a precondition for successful tumor treatment due to additional adjuvant effects compared to a library from an autologous self-source. Again, the antitumoral effect was correlated with a tumor-specific IL-17 response, which was in turn utilized to screen for cDNA-viruses that induced IL-17 memory for identification of tumor rejection antigens. After validation of IL-17 inducing clones, three VSV-encoded tumorantigens were tested to treat established B16 tumors. Intriguingly, injection of a single VSV-clone or a pool of two VSV-clones did not show a therapeutic response, only the combination of all three VSV-clones cured melanoma tumors to a similar extent as the whole melanoma-library did. Although, it remains unclear why only all three different TAA-coding VSVs contribute to therapeutic effects, this finding suggest that applications targeting multiple antigens at the same time should be preferred in immunotherapeutic strategies. These studies establish a rational approach to identify novel tumor-targets for immunotherapy and establish an effective generic virus-based ASEL-vaccine for defined tumor entities.

To date, identification of novel tumor-antigens that can be addressed by targeted therapeutics appears to be a crucial step toward the establishment of clinically effective immunotherapies and toward induction of sustained adaptive T cell responses. In the past, antitumoral vaccine research has focused on finding non-mutated, tumor-associated antigens such as telomerase or MAGE, which can be found either in a relevant numbers of patients and/or across several entities to promise broad applicability. Disappointingly, corresponding vaccination approaches have so far delivered insufficient effects in the clinic (125). A limiting factor is that non-mutated tumor-antigens may not reflect essential molecular functions required for tumor cell survival promoting the generation of escape variants (126). Furthermore, T cell precursors against this type of antigens are subject to thymus selection and self-tolerance mechanisms thus limiting the number of required high-affinity T cell precursors that are essential for effective antitumoral T cell responses. In this regard, triggering T cells that recognize immunogenic neoepitopes reflecting tumor-associated mutated proteins could be a more promising alternative. Data from melanoma patients indicate that autologous T cell responses to tumors are predominantly directed to neoantigens (127). In murine models as well, tumor rejection responses were also primarily induced by altered-self antigens (128, 129). However, this would require individualized (personalized) molecular diagnosis and therapy. Individual (solid) human cancers usually harbor about 30 to more than hundred of proteinencoded mutations referred to as mutanome (129–131), which can be nowadays rapidly and cost-effectively analyzed by Next Generation Sequencing (NGS) technology. Using this method, non-synonymous single nucleotide variants (SNV) can be identified, representing promising candidates for immunotherapies, since single amino acid variations in corresponding epitopes can be processed and presented by MHC to T cells.

In a pioneering study targeting the mutanome by vaccinations, NGS was used for immunoepitope identification in B16F10 melanoma cells. Selected from 563 non-synonymous SNV candidates, the immunogenicity of 50 validated mutations was determined using corresponding peptide immunizations in mice. The authors showed that immune responses could be raised against 60% of these epitopes and the vaccinations against these predicted and validated epitopes successfully raised antitumoral adaptive immune responses and significantly slowed tumor-growth (132). This illustrates the great potential of this method in identification of neoepitopes. However, the observation is also astonishing since those epitopes should be per definition of low immunogenic nature. In clinically manifest tumors, the remaining epitope spectrum is the result of a dynamic process termed cancer immunoediting, which acts on nascent tumors via different immune cell types to protect against cancer development and shapes the tumor at the same time toward decreased immunogenicity (129). In the study by Castle and colleagues, the key for successful induction of immune responses to immunoedited tumor-epitopes by DCvaccination is most likely attributable to the use of adjuvants, i.e., poly(I:C) in the B16F10 model. Oncolytic virotherapy is likewise a potent trigger of innate immune receptors and inflammation and could be an interesting tool that enables identification of inflammation induced neoepitope-directed T cell responses and to cooperate with tailored neoepitope-directed DC-vaccines. However, it will be a challenging task to identify neoepitope-specific T cell reactivities that are involved in tumor responses induced by oncolytic virotherapy.

#### **ONCOLYTIC VIROTHERAPY AND IMMUNE CHECKPOINT BLOCKADE**

The recent clinical success of immune checkpoint blockade (133) has confirmed the curative potential of tumor immunotherapies. Checkpoint blockade using ipilimumab, a CTLA-4-blocking monoclonal antibody, has shown promising results in a phase III study (134). Remarkably, responses seemed to include even complete cures, but only a small proportion of patients benefited from therapy. In a case study which described a patient with

advanced melanoma experiencing tumor response under ipilimumab, neoepitope analysis by NGS and epitope prediction led to identification of a single ipilimumab-responsive neoepitopespecific CD8 T cell that increased fivefold under therapy and remained stable for a 10-month period (135). The fact, that only one epitope was triggered in a tumor displaying 448 potential T cell neoepitopes is remarkable but reflects that natural and thus immunoedited tumors are low immunogenic despite harboring a high number of mutations and may also explain why only small subgroups of patients respond to certain immunotherapies. Oncolytic viruses can serve as an ideal tool to augment tumor immunogenicity and could be ideally combined with immune checkpoint blockade. Gao and colleagues have investigated the application of a Her2/neu targeted oncolytic VSV in combination with a CTLA-4 antibody in mice bearing Her2/neu transgenic murine mammary tumors. This combination achieved cure in the majority of mice whereas the virotherapy alone only prolonged survival (136). Additionally, it has been tried to include an expression cassette for a CTLA-4-specific antibody into the backbone of an oncolytic Ad to enhance local concentrations and to avoid adverse events by systemic CTLA-4 inhibition (137). Recently, it has been reported that injection of oncolytic NDV in a preclinical model of B16 melanoma under CTLA-4 antibody treatment induces an inflammatory response in tumor tissue, leading to lymphocytic infiltration and antitumor effect in distant, non-virally injected tumors (138). Effective treatment induced activated CD4 and CD8 T cell infiltration in distant tumors and was dependent on CD8+ cells, natural killer cells, and type I IFNs. Overcoming systemic resistance to immune checkpoint blockade by oncolytic virotherapy moreover led to protection from tumor rechallenge in poorly immunogenic tumors, even in a cell line refractory to NDVmediated lysis. An alternative to checkpoint blockade is the direct activation of costimulation using oncolytic viruses expressing the costimulatory CD40L (53). Further approaches used oncolytic vaccinia viruses expressing the ligand for the costimulatory receptor 4-1BB (CD137) that achieved maximum antitumoral efficacy in lymphodepleted hosts (139). Strong antitumoral immune responses were also elicited by combining oncolytic vaccinia virus with systemic application of a 4-1BB agonistic antibody (140). An interesting immune checkpoint that has not yet been investigated

with virotherapy is PD-1/PD-L1. PD-1/PD-L1-blocking antibodies are in a very promising clinical development (141). PD-1/PD-L1 inhibition primarily activates antigen-experienced T cell responses in the periphery, thus providing a mechanism that could be promising to combine with virotherapeutic treatments.

#### **PERSPECTIVE: ONCOLYTIC VIROTHERAPY IN MULTIMODALE THERAPIES**

There is increasing evidence that oncolytic virotherapy shows antitumoral efficacy in clinical application even as monotherapy. However, most preclinical data suggest that virotherapy can be ideally combined with other treatment options to raise significant therapeutic synergies on several levels (see also an overview in **Figure 2**). First of all, oncolytic virus treatment needs to be integrated in combined tumor-treatments leading to optimized induction of ICD. Excellent reviews already exist on this aspect (16, 142, 143). Next step should be additional measures that amplify, and prolong antitumoral immune responses. First data obtained in humans and in murine melanoma models suggest significant synergies when systemic immunotherapies, such as ipilimumab and virotherapy are combined in a well-coordinated manner (138, 144). A very promising but clinically challenging point will be the combination of viral oncolysis with surgical removal of the tumor. Finally, it still needs further investigations to establish follow-up therapies that work like classical boost strategies and may also pick up personalized approaches such as NGS of tumors, epitope prediction and and immunoanalysis in treated patients. Multimodal therapy schemes will be a clue to establish virotherapy in the clinic.

#### **ACKNOWLEDGMENTS**

This work and the experimental work in our Laboratory we referred to was supported by funding from the Wilhelm-Sander-Stiftung, the Deutsche Krebshilfe e.V. and the German Research Council (DFG, SFB/TRR77)

#### **REFERENCES**

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antibody eliminates implanted mammary tumors. *Cancer Gene Ther* (2009) **16**:44–52. doi:10.1038/cgt.2008.55

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 10 June 2014; paper pending published: 26 June 2014; accepted: 06 July 2014; published online: 21 July 2014.*

*Citation: Woller N, Gürlevik E, Ureche C-I, Schumacher A and Kühnel F (2014) Oncolytic viruses as anticancer vaccines. Front. Oncol. 4:188. doi: 10.3389/fonc.2014.00188*

*This article was submitted to Tumor Immunity, a section of the journal Frontiers in Oncology.*

*Copyright © 2014Woller, Gürlevik, Ureche, Schumacher and Kühnel. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

### Oncolytic virotherapy as emerging immunotherapeutic modality: potential of parvovirus H-1

#### **Markus Moehler 1\* † , Katrin Goepfert 1† , Bernd Heinrich1 , Caroline J. Breitbach2 , Maike Delic 1 , Peter Robert Galle1 and Jean Rommelaere3**

1 1st Department of Internal Medicine, University Medical Center of the Johannes Gutenberg, University of Mainz, Mainz, Germany

2 Jennerex Biotherapeutics, Inc., San Francisco, CA, USA

3 Division of Tumor Virology, German Cancer Research Center (DKFZ), Heidelberg, Germany

#### **Edited by:**

Philippe Fournier, DKFZ, Germany

#### **Reviewed by:**

Valery Grdzelishvili, University of North Carolina at Charlotte, USA Volker Schirrmacher, DKFZ, Germany Yong Lu, Cleveland Clinic Foundation, USA

William L. Redmond, Earle A. Chiles Research Institute, USA

#### **\*Correspondence:**

Markus Moehler, 1st Department of Internal Medicine, University Medical Center of the Johannes Gutenberg, University of Mainz, Building 601, Langenbeckstrasse 1, Mainz 55131, Germany

e-mail: markus.moehler@ unimedizin-mainz.de

†Markus Moehler and Katrin Goepfert have contributed equally to this work.

Human tumors develop multiple strategies to evade recognition and efficient suppression by the immune system. Therefore, a variety of immunotherapeutic strategies have been developed to reactivate and reorganize the human immune system. The recent development of new antibodies against immune check points may help to overcome the immune silencing induced by human tumors. Some of these antibodies have already been approved for treatment of various solid tumor entities. Interestingly, targeting antibodies may be combined with standard chemotherapy or radiation protocols. Furthermore, recent evidence indicates that intratumoral or intravenous injections of replicative oncolytic viruses such as herpes simplex-, pox-, parvo-, or adenoviruses may also reactivate the human immune system. By generating tumor cell lysates in situ, oncolytic viruses overcome cellular tumor resistance mechanisms and induce immunogenic tumor cell death resulting in the recognition of newly released tumor antigens. This is in particular the case of the oncolytic parvovirus H-1 (H-1PV), which is able to kill human tumor cells and stimulate an anti-tumor immune response through increased presentation of tumor-associated antigens, maturation of dendritic cells, and release of pro-inflammatory cytokines. Current research and clinical studies aim to assess the potential of oncolytic virotherapy and its combination with immunotherapeutic agents or conventional treatments to further induce effective antitumoral immune responses.

**Keywords: immunotherapy, autonomous parvovirus, H-1PV, talimogene laherparepvec, T-VEC, JX-594, dendritic cells, CTLA-4**

#### **INTRODUCTION**

Human tumors develop complex strategies to circumvent the human immune system and to become resistant to classical therapies like radiotherapy or chemotherapy (1). Besides the low immunogenicity of tumors, tumor-induced dysregulation of the immune response leads to loss of effective immune defense and uncontrolled tumor growth. Even though many classical chemotherapy or radiation strategies induce some extent of tumor surveillance (1), new approaches should be tested to overcome early tumor resistance and recurrence. Thus, the basic challenge of molecular immune targeting is to conquer local regulatory mechanisms in order to re-introduce tumor immune recognition and promote tumor cell apoptosis and immunogenic cell death (ICD) (2). Recently, loss of immune defense has been shown to be caused by expression of different immune suppressive receptors also called immune checkpoint pathways, such as cytotoxic T-lymphocyte antigen-4 (CTLA-4) (3). Its ligation is crucial to preventing immune overreaction by inhibiting T-cell activation (4). The inhibitory CTLA-4 antibody ipilimumab [Yervoy, Bristol Myers Squibb (BMS)], approved for the treatment of metastatic melanoma patients, blocks this negative immune stimulatory receptor, thereby preventing downregulation of T-cell activation (5).

