Poxvirus Oncolytic Virotherapy

Expert Opinion on Biological Therapy

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Poxvirus oncolytic virotherapy

Lino E. Torres-Domínguez & Grant McFadden

To cite this article: Lino E. Torres-Domínguez & Grant McFadden (2019): Poxvirus oncolytic virotherapy, Expert Opinion on Biological Therapy, DOI: 10.1080/14712598.2019.1600669 To link to this article: https://doi.org/10.1080/14712598.2019.1600669

Accepted author version posted online: 28 Mar 2019. Published online: 04 Apr 2019.

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Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=iebt20 EXPERT OPINION ON BIOLOGICAL THERAPY https://doi.org/10.1080/14712598.2019.1600669

REVIEW Poxvirus oncolytic virotherapy

Lino E. Torres-Domínguez and Grant McFadden Biodesign Center for Immunotherapy, Vaccines and Virotherapy, Arizona State University, Tempe, AZ, USA

ABSTRACT ARTICLE HISTORY Introduction: Over the last decade, advances in biological therapies have resulted in remarkable clinical Received 14 December 2018 responses for the treatment of some previously incurable cancers. Oncolytic virotherapy is one of these Accepted 25 March 2019 promising novel strategies for cancer therapy. A successful oncolytic virus promotes tumor cell onco- KEYWORDS lysis and elicits a robust long-term anti-tumor immunity. Myxoma virus; oncolytic Areas covered: Oncolytic poxviruses (Vaccinia virus and Myxoma virus) demonstrated encouraging virus; oncolytic; Vaccinia results in multiple pre-clinical tumor models and some clinical trials for the treatment of various virus; virotherapy cancers. This review summarizes the advances made on poxvirus oncolytic virotherapy in the last five years. Expert opinion: Many challenges remain in poxvirus oncolytic virotherapy. Two key goals to achieve are enhancing the efficiency of viral delivery to tumor sites and overcoming local tumor immune- evasion. Additional efforts are necessary to explore the best combination of virotherapy with standard available treatments, particularly immunotherapies. By addressing these issues, this new modality will continue to improve as an adjunct biotherapy to treat malignant diseases.

1. Introduction makes them a powerful immunotherapeutic agent to assist the treatment of cancer (Figure 1)[6,7]. The increased understanding of the critical role of the adap- Among the different OVs that have been tested, several tive immune system in cancer development and progression belong to the Poxviridae family. Poxiruses are double- has led to novel interventions using several immunotherapies, stranded DNA viruses possessing a large and well- which could in theory overcome the disadvantages of classical characterized genome (130–300 kb). Poxvirus particles are anti-cancer therapies. Among these biological strategies, exceptionally large; their size is around 250 nm in diameter oncolytic virotherapy has emerged as a novel promising mod- and 360 nm in length, characterized by having a round ality, with encouraging results in both preclinical models brick-shape. There are two different infectious particles: (Table 1) and clinical trials with human patients [1, 2] The intracellular mature virus (IMV) and extracellular enveloped first oncolytic virus (OV) was approved in 2015 by the FDA virus (EEV), they are structurally similar, however EEV carry to treat melanoma (T-Vec or Talimogene laherparepvec), an additional outer lipid membrane containing proteins a genetically modified Herpes virus, and currently many absent from IMV [8]. The precise mechanism of poxvirus other candidates are being tested in clinical trials [3]. In entry is not fully understood, but it has been proposed China, the clinical use of H101 (Oncorine), a recombinant that a macropinocytosis process is involved. While pox- adenovirus, was approved in 2005 for the treatment of squa- viruses are capable of attaching to and entering a wide mous cell carcinoma of head and neck [4]. variety of mammalian cells, post entry factors (such as OVs are capable of selectively replicating and killing trans- induced antiviral signaling pathways) are thought to define formed cells with a malignant phenotype, while sparing normal cellular and host tropism [9]. The poxvirus replication cycle cells and tissues (Figure 1). The anticancer activities of OVs are takes place exclusively in the cytoplasm, which makes them derived from multiple cancer-killing mechanisms: the direct attractive candidates as OVs, since there is no risk for viral infection and oncolysis of tumor cells by the virus, the infection DNA integration into the host genome [8,9]. Poxviruses are of tumor-associated endothelial cells that leads to vascular also highly immunogenic, expressing multiple viral antigens, collapse, and the cytotoxity mediated by the activation and and exhibit a strong capacity to activate acquired antitumor recruitment of immune cells into the tumor microenvironment immunity following replication within cancer tissues [10]. To (TME) [5]. The immunogenic potential of OVs relies on their date, six poxviruses from four different genera have been capability to selectively infect and lyse cancer cells, releasing investigated as potential OV: Vaccinia virus (VV), Racoonpox tumor-associated antigens andtriggeringstronginnateand virus and Cowpox virus (Orthopoxviruses), Myxoma virus adaptive immune responses against cancer-specific epitopes. (MYXV) (Leporipoxvirus), Yaba monkey tumor virus Therefore, their ability to elicit robust anti-tumor immunity, (Yatapoxvirus), and Squirrelpox virus (Unassigned)[11]. This particularly against neoantigens and cancer-specific markers,

CONTACT Grant McFadden [email protected] Biodesign Center for Immunotherapy, Vaccines and Virotherapy, Arizona State University, Tempe, AZ 85281, USA © 2019 Informa UK Limited, trading as Taylor & Francis Group 2 L. E. TORRES-DOMÍNGUEZ AND G. MCFADDEN

in 1977. Consequently, this virus has a well-established safety Article highlights record in humans based on its extensive use [12]. While VV

