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The heterogeneity of the solid tumor microenvironment (TME) impairs the therapeutic efficacy of standard therapies and also reduces the infiltration of antitumor immune cells, all of which lead to tumor progression and invasion. In addition, self-renewing cancer stem cells (CSCs) support tumor dormancy, drug resistance, and recurrence, all of which might pose challenges to the eradication of malignant tumor masses with current therapies. Natural forms of oncolytic viruses (OVs) or engineered OVs are known for their potential to directly target and kill tumor cells or indirectly eradicate tumor cells by involving antitumor immune responses, including enhancement of infiltrating antitumor immune cells, induction of immunogenic cell death, and reprogramming of cold TME to an immune-sensitive hot state. More importantly, OVs can target stemness factors that promote tumor progression, which subsequently enhances the efficacy of immunotherapies targeting solid tumors, particularly the CSC subpopulation. Herein, we describe the role of CSCs in tumor heterogeneity and resistance and then highlight the potential and remaining challenges of immunotherapies targeting CSCs. We then review the potential of OVs to improve tumor immunogenicity and target CSCs and finally summarize the challenges within the therapeutic application of OVs in preclinical and clinical trials., (© 2024 John Wiley & Sons Ltd.)

Cytokines are small proteins that regulate the growth and functional activity of immune cells, and several have been approved for cancer therapy. Oncolytic viruses are agents that mediate antitumor activity by directly killing tumor cells and inducing immune responses. Talimogene laherparepvec is an oncolytic herpes simplex virus type 1 (oHSV), approved for the treatment of recurrent melanoma, and the virus encodes the human cytokine, granulocyte-macrophage colony-stimulating factor (GM-CSF). A significant advantage of oncolytic viruses is the ability to deliver therapeutic payloads to the tumor site that can help drive antitumor immunity. While cytokines are especially interesting as payloads, the optimal cytokine(s) used in oncolytic viruses remains controversial. In this review, we highlight preliminary data with several cytokines and chemokines, including GM-CSF, interleukin 12, FMS-like tyrosine kinase 3 ligand, tumor necrosis factor α, interleukin 2, interleukin 15, interleukin 18, chemokine (C-C motif) ligand 2, chemokine (C-C motif) ligand 5, chemokine (C-X-C motif) ligand 4, or their combinations, and show how these payloads can further enhance the antitumor immunity of oHSV. A better understanding of cytokine delivery by oHSV can help improve clinical benefit from oncolytic virus immunotherapy in patients with cancer., Competing Interests: Competing interests: SDR is a co-inventor on patents relating to oncolytic herpes simplex viruses, owned, and managed by Georgetown University and Massachusetts General Hospital, which have received royalties from Amgen and Acti\Vec Inc., and acted as a consultant and received honoraria from Replimune, Cellinta, and Greenfire Bio, and honoraria and equity from EG 427. HLK is an employee of Ankyra Therapeutics and has received honoraria for participating on advisory boards for Castle Biosciences, Midatech Pharma, Marengo Therapeutics, and Virogin. The remaining 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., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Newcastle disease virus (NDV) is an avian pathogen with an unsegmented negative-strand RNA genome that belongs to the Paramyxoviridae family. While primarily pathogenic in birds, NDV presents no threat to human health, rendering it a safe candidate for various biomedical applications. Extensive research has highlighted the potential of NDV as a vector for vaccine development and gene therapy, owing to its transcriptional modularity, low recombination rate, and lack of a DNA phase during replication. Furthermore, NDV exhibits oncolytic capabilities, efficiently eliciting antitumor immune responses, thereby positioning it as a promising therapeutic agent for cancer treatment. This article comprehensively reviews the biological characteristics of NDV, elucidates the molecular mechanisms underlying its oncolytic properties, and discusses its applications in the fields of vaccine vector development and tumor therapy.