Oncolytic virotherapy represents an emerging therapeutic modality that has achieved tumor regression in several pre-clinical models and in clinical trials (6). Preferential depletion of cancer cells by oncolytic viruses (OV) is based on the fact that more aggressive tumor cells show both impaired antiviral responses and higher permissiveness for virus replication. Therefore, these agents open up new horizons for the treatment of cancer types that commonly display poor prognosis (7, 8). Cancer virotherapy is an old concept that arose from observations of unexpected tumor regressions coinciding with virus infections. This can be exemplified by a report on Newcastle disease virus (NDV) in gastric cancer dating back to 1971 (9). It should be stated that viruses with natural or engineered effects on the immune system are highly potent candidates for cancer therapy (**Table 1**). Herein, oncolytic viruses can be engineered to deliver therapeutic transgenes to cancer cells, causing additional anti-tumor effects through cytokine secretion and induction of anti-tumor immune responses (10–14). For example, the oncolytic vaccinia virus pexastimogene devacirepvec (Jennerex, Inc., and Transgene SA; Pexa-Vec, JX-594) and herpes simplex virus (HSV) talimogene laherparepvec (T-VEC, Amgen) were "armed" with GM-CSF-expressing genes (15, 16) to initiate local and systemic immune responses. Recently a randomized, Phase III trial of talimogene laherparepvec or GM-CSF in patients (pts)

with unresectable melanoma with regional or distant metastases (OPTiM) met its primary endpoint by improving durable response rates versus GM-CSF alone, and showed a tolerable safety profile (17). A Phase II study of Pexa-Vec in primarily first-line liver cancer (HCC) patients demonstrated survival improvement in patients receiving intratumoral (it) injections of high-dose Pexa-Vec (18). The following randomized Phase IIb study in second-line HCC patients did not meet its primary endpoint of survival improvement for Pexa-Vec compared to best supportive care (BSC) (19). However, this trial was comprised primarily of patients with endstage disease and significant comorbidities such as liver cirrhosis, therefore likely not the optimal population for successful OV therapy. Therefore, further studies of Pexa-Vec in a less advanced HCC population as well as other indications are warranted. Besides above-mentioned agents, various other viruses were shown to have oncolytic and/or immunostimulating properties, and are presently used in clinical trials. These include Parvovirus, Adenovirus,Vesicular Stomatitis Virus, Reovirus, NDV, Measles Virus, Seneca Valley Virus, Poliovirus, and Coxsackie Virus (**Table 1**).

The aim of this article is to provide an overview of upcoming oncolytic viruses and their potential immunogenic therapeutic effects. A first insight into this issue is provided through our pioneer studies showing that infection with the autonomous parvovirus H-1 (H-1PV) generated immunogenic tumor cell lysates (TCLs) (14). H-1PV-infected TCLS proved able to induce maturation of dendritic cells (DCs), release of pro-inflammatory cytokines, tumor-associated antigens (TAA) cross-presentation, and T-cell stimulation in an *ex vivo* human melanoma model (see **Figures 1** and **2**) (7, 14, 55, 56). On the basis of these observations, we present the prospects of H-1PV and other OVs activating the human immune system either alone or in combination with immunomodulators, such as antibodies blocking immune suppressive receptors.

#### **METHODS**

The human *ex vivo* melanoma model (**Figure 2**) represents a system that mimics the *in vivo* situation (14). Thus, it was used to investigate effects of H-1PV-infected or tremelimumab-treated tumor cells on immune activation. The human melanoma cells MZ7-Mel, SK29-Mel-1, and SK29-Mel-1.22 used were a gift from T. Woelfel (Mainz, Germany) (57). The SK29-Mel-1.22 cell line (A2−) is an *in vitro* selected HLA-A2-loss variant of HLA-A2 positive SK29-Mel-1 (A2+) line (58, 59). The cytotoxic T-cell clones CTL2/9 and CTL IVSB recognize different antigens of SK29-Mel-1 cells in association with HLA-A2 (57, 58), lyse SK29- Mel cells, and release interferon γ (IFNγ) upon specific recognition of SK29-Mel-specific TAA (58).

Peripheral blood mononuclear cells (PBMCs) were derived from buffy coats of healthy blood donors. Monocytes were isolated via adherence, and differentiation into immature DCs (iDCs) was achieved by stimulation with GM-CSF and interleukin-4. Matured DCs (mDCs) were generated by stimulation with a cytokine cocktail for 2 days (60). For coculture experiments, melanoma cells were kept in FCS-free medium. For induction of maturation and phagocytosis, tumor cells were co-cultured with iDCs at a ratio of 1:3 for 2 days. CTL-Coculture with DC was performed at 1:10 ratio (60).

#### **RESULTS: ONCOLYTIC VIRUSES ARE ABLE NOT ONLY TO KILL HUMAN TUMOR CELLS BUT ALSO TO STIMULATE ANTI-TUMOR IMMUNE RESPONSES: THE CASE OF PARVOVIRUS H-1PV**

Over the last years, OV therapy has shown promising results in both pre-clinical and clinical studies against various solid tumors (61). It is worth noting that besides their own anti-tumor efficiency, OVs can resensitize resistant tumors to chemotherapeutics, thereby highlighting the potential of OVs in multimodal treatments (12, 13). We were particularly interested in the oncolytic parvovirus H-1PV [for reviews, see Ref. (20, 62)]. The mode of action of H-1PV involves both direct oncolytic and immunemediated components, making this virus an attractive candidate for inclusion in the cancer immunotherapy armamentarium (60). H-1PV is a small nuclear-replicating DNA virus, which preferentially multiplies in oncogene-transformed and tumor-derived cells (7). This oncotropism results at least in part from the dependence of H-1PV on proliferation and differentiation factors that are dysregulated in neoplastic cells (20). In consequence, H-1PV exerts oncolytic effects, which were documented in human cells from various tumor entities including melanoma, pancreatic (PDAC), hepatocellular (HCC), colorectal or gastric carcinomas, sarcoma, glioma, and other neuroectodermal tumors (7, 20, 21, 62–64). Most interestingly, the death mechanisms activated by parvoviruses allow them to overcome resistance of tumor cells to conventional cytotoxic agents (22, 65). Another intriguing aspect of H-1PV-mediated OV lies in the possibility of combining H-1PV with conventional cytotoxic drugs to achieve synergistic tumor cell killing effects, as demonstrated for instance in the PDAC system (13, 21, 22, 66).

Though not or poorly infectious for humans under natural conditions, H-1PV can be administered experimentally to patients, resulting in viremia and seroconversion (67). Infections with H-1PV appear to be clinically silent (68). It should also be stated that recombinant parvoviruses can be constructed, for example to transduce immunostimulatory cytokines (62). This arming strategy was found to increase the anti-tumor effects of parvoviruses in certain models (69–71).

#### **BRINGING H-1PV FROM THE BENCH TO THE BEDSIDE**

Recent work using an immunocompetent rat glioma model showed that H-1PV was able to efficiently cure gliomas, while raising an anti-tumor memory immune response. This oncosuppressive effect appears to rely on both the direct oncolytic activity of H-1PV and its handover to the host immune system (23). These pre-clinical data led to the current clinical evaluation of H-1PV it and intravenous (iv) administration to patients with recurrent resectable GBM progressing in spite of conventional therapies (27).

#### **H-1PV-INDUCED TUMOR CELL LYSATES TRIGGER MATURATION OF iDCs AND EXERT IMMUNOSTIMULATING EFFECTS**

H-1PV had little direct killing activity on human immune cells *in vitro*, in particular APCs and CTLs. Interestingly, the analysis of infected PBMCs revealed the induction of markers of both macrophage and Th1cell activation (**Table 2**). This Th1 bias is indicative of a possible direct immunostimulating capacity

#### **Table 1 | Oncolytic viruses**.

(Continued)

**Table 1 | Continued**

Oncolytic viruses in clinical trials (ds, double stranded; ss, single stranded).

of the parvovirus. Nevertheless, a major impact of H-1PV on the immune system appears to be indirect, i.e., mediated by infected tumor cells, as discussed in the following sections. H-1PV caused the death of human melanoma cells in culture, including the above-mentioned SK29-Mel-1 and SK29-Mel-1.22 lines. The extent of cell killing varied between tested lines, was dependent on the multiplicity of infection (MOI) and

correlated with expression of the replicative viral non-structural protein NS1. In this system, H-1PV induced an apoptotic cell death, which was accompanied with the release of immunogenic HSP72 (63).

In further experiments it was shown that H-1PV-infected melanoma TCLs were phagocytosed by iDCs and induced their maturation, in particular the secretion of pro-inflammatory

cytokines such as TNFα and IL-6 (13, 63). Lysates of infected SK29-Mel-1.22 and MZ7-Mel cells were both competent for inducing DC maturation, although the former were more potent than the latter in this regard (13, 14). Primary immune cells were not permissive for H-1PV infection.

We also demonstrated that human DCs coincubated with H-1PV-induced melanoma TCLs showed enhanced expression

#### **Table 2 | Direct immunostimulating effects of parvovirus H-1PV**.

of TLR3, TLR9, and other maturation markers. This suggested that virus-induced TCLs contained molecular patterns triggering TLR signaling in DCs, as further evidenced by increased NFκB levels and production of pro-inflammatory cytokines (12). Some of these immunostimulating patterns may consist of viral constituents, given the known ability of TLR3 and TLR9 for sensing viral determinants.

Combination of the oncolytic virus with cytostatic (cisplatin, vincristine) or targeted (sunitinib) drugs resulted in a further increase in melanoma cell apoptosis but failed to strengthen maturation of DCs. It was verified that the cytotoxic or targeted drug regimen used did not interfere with H-1PV infection (13). Interestingly, the interleukin profile of DCs was altered upon exposure to H-1PV plus sunitinib-cotreated TCLs. It therefore appears that H-1PV combination with this antiangiogenic drug may reinforce its capacity not only for jeopardizing tumor cell survival but also for modulating the immune system.

#### **H-1PV INDUCE ACTIVATION OF ANTIGEN-SPECIFIC CYTOTOXIC T-CELLS AND OTHER ANTI-TUMOR IMMUNE EFFECTORS**

To further assess whether phagocytosis of H-1-infected TCLs by DCs induces cross-presentation of TAAs to antigen-specific CTLs in an HLA-class I-restricted manner, the above-mentioned human melanoma *in vitro* model was used (58, 72). Both melanoma-specific CTL clones tested were found to release increased levels of IFNγ after being co-cultured with DCs preincubated with H-1PV-infected SK29-Mel-1 or HLA-negative SK29-Mel-1.22 cells (14). Thus, H-1PV-induced TCLs stimulated cross-presentation of TAAs by DCs. This effect may contribute to reinforce the anti-tumor immune response by generating tumor-specific CTLs (14). In addition, several H-1PV-infected tumor cells were recently found to acquire an enhanced capacity for activating NK cells and getting killed by these cells (73, 74). The adjuvant effect of H-1PV was also evidenced *in vivo* by the virus-enhanced efficacy of an autologous tumor cell vaccine (24) and the adoptive transfer of antitumor immune cells from animals undergoing oncolytic H-1PV therapy (75).

#### **ONCOLYTIC H-1PV VIROTHERAPY CAN BE COMBINED WITH IMMUNOTHERAPEUTIC AGENTS TO ENHANCE TREATMENT EFFICACY**

Recent evidence for the expression of the immunosuppressing molecule CTLA-4 on regulatory T-cells (Tregs) and tumors generated widespread interest in the role of CTLA-4 in tumor escape and peripheral tolerance (3, 58). In particular, the human colon adenocarcinoma line SW480 was found to express CTLA-4 on the cell surface. This prompted us to extend the analysis of H-1PV anti-tumor effects to the SW480 system in combination with the anti-CTLA-4 antibody tremelimumab. When applied alone, this antibody had no detectable effect on SW480 cell viability and DC maturation. On the other hand, H-1PV alone was able to kill SW480 cells in a MOI-dependent manner. H-1PV-induced SW480 TCLs triggered iDC maturation in coculture experiments, as revealed in particular by increased release of the pro-inflammatory cytokines IFNγ, TNFα, and IL-6 (64). The secretion of IFNγ was stimulated to a low extent by treatment of the coculture with tremelimumab, recommend the use of the H-1PV/tremelimumab combination treatment to enhance tumor immunogenicity through both DC activation and CTLA-4 masking. It should also be stated that other (immuno)modulators, namely IFNγ (75) and HDAC inhibitors (76), were recently reported to cooperate with H-1PV for tumor suppression in human carcinoma animal models.