● Multiple preclinical studies and clinical trials have validated the safety naturally displays preferential growth kinetics in cancer cells, it and efficacy of Poxviruses (VV and MYXV) as platforms for oncolytic can productively infect a wide variety of animals and cell virotherapy. types, including non-dividing cells [10]. Hence, only geneti- ● The optimization of OV-delivery to the tumor site is critical to improve the success rate of virotherapy in clinical trials. cally modified VV constructs selectively target tumor cells. The ● The use of Poxviruses and other OVs to enhance the anti-tumor extensive success of VV as an OV in multiple preclinical models immune response is key for their success as oncotherapy. (Table 1) has led to the translation to clinic (Table 2)[10,13]. ● Engineering poxviruses with multiple therapeutic transgenes offers innumerable possibilities to design vectors with improved properties Currently, there are three oncolytic VV variants being tested in (immune stimulators, imaging, etc.). clinical trials, with promising outcomes for the treatment of ● The most successful virotherapeutic approaches are those that act several malignant diseases [10,11]. synergistically when combined with other anti-cancer therapies, particularly with immunotherapies. MYXV has a natural narrow host range within the lago- morphs and is only pathogenic for European rabbits This box summarizes key points contained in the article. (Oryctolagus cuniculus). This restrictive tropism in nature makes it a very safe agent for oncotherapy [9]. Although MYXV is nonpathogenic to humans and mice, the virus exhibits natural tropism for a wide spectrum of human cancers Table 1. Summary of Poxvirus virotherapy in preclinical animal models. [14]. The selective replication in human tumor cells is based on Poxvirus Strain Type of cancer Ref. multiple dysregulated intracellular pathways found in trans- VV VVtk Liver cancer 84 formed cells. These antiviral pathways, on the other hand, VVtk Multiple myeloma 85 effectively abort MYXV replication in most normal primary VG9 Melanoma 25 somatic human and mouse cells. Oncolytic MYXVs have been VG9-GMCSF Melanoma 86 VVGMCSF Mammary carcinoma 87 tested in a wide variety of animal tumor models, proving its VV-GMCSF-Lact Breast cancer 33 effectiveness as virotherapy against many types of can- VV-ING4 Pancreatic cancer 37 cers [14]. GLV-2b372 Hepatocellular carcinoma 88 GLV-1h153 Triple-negative breast cancer 89 The topic of poxvirus oncolytic virotherapy has been CVV Metastatic liver cancer 18 reviewed before [10, 11, 14−16]. Here, we intend to update oncoVV-TTL Hepatocellular carcinoma 36 the progress made in the last five years and to discuss the GLV-1h68 Melanoma 68 Thyroid carcinoma 17 current challenges the field still faces. Cholangiocarcinoma 90 Pancreatic adenocarcinoma 70 Lung adenocarcinoma 91 JX-594 Melanoma 34 2. Virus delivery: systemic vs intratumoral Pancreatic neuroendocrine tumors 84 2.1. Vaccinia virus vvDD-CXCL11 Colon or ovarian cancer 46 vvDD-IL-15 Colon and ovarian cancer 44 vvDD Peritoneal carcinomatosis 27 The successful delivery of virions to the TME is among the key Renal and colorectal cancer. 74 challenges faced by virotherapy in general (Figure 1). Most of Oncopox-Trail Lung cancer 38 the preclinical and clinical trials have exploited a systemic OVV-CXCR4-A-Fc Neuroblastoma 92 Ovarian cancer 50 delivery of the purified virus, mainly by direct intravenous MYXV MYXV Glioma 80 (IV) injection route [10]. This route in theory allows delivery Ovarian cancer 76 to both the primary tumor and any overt or undiagnosed Gallbladder carcinoma 22,79 Lymphoma 93 metastatic sites accessible to the circulatory system simulta- Pancreatic cancer 77 neously. Data from clinical trials have shown that OVs can be Multiple myeloma 66 delivered systemically safely and with limited toxicity, however MYXV-135KO-GFP Multiple myeloma 20 MYXV-IL15 Melanoma 94 the efficacy has not always met expectations [15]. A possible vMyx -IL15R Melanoma 54 reason is that the injected free virions can be cleared very vMyx- sPD1 Melanoma 55 quickly from circulation through neutralizing antibodies, com- vMyx-M011L-KO Glioblastoma 40 vMYX- CRISPR/Cas9 Embryonal rhabdomyosarcoma 95 plement activation, antiviral cytokines, and tissue-resident vMYX-FRP Embryonal rhabdomyosarcoma 96 macrophages, as well as nonspecific filtering uptake by other vMYX-Δserp2 Canine tissue sarcoma 97 tissues such as the lung, liver and spleen. Thus, this limits the GMCSF Granulocyte-macrophage colony-stimulating factor, MYXV, Myxoma amount of OV that can effectively reach the TME [16]. Another virus; VV, Vaccinia virus, route extensively used in preclinical models and clinical trials with VV is the intratumoural (IT) delivery route, which has proved to be well tolerated and safe in humans [10,15]. review focuses on the two candidates at the furthest state Local delivery means less systemic toxicity while focusing the of development: VV and MYXV. immune response on the TME and the affected lymph nodes. VV is the prototypic and best-characterized member of the Nevertheless, this IT injection method is limited to easily Orthopoxvirus genus. VV was the live vaccine used in the reachable solid tumors and is more invasive for the patient. vaccination program coordinated by the World Health Other delivery strategies for specific types of cancer have been Organization that led to the eradication of human smallpox intraperitoneal (for peritoneal carcinomatosis) and intrapleural EXPERT OPINION ON BIOLOGICAL THERAPY 3

Figure 1. Therapeutic features that mediate Oncolytic poxvirus potency. There are different aspects that influences the efficacy of poxvirus oncolytic virotherapy. First, the OV must be delivered efficiently to the tumor microenvironment. Second, it must exhibit a strict tropism for cancerous cells, being safe and non-pathogenic for the patient. Third, the OV replicates and spread efficiently in tumor cells, promoting a cancer-specific oncolysis. Fourth, it must act as an immunotherapeutic agent, eliciting a strong innate and adaptive anti-tumor immune response. Fifth, it needs to exhibit synergistic effects in combination regimens with standard treatments and novel immunotherapies, improving the final therapeutic outcome. DAMPs, damage-associated molecular patterns; NK, natural killer; CARs, chimeric antigen receptors.