#### **CLINICAL EVIDENCE OF OV-MEDIATED ACTIVATION OF IMMUNE RESPONSES IN HUMANS**

Extensive analyses were performed to evaluate mechanisms-ofaction of the oncolytic and immunotherapeutic vaccinia virus Pexa-Vec in patients. These include oncolysis (15, 77, 78), acute vascular disruption (29) as well as anti-tumor immune response induction. Pexa-Vec was engineered to express GM-CSF to stimulate white blood cell production and activate DCs. Detectable concentrations of GM-CSF in plasma were measured 4–15 days after treatment and associated with increased neutrophil, monocyte, and eosinophil production in patients receiving iv or it iPexa-Vec (77, 78). Inflammatory cell recruitment to tumors was confirmed on biopsy following Pexa-Vec administration in patients with melanoma (79, 80). Furthermore, functional anti-cancer immunity of Pexa-Vec treatment was demonstrated in patients by measuring induction of antibody-mediated complement-dependent cytotoxicity (CDC) utilizing a panel of tumor cell lines of different histologies (30). Low concentrations of serum *ex vivo* incubated with tumor cells resulted in a dramatic reduction in tumor cell viability; when normal cells did not exhibit decreased viability. This activity was shown to be dependent on both active complement as well as IgG antibody. Reproducible CDC activity was also observed in a Phase II study in HCC patient (18). Furthermore, T-cell responses to β-galactosidase peptides were detected in HCC patients treated with Pexa-Vec, as shown by ELISPOT analysis. In that way, the proof-of-concept provides that T-cell responses can be induced to transgenes encoded by oncolytic vaccinia viruses (18).

Talimogene laherparepvec is an oncolytic immunotherapy comprising a modified HSV type 1 engineered to selectively replicate in tumor cells and to express the immune-stimulating cytokine GM-CSF, while retaining sensitivity to antiherpetic agents (16). Local effects after intralesional injection include selective lysis of tumor cells and subsequent release of tumor antigen, as well as secretion of GM-CSF into the local environment, which results in the stimulation and maturation of DCs (32, 81). Antigen presentation by stimulated DCs to CD4+ and CD8+ cells may induce an adaptive systemic immune response (16, 82, 83). Recently a randomized, Phase III trial of talimogene laherparepvec in patients (pts) with unresected melanoma with regional or distant metastases (OPTiM) met its primary endpoint, demonstrating a significant improvement in durable response rate (defined as partial or complete responses that were maintained for ≥6 months starting within 12 months) versus GM-CSF alone (16 versus 2%, *p* < 0.0001) (17). Overall response rate was also higher in the talimogene laherparepvec arm (26.4 versus 5.7%, *p* < 0.0001). Subjects treated with talimogene laherparepvec showed a tolerable safety profile with the only grade 3/4 adverse event that occurred in >2% of patients being cellulitis (2.1%). A trend toward improved overall survival was seen based on a planned interim analysis (17). The primary overall survival results are pending. Evidence of durable responses together with the safety profile of talimogene laherparepvec supports evaluation of combinations with other immunotherapies, such as high-dose IL-2 or immune checkpoint blockade and with radiation therapy, chemotherapy, and/or targeted therapies that might amplify the anti-tumor response generated by talimogene laherparepvec (32).

#### **DISCUSSION: POTENTIAL OF THE IMMUNOVIROTHERAPY CONCEPT**

Despite recent improvements in surgical, locoregional, and systemic therapies, the prognosis of patients with gastrointestinal, hepatobiliary, and pancreatic cancers remains dismal, and treatment is limited to palliation in the majority of patients. These limitations indicate an urgent need for novel therapeutic strategies (13, 64, 66, 84). Combinations of oncolytic viruses with new targeted therapies draw much attention. It is however necessary to proceed with caution, as these therapies may interfere with pathways, which are needed for replication of genetically modified viruses. It was demonstrated that by interacting with the EGFR/RAS/RAF pathway, sorafenib inhibits replication of Pexa-Vec in liver cancer, when applied in combination. This is not surprising as Pexa-Vec replication is in part dependent on the EGFR/RAS/RAF pathway (85). Nevertheless, sequential therapy with Pexa-Vec followed by sorafenib resulted in decreased tumor perfusion and was associated with objective tumor responses for HCC (85). It is noteworthy that some oncolytic viruses such as parvovirus H-1PV also have potential to inhibit neo-angiogenesis. Therefore, OV-based combination treatments targeting both tumor cell proliferation and tumor angiogenesis represent a promising strategy for impeding the growth of various cancers (25).

Besides their low expression of TAA and low immunogenicity, tumors can induce an immune tolerance milieu by releasing anti-inflammatory cytokines such as IL-10 or TGF-β or recruiting Tregs to their microenvironment (86). T-cell activation relies on both, recognition of major histocompatibility complex (MHC) molecules by the T-cell receptor (TCR), and on costimulatory signals. Depending on the type of costimulatory receptor, T-cells can be activated or become anergic. For example, T-cell activation was prevented by engagement of CTLA-4 receptors with CD80 or CD86. In contrast, engagement of CD80 or CD86 with CD28 induced T-cell activation, often with a low affinity (87). Thus, a promising therapeutic option to achieve strong anti-tumor immune responses is the use of monoclonal antibodies against CTLA-4 and PD-1 alone or in combination. Herein, the constitutive expression of CTLA-4 and PD-1 on Tregs may play a crucial role in inhibiting anti-tumor T-cell responses. Tregs are often found in the peripheral blood of cancer patients and in the tumor microenvironment. These cells suppress an optimal anti-tumor immune response by preventing infiltrating CD8+ T-cells from proliferating and producing cytolytic granules (88). BMS developed an anti-CTLA-4 monoclonal antibody named ipilimumab and an anti-PD-1 monoclonal antibody named nivolumab. Both antibodies were already tested in Phase III trials and found to achieve clinically significant benefits in median overall survival (89, 90). First pre-clinical studies of the combination of these antibodies to achieve blockade of both CTLA-4 and PD-1 showed increased tumor infiltration by CD4+ and CD8+ T-cells, enhanced IFNγ and TNFα production, and reduced amounts of Tregs (91). A Phase I study of nivolumab and ipilimumab combination in advanced melanoma patients showed an outstanding activity in 65% of patients with an objective response rate of 40% (92). As part of their further development and mechanistic understanding, these antibodies against immune check points would certainly deserve to be combined with OV in order to optimize anti-tumor immune responses. Preliminary data from a Phase Ib trial combining talimogene laherparepvec with ipilimumab indicated that the combination was tolerable and devoid of unexpected toxicities (93). Exploiting these combinations represents a promising strategy to bring oncolytic viruses from bench to bedside and to establish oncolytic virotherapy as a new effective immunotherapeutic approach.

#### **KEY CONCEPTS**

#### **ACKNOWLEDGMENTS**

The authors wish to thank Christiane Dinsart from the DKFZ for her support. Eduard Gasal (Amgen Inc., Thousand Oaks, CA, USA) provided the text relating to talimogene laherparepvec clinical development and data.

#### **REFERENCES**

combined with the oncolytic parvovirus H-1. *BMC Cancer* (2011) **11**:464. doi:10.1186/1471-2407-11-464

factor-encoding, second-generation oncolytic herpesvirus in patients with unresectable metastatic melanoma.*J Clin Oncol* (2009) **27**(34):5763–71. doi:10.1200/ JCO.2009.24.3675

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 19 February 2014; accepted: 14 April 2014; published online: 01 May 2014. Citation: Moehler M, Goepfert K, Heinrich B, Breitbach CJ, Delic M, Galle PR and Rommelaere J (2014) Oncolytic virotherapy as emerging immunotherapeutic modality: potential of parvovirus H-1. Front. Oncol. 4:92. doi: 10.3389/fonc.2014.00092*

*This article was submitted to Tumor Immunity, a section of the journal Frontiers in Oncology.*

*Copyright © 2014 Moehler, Goepfert, Heinrich, Breitbach, Delic, Galle and Rommelaere. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

### How informative is the immune response against surrogate tumor antigens to assess antitumor immunity?

#### **Valérie Janelle1,2 and Alain Lamarre1,2\***

1 Immunovirology Laboratory, Institut National de la Recherche Scientifique, INRS-Institut Armand-Frappier, Laval, QC, Canada

2 Department of Biology, Biomed Research Center, Université du Québec à Montréal, Montréal, QC, Canada

\*Correspondence<EMAIL_ADDRESS>
#### **Edited by:**

Philippe Fournier, German Cancer Research Center (DKFZ), Germany

#### **Reviewed by:**

Volker Schirrmacher, German Cancer Research Center (DKFZ), Germany John Cameron Bell, Ottawa Hospital Research Institute, Canada

**Keywords: oncolytic viruses, tumor antigens, CD8-positive T-lymphocytes, B16F10, VSV**

#### **A commentary on**

**The strength of the T cell response against a surrogate tumor antigen (TA) induced by oncolytic vesicular stomatitis virus (VSV) therapy does not correlate with tumor control**

*by Janelle V, Langlois M-P, Lapierre P, Charpentier T, Poliquin L, Lamarre A. Mol Ther (2014). doi: 10.1038/mt.2014.34*

The last decade has seen the development of numerous antitumor therapeutic approaches. Concomitantly, the interest for using oncolytic viruses (OV) against cancer has grown tremendously and a number of promising candidates are now in preclinical and clinical studies. Tumor regression *in vivo* following viral infection has been shown to be a multifactorial process (1). The reductionist view of viruses simply causing direct lysis of infected cancer cells has now been replaced by a view including the complex interplay between viruses and the tumor environment. The important role of the immune response in either limiting or enhancing OV therapy is also now well recognized (2, 3). The prototypic *Rhabdoviridae* VSV has generated encouraging results in various experimental tumor models and is now used in a phase I clinical trial in patients with liver cancer (www.clinicaltrials.gov; #NCT01628640). VSV possesses intrinsic oncolytic properties as it replicates more efficiently in type-I interferon (IFN)-defective cells, a pathway frequently impaired during tumorigenesis (4). Cancer therapy using VSV has been shown to generate a variety of immune responses including tumor-specific CD8+ T cells that are induced following the release of TA by infected cells (5). However, the tumor-specific immune response generated following VSV treatment is usually weak and often only leads to transient tumor control. Experimental tumor models expressing various surrogate nonself-TA have been developed over the years to more easily assess the magnitude and quality of immune responses generated against tumors. However, whether these responses are always representative of physiological antitumor immune responses is unclear.

Recently, our group characterized various VSV glycoprotein (G) mutants capable of interfering with host cell metabolism by inhibiting cellular transcription and translation in a kinetic similar to WT VSV as opposed to the prototypic matrix (M) mutant (MM51R) that is slightly attenuated *in vitro* (6). Furthermore, VSV G mutants proved to be more cytolytic for B16 melanoma cells *in vitro* than the M mutant. To analyze their oncolytic potential *in vivo*, we used an immunocompetent mouse model implanted with B16 tumors transfected with a DNA minigene encoding the immunodominant CD8+ T cell epitope of the lymphocytic choriomeningitis virus (glycoprotein aa 33–41) (7) as a surrogate non-self-TA (B16gp33) (8). Mice were injected subcutaneously into the flank with B16gp33 cells and when tumors reached a palpable size (day 7), animals were treated intratumorally every second day with three doses (days 7, 9, and 11) of WT VSV or of the G or M mutants. Tetramer and intracellular cytokine staining analysis revealed that CD8+ T cells harvested from mice treated with WT VSV or the G mutants developed a polyfunctional gp33 specific immune response. Surprisingly however, the strength of the gp33-specific immune response generated did not correlate with the ability of a particular strain of VSV to slow down parental B16 growth and improve mice survival. Treatment with WT VSV was the poorest at controlling B16 tumor progression even though it induced a strong CTL response against gp33. On the other hand, MM51R was more efficient than WT VSV at slowing down B16 growth despite the fact that this virus induced the lowest gp33-specific T cell response. We therefore determined whether CD8+ T cell responses directed against endogenous self-TA were involved in limiting tumor progression. CTL responses against self-TA, such as TRP-1 and gp100, were barely detectable *ex vivo* when analyzed separately. However, adoptive transfer of purified CD8+ T cells harvested from MM51Rtreated B16gp33 melanoma-bearing mice into naive mice provided better protection against parental B16 tumor implantation compared to CTLs taken from WT or G mutant-treated mice. These results suggest that the M mutant, despite being the weakest at inducing a T cell response against the surrogate non-self-TA gp33, induces the broadest antitumoral CTL response.