Table 2. Summary of Vaccinia virotherapy in clinical trials. Poxvirus Strain Type of cancer Phase Status Sponsor Ref. VV Wyeth Pexa-Vec Hepatocellular Carcinoma Phase III Recruiting Sillajen 15 (JX-594) Hepatocellular carcinoma Phase I/II Completed Sillajen 60 Colorectal Cancer, Phase I/II Recruiting Sillajen 15 Colorectal Carcinoma Colorectal Adenocarcinoma Solid Tumors Phase I/II Recruiting Institut Bergonie 15 Soft-tissue Sarcoma Breast Cancer Renal Cell Carcinoma Phase Ib Recruiting Sillajen 15 Melanoma Phase I Recruiting Sillajen 15 Metastatic Tumor Phase I Recruiting Centre Leon Berard 15 Advanced Tumor Colorectal cancer Phase I Completed Sillajen 58 Solid tumors in pediatric patients Phase I Completed Sillajen 57 (Neuroblastoma, Rhabdomyosarcoma, Lymphoma) Melanoma, Lung Cancer Phase I Completed Sillajen 15 Renal Cell Carcinoma Squamous Cell Carcinoma of the Head and Neck VV Lister GL-ONC1 Ovarian Cancer Phase I/II Recruiting Genelux 15 Peritoneal Carcinomatosis Fallopian Tube Cancer Lung Cancer Phase I Active, no Recruiting Genelux 15 Advanced head and neck carcinoma Phase I Completed Genelux 62 Peritoneal carcinomatosis Phase I Completed Genelux 61 Advanced Cancers (Solid Tumors) Phase I Completed Genelux 15 VV WR vvDD-CDSR Solid tumors Phase I Completed University Pittsburgh 26,63 MYXV, Myxoma virus; VV, Vaccinia virus