B16 melanoma is a highly aggressive tumor model in part because major histocompatibility complex class I (MHC-I) surface expression is very low on these

cells. Strikingly, B16 infection with VSV M mutant induced the upregulation of surface MHC-I both *in vitro* and *in vivo*, a phenomenon that was not observed for WT VSV of the G mutants (8). The matrix protein of VSV was previously shown to alter trafficking of a molecule structurally similar to MHC-I, namely CD1d (9, 10). This leads to inhibition of antigen presentation to natural killer T (NKT) cells (11). Thus, VSV matrix protein could participate in the retention of MHC-I molecules within infected cells while the mutated protein in MM51R may lack this ability. Thus, surface MHC-I upregulation following MM51R treatment likely explains the significantly improved CD8+ T cell-dependent survival despite the poor gp33-specific CTL response induced by this mutant. This may subsequently lead to presentation of a broader pool of B16 TA proportionally reducing the response against gp33 (see **Figure 1** for model).

In a recent study, Pedersen et al. compared vaccine-induced CD8+ T cell responses directed against self and nonself-TA and showed that vaccination with adenoviral vectors encoding endogenous TA had little or no effect on the growth of B16 melanomas whereas vaccination with a similar vector construct expressing a surrogate non-self-TA induced efficient tumor control (12). Although vaccination against both self and non-self-TA induced comparable CD8+ T cell responses in terms of cell numbers and effector functions, CTLs directed against self-TA were of lower functional avidity. These results are in agreement with our study and provide a potential mechanism explaining why T cell responses against self and nonself-TA are different and might not be induced at proportional levels during OV therapy.

Taken together, these results highlight a considerable limitation of many experimental systems used to assess antitumor immunity and warrant caution when extrapolating responses against surrogate TA to the overall antitumoral immune response. This may prove critical for the development of novel or improved OV, which may be biased by incorrectly estimating immune response correlates using such experimental systems. Therefore, great efforts will need to be made to develop improved methods for analyzing the antitumoral immune response induced by OVs against a broader array of TA in order to better appreciate their full therapeutic potential.

#### **ACKNOWLEDGMENTS**

This work was supported by the Canadian Institutes of Health Research and by the J.-Louis Lévesque Foundation.

#### **REFERENCES**

signaling pathways. *Expert Opin Biol Ther* (2009) **9**(9):1163–76. doi:10.1517/14712590903170653

correlate with tumor control. *Mol Ther* (2014). doi:10.1038/mt.2014.34

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 13 March 2014; accepted: 21 May 2014; published online: 04 June 2014.*

*Citation: Janelle V and Lamarre A (2014) How informative is the immune response against surrogate tumor antigens to assess antitumor immunity? Front. Oncol. 4:135. doi: 10.3389/fonc.2014.00135*

*This article was submitted to Tumor Immunity, a section of the journal Frontiers in Oncology.*

*Copyright © 2014 Janelle and Lamarre. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

### Armed therapeutic viruses – a disruptive therapy on the horizon of cancer immunotherapy

#### **Maxine Bauzon and Terry Hermiston\***

Bayer HealthCare, US Innovation Center, Biologics Research, San Francisco, CA, USA

#### **Edited by:**

Volker Schirrmacher, German Cancer Research Center, Germany

#### **Reviewed by:**

Can Peng, Tongji University, China Yong Lu, Cleveland Clinic Foundation, USA

#### **\*Correspondence:**

Terry Hermiston, Bayer HealthCare, US Innovation Center, Biologics Research, 455 Mission Bay Blvd. South, San Francisco, CA 94158, USA e-mail<EMAIL_ADDRESS>
For the past 150 years cancer immunotherapy has been largely a theoretical hope that recently has begun to show potential as a highly impactful treatment for various cancers. In particular, the identification and targeting of immune checkpoints have given rise to exciting data suggesting that this strategy has the potential to activate sustained antitumor immunity. It is likely that this approach, like other anti-cancer strategies before it, will benefit from co-administration with an additional therapeutic and that it is this combination therapy that may generate the greatest clinical outcome for the patient. In this regard, oncolytic viruses are a therapeutic moiety that is well suited to deliver and augment these immune-modulating therapies in a highly targeted and economically advantageous way over current treatment. In this review, we discuss the blockade of immune checkpoints, how oncolytic viruses complement and extend these therapies, and speculate on how this combination will uniquely impact the future of cancer immunotherapy.

**Keywords: oncolytic virus, cancer immunotherapy, immune-checkpoint inhibitors, CTLA-4, PD1, PDL1, PDL2, blockade of checkpoint inhibitors**

#### **INTRODUCTION**

Tumors are difficult to treat and in many instances lethal. The treatment challenge is not surprising as they are genetically unstable and complex biological systems with an ability to adapt to and thrive in often harsh and changing environments. Furthermore, this plasticity increases the probability that subpopulations will acquire resistance to any one therapy. Thus one could argue that a disease with such a complex etiology must be met with an equally complex therapeutic approach. Appropriately, oncologists have for some time combined chemotherapy, radiation and surgery and complemented these strategies with more targeted approaches such as tumor selective antibodies and/or small molecule kinase inhibitors (1). More recently, two alternative therapeutic approaches, cancer immunotherapy and oncolytic viruses, have begun to show promise that should further complement the oncologist's repertoire of anti-cancer agents.

The area of cancer immunotherapy has had a long and complex history (2, 3). The idea that a patient's own immune system could remove a tumor in much the same way it so efficiently removes invading microbes has been around for more than a century. Through the years, however, this concept of immunosurveillance has fallen in and out of favor perhaps appropriately given the complex and dynamic role, it is now believed to play in cancer, acting anywhere from anti to pro-tumorigenic (4–6). Research is beginning to elucidate the mechanisms by which tumors evade the immune system and in some instances how tumors use it to their advantage. From this research several promising immunecheckpoint inhibitor targets that are now translating into exciting clinical trial results have emerged (7–9).

Like cancer immunotherapy, the concept of oncolytic viruses is not new dating back to at least the beginning of the twentieth century when it was observed that on occasion tumor regression would follow a viral infection (10, 11). Although over 100 years have passed since these initial observations, the idea of using a replicating virus to selectively infect and kill tumor cells remains understandably appealing.  Furthermore, the progeny released from the lysed cancer cells would result in a self-perpetuating and amplifying therapy. Adding to their appeal is the ability of such agents to deliver exogenous genetic material whose product or products could augment the oncolytic viral treatment (12–14). Despite their theoretical promise, the reality is that oncolytic viruses have had limited clinical success as monotherapies perhaps due to an imbalanced focus on safety over potency. Recently however, there are several late-stage clinical trials showing promise which may eventually lead to clinical acceptance (15, 16).

Here, we suggest merging immune-checkpoint blockers with oncolytic viruses. We will discuss not only how these approaches could complement one another biologically for increased therapeutic benefit, but also how they may represent a unique opportunity to employ alternative biological formats not normally utilized commercially (e.g., Fabs, scFv) to increase both the safety and therapeutic profile of these agents. Finally we will touch upon how, together, these attributes might translate into a more economically appealing and clinically active therapy resulting in a truly new and disruptive treatment for malignancies.

#### **CANCER IMMUNOTHERAPY-BLOCKADE OF IMMUNE CHECKPOINTS**

Immunotherapy works to direct the extensive repertoire of the host immune system to fight cancer. This approach strives to stimulate tumor suppression by (a) boosting the patient's immune system, (b) decreasing the cancer-induced immunosuppression, and/or (c) increasing the immunogenicity of the tumor itself. If the immune system's ability to rapidly respond to and clear invading microorganisms could be extended to malignant cells then a powerful therapeutic may be realized. Such an approach may hold greater potential than current treatment approaches as it may prove to be more potent, benefit many more cancer types, offer long-lasting protection against the disease, and come with fewer off-target effects. Advances in cellular and molecular immunology have provided enormous insight into the inter-play between tumors and immune cells and from this research have come strategies by which the immune system might be harnessed to fight cancer (7).

The blockade of immune checkpoints is a more recent approach taken to decrease cancer-induced immunosuppression. Immune checkpoints refer to a number of inhibitory pathways that play crucial roles in maintaining self-tolerance and immune homeostasis. Their function is to down-regulate T-cell signaling in order to prevent uncontrolled T-cell proliferation thereby protecting tissues from auto-immune damage while maintaining tolerance to self-antigens. It is becoming increasingly clear that tumors commandeer certain immune-checkpoint pathways particularly against T cells that are specific for tumor antigens. Preclinical and clinical data have demonstrated that this is a major mechanism utilized by the tumor to evade the immune system. If this could be reversed, the resulting amplification of T cells and their activity would be highly beneficial to the patient given the central role T cells play in cell-mediated immunity. The immune checkpoints are controlled by ligand–receptor interactions, which can be readily blocked by antibodies or disrupted by recombinant forms of ligands or receptors making them appealing therapeutic targets. For a list of immune-checkpoint targeting antibodies that are currently in clinical trial see **Table 1**.

The inhibitory receptor, Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), was the first checkpoint receptor to be extensively and successfully pursued as an anti-cancer target (32). The primary function of CTLA-4 is to regulate the magnitude of Tcell activation. It is expressed solely on T cells where it offsets the actions of CD28, a T-cell co-stimulatory receptor. Because CTLA-4 has a higher affinity for the CD28 ligands B7.1 and B7.2 it, in effect, out-competes CD28 for ligand binding resulting in an attenuated T-cell response (33–37). The lethal systemic immune hyperactivation phenotype of *Ctla4*-knockout mice clearly shows the importance of CTLA-4 and the need to keep T cells in check (38, 39). In 2011, an antibody against CTLA-4 (ipilimumab) was given FDA approval for the treatment of metastatic melanoma (20, 40–42). In a pivotal phase III randomized three-arm clinical trial, melanoma patients were treated with a glycoprotein 100 (gp100) peptide vaccine alone, ipilimumab alone, or the gp100 peptide and ipilimumab. Both ipilimumab groups demonstrated an increased survival of 3.5 months compared with the group receiving the gp100 peptide alone. Moreover, long-term survival was greatly increased with 18% of patients receiving ipilimumab surviving for greater than 2 years as compared with only 5% for the gp100 peptide alone cohort (17). Although ipilimumab treatment was relatively brief, spanning only 3 months, the finding of long-term progression-free survival supports the idea that immune-based

**Table 1 |The most advanced clinically evaluated immune-checkpoint blocking antibodies.**

Above trial information from ClinicalTrials.gov.

therapies may actually result in a reprogramed immune system which can confer long-term antitumor immunity. Clinical trials are on-going evaluating the use of anti CTLA-4 antibodies in other cancer indications including lung, colorectal, renal, and ovarian (43).

The immune-checkpoint receptor, programed cell death 1 (PD1) and its ligands PDL1 and PLD2, are also emerging as promising targets. PD1 like CTLA-4 plays a role in regulating and maintaining the balance between T-cell activation and tolerance (44, 45). However, unlike CTLA-4, PD1 is more broadly expressed and can be found on other activated non-T-lymphocyte subsets including B cells and natural killer (NK) cells. Additionally while CTLA-4 primarily regulates T-cell activation, PD1 principally controls T-cell activity (46). The ligands PDL1 and PDL2 are commonly upregulated on the surface of many different human tumors with PDL1 being the predominant PD1 ligand on solid tumors. High expression levels of PDL1 have been shown on melanoma, lung, ovarian, and other human cancers (47, 48). PDL1 is also expressed on myeloid cells in the tumor microenvironment. *Pdl*, *Pdl1*, and *Pdl2*-knockout mice demonstrate a milder auto-immune phenotype than *Ctla4*-knockout mice (49–52). Preclinical studies have shown that blocking PD1 or its ligand PDL1 enhances immunity *in vitro* and mediates antitumor activity in preclinical models (53–55). Although the development of PD1 targeting antibodies is not as mature as that of CTLA-4 antibodies, preliminary clinical results look encouraging. In phase I trials of an anti-PD1 antibody (nivolumab), objective responses (complete or partial responses) were observed in those with non-small-cell lung cancer, melanoma, or renal-cell cancer with cumulative response

rates ranging from 18 to 28%. Responses were durable with 20 of 31 responses lasting 1 year or more (56). In a separate phase I trial of patients with various advanced cancers, an anti-PDL1 antibody (MDX-1105) also induced durable tumor regression (objective response rate, 6–17%) and prolonged stabilization of disease (12–41% at 24 week) (57).

Beyond CTLA-4 and PD1, molecular immunology has begun to reveal additional receptors and ligands that serve an inhibitory immune function. These include B and T-lymphocyte attenuator (BTLA), T-cell membrane protein 3 (TIM3), Lymphocyte activation gene 3 (LAG3), adenosine A2a receptor (A2aR), and the B7 family of inhibitory ligands (58–66). Each has been associated with the inhibition of lymphocyte activity in preclinical models and consequently antibodies against a number of these targets are being actively pursued (58–66). Additionally, because multiple inhibitory ligands and receptors contribute to the tumor's evasion of the immune system and appear to be non-redundant, there remains the possibility of further enhancing antitumor immunity by blocking multiple immune checkpoints. Currently several preclinical and clinical studies are on-going testing the effects of blocking a combination of immune checkpoints (**Table 2**) (67–73). In fact, a recently published phase I study in patients with melanoma that combined anti-CTLA-4 (ipilimumab) and anti-PD1(nivolumab) mAbs resulted in a rapid and deep tumor regression in a substantial proportion of patients (53% of patients had an objective response, all with tumor reduction of 80% or more) (74). These objective response rates exceeded the previously reported results with either mAb alone (17, 56).

#### **ONCOLYTIC VIRUSES AS (IMMUNO)THERAPIES**

Oncolytic viruses can be RNA or DNA based and derived from human (e.g., herpes simplex virus, adenovirus, measles virus) or animal [e.g., vesicular stomatitis virus (VSV), Newcastle disease virus, myxoma virus] viruses.  This selectivity can be a natural property of the virus or an engineered trait (75–81). Oncolytic viruses can also be genetically armed to improve or generate more tumor selective cell killing. For example, cell death can be induced by delivering tumor-suppressors (e.g., p53, p16), pro-apoptotic proteins (e.g., TRAIL, IL-24), or small hairpin RNA targeting cell survival or proliferation factors (e.g., hTERT, survivin) (82–87). Arming can also sensitize the tumor to chemo or radiotherapy (Prodrug enzymes, NIS) (88–90).

Although direct oncolysis was envisioned as the primary desired outcome of this therapeutic approach, research and clinical data is supporting the assertion that these productive tumor-specific infections can elicit additional antitumor effects. For example there is evidence that oncolytic viral therapy can induce tumor vasculature shutdown resulting in tumor necrosis (91, 92). Data also suggests that because oncolytic viruses result in highly proinflammatory and immunogenic events (tumor cell death and the release of tumor-specific antigens) (93–95) they can elicit a tumorspecific immune response (96). Additionally, viruses encode products that can be recognized by immune and non-immune cells as Pathogen-associated molecular patterns (PAMPs) and can also cause the release of Damage-associated molecular pattern molecules (DAMPs) (97). PAMPs are structural motifs which serve

Above trial information from ClinicalTrials.gov.

as "danger" signals to the host indicating the presence of virus that trigger host defenses. These danger signals can be structural proteins and glycolipids but are mainly nucleic acids including double-stranded RNA (dsRNA), viral single-stranded RNA, and CpG DNA (98, 99). DAMPs are host nuclear or cytosolic proteins with defined intracellular function that activate effector cells from the innate immune system when they are released outside the cell (100). Virus-induced changes such as an increase in pro-inflammatory cytokines and chemokines, a decrease in immunosuppressive cytokines, and the release of PAMPs and DAMPs at the site of the tumor may diminish or reverse the established immunosuppressive microenvironment and initiate antitumor immunity.

Several oncolytic virus classes are currently in late-stage clinical trials (**Table 3**). The most advance of these, Talimogene laherparepvec (T-VEC, formerly OncoVex or JS1/ICP34.5-/ICP47- /GM-CSF; an HSV isolate selected for its potency over laboratory strains, it is deleted in both the ICP34.5 and ICP47 genes to further increase viral replication and tumor cell killing, it also expresses human GM-CSF for immune stimulation) has demonstrated some very promising clinical data. From recently announced results of a phase III trial in unresectable stage IIIB-IV melanoma receiving either T-VEC injected into the lesion or GM-CSF administered subcutaneously, the overall durable response rate (DRR) was 16.3% for T-VEC treated patients as compared to 2.1% for GM-CSF treated individuals (101). The objective overall response rate (ORR) was 26.4% for the T-VEC group (including 10.8% complete responders) compared to an ORR of 5.7% and a complete response rate of 0.7% in the GM-CSF alone group (101). Importantly, in a phase II trial, tumor shrinkage was noted in non-injected lesions, demonstrating that systemic immunity was induced (102). In addition, and across a number of viruses, studies have shown that both innate and adaptive immune responses are generated following viral tumor lysis (92, 103–111). This antitumor immunity is an important outcome of oncolytic viral therapy as it would lead to the destruction of tumor cells that escaped the initial viral lysis.

Above trial information from ClinicalTrials.gov.

#### **MERGING ONCOLYTIC VIRUSES AND IMMUNE-CHECKPOINT BLOCKING**

The realization that oncolytic viral therapy can itself be an immunotherapy has in many ways reinvigorated the field and expanded the possible approaches that can be taken to treat cancer. Similarly, the discovery and targeting of immune checkpoints has opened a new immuotherapeutic avenue generating very promising clinical results. The potential to combine oncolytic viruses with a blockade of immune checkpoints is a very exciting strategy that may be beneficial on many levels and help overcome current shortcomings associated with either approach alone. To date, there have been only a few preclinical studies combining oncolytic viruses and immune-checkpoint blockers (anti-CTLA-4 mAb) (144, 145). However, results have been promising with one study showing that replication competent VSV in combination with anti-CTLA-4 mAb resulted in the elimination of macroscopic tumor implants in the majority of test animals, an outcome that could not be achieved by either treatment alone (145). The study went on to show that the response was CD4 and CD8 T-cell mediated (145). When combining these two approaches, the exact virus/checkpoint combination will likely need to be determined empirically with many factors including indication and immune status of patient playing a role. However, in general an argument can be made that the greatest synergies between these strategies would be realized by delivering

#### **Table 4 |The benefits of using an oncolytic virus to deliver immune-checkpoint blockers.**

the immune-checkpoint therapy directly from the oncolytic virus (**Table 4**).

#### **INCREASED PRIMING AND GREATER IMMUNE POTENCY**

Preclinical studies have shown that in mice bearing partially immunogenic tumors, treatment with CTLA-4 antibodies could elicit significant antitumor responses whereas poorly immunogenic tumors were refractory to anti-CTLA-4 administration (32, 146). However, these refractory tumors could be made more responsive by administering granulocyte-macrophage colonystimulating factor (GM-CSF) in combination with the anti-CTLA-4 (146). These findings suggested that a CTLA-4 blockade enhances an already existing endogenous antitumor response resulting in tumor regression. But when the tumor is poorly immunogenic and does not induce a robust enough immune response the anti-immune checkpoint is not as efficacious. Similar results have been found in the clinic where analysis of pre-treatment tumors indicated that patients with high baseline expression levels of immune-related genes were more likely to respond favorably to ipilimumab (147). Just as the GM-CSF is used to help boost the initial innate immune response, oncolytic viruses could have a similar effect as it is clear that the oncolytic viral infection has pro-inflammatory properties, eliciting both an innate and adaptive immune response.

#### **ENHANCED SAFETY AND EFFICACY BY EXPRESSING IMMUNE-CHECKPOINT BLOCKERS FROM THE ONCOLYTIC VIRUS**

The oncolytic virus and the immune-checkpoint blocker could be administered as two separate therapeutics but one of the most appealing aspects of the oncolytic viral approach is that it is localized to the tumor. This localization confers several advantages for both safety and potency. Clinical and preclinical data strongly suggest that a blockade of immune checkpoints is a very potent antitumor therapy. However, there are, in some cases, unwanted side effects. Given the importance of the immune checkpoints in maintaining immune homeostasis there is concern that a blockade of these receptors and/or ligands could lead to a break in immune self-tolerance resulting in autoimmune/autoinflammatory side effects (148). Blocking CTLA-4 as a therapy was initially questioned given its crucial role in the regulation of T-cell amplification. The phenotype of *Ctla4*-knockout mice also hinted at the possibility of a high number of unwanted immune-related effects. In the pivotal phase III trial of ipilimumab, Grade 3 or Grade 4 immune-related adverse events (including rash, colitis, hepatitis, and endocrinopathies) occurred in 10–15% of patients

treated with the anti-CTLA-4 antibody as compared to 3% of those treated with gp100 alone. During this trial, there were 14 deaths related to ipilimumab (2.1%), 7 of which were due to immunerelated adverse events (17). Delivering the immune-checkpoint blocker (Ab,Ab derivative or modified ligand or receptor) from the oncolytic virus would localize the treatment and mitigate the risks inherent in systemic delivery. In preclinical studies of a replication competent adenovirus armed with the coding region of a full length CTLA-4 antibody a 43-fold higher antibody concentration in the tumor as compared to the plasma was noted (144). Moreover, plasma levels in treated mice remained below the reported human safety threshold (144).

It is also possible to make expression of these immunecheckpoint blockers contingent upon a productive viral infection (i.e., selective replication that is restricted to the tumor cell) further increasing the safety of the therapeutic. This can be done by utilizing endogenous late viral promoters that are dependent upon the uptake and replication within the target tumor cell to express exogenous genes and has been described for human adenovirus (12, 13, 149). In the normal cell, this expression would be blocked as replication would not be achieved consequently confining expression to target cancer cells. Potency, like safety also benefits from this localized delivery, concentrating the therapeutic to the tumor and its microenvironment. Accumulation of virally delivered transgenes (including reporter genes, prodrug converting enzyme, anti-angiogenic factors, immunostimulatory factors) at the site of the infected tumor has been shown in numerous studies (97, 115, 132,150–153). For example, PET imaging experiments have dramatically demonstrated the tumor localized expression of thymide kinase following infection with an oncolytic virus armed with the enzyme (154, 155). This accumulation was translated into efficacy upon administration of the prodrug Ganciclovir (154). Additionally, the self-perpetuating nature of an oncolytic infection results in sustained transgene expression (156) that will continue until tumor regression is complete and the virus is eliminated from the tumor site by the immune system (157). Therefore the amount of material produced would be directly related, in theory, to the tumor load, personalizing the respective dose to the individual and their tumor burden. It is also appealing to consider that this may eliminate peaks and valleys associated with the intravenous administration of the therapeutic as the virus expressed molecule would be generated on a more constant basis that might also benefit the patient.

#### **ENABLEMENT OF ALTERNATIVE THERAPEUTICS**

Although viruses can be used to deliver an intact IgG, their focused delivery to the tumor site and their self-perpetuating nature allow for the use of alternative antibody formats such as diabodies, Fabs, and scFvs (144, 158). This could have a profound impact on any mAb-based antitumor therapeutic particulary immunecheckpoint blockers. From a safety standpoint, the use of these alternative Ab formats could be beneficial because IgGs, due to their size (150 kDa), have prolonged serum half-lives (>10 days) and are therefore more likely to have associated toxicities. If these alternative formats were to escape the tumor site their faster clearance reduces the risk for off-target events. For immune-checkpoint blockers, this could help to decrease the immune-related adverse

events that have been associated with this therapeutic approach (148, 159). Additionally, smaller formats would potentially penetrate the tumor to a greater extent than a full length antibody. Studies have shown that an intact IgG molecule takes 54 h to move 1 mm into a solid tumor, whereas a Fab fragment travels the same distance in only 16 h (160). This enhanced penetration could increase overall efficacy. The diabodies in particular have been shown to provide rapid tissue penetration, high target retention, and rapid blood clearance presumably as a result of their multi-valent nature and intermediate size (55 kDa) (161). The use of alternative antibody formats also opens up the possibility of delivering multiple therapies from one oncolytic virus. This may have broad implications for the blockade of immune-checkpoint approach as studies are beginning to show that targeting multiple checkpoints may be more efficacious (67–71, 74). Without localized delivery, the use of these alternative formats would likely not be feasible as they would clear too rapidly (on the order of a few hours or minutes dependent upon the format) (162). This may necessitate the need for higher input doses or multiple injections of the Ab, which could potentially be cost prohibitive. Having localized delivery via the virus would avoid the need for full length Abs and make the smaller, faster-clearing formats viable therapies that are still capable of efficacious outcomes.

#### **ECONOMICALLY ADVANTAGEOUS**

Expression of immune-checkpoint blockers from an Oncolytic virus is economically appealing. If one assumes that the initial promising results seen with combination checkpoint blockers are maintained in larger phase II and III trials, the delivery of a combination of blockers from a virus would eliminate the need to commercially manufacture the molecules separately. This approach utilizes a single entity (the virus) to exploit the natural machinery of the virus and the tumor cell to continuously produce the therapeutic agents so long as the tumor cells continue to exist. Moreover, it has been demonstrated that multiple exogenous proteins can be delivered from a single virus (149). Due to their tumor selective localization, as mentioned previously, they would not need to express a full length antibody, making this approach potentially attractive and novel for delivering multiple-checkpoint inhibitors to the site. In addition, this therapy would have the potential added benefit of increased immunogenicity and/or direct tumor cell lysis offered by the oncolytic virus. Thus expressing a single biological agent with the ability to deliver multiple-checkpoint inhibitors that itself has anti-cancer activity is an interesting possibility. However, it should be kept in mind that the commercial manufacture of oncolytic viruses is behind that of antibodies and thus may be only a true economic advantage in the future with additional optimization.