(for lung cancer). Both strategies were safe for these malignant infectious viral particles, facilitates sustained viral delivery diseases [15]. and transgene expression, and improves tumor control. Such To address these delivery limitations in preclinical and optimization of methods of OVs delivery may enhance ther- clinical trial, original methods are being explored to increase apeutic outcomes [17]. Badrinath et al. used VV coated with the efficacy of VV delivery to the tumor bed. A study reported poly lactic-co-glycolic acid nanofiber for tumor delivery in that the use of silk-elastin-like protein polymer matrix for a colon carcinoma mouse model and confirmed the therapeu- intraoperative VV delivery retards the degradation of tic efficacy of this approach [18]. Although in the past some 4 L. E. TORRES-DOMÍNGUEZ AND G. MCFADDEN preclinical studies tested the possibility of exploiting carrier poxvirus antibodies, increasing virus spread and is highly cyto- cells for viral delivery of VV vectors, none of these studies have toxic to cancer cells [27]. been pursued or translated to the clinic [19]. Recently, it has been proposed that the deletion of two virus-encoded decapping enzymes (D9 and D10) could generate an effective oncolytic VV. D9 and D10 remove protective 2.2. Myxoma virus cap structures from mRNA 5‘-termini, accelerates mRNA decay The intracranial injection of MYXV in either immunocompetent and limits activation of host cell antiviral defenses. Therefore, or nude mice was nontoxic, demonstrating the safety of this these VV mutants are highly attenuated, fail to counter cellular vector even in immunocompromised animals [10,14]. On the double-stranded RNA-responsive innate immune effectors, other hand, Lilly et al. reported that unfractionated leukocyte and thus hyperactivate the host anti-viral enzyme PKR in non- populations from bone marrow (BM) transplant samples, and tumorigenic primary cells compared to wild-type virus. In particularly shown for purified T cells and neutrophils, could addition, they displayed anti-tumor activity against syngeneic act as carrier cells for MYXV to cancer cells located in the bone mouse tumors of different genetic backgrounds [28]. marrow or spleen. Thus, ex vivo MYXV-treated allogeneic BM transplantation efficiently ablated pre-seeded residual multi- 3.2. Myxoma virus ple myeloma (MM) in an immunocompetent murine model [20]. This novel leukocyte delivery approach has the potential MYXV has a strict natural tropism for rabbits and hares and is to circumvent the typical problems associated with the sys- totally non-pathogenic for humans and other mammals; how- temic delivery of OVs [20]. Human T cells can also be armed ex ever, the virus is able to productively infect a wide variety of vivo with MYXV and then can transfer MYXV to MM cells via human and murine cancer types [14]. In an attempt to further cell-cell contact in vitro [21]. A similar strategy using BM- attenuate MYXV to make it safe for all potential vertebrate derived stem cells (BMSCs) as MYXV carriers was explored in hosts, several immunomodulatory proteins have been deleted a gallbladder carcinoma murine model [22]. Significantly, IV from the viral genome. Some of the resultant MYXV mutants injection of MYXV-infected BMSCs dramatically improves the were host-range restricted even in rabbits, but still retained oncolytic effect of MYXV alone, almost at the same level as the capability to infect and kill cancer cells in vitro and in vivo. intratumoral (IT) injection [22]. In addition, administration of In particular, the recombinant M135R-knockout MYXV was adipose-derived mesenchymal stem cells pre-loaded with more attenuated than wild-type MYXV in rabbits [29]. For MYXV led to long-term survival of mice bearing gliomas, indi- example, the M135-knockout MYXV is fully oncolytic against cating that those cells may also be suitable for use as MYXV- human cancer cells yet completely nonpathogenic for all delivery vehicles to tumor sites [23]. known vertebrate hosts [14]. The selective replication of MYXV in human tumor cells is based on multiple dysregulated intracellular pathways com- 3. Oncotropism: preferential infection of cancer cells monly found in transformed cancer cells, for example the failure 3.1. Vaccinia virus to induce type I interferons (IFN) and/or tumor necrosis factor (TNF) antiviral responses or upregulation of the cellular serine/ To target specifically tumor cells without infecting healthy tis- threonine kinase (Akt), all of which support MYXV permissiveness sues is a critical aspect for the development of all OVs (Figure [10]. Indeed, the screening of human cancers for the downregu- 1). Several VVs possess inherent affinity for cancer cells, and lation of IFN/TNF response pathways or the upregulation of additional genetic modifications have been introduced to cellular Akt activation might prove to be useful biomarkers for further promote cancer cell specificity. The mutation of thymi- future MYXV virotherapy in patients. Recently, several eukaryotic dine kinase gene (TK) from the VV genome is the most common dead-box RNA helicases that regulate cellular tropism of MYXV in mutation for achieving more selective tumor cell restriction human cancer cells were identified. The modulation of these self- [24]. TK is involved in nucleotide biosynthesis pathways and defense host helicases in future studies may improve the capa- the deletion of this gene limits viral replication to cells with city of MYXV to infect an even broader spectrum of tumor high levels of TK activity and high intracellular nucleotide pools, cells [30]. typically seen in most cancer cells. In fact, the three VV candi- F11L is a VV gene product that promotes virus exit and rapid dates currently in clinical trials were all engineered by deletion spread by inhibiting Rho signaling. When the VV F11L gene was of the viral TK gene, which engenders more selective infection inserted in a recombinant MYXV, it considerably improved the of cancer cells [19]. The same strategy is being exploited for cell-cell spread of the virus in natural host cells and replicated other OVs in preclinical studies [25]. Other VV genes related to to higher levels in various human cancer cells [31]. nucleotide metabolism might be candidates to further improve viral restriction to cancerous cells [19]. Vaccinia virus WR ‘double-deleted’ virus (vvDD) combines a TK deletion with 4. Oncolysis: killing of poxvirus-infected cancer cells a mutation in the VV growth factor (VGF) gene: this vector 4.1. Vaccinia virus showed little ability to replicate outside of tumors and exhibits increased cancer cell selectivity compared to TK− mutants Although most cancer cells have acquired the ability to evade in vitro [26]. Genetically engineered vvDD with a mutated apoptosis and other programmed cell death pathways, OVs A34R gene produces more total progeny virus and EEV, and can often induce in virus-infected cancer cells an apoptosis- can thus more effectively evade neutralization from anti- like death or other types of virus-mediated cell death such as: EXPERT OPINION ON BIOLOGICAL THERAPY 5 necrosis, senescence and/or autophagy [5]. Traditionally, one be independent of extrinsic death ligands and instead corre- of the main aims in the oncolytic virotherapy field is to lates with depletion of cellular inhibitors of apoptosis. improve and optimize the cancer cell killing capacity of the Consequently, MYXV might eliminate human MM cells virus. In recent years, however, the tendency is to focus not through a pathway unique to oncolytic poxviruses [39]. A pro- only on increasing cytotoxicity but also on the release of apoptotic MYXV was constructed, by deletion of M011L, an cellular tumor-associated antigens, neoantigens and further- anti-apoptotic viral protein. vMyx-M011L-KO induced apopto- more the induction of immunogenic cell death (ICD) to pro- sis in patient-derived brain tumor initiating cells and in vivo it mote acquired antitumor immune responses (Figure1). The significantly prolonged survival in an immunocompetent precise type of virus-induced cancer cell death is thought to glioma mouse model, making this proapoptotic OV a strong be critical in order to prime a robust antitumor immunity [5]. candidate for clinical translation [40]. VV vectors have been armed with apoptosis-inducing proteins like apoptin (VV-GMCSF-Apo) or lactaptin (VV-GMCSF- Lact). Both of these recombinant VV vectors activated a set 5. Immune-stimulation: triggering anti-tumor of critical cellular apoptosis markers in virus-infected cells, immune responses proving that the apoptosis-inducing proteins expressed from 5.1. Vaccinia virus the VV genome can serve as an efficient tool for altering the death pathway of infected tumor cells. Nevertheless, these VV- Historically, the concept of oncolytic virotherapy implied expressing apoptotic proteins were not better at inducing a virus-mediated lysis of the tumor cells, but as multiple evi- classic ICD markers in infected cancer cells than the parental dences revealed the significant immunotherapeutic potential of virus [32,33]. On the other hand, VV JX-594-GMSCF and OVs, the field gradually shifted to a more virus-enhanced immu- TG6002 (genetically modified to permit transforming 5-fluor- notherapeutic perspective [6]. Hence, harnessing the immuno- ocytosine (5-FC) into 5-fluorouracil (5-FU)) showed promising genic capacity of OVs has broadly become a priority. Indeed, results to partially induce ICD markers and also to stimulate those OVs that exploit their immunotherapeutic abilities are the dendritic cells (DCs) as a central part of the adaptive immune most effective in promoting long-term tumor regression system in a human melanoma model [34]. These results sup- [5,6,41]. In this direction, it was determined that a WR-Δ4 port the need to search for better alternatives to increase the virus, with the combined deletion of four specific viral genes capacity of all OVs to induce ICD in target cancer cells, in order that act on metabolic, proliferation, and immune signaling to optimally promote a robust anti-tumoral immune response. pathways (A48R, B18R, C11R, and J2R), exhibits effective anti- By using a directed evolution selection process, by pooling tumor capabilities in vivo [42]. different strains of VV, including Copenhagen, Western Most DNA-genome OVs, and some RNA-genomeOVs, have Reserve (WR) and Wyeth strains and the attenuated modified been genetically engineered, by inserting a myriad of trans- vaccinia virus Ankara (MVA), a new recombinant poxvirus was genes, to further improve their immunogenic and/or cancer generated with increased oncolytic properties. Through selec- cell killing properties (Figure 2)[6,13,41]. Accordingly, OVs tive pressure in cancer cells, the chimeric VV deVV5 possessed have been armed with a wide variety of immunostimulatory increased cancer cell killing capacity and tumor selectivity genes, including cytokines, chemokines, co-stimulatory pro- in vitro[35]. In another report, a VV (oncoVV) was engineered teins, or receptor-inhibitors. To date, the most successful to express a gene encoding a Lectin protein which showed example is the insertion of granulocyte-macrophage colony- significant antitumor activity in a hepatocellular carcinoma stimulating factor (GM-CSF), a molecule expressed by T-Vec mouse model. Lectins play diverse roles in physiological pro- (the only OV approved by FDA) [3] and also Pexa-vec, the most cesses as biological pattern recognition molecules. Moreover, advanced VV strain in human clinical trials [24]. GM-CSF pro- Lectin enhanced oncoVV replication by suppressing expres- motes recruitment and maturation of many immune cell types sion of antiviral factors such as mitochondrial antiviral signal- such as: DCs, macrophages and T cells [24,43]. A vvDD strain ing protein (MAVS) and interferon-beta (IFN-β)[36]. expressing a superagonist IL-15 protein elicited a potent adap- The construction of a novel oncolytic VV harboring the inhi- tive antitumor immunity, which led to significant tumor bitor of growth family member 4 gene (VV-ING4) displayed regression and extended survival in murine models of colon greater cytotoxic efficiency, and induced cell apoptosis in pan- and ovarian cancer [44]. The construction of a membrane- creatic cancer cell lines, in vitro and in vivo [37]. In addition, Hu anchored IL-2 expressed from VV maintained this cytokine et al. constructed a VV recombinant that expresses the human within the TME and reduced the systemic toxicity associated membrane-binding TNF-related apoptosis-inducing ligand with the soluble ligand. VV-IL2-anchored effectively modified (TRAIL). TRAIL protein mainly induced apoptosis and inhibited the cancer-immune set point and showed no additional toxic necrosis in the virus-infected cells. In vitro this vector displayed side effects in a variety of murine tumor models [45]. Several a more robust cytotoxic effect in a lung cancer cell line than other cytokines have been inserted into VV and tested in parental virus while in vivo it exhibited enhanced anti-cancer preclinical models, such as: IFN-β and TNF-α but these have effects in two transplanted tumor models of lung cancer [38]. not yet been translated to the clinic, perhaps due to their capacity to trigger direct anti-viral effects [6]. Liu et al. 4.2. Myxoma virus reported that a VV armed with the chemokine CXCL11 (vvDD- CXCL11) enhanced therapeutic efficacy when it was delivered In MM cell lines, MYXV infection initiates apoptosis through via IT injection in a murine AB12 mesothelioma model. activation of the extrinsic initiator caspase-8, which appears to Moreover, vvDD-CXCL11 induced a potent antitumor 6 L. E. TORRES-DOMÍNGUEZ AND G. MCFADDEN

Figure 2. Arming oncolytic Poxviruses with improved properties. The bioengineering of poxviruses constitutes a powerful tool for the development of next generation OVs. Many transgenes have been inserted into oncolytic poxvirus genomes, to further improve their immunogenic and/or cancer cell killing capacity. The local expression of these virus-encoded therapeutic proteins into the TME suggests a low toxicity than their secreted systemic counterparts. Thus, oncolytic poxviruses have been armed with a wide variety of immunostimulatory genes, including cytokines, chemokines or receptor-inhibitors. BiTEs (bi- specific T cell engagers), CTLA4 (cytotoxic T-lymphocyte-associated protein 4), reporters such as GFP (green fluorescent protein) or NIS (sodium-iodide transporter), GM-CSF (granulocyte- macrophage colony-stimulating factor), various other ILs (interleukins), PD1 (programmed cell death protein 1), TRAIL (TNF-related apoptosis-inducing ligand), and TNF (tumor necrosis factor). immunity, due to the migration of higher numbers of tumor- improved overall survival compared with oncolysis alone specific T cells and reduction of suppressive chemokines in the [49]. Subsequent studies also demonstrated that VV-CD44 TME [46]. Other chemokines that proved to be effective as inhibited metastatic growth of murine and human ovarian anti-tumoral transgenes in VV are CCL5 and CCL19 [19]. tumor variants that were resistant to paclitaxel and carbopla- A very innovative approach has been the insertion of innate tin. The tumors described were more prone to ICD and shown immune adaptor genes into the VV genome, such as: TIR-domain- to have reduced immunosuppression in the TME [50]. containing adapter-inducing IFNs (TRIF) and DNA-dependent acti- In addition, Yu et al. constructed an OV expressing a Bi-specific vator of IFN-regulatory factors (DAI). The authors attempted to T cell engager (BiTe), which are a fusion of two modified antibodies activate toll-like receptors (TLR3)-signaling pathways, which is designed to transiently engage cytotoxic T-cells for contact- associated with boosting a cytotoxic T lymphocyte (CTL) response. mediated lysis of selected target cells. VV encoding a BiTe against The results demonstrated that both transgenes significantly the tumor cell surface antigen EphA2 induced a strong T cell enhanced the immunotherapeutic activity of VV [47]. In activation that resulted in potent antitumor activity in a lung a subsequent study, the treatment with DAI-armed VV resulted in cancer murine xenograft model [51]. The link of OVs with immu- significant reduction in the size of syngeneic melanoma tumors in notherapieswasshownalsoonaVVarmedwithanAbblocking mice, and the engineered virus triggered a long-lasting adaptive the murine programmed cell death protein 1 (mPD1), where the IT immunity against that cancer [48]. injection of VV-antiPD1 on a MCA 205 tumor murine model Arming OVs to counteract tumor-mediated immune eva- induced a massive infiltration of immune cells including activated sion has been also explored, by inserting genes that block lymphocytes, supporting the synergistic effect of this combination suppressive cytokines (eg TGF-β, IL-10) and regulatory cells [52]. However, the strain of VV-GLV-1h376 encoding for a secreted (Tregs, myeloid-derived suppressor cells, MDSCs) in the human Cytotoxic T-lymphocyte-associated protein 4-blocking sin- tumor bed [6,19]. For instance, signals mediated by the che- gle-chain antibody (CTLA4 scAb) failed in increasing cytotoxic mokine CXCL12 and its receptor CXCR4 are involved in the T cells at the tumor site, proving the complexity of the interaction progression of ovarian cancer through enhancement of an between OVs, the immune system and the TME [53]. immunosuppressive state and tumor angiogenesis within the TME. Consequently, a VV that was armed with hyaluronan 5.2. Myxoma virus receptor CD44, a CXCR4 antagonist, was tested in a murine epithelial ovarian cancer model. Intraperitoneal delivery of VV- The co-expression of IL15 fused with the α subunit of IL15 CD44 led to reduced metastatic spread of tumors and receptor (IL15Rα) greatly enhances IL15 stability and EXPERT OPINION ON BIOLOGICAL THERAPY 7 bioavailability. Hence, a recombinant MYXV (vMyx-IL15Rα- than at low dose (14.1 vs 6.7 months; p = 0.08) in a Phase II study tdTr) was engineered to expresses an IL15Rα-IL15 fusion [59]. However, in a Phase IIb clinical trial with HCC patients who protein plus tdTomato red fluorescent reporter. In vivo failed sorafenib therapy, the virotherapy did not achieve the experiments with immunocompetent C57BL/6 mice primary endpoint of prolonging overall survival in treated showed that subcutaneous B16-F10 tumors treated with patients when compared to patients treated with standards vMyx-IL15Rα-tdTr exhibited delayed tumor growth and therapies [43,60]. These observations support the current view a significant survival benefit as compared with parental that drug pre-treated (ie standard therapy failures) and therapy- virus, and remarkably stimulates both innate and adaptive induced immunocompromised patients respond less well to antitumor immune responses [54]. treatments with OVs in general, re-enforcing the concept that Bartee et al. inhibited the PD1/PDL1 pathway within the OV responses lead to long term tumor regression more fre- TME by designing a MYXV contruct expressing a soluble quently when the host immune system is as intact as possible. ectodomain form of PD1 (sPD1) from virus-infected cells. Additional Phase II studies evaluating single-agent Pexa- This sPD1-expressing virus induced and maintained antitu- Vec treatment administered by multiple IV infusions are cur- mor cytotoxic T-cell responses within directly treated tumors rently undergoing in treatment-refractory patients with renal and proved safer and more effective than combination ther- cell carcinoma and colorectal cancer [15]. There are also sev- apy using unarmed MYXV in combination with more tradi- eral Phase I/II trials currently ongoing on distinct types of solid tional systemic αPD1 antibody. This study demonstrated that cancers for evaluating the efficacy of Pexa-Vec in combination tumor-localized inhibition of the PD1/PDL1 pathway could with checkpoint inhibitors. Moreover, a randomized Phase III significantly improve outcomes of oncolytic virotherapy with study is undergoing, to compare treatment of Pexa-Vec fol- poxvirus vectors [55]. lowed by sorafenib vs treatment with sorafenib alone in patients with advanced hepatocellular carcinoma [15]. A second VV candidate in clinical trials is GL-ONC1, based on 6. Poxvirus oncolytic virotherapy: preclinical models vaccinia strain Lister. This OV was generated by insertion of three and human clinical trials expression cassettes (encoding Renilla luciferase-Aequorea 6.1. Vaccinia virus green fluorescent protein fusion, beta-galactosidase, and beta- glucuronidase) replacing the genes F14.5L (virulence factor), J2R The armed oncolytic vector VV Pexa-Vec (pexastimogene (encoding TK) and A56R (encoding hemagglutinin) in the viral devacirepvec, JX-594) expressing GM-CSF, licensed originally genome [19]. GL-ONC1 has been evaluated in four completed by Jennerex Inc. and now developed by SillaJen, is the most Phase I/Ib human clinical trials with 89 cancer patients (Table 2) clinically advanced VV with the potential to become the first [15]. GL-ONC1 was tested against a wide range of cancer types, OV of the Poxviridae family approved by regulatory agencies with various methods of administration (i.e. IV or regionally), for treating human cancers [43]. Pexa-Vec is derived from the either as a single agent therapy or in combination with conven- VV vaccine strain VV-Wyeth but with disruption of the viral TK tional therapy. In all of these clinical trials, GL-ONC1 was well- gene and expression of the human GM-CSF and β- tolerated, without showing significant adverse side effects galactosidase transgenes under control of the synthetic [61,62]. This OV achieved the expected goal of reaching, infecting early/late and p7.5 promoters, respectively. The mechanisms and selectively killing tumor cells as well as triggering anti- of action for Pexa-Vec, which has been shown in preclinical tumoral immune responses. Furthermore, these Phase I studies models and patients, includes selective tumor cell infection indicate encouraging evidence of efficacy and clinical benefit on and lysis, antitumor immune response induction and tumor the treated patients [61,62]. vascular disruption [24,43]. Pexa-Vec has been evaluated in 17 The last VV-based vector to undergo clinical trials is vvDD, completed and ongoing clinical trials to date (Table 2). Over derived from vaccinia strain Western Reserve, the more virulent 300 patients have been treated by IV infusion and/or IT injec- parent strain of VV in animal models, but which has been tion [15,43]. In general, Pexa-Vec treatment was well-tolerated engineered for less pathogenicity and improved tumor selectiv- up to a dose of 3 × 107 pfu/kg per patient, with transient flu- ity through two targeted gene deletions (TK deletion and like symptoms (including fever and chills) being the most amutationtheVGFgene)[63]. Two Phase I clinical trials have commonly reported adverse event. Pexa-Vec–related skin pus- been performed: an IT dose escalation clinical trial of vvDD in 16 tules were observed in a minority of patients following IV patients with advanced solid tumors and a Phase I study of IV infusion [56]. Therefore, immunocompromised individuals delivery on patients with advanced colorectal or other solid and those with inflammatory skin conditions were excluded cancers [15]. In both trials the treatment with vvDD was well- from subsequent enrollment on trials, since these individuals tolerated in all patients and resulted in selective infection of would have a higher risk of complication from VV vaccina- injected and noninjected tumors and antitumor activity [26,63]. tion [43]. Notably, Pexa-Vec was associated with dose-dependent deliv- 6.2. Myxoma virus ery to multiple solid tumor types (including colorectal cancer, lung cancer, pancreatic cancer, and mesothelioma) and resulted Oncolytic MYXV has been tested in a wide variety of animal in antitumor activity at high doses [57,58]. Some patients models for cancers including: gallbladder cancer, melanoma, responded to treatment based on modified response evaluation gliomas, medulloblastomas, acute myeloid leukemia, pancrea- criteria in solid tumors. JX-594 improved median survival in tic cancer and hematological malignancies (Table 1)[10,14]. patients with local/metastatic HCC more strongly at high dose Overall, these preclinical studies have demonstrated the 8 L. E. TORRES-DOMÍNGUEZ AND G. MCFADDEN effectiveness of MYXV as an oncolytic therapeutic agent Chemotherapy is another classical therapy that has been exten- against a variety of cancers for which there are no current sively combined with oncolytic VV in preclinical models [10]. For therapies. Notably, MYXV has the unique ability of infecting example, the combination of VV oncolytic candidates with che- and deleting contaminating human myeloma or leukemia cells motherapeutic agents such as gemcitabine, paclitaxel, cisplatin, from autologous bone marrow transplant samples ex vivo cyclophosphamide and sorafenib, significantly improved the while at the same time sparing the normal CD34+ hemato- tumor regression in different animal models [10,13,69,70]. poietic stem cells needed for immune reconstitution after Currently, there are several clinical trials ongoing that combine immunoablative therapy. In fact, MYXV functionally different VV candidates with chemotherapy [15]. A phase III clinical eliminated MM-CD138+ cells from patient BM samples within trialisinprogressusingthecombination of Pexa-Vec followed by 24 h of treatment ex vivo [64]. Thus, MYXV is currently being sorafenib vs treatment with sorafenib alone in patients with explored as an oncolytic therapy for clinical trials with patients advanced hepatocellular carcinoma [15]. Also, a Phase I Trial of GL- receiving autologous BM transplants [14]. When investigated ONC1 proved its safety when applied simultaneously with cisplatin in a xenograft murine model of pre-implanted human multiple and radiotherapy in patients with advanced head and neck carci- myeloma, the pre-infection of a human BM transplant sample noma [62]. with MYXV for just 1 h prior to transplantation greatly reduced Immune checkpoint blockade has become one of the most the posttransplant mortality in immunodeficient NSG mice successful of the novel immunotherapies and has demon- [65]. The control mice were subsequently shown to have strated tremendous promise for the treatment of diverse died from acute graft-versus-host disease (GVHD), caused by types of cancer. Immune checkpoint inhibitors (ICIs), such as donor T cells resident in the human BM transplant, whereas anti–programmed cell death 1 (PD-1) and anti–programmed the ex vivo pretreatment of donor BM with MYXV prevented cell death ligand 1 (PD-L1) antibodies, are now widely used in the disease. These observations were further extended by multiple anti-tumor interventions [71]. Several preclinical stu- testing ex vivo MYXV virotherapy against residual dies have investigated the combination of oncolytic VV and murine MM in immunocompetent mice using an allogeneic ICIs. Rojas et al. tested VV-WR in combination with anti-CTLA4 mouse-mouse transplant model [20]. Importantly, despite the antibodies in mouse models. The authors concluded that the resistance to direct MYXV infection and oncolysis of this interaction between ICIs and VV is complex, with correct particular MM cell line in vitro, the ex vivo MYXV-armed allo- selection of viral strain, antibody and timing of the combina- geneic BM transplantation dramatically ablated pre-seeded tion being critical for synergistic effects [72]. Furthermore, residual MM in the BM and spleen in vivo [20]. Comparably, some combinations produced antagonistic effects and loss of systemic delivery of MYXV in a syngeneic mouse model of MM therapeutic activity[72]. Another report showed that a PD-L1– significantly reduced tumor burden and prolonged survival inducing VV combined with PD-L1 blockade was able to time, while leaving the healthy BM niche intact [66]. Despite reduce tumor burden and induce long-term survival in anti– numerous preclinical studies supporting MYXV as an excep- PD-L1 resistant murine cancer models. This approach demon- tionally safe and effective OV, it has not yet been translated to strates how the two therapies can synergize in order to sur- humans. Before being tested in clinical trials, some issues need pass the clinical limitations of each alone [73]. Similar results to be addressed such as: good manufacturing practice (GMP) were obtained by Liu et al., who observed that VV attracted production of the virus, pharmacology/toxicology of the con- effector T cells and induced PD-L1 expression on both cancer struct chosen for clinical development, FDA approval for the and immune cells in the tumor, leading to more susceptible use of MYXV in humans, and potentially additional approval of targets for anti-PD-L1 immunotherapy in colon and ovarian leukocyte-mediated delivery of the virus. murine cancer models [74]. The positive combinations of OV and ICIs have been successful in murine models that used armed-VV expressing immunostimulatory transgenes like IL- 7. Combination therapies: who does what with 15 [44] and Il-2 [45], by further eliciting a potent antitumor whom? immunity. These results indicate that OVs may promote the 7.1. Vaccinia virus activation and migration of effector T cells to the TME, whereas the ICI may liberate the anti-tumoral immune In the oncology research field, the use of combination therapies, response by relieving T cell inhibition. Currently, there are a modality that combines two or more distinct therapeutic agents, several clinical trials ongoing to test the combination of VV is becoming crucial in the design of more effective anti-cancer with ICIs (Table3)[15]. interventions. Particularly with VV, many studies have been con- Combining two OVs is an innovative alternative that ducted preclinically and in clinical trials to determine its efficacy remains to be more extensively investigated. For example, when combined with other therapies (Table 3)[10,11,19]. VV and vesicular stomatitis virus (VSV) synergistically In preclinical models, the combination of VV and radiotherapy enhanced each other, promoting better tumor penetration strongly suppressed tumor growth and prolonged survival, as and prolonged survival in a cancer murine model [75]. compared to the single treatments [67,68]. The authors suggested that the virally-mediated down-regulation of anti-apoptotic pro- 7.2. Myxoma virus teins may increase the sensitivity of cancer cells to the cytotoxic effects of ionizing radiation [67]. VV expressing the human sodium- Multiple interventions have preclinically tested the combina- iodide transporter protein (hNIS) have been found to have anti- tion of MYXV with standard treatments, particularly, che- tumor effect when combined with systemic radiation [13]. motherapeutic agents (Table 3). For instance, pretreatment EXPERT OPINION ON BIOLOGICAL THERAPY 9