#### **CONCLUSION**

In the fight against cancer, no single magic bullet has emerged. Despite several improvements in diagnostics and therapies nearly 7 million cancer-related deaths still occur every year worldwide (163). One reason is that cancer is complex and can evolve to thrive under harsh conditions and to evade the body's natural defenses. Two promising therapeutic strategies have emerged; the blockade of immune checkpoints and oncolytic viruses and we

believe that an argument can be made that the greatest potential for both of these therapies lies in the synergies that would be realized by delivering the immune-checkpoint therapy directly from the oncolytic virus (**Table 5**). We look forward to the continued evolution of these agents and to the exciting years ahead as we begin to see these agents come forward pre-clinically and clinically.

#### **REFERENCES**

chromosome technology for their construction. *Curr Opin Mol Ther* (2007) **9**(5):447–66.

microenvironment through upregulation of the inhibitory receptors BTLA and PD-1. *Cancer Res* (2012) **72**(4):887–96. doi:10.1158/0008-5472.CAN-11-2637

targeting hTERT gene. *Cancer Biol Ther* (2009) **8**(1):84–91. doi:10.4161/cbt.8. 1.7204

monoclonal antibody specific for CTLA-4. *Gene Ther* (2012) **19**(10):988–98. doi:10.1038/gt.2011.176

**Conflict of Interest Statement:** Dr. Terry Hermiston serves on the following boards: Scientific Advisory Board for Psioxus Therapeutics and Board of Directors for Jennerex Biotherapeutics. The other co-author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 13 December 2013; paper pending published: 25 January 2014; accepted: 11 February 2014; published online: 24 February 2014.*

*Citation: Bauzon M and Hermiston T (2014) Armed therapeutic viruses – a disruptive therapy on the horizon of cancer immunotherapy. Front. Immunol. 5:74. doi: 10.3389/fimmu.2014.00074*

*This article was submitted to Tumor Immunity, a section of the journal Frontiers in Immunology.*

*Copyright © 2014 Bauzon and Hermiston. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

 This paper summarizes (i) the peculiarities of this virus as an anti-cancer and immune stimulatory agent and (ii) the approaches to further harness this virus as a vector to combat cancer. Special emphasis is given on combining virus therapy with cell therapy and on improving tumor targeting. The review will include some of the authors work on NDV, bi-specific antibodies, and cell therapy as building blocks for a new perspective of multimodal cancer therapy. The broad anti-tumor immune reactivation includes innate and adaptive, tumor antigen (TA) specific and TA independent activities

**Keywords: Newcastle disease virus,T-cells, dendritic cells, bi-specific antibodies, tumor targeting, cellular therapy, hyperthermia**

#### **INTRODUCTION**

Paramyxoviruses are a family of viruses that infect a diverse range of hosts. Animal pathogens, such as Newcastle disease virus (NDV), SV-5, and Sendai virus (SV), have been major subjects for basic research by virologists, immunologists, and molecular biologists. Previously, genetic manipulation of paramyxoviruses was not possible because the genome is not infectious alone and RNA recombination is essentially non-existent. During the last 15 years, methods of producing infectious paramyxoviruses from c-DNA clones (reverse genetics) have been developed. This review will focus on NDV, an avian paramyxovirus, because this has a number of very interesting anti-neoplastic and immune stimulating properties in mammalian cells, including human being, because it has a high safety profile for clinical application and because it can be harnessed by therapeutic transgenes.

 NDV wildtype (wt NDV) virus shows naturally tumor selective replication behavior (2). An abortive replication cycle by lentogenic strains leads eventually to tumor cell death. A lytic replication cycle by mesogenic or velogenic strains leads to fast tumor cell death (oncolysis) and further spread of the virus in the tumor tissue. The strong interferon (IFN) response of normal cells (2) prevents virus replication and cell death thus explaining the high safety record of NDV in cancer patients (3).

There are additional properties that make NDV a particularly interesting anti-neoplastic agent. It replicates and destroys in particular cancer cells that are resistant to certain types of chemotherapy (4–6) and apoptosis-resistant tumor cells from hypoxic tumor tissue (7). The oncogenic protein Rac1 was reported as a link between tumorigenesis and sensitivity of cells to oncolytic NDV (8). Furthermore, NDV triggers autophagy in glioma cells (9) and promotes Bax redistribution to mitochondria and cell death in HeLa cells (10). A time-course analysis revealed that NDV-induced apoptosis involved an early extrinsic pathway with TRAIL expression (peak at 24 h *p.i.*) and a later intrinsic mitochondrial pathway (peak at 48 h *p.i.*) (11).

NDV was reported to repress the activation of human hepatic stellate cells and reverse the development of hepatic fibrosis in mice (12). Liver fibrosis is a major health problem and the 12th most common cause of death in the United States (13).

#### **HARNESSING NDV BY TRANSGENES AND BI-SPECIFIC ANTIBODIES**

Recombinant NDV strains (rNDV) could be harnessed by transgenes to show enhanced oncolytic potential. This was achieved by F gene mutations (14, 15) or by addition of the NS1 (16) or Apoptin (17) gene. It could also be harnessed by genes coding for cytokines, such as IL-2 (18), GM-CSF (19), IL-15 (20), or IFN-γ (21) to express enhanced immune stimulatory properties. Other transgenes conferred resistance to complement (22). NDV was also capable of incorporating two transgenes, one coding for the light chain and the other for the heavy chain of a monoclonal antibody interacting with angiogenesis (23). The transfer of a gene coding for a tumor antigen (TA) created a vector with which the immune response could be targeted to a specific TA in order to compete with the usually stronger response to viral antigens (VA) (24). A recombinant oncolytic MV (MV-AC133) could be targeted to CD133+ cancer-initiating cells causing their specific elimination (25).

To augment the immune stimulatory properties of NDV infected tumor cells, another elegant approach was successful. It consists of the attachment of single-chain variable fragment (scFv) bi-specific antibodies (bsAbs). These attach with one arm to a VA and with the other arm to a target on immune cells. In case of T-cells, such targets were CD3 (26), CD25 (27), and CD28 (28). The VAs of NDV were either HN or F. These served as universal anchor molecules through which T-cell co-stimulatory molecules could be attached to any type of tumor cell infectable by NDV (29).

In the following paragraph, we will present a perspective how such bsAbs can be further used in a multimodal approach for improvements of cancer therapy.

#### **FUTURE PERSPECTIVE: COMBINING NDV WITH bsAbs AND WITH ADOPTIVE CELLULAR THERAPY**

#### **TUMOR TARGETING OF NDV**

A major problem with the clinical application of oncolytic viruses is a proper targeting of tumor tissue. This can be achieved by intratumoral application (30) but metastases are often not accessible by this approach. Nevertheless, localized oncolytic virotherapy was reported to overcome systemic tumor resistance to immune checkpoint blockade immunotherapy (31).

Locoregional application (e.g., via the hepatic vain) was reported to be superior to systemic tail vain inoculation (32). Locoregional virotherapy was effective even against oncolysisresistant tumor cells, thus suggesting that the anti-tumor effect was host mediated (32). Inhalation is another way of locoregional application. Inhalation of oncolytic NDV was applied to 33 advanced chemorefractory patients in a Phase II clinical study in Hungary as a means to affect their lung metastases (33). Virus inoculation into body cavities in case of tumor ascites is another way of locoregional application. For instance, intraperitoneal NDV virotherapy was effective against peritoneal carcinomatosis from human gastric cancer in a xenograft model (34) and intrapleural NDV virotherapy induced sustained remission of malignant pleural mesothelioma in an orthotopic model (14).

Upon systemic administration of NDV, its binding to normal cells could prevent it from reaching the tumor tissue and could cause undesired side effects. Since efficient distribution at the tumor site may be a very critical parameter for tumor selective gene delivery and for anti-tumor efficacy of oncolytic virotherapy (35), we have developed adaptor molecules that redirect the virus to tumor tissue (36). The targeting molecule used, anti-HN-IL-2, contains a scFv antibody cloned from a neutralizing HN specific hybridoma linked to the human gene for the cytokine IL-2. Selective virus entry was observed *in vitro* in a mixture of IL-2 receptor positive and negative human tumor cells (37). Retargeted virus infection of tumor cells required specific binding via the bi-specific fusion protein and membrane fusion via the viral F-protein. After systemic virus inoculation into tumor-bearing mice, the modification of NDV by the adaptor protein did not compromise the efficiency of gene delivery into target positive tumors but greatly reduced viral gene expression in target negative tumors and in normal tissues thereby reducing side effects (38).

#### **UNIVERSAL ACTIVATION OF CANCER PATIENTS T-CELLS (NAÏVE AND MEMORY) VIA TUMOR CELL-BOUND BI-SPECIFIC ANTIBODIES**

Infection of tumor cells by NDV leads to increase in tumor cell immunogenicity (39). A prospective, randomized, controlled clinical study of post-operative immunization with the autologous tumor vaccine ATV-NDV revealed evidence for clinical effectivity and long-term survival for colon cancer patients (40). Further augmentation of T-cell stimulatory capacity of the ATV-NDV vaccine was achieved by attachment of specifically designed bsAbs binding to viral HN or F on the infected tumor cells and to CD3 or CD28 on T-cells (41). The optimized vaccine ATV-NDV/bsHNxCD3/bsHNxCD28 appeared to be able to revert unresponsiveness of partially anergized TAspecific T-cells (42). It was also capable of *de novo* activation of anti-tumor activity from naïve T-cells, independent of TA recognition (**Figure 1A**) (42). The strongest potentiation of the T-cell stimulatory capacity of the ATV-NDV vaccine was observed upon attachment of a suboptimal amount of bsHNCD3 together

**FIGURE 1 | Activation of naïve human T-cells by co-incubation with NDV infected irradiated tumor cells modified with bi-specific or tri-specific antibodies**. **(A)** Time course of the induction of T-cell activation and proliferation by a stimulatory cell (NDV infected and y-irradiated tumor cells) optimized for co-stimulation by attachment of the bi-specific fusion proteins anti-CD3 (anti-HNxanti-CD3) and anti-CD28 (anti-HNxantiCD28). Purified and CFSE-labeled naïve human T-cells were cocultivated for 5 or 7 days with the stimulatory cells. The CFSE signal intensities were compared with unstimulated cells by FACS analysis. We also followed by the FACS analysis the expression of the IL-2 receptor α chain (CD25) and of the memory marker CD45RO. **(B)** Diagram of the components of a tumor vaccine infected by NDV and modified by a bi-specific antibody (anti-HNxanti-CD3, suboptimal amount for signal 1) and a tri-specific immunocytokine (anti-HNxIL-2xanti-CD28, for delivery of two T-cell co-stimulatory signals via CD28 and CD25).

with the tri-specific (ts) fusion protein tsHNxIL-2xCD28. The latter delivers two co-stimulatory signals to T-cells, one via CD28 and the other via CD25 (26). **Figure 1B** illustrates the modular concept of the tumor vaccine infected by NDV and modified by bsAbs.

We suggest to use T-cell activation one universal GMP tumor cell line for patients. This will be modified by infection with NDV and by attachment of the above bsAbs and tsAbs. This universal T-cell stimulatory cell can be applied for non-specific activation of anti-tumor activity of T-cells from any type of cancer patient and is independent from a TA.

#### **PROGRAMING OF CANCER PATIENTS DENDRITIC CELLS TOWARD DC1 VIA INFECTION BY NDV**

We reported on polarization of human monocyte-derived DCs to DC1 by *in vitro* stimulation with NDV (43). Also, murine DCs upon infection by NDV differentiate into the immunogenic phenotype DC1 characterized by secretion of pro-inflammatory cytokines,in particular IL-12 and IFN-α and -β (44). Two receptorinitiated signaling cascades were involved: the first one is induced by triggering and upregulation of the intra-cellular cytoplasmic receptor RIG-1 upon recognition of viral non-capped RNA as ligand (45). The second signal cascade involves cell-surface expressed type I IFN receptor (IFNAR), which initiates a feedback loop cell activation upon interaction with extra-cellular type I IFN as ligand (31, 44). RIG-1/RNA ligand interaction not only activates type I IFN, but also induces inflammasome activation for IL-1β production (46). Type I IFN and IL-12 are critical mediators of cross-priming and Th1 polarization of CD8 T-cell responses (47) while IL-1β is critical for Th1 polarization of CD4 T-cells (48).

DCs can also be pulsed with NDV oncolysate. Such cells were superior in stimulating patients T-cells in ELISPOT assays compared to DCs pulsed with tumor lysate without NDV (49).

#### **GRAFTING OF AUTOLOGOUS ACTIVATED T-CELLS AND DC1 BACK TO THE PATIENT**

Our proposal for a multimodal cancer therapy involves the transfer of immune T-cells and of DC1 as professional antigen-presenting cells back to the patient. Activation of the tumor microenvironment by low dose irradiation (LDI) (50) or by local hyperthermia (LHT) (51) should improve tumor targeting of virus, T-cells, and DCs (52). Tumor destruction by the activated T-cells should release TAs, which would be taken up by co-injected DC1 to be then cross-presented to naïve or memory T-cells.