Table 3. Summary of Poxvirus virotherapy with combination therapies in clinical and preclinical studies. Poxvirus Strain Type of cancer Combination Status Ref. VV Pexa-vec Colorectal Cancer ICIs (Anti-PD-L1 and Anti-CTLA-4) Clinical, Recruiting 15 (Jx-594) Metastatic Tumor ICIs (Anti-CTLA-4) Clinical, Recruiting 15 Hepatocellular Carcinoma ICIs (Anti-PD1) Clinical, Recruiting 15 Renal cell cancer ICIs (Anti-PD1) Clinical, Recruiting 15 Hepatocellular carcinoma Sorafenib Clinical, Recruiting 15 GL-ONC1 Ovarian Cancer Chemotherapeutic agents Clinical, Recruiting 15 (GLV-1h68) Peritoneal Carcinomatosis Advanced Head and Neck Carcinoma Cisplatin Clinical, completed 62 Sarcoma Radiotherapy Preclinical 67 Pancreatic adenocarcinoma Paclitaxel and Gemcitabine Preclinical 70 Lung adenocarcinoma Cyclophosphamide Preclinical 91 Melanoma Radiotherapy Preclinical 68 JX-594 Pancreatic neuroendocrine tumors Sunitinib Preclinical 98 vvDD-CXCL11 Colon or ovarian cancer ICIs (Anti-PD1) Preclinical 46 vvDD Colon and ovarian cancer ICIs (Anti-PD-L1) Preclinical 73 Renal and colorectal cancer. ICIs (Anti-CTL4) Preclinical 72 MYXV vMyx-GFP Metastatic pancreatic carcinoma Gemcitabine Preclinical 77 Gallbladder cancer Rapamycin Preclinical 79 Glioblastoma Rapamycin Preclinical 79 Glioma Cyclophosphamide Preclinical 80 Ovarian cancer Cisplatin Preclinical 76 Melanoma ICIs (Anti-PD-L1) Preclinical 55 CTLA4, Cytotoxic T-lymphocyte-associated protein 4; ICI, Immune checkpoint inhibitor; MYXV, Myxoma virus; PDL, Programmed cell death ligand 1; VV, Vaccinia virus.

with replication-competent MYXV-sensitized tumor cells to therapy, poxviruses do not need to overcome any pre-existing subsequent cisplatin treatments and drastically improve sur- anti-viral immunity that affects many other OVs, making them vival in a murine syngeneic ovarian cancer [76]. Similar more suitable for systemic delivery, while others are highly vulner- observations were made for models of disseminated pan- able to host defense mechanisms like neutralizing antibodies. creatic cancer, in which MYXV sensitized cancer cells to Moreover, the unique ability of MYXV to infect BM-derived leuko- gemcitabine treatment, with the combination interventions cytes or PBMCs ex vivo and then co-migrate with diverse classes of showing higher efficacy than either of the monotherapies in leukocytes to tumor sites after infusion in vivo, make it an ideal promoting survival [77]. Additionally, Zemp et al. observed candidate for the treatment of hematological cancers or metastatic that MYXV in combination with rapamycin infects and kills cancers that lie within the patrolling reach of migratory leukocytes. brain tumor-initiating cells, significantly prolonging the sur- Also, the extensive track record of vaccination makes VV a very safe vival of tumor-bearing mice [78]. The same synergistic effect agent for oncotherapy, MYXV’s highly restrictive natural rabbit- of MYXV plus rapamycin was described in a gallbladder specific tropism in nature suggests a high biosafety profile in cancer-bearing mouse model [79]. Additionally, the applica- humans. Moreover, poxviral DNA cannot integrate into the host tion of cyclophosphamide increased MYXV infection of can- genome, which could happen for viruses that need to exploit the cer cells in a glioma murine model, the combined approach nucleus as part of their replication cycle. Poxviruses like VV and being more effective than the monotherapies [80]. MYXV also replicate rapidly and efficiently in tumor cells, a key Interestingly, MYXV exhibited synergistic effects with multi- feature for oncotherapy use. They are highly immunogenic, being ple small molecule inhibitors against human brain tumor- able to subvert the immune-evasion associated with the TME. initiating cells in vitro, supporting the fact that MYXV can Interestingly, when tested in primary human or mouse dendritic potentiate the anti-cancer effects of many cancer drugs. cells, MYXV was more stimulatory than VV at inducing cell activa- Some of these compounds have not been previously tion associated with antigen presentation pathways [82,83], but showntosynergizewithOVsinvitro[81]. the long history of VV as a vaccine platform indicates that this virus Bartee et al. reported that the application of MYXV with anti-PD can also robustly induce long-term immunity against a wide vari- -1 Ab improved overall survival in a murine model of melanoma. ety of virus-encoded antigens. Finally, the large dsDNA genomes of Almost 30% of mice receiving combination therapy exhibited poxviruses also allow them to be engineered with a high number complete responses, whereas either monotherapy only delayed of exogenous transgenes, being suitable to construct multiple tumor growth. Note that the combined therapy produced an armed OVs with enhanced immunostimulatory or other desired adverse side effect, as it led to an autoimmune related-disorder properties. Combined, all of these characteristics suggest that of resulting hair loss, indicating that caution needs to be exercised poxviruses like VV and MYXV should be included among the in controlling the known autoimmune complications of ICIs [55]. most optimal platforms for oncolytic virotherapy.