#### **HITCHHIKING OF NDV ON ACTIVATED T-CELLS: COMBINING CELL THERAPY WITH VIRUS THERAPY**

One way of further enhancement of the efficacy of this multimodal therapy concept consists in the loading of the activated T-cells with oncolytic NDV before grafting the cells back to the patient. In a tumor neutralization assay *in vitro*, monolayers of human tumor cells could be completely and effectively destroyed by the addition of polyclonally activated human T-cells loaded with oncolytic NDV (53). In this process, synergistic effects between cytotoxic T-cells and oncolytic virus in the tumor contact zone were apparent (53).

If activated T-cells are not available, a multimodal therapy could also consist of the combination of LHT, systemic application of oncolytic NDV and of DC1. Such approach resulted in long-term remission of metastatic prostate cancer (52).

#### **TARGETING AN INTRODUCED VIRAL ANTIGEN IN TUMOR TISSUE BY GRAFTED T-CELLS AND DCs VIA CELL-BOUND TRI-SPECIFIC ANTIBODIES**

**Table 1** summarizes five steps that are essential for a new adoptive cellular cancer therapy strategy. Oncolytic NDV can be introduced into tumor tissue of the patient by various means as discussed before. The patients T-cells and DCs would be activated and polarized also as discussed before. The tsAbs have three different binding sites, each of which is only monovalent. To increase the avidity and stability of the cell surface attached ts fusion protein, we propose that two of the binding sites should bind to well-defined targets on T-cells or DCs. The addendum of the table lists some of the potential targets.

This approach is only meant as a perspective for the future and has not been tested experimentally or clinically. There should be a proper timing between virus-pretargeting of tumor tissue (including metastases) and the cell therapy. We envisage that 24– 48 h after virus inoculation should be a good time period for grafting the cells for a VA targeted therapy. Excessive virus should

#### **Table 1 | Adoptive cellular cancer therapy: targeting a viral antigen (e.g., HN) by grafted T-cells and DCs via cell-bound tri-specific antibodies**.

**Addendum:**The tri-specific single-chain antibodies should bind with two arms to targets on T cells or targets on DCs and expose the third arm anti-HN

be cleared by then and the tumor tissue should be infected and expressing cell-bound VAs.

#### **CONCLUDING REMARKS**

We propose a multimodal approach for effective cancer therapy because previous monomodal approaches of chemo- or radiotherapy faced problems of tumor resistance mechanisms. Specific immunotherapies targeted to specific TA faced similar problems of tumor escape and resistance mechanism. There may be a long way to get a multimodal therapy such as the one proposed and established but we believe it is important to propose a viable perspective for future orientation. Oncolytic viruses, T-cells, dendritic cells, and bi-specific antibodies are all promising biologics whose intelligent combination holds a lot of promiseforfuture cancer therapy.

#### **REFERENCES**

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 10 June 2014; paper pending published: 03 July 2014; accepted: 05 August 2014; published online: 11 September 2014.*

*Citation: Schirrmacher V and Fournier P (2014) Multimodal cancer therapy involving oncolytic Newcastle disease virus, autologous immune cells, and bi-specific antibodies. Front. Oncol. 4:224. doi: 10.3389/fonc.2014.00224*

*This article was submitted to Tumor Immunity, a section of the journal Frontiers in Oncology.*

*Copyright © 2014 Schirrmacher and Fournier. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Attacking postoperative metastases using perioperative oncolytic viruses and viral vaccines

#### **Lee-Hwa Tai 1 and Rebecca Auer 1,2\***

1 Centre for Innovative Cancer Research, Ottawa Hospital Research Institute, Ottawa, ON, Canada

2 Department of Surgery, University of Ottawa, Ottawa, ON, Canada

#### **Edited by:**

Philippe Fournier, DKFZ, Germany

#### **Reviewed by:**

Volker Schirrmacher, DKFZ, Germany Zong Sheng Guo, Harvard University, USA

#### **\*Correspondence:**

Rebecca Auer, Ottawa General Hospital, 1617 CCW, Box#134, 501 Smyth Road, Ottawa, ON K1H8L6, Canada e-mail<EMAIL_ADDRESS>
Surgical resection of solid primary malignancies is a mainstay of therapy for cancer patients. Despite being the most effective treatment for these tumors, cancer surgery has been associated with impaired metastatic clearance due to immunosuppression. In preclinical surgery models and human cancer patients, we and others have demonstrated a profound suppression of both natural killer (NK) andT cell function in the postoperative period and this plays a major role in the enhanced development of metastases following surgery. Oncolytic viruses (OV) were originally designed to selectively infect and replicate in tumors, with the primary objective of directly lysing cancer cells. It is becoming increasingly clear, however, that OV infection results in a profound inflammatory reaction within the tumor, initiating innate and adaptive immune responses against it that is critical for its therapeutic benefit. This anti-tumor immunity appears to be mediated predominantly by NK and cytotoxic T cells. In preclinical models, we found that preoperative OV prevents postoperative NK cell dysfunction and attenuates tumor dissemination. Due to theoretical safety concerns of administering live virus prior to surgery in cancer patients, we characterized safe, attenuated versions of OV, and viral vaccines that could stimulate NK cells and reduce metastases when administered in the perioperative period. In cancer patients, we observed that in vivo infusion with oncolytic vaccinia virus and ex vivo stimulation with viral vaccines promote NK cell activation.These preclinical studies provide a novel and clinically relevant setting for OV therapy. Our challenge is to identify safe and promising OV therapies that will activate NK andT cells in the perioperative period preventing the establishment of micrometastatic disease in cancer patients.

**Keywords: metastasis, postoperative period, oncolytic viruses, viral vaccines, cancer, perioperative immunostimulation, natural killer cells, surgical stress**

#### **SURGICAL STRESS PROMOTES THE FORMATION OF METASTASES**

Surgical resection is the mainstay of therapy for most solid malignancies but, even with complete resection, many patients harbor microscopic residual disease and ultimately die of a recurrence (1). Our group (2, 3) and others have clearly demonstrated, using different animal and tumor models, that surgery promotes the formation of metastatic disease (4–11) and the number of metastatic deposits is directly proportional to the magnitude of surgical stress (6, 12). In clinical studies, a complicated postoperative course correlates with inferior cancer survival and increased incidence of metastases (13, 14). A number of perioperative changes have been proposed to explain the promotion of metastases formation following surgery including (1) dissemination of tumor cells during the surgical procedure (15–20), (2) local and systemic release of growth factors, such as vascular endothelial growth factor (VEGF) (21, 22), and (3) cellular immune suppression. The cellular immune suppression following major surgery appears to peak at 3 days (23) following surgery but may persist for weeks (7, 23–25). It is hypothesized to be mediated by secretion of stress hormones, such as glucocorticoids (26, 27), catecholamines (27– 29), and prostaglandins (26). It is characterized by both plasma cytokine changes [a decrease in IL-2 (30), IL-12 (31) and an increase IL-6 (27,30,32,33),IL-10 (34)] and a decrease in the number and function of circulating lymphocytes [cytotoxic T cells (35), dendritic cells (DC) (36) and natural killer (NK) cells (2, 3, 37)].

The postoperative stress response represents a diverse set of physiological changes that have evolved to ensure that the host can heal following major tissue trauma. These changes, however, involve pathways and mediators that can be exploited by cancer cells to facilitate metastatic spread. While a number of correlative studies have demonstrated an association between some of these changes and the enhanced formation of metastases following surgery, few mechanistic studies have been undertaken to understand it. This review will focus on the importance of both innate and adaptive postoperative cellular immune suppression, specifically NK and cytotoxic T cell postoperative dysfunction and make the case for the use of preoperative oncolytic viruses (OV) and viral vaccines to prevent the promotion of cancer metastases following surgery.

#### **SURGICAL STRESS INHIBITS NK CELL FUNCTION AND ANTIGEN-SPECIFIC CD8**+ **T CELL FUNCTION**

Both the innate and adaptive immune system play a significant role in anti-tumor immunity. As integral members of the innate immune system, NK cells are involved in the direct killing of cells displaying abnormalities linked to infection, malignancy, or transplantation (38, 39). Immunosurveillance of the host by NK cells for malignant cells results in direct cytotoxicity and the production of cytokines to enhance the immune response (39).

Natural killer cell dysfunction following surgery, as measured in a standard [51-Cr]-release assay, has been documented in both human patients (3, 25, 40–42) and animal models (3, 5, 7, 40, 41, 43). Postoperative NK cell suppression correlates with increased metastases in animal models of spontaneous (3, 9) and implanted (3, 10, 11) metastases, while in human studies low NK activity during the perioperative period is associated with a higher rate of cancer recurrence and mortality in a number of different cancer types (44–46). Despite the large number of studies that have documented postoperative NK cell dysfunction, very few studies have thoroughly characterized and directly explored the mechanism of this suppression (9, 11, 47). Our laboratory has clearly defined a role for NK cells in the development of postoperative metastases (2). Using several reproducible mouse models of surgical stress, including B16 melanoma, CT26 colon cancer and 4T1 breast cancer, our laboratory has demonstrated a consistent and significant (two- to fourfold) increase in the formation of experimental and spontaneous pulmonary metastases following surgery. In these experimental models, surgery markedly reduced NK cell total numbers in the spleen and affected NK cell migration. Further, *ex vivo* and *in vivo* tumor cell killing by NK cells were significantly reduced in surgically stressed mice. To establish that NK cells play the crucial mediating role in clearing tumor metastases following surgery, we transferred surgically stressed NK cells into NK-deficient mice (IL-2γR-knock out) and observed enhanced lung metastases in tumor-bearing mice compared to mice who received untreated NK cells (3). Transfer of NK cells labeled with the NK specific marker DX5 from surgically stressed and no surgery control donors into naive recipient mice represents the first *in vivo* evidence that links surgery to the spread of cancers via NK cells (3). In human studies, we have also confirmed that postoperative cancer surgery patients had markedly reduced NK cell cytotoxicity (3).

The adaptive immune system and more specifically CD8+ T cells responses have received the majority of the attention from the cancer immunity field. Of recent interest in our lab is the impact of surgical stress on the development and maintenance of an acquired T cell-mediated anti-tumor immune response. A global reduction in T cell numbers and function post-surgery has been documented in preclinical studies and cancer patients (35). However, the effects of tumor-associated antigen (TAA)-specific T cells have not been evaluated and represent a current focus of research interest in our lab.

#### **POSTOPERATIVE CELLULAR IMMUNE SUPPRESSION IS REVERSIBLE**

Fortunately postoperative immune suppression is reversible, so while the postoperative period provides a window of opportunity for cancer cells to metastasize and grow, it also provides a window of opportunity to intervene**,** by supporting or further stimulating the immune system, and, in doing so, attenuate the development of cancer recurrences (48, 49). Based on promising preclinical results (8, 50, 51), clinical trials of preoperative non-specific immune stimulation with low-dose recombinant IFNα (52) or IL-2 (53– 58) have demonstrated less NK and T cell suppression following surgery. In two randomized studies of patients undergoing resection of colorectal cancer (CRC) primary tumors (58) and hepatic metastases (57), preoperative low-dose subcutaneous (s.c.) IL-2 was associated with an improved prognosis. In the first study, 86 CRC patients with stage II or III disease were randomized to receive low-dose IL-2 twice a day for 3 consecutive days prior to surgery or no preoperative treatment. At a median follow-up of 54 months, there were significantly few recurrences in the IL-2 group (21.4 vs. 43.1%, *p* = 0.03) and a trend toward improved overall survival (OS). In the second study, 50 CRC patients with Stage IV disease, undergoing curative or palliative surgery, were randomized to the same two treatment arms. The median progression-free survival (PFS) and OS were significantly longer in the preoperative IL-2 group. While these studies were not designed to evaluate cancer outcomes, a Phase II trial in 120 patients undergoing resection for renal-cell carcinoma has demonstrated a significant improvement in 5-year PFS with preoperative IL-2 (74 vs. 62%, *p* = 0.02) (54). Moreover, in all of these studies, preoperative IL-2 was safe and well tolerated with adverse events limited to pyrexia (Grade I– III). A few other non-conventional immunomodulators have been evaluated for their ability to boost cellular immunity in the perioperative period including cimetidine (59, 60), mistletoe extract (61, 62), and granulocyte colony-stimulating factor (GMCSF) (63). Despite the paucity, the data are promising and perioperative treatment strategies, aimed at stimulating the cellular immune system warrants further study. As outlined in the remainder of this review, OV are an attractive agent to reverse perioperative immune suppression.