8. Conclusions 9. Expert opinion Poxviruses possess many advantages that make them excellent Despite the progress obtained during the last few years in the candidates for oncolytic virotherapy. For instance, with the notable field of oncolytic virotherapy, there are several key challenges exception of previously smallpox-vaccinated patients receiving VV that still need to be overcome for these OVs to succeed in the 10 L. E. TORRES-DOMÍNGUEZ AND G. MCFADDEN clinic. One important goal is to develop more optimal and other independent modalities or immunotherapy regimens efficient delivery systems of OVs to the sites of disease. Many (ICIs, CAR-T cells, etc.). Also, the sequential use of two immu- OVs have shown modest results when translated to the clinic, nologically distinct OVs may help to overcome the weaknesses due to the limited ingress of virions into the tumor sites. of each the vectors. As discussed, combining two anti-cancer Therefore, the search for novel and more effective routes for strategies can enhance efficacy compared to either mono- OV delivery is a priority, in order to increase their success as therapy approach, if they work in a synergistic or even an oncotherapy. For example, the use of MYXV-armed carrier cells additive manner. Further studies are necessary to define the might considerably increase the success of virus delivery to optimal combinations with these anti-cancer therapies, which the TME, by overcoming the natural physiological barriers that will serve to increase the positive effects of clinical outcomes. limit virus access to tumor beds in hard to reach niches and Without doubt, with so many OVs awaiting to be validated in metastatic sites. Particularly for MYXV, due to his natural ability clinical trials and multiple challenges to be addressed, this is to be disseminated by virus-infected leukocytes in rabbits, it is an exciting time for oncolytic virotherapy. an ideal candidate to be tested in this alternative systemic delivery method. Moreover, the approval of oncotherapies Funding using patient-modified leukocytes (CAR-T cells) lessen the risk for the exploitation of ex vivo MYXV-loaded leukocytes This work was supported by Arizona State University start-up funds to in clinical trials. G McFadden. For poxviruses, their tumor cell oncotropism (artificially rendered by genetic engineering for VV, natural for the Declaration of interest MYXV platform) makes them very safe therapeutic candidates, although in the case of VV some immunocompetent patients G McFadden is a co-founder of OncoMyx Therapeutics. The authors have no might be contra-indicated. For MYXV, properly designed other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or human clinical trials must be conducted to investigate the materials discussed in the manuscript apart from those disclosed. biosafety and potential of this virus in cancer patients. There is also a need to identify patient biomarkers to predict which OV, not just poxviral candidates, will be appropriate to treat Reviewer Disclosures certain tumors in specific patients. Peer reviewers on this manuscript have no relevant financial relationships Although the most desirable feature for OVs may be the or otherwise to disclose. targeted death of the tumor cells, the concept of ICD as a desired end point of OV-infected cancer cells has changed this perspective. Thus, now researchers must focus not only on References increasing the oncolytic capacity of the poxvirus vectors, but Papers of special note have been highlighted as either of interest (•)orof also on inducing ICD in the target cancer cells, thus promoting considerable interest (••) to readers. a more robust antitumor immune activation. The next genera- 1. Seymour LW, Fisher KD. Oncolytic viruses: finally delivering. Br tion of genetically modified poxvirus-based OVs (presumably J Cancer. 2016 Feb 16;114(4):357–361. achieved by mutations of viral genes and/or inserting exogen- 2. Jhawar SR, Thandoni A, Bommareddy PK, et al. Oncolytic viruses-natural and genetically engineered cancer ous transgenes) may be more efficient in inducing ICD in cells immunotherapies. Front Oncol. 2017;7:202. with a malignant phenotype. 3. Rehman H, Silk AW, Kane MP, et al. 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