#### **WHY USE PERIOPERATIVE ONCOLYTIC VIRUSES FOR IMMUNE STIMULATION? A MULTIPRONGED APPROACH FOR A MULTIFACTORIAL PROBLEM**

Oncolytic viruses are not considered a "traditional" immunotherapy but their multiple mechanisms of action provide several advantages over traditional cytokine immune stimulants in the complex postoperative period. First, the immune stimulation provided by an OV is a more "physiological" immune stimulus, engaging and maturing DC, which in turn activates NK and T cells. The multitude of cytokines and chemokines, stimulate the appropriate picomolar concentration, by a systemic virus infection would be impossible to replicate even with the most carefully designed cytokine cocktail. Second, the OV will selectively replicate in and kill residual cancer cells, providing a direct cytolytic effect to remaining micrometastases, but also delivering the immune response to the tumor selectively. Finally, there is strong rationale to hypothesize that OV could infect and replicate better in the postoperative state because of the surge of growth factors such as VEGF, providing a therapeutic advantage for OV in postoperative cancer patients.

#### **PRECLINICAL EVIDENCE FOR NK CELL ACTIVATION WITH PERIOPERATIVE ONCOLYTIC VIRUSES**

Viruses, in general, are known to activate NK cells (64, 65) and OV are no exception. One of the first reports to support the anti-tumor activation of NK cells in response to OV therapy was reported by Diaz et al. in which depletion experiments were performed to demonstrate that B16 melanoma tumor regression was achieved in a CD8+ T and NK cell-dependent manner following vesicular stomatitis virus (VSV) intratumoral (i.t.) injection (66). Supporting these findings, oncolytic Reovirus treatment of prostate cancer produced an anti-tumor CD8+ T cell response along with prominent NK cell infiltration (67, 68). Miller et al. also observed that i.t. therapy with oncolytic herpes simplex virus (HSV) for B16 melanoma was abrogated in syngeneic models lacking NK and T cell subsets (69). In mechanistic studies with oncolytic new castle disease virus (NDV), Jarahian et al. demonstrated enhanced NK cytotoxicity against human tumor cell lines infected with NDV. Further, soluble receptor binding and blocking assays suggest that NKp44 and NKp46 recognition of viral ligand hemagglutinin-neuraminidase on NDV infected tumor cells mediated NK anti-tumor activity (70). We have demonstrated that oncolytic ORF virus (ORFV) has a profound effect on NK cells following i.v. delivery and that this NK cell activation is the main mechanism by which ORFV exerts its anti-tumor effect (71). It is very likely that stimulation of NK cells play an important role in the therapeutic effect of many OV, not only by enhancing NK cell-mediated killing of tumor target cells but also by triggering a robust, T cell-mediated, anti-tumor immune response (72).

Given that surgery suppresses NK cell activity and OV activate NK cells, we explored the ability of preoperative OV to prevent postoperative NK cell suppression, and in turn prevent the development of postoperative metastases. Using our established murine model of surgical stress, we demonstrated that perioperative administration of novel oncolytic ORF and vaccinia viruses can reverse NK cell suppression following surgery and this correlates with a reduction in the postoperative formation of metastases (3). Similar effects were observed in 4T1-tumor bearing surgically stressed mice treated with perioperative OV. When NK cells were depleted, the effect was no longer present, suggesting that suppression of tumor metastases in a surgical stress model is mainly mediated through OV activation of NK cells and subsequent NK cell-mediated tumor lysis (3).

We demonstrated a similar effect with the novel oncolytic rhabdovirus, Maraba (MG1) and used this model to explore the mechanism of NK cell activation further. MG1 is a double mutant rhabdovirus with deletion in the G and M proteins (73). It is a clinical candidate OV that is scheduled to begin a Phase I clinical trial in 2014. MG1 infection in immune competent mice resulted in an immediate (24 h) and intense activation of NK cells, as evidenced by significantly increased NK cell cytotoxicity and cytokine secretion. Moreover, preoperative i.v. administration of MG1 overcame surgery-induced NK suppression and attenuated the development of postoperative metastases in the B16lacZ model of implanted lung metastases, as well as in the breast 4T1 model of spontaneous lung metastases (74).

Mechanistically, we demonstrated that MG1 activates NK cells through conventional DC (cDC) (**Figure 1**). Using an *ex vivo* NK:DC co-culture system, we showed lack of NK infection, activation, and cytotoxicity in the absence of cDC. Further, in cDC ablated mice (CD11c-Diphtheria Toxin Receptor Transgenic mice), NK cell cytotoxicity was significantly reduced following MG1 administration (74). While we demonstrated that MG1 does

not directly infect or activate NK cells, this is not the case for other OV. For instance, vaccinia virus has been shown to interact directly with NK cells through Toll-like-receptor-(TLR)-2 (75).

As the interplay between OV and immune cells in the perioperative period is critically important for the eradication of tumors, we further explored these interactions in our preclinical models of tumor and surgical stress. In both B16 melanoma and 4T1 breast tumor models, we observed postoperative expansion of myeloidderived suppressor cells (MDSC) (3), which are known regulatory cells that have been shown to expand following various pathologies to suppress innate and adaptive immunity (76–80). The role of MDSC on surgery-induced dysfunction of NK cells and antigenspecific T cells and its potential interaction with OV is part of ongoing research in our lab (**Figure 1**).

#### **PRECLINICAL EVIDENCE OF TAA-SPECIFIC T CELL ACTIVATION WITH PERIOPERATIVE ONCOLYTIC VACCINE**

Oncolytic vaccines (OVax) are OV that express TAA that can direct the host immune response toward the TAA while simultaneously performing viral oncolysis and creating an inflammatory tumor microenvironment (81, 82). Dr. Brian Lichty has pioneered this prime-boost OVax platform and demonstrated remarkable efficacy in the B16 model (82–86). B16 cells express the TAA, dopachrome tautomerase (DCT), which is a protein involved in melanogenesis and is present in normal melanocytes and melanoma. As previous studies have demonstrated, Ad-DCT is able to prime a DCT specific T cell immune response and protect mice from a B16 tumor challenge or tumor re-growth (87, 88), but has limited efficacy in a therapeutic model of lung metastases (89). Dr. Lichty's group engineered MG1, to express DCT upon productive infection and used these two viruses in a prime-boost strategy in tumor-bearing animals. They found that when Ad-DCT was allowed to prime an immune response, followed 9 days later by an MG1-hDCT boost, the results were remarkable, leading to a significant reduction in lung metastases with durable cures in >20% of mice, something not seen when MG1 expressing an irrelevant transgene (MG1-GFP, green-fluorescent protein) was used. Strikingly, ~27% of CD8+ T cells were directed against DCT. Selective depletion of cytotoxic T lymphocytes (CTL) at the time of the boost abrogates the therapeutic efficacy, underscoring their central role. In the near and longer term, we will focus on using OVax, such as MG1-DCT in preclinical mouse tumor models of surgical stress to perioperative boost adaptive immune functions.

#### **CLINICAL EXPERIENCE WITH PERIOPERATIVE OV IN CANCER SURGERY PATIENTS**

The compelling preclinical and clinical data with oncolytic vaccinia virus, in particular the evidence that it can stimulate a potent anti-tumor immune response (90) led us to hypothesize that perioperative treatment with this OV could improve recurrence-free survival following surgical resection. We designed a single center Phase II clinical trial where patients with metastatic colorectal tumors within the liver were treated with a single i.v. dose of oncolytic vaccinia virus prior to surgical resection (91). This trial explored the mechanisms of action of oncolytic vaccinia virus through a series of correlative blood and tissue studies collected from patients pre- and post-OV treatment and surgery. In this

study, we confirmed that NK cell cytotoxicity improved in the setting of pre-operative oncolytic vaccinia virus compared to baseline control blood (3). Further, we detected genome copies of vaccinia virus in the tumors of patients following resection (unpublished data), which suggests that viral targeting of the tumor by i.v. injection may elicit an immune response in the tumor. These results demonstrated for the first time that oncolytic vaccinia virus markedly increases NK activity in cancer surgery patients.

In the same patient population of CRC, systemic delivery of oncolytic reovirus prior to planned surgical resection of liver metastases was undertaken by researchers in the UK (92) In this "window of opportunity" trial of 10 patients, Adair et al. was able to recover live reovirus from the blood cells, but not from plasma removed from these patients. In addition, reovirus protein was identified preferentially in resected tumor tissue, but not in normal liver tissue. Their results suggest that immune cells in the blood may protect virus from neutralizing antibodies, thus providing targeted delivery of OV to tumors. Importantly, preoperative treatment with oncolytic reovirus was well tolerated, with the most common side effects being flu-like symptoms and no reported grade 3 or 4 toxicities in any patients (92). In a study of perioperative oncolytic HSV delivery, virus was injected intratumorally preand post-surgical resection into patients with recurrent glioblastoma multiforme (93). Evidence of immune cell infiltration and viral replication in the resected tumors was reported by the authors. Notably, no patients developed HSV related encephalitis or required antiviral treatment (93). In a series of clinical trials using NDV-modified autologous tumor cell vaccine (NDV-ATV) for treatment of colorectal, renal cell, and glioblastoma cancer patients, researchers detected a significantly improved survival advantage compared to unvaccinated and historical controls. However, NDV-ATV was mostly administered postoperatively and not preoperatively to prevent surgery-induced immunosuppression, which might further improve upon the survival advantage. Similar to the above studies, NDV-ATV was well tolerated, with the most common side effects being mild-fever/headache and no associated autoimmunity (70, 94–99). These reports demonstrate the feasibility of perioperative OV administration into cancer surgery patients.

#### **THE IMPORTANCE OF TIMING FOR OV ADMINISTRATION IN THE PERIOPERATIVE PERIOD**

While the postoperative period provides a window of opportunity for cancer cells to metastasize and grow, it also provides a window of opportunity to intervene, by strengthening the immune system and reducing recurrence of cancer following surgery in cancer patients. In our preclinical perioperative vaccine studies, we hypothesized that neoadjuvant delivery of vaccine immediately prior to surgery will allow for maximal NK cell stimulation to counteract surgery-induced NK cell suppression (100, 101). Indeed, we observed that influenza vaccine administered on the same day, immediately prior to surgery, reduced metastases most effectively. The results from NK cells isolated from cancer surgery patients also confirm that the timing of influenza administration is critical for its effect. In four out of four cancer surgery patients, NK cells isolated prior to surgical resection demonstrated enhanced cytotoxicity and IFNγ secretion following *ex vivo* pulsing with influenza vaccine, while in only one of these patients was similar activation demonstrated in NK cells isolated 1 day following surgery, suggesting that surgery-induced NK cell dysfunction can be prevented but not reversed by influenza. In humans receiving a flu shot as part of a vaccination campaign, NK cell activation peaked at 1–2 days following immunization (101). Based on this, it appears that a cancer vaccination strategy is probably best delivered the day before cancer surgery, in order to allow sufficient time to maximally activate NK cells prior to surgical stress.

Equally important for a replicating virus is the growth advantage that the postoperative state may provide, increasing oncolysis, viral replication, and spreading. Surgical stress results in a surge of VEGF with resulting angiogenesis to facilitate wound healing (21). Kottke et al. (102, 103) have previously demonstrated that a VEGF surge improved viral replication, viral cell lysis, and an innate immune mediated attack, in particular by NK cells, by allowing tumor-associated endothelial cells to transiently support viral replication during the VEGF surge. The sequential combination of oncolytic vaccinia virus and the small molecule B-raf and VEGF inhibitor, sorafenib, has also demonstrated efficacy in preclinical models and a few patients (104), further supporting the concept that OV and VEGF may act synergistically if the timing of viral administration is considered.

#### **BARRIERS TO PERIOPERATIVE OV THERAPY AND STRATEGIES TO OVERCOME THEM**

While these data are exciting, the perioperative use of OV is in preclinical and early stages of clinical investigation. In the design of our preoperative OV trial, we were confronted with multiple concerns associated with the use of a live virus immediately prior to surgery in cancer patients. In particular, concerns were raised about the potential for an overwhelming postoperative systemic inflammatory response, the risk of spread to members of the operating room team, and risk of meningitis with epidural analgesia. These safety concerns present real barriers to the development of perioperative OV. In their recent publication, Adair et al. demonstrated the feasibility and safety of perioperative live reovirus infusion prior to surgery in CRC patients. However, OV infusion was administered 6–28 days prior to surgery and not immediately before surgery. Further, three patients received fewer than their planned five doses of reovirus. In one patient, this was due to a decline in white blood cell count, while the remaining two patients opted to not receive their last doses of OV prior to surgery because of their own concerns that flu-like symptoms might interfere with the planned surgery, highlighting a strongly held belief that remains a theoretic barrier to immediate preoperative delivery of a replicating virus (92).