Dendritic cell (DC)-based vaccines have emerged as a promising strategy in cancer immunotherapy due to low toxicity. However, the therapeutic efficacy of DC as a monotherapy is insufficient due to highly immunosuppressive tumor environment. To address these limitations of DC as immunotherapeutic agent, we have developed a polymeric nanocomplex incorporating (1) oncolytic adenovirus (oAd) co-expressing interleukin (IL)-12 and granulocyte-macrophage colony-stimulating factor (GM-CSF) and (2) arginine-grafted bioreducible polymer with PEGylated paclitaxel (APP) to restore antitumor immune surveillance function in tumor milieu and potentiate immunostimulatory attributes of DC vaccine. Nanohybrid complex (oAd/APP) in combination with DC (oAd/APP+DC) induced superior expression level of antitumor cytokines (IL-12, GM-CSF, and interferon gamma) than either oAd/APP or DC monotherapy in tumor tissues, thus resulting in superior intratumoral infiltration of both endogenous and exogenous DCs. Furthermore, oAd/APP+DC treatment led superior migration of DC to secondary lymphoid organs, such as draining lymph nodes and spleen, in comparison with either monotherapy. Superior migration profile of DCs in oAd/APP+DC treatment group resulted in more prolific activation of tumor-specific T cells in these lymphoid organs and greater intratumoral infiltration of T cells. Additionally, oAd/APP+DC treatment led to lower subset of tumor infiltrating lymphocytes and splenocytes being immunosuppressive regulatory T cells than any other treatment groups. Collectively, oAd/APP+DC led to superior induction of antitumor immune response and amelioration of immunosuppressive tumor microenvironment to elicit potent tumor growth inhibition than either monotherapy., Competing Interests: C-OY and JH are CEO and employee of GeneMedicine Co., Ltd. The remaining 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., (Copyright © 2024 Kim, Yoon, Hong, Kasala and Yun.)

Background: Metastatic breast cancer is a leading cause of cancer death in woman. Current treatment options are often associated with adverse side effects and poor outcomes, demonstrating the need for effective new treatments. Immunotherapies can provide durable outcomes in many cancers; however, limited success has been achieved in metastatic triple negative breast cancer. We tested whether combining different immunotherapies can target metastatic triple negative breast cancer in pre-clinical models., Methods: Using primary and metastatic 4T1 triple negative mammary carcinoma models, we examined the therapeutic effects of oncolytic vesicular stomatitis virus (VSVΔM51) engineered to express reovirus-derived fusion associated small transmembrane proteins p14 (VSV-p14) or p15 (VSV-p15). These viruses were delivered alone or in combination with natural killer T (NKT) cell activation therapy mediated by adoptive transfer of α-galactosylceramide-loaded dendritic cells., Results: Treatment of primary 4T1 tumors with VSV-p14 or VSV-p15 alone increased immunogenic tumor cell death, attenuated tumor growth, and enhanced immune cell infiltration and activation compared to control oncolytic virus (VSV-GFP) treatments and untreated mice. When combined with NKT cell activation therapy, oncolytic VSV-p14 and VSV-p15 reduced metastatic lung burden to undetectable levels in all mice and generated immune memory as evidenced by enhanced in vitro recall responses (tumor killing and cytokine production) and impaired tumor growth upon rechallenge., Conclusion: Combining NKT cell immunotherapy with enhanced oncolytic virotherapy increased anti-tumor immune targeting of lung metastasis and presents a promising treatment strategy for metastatic breast cancer., (© 2024. The Author(s).)

Resistance to treatment poses a major challenge for cancer therapy, and oncoviral treatment encounters the issue of viral resistance as well. In this investigation, we introduce deterministic differential equation models to explore the effect of resistance on oncolytic viral therapy. Specifically, we classify tumor cells into resistant, sensitive, or infected with respect to oncolytic viruses for our analysis. Immune cells can eliminate both tumor cells and viruses. Our research shows that the introduction of immune cells into the tumor-virus interaction prevents all tumor cells from becoming resistant in the absence of conversion from resistance to sensitivity, given that the proliferation rate of immune cells exceeds their death rate. The inclusion of immune cells leads to an additional virus-free equilibrium when the immune cell recruitment rate is sufficiently high. The total tumor burden at this virus-free equilibrium is smaller than that at the virus-free and immune-free equilibrium. Therefore, immune cells are capable of reducing the tumor load under the condition of sufficient immune strength. Numerical investigations reveal that the virus transmission rate and parameters related to the immune response significantly impact treatment outcomes. However, monotherapy alone is insufficient for eradicating tumor cells, necessitating the implementation of additional therapies. Further numerical simulation shows that combination therapy with chimeric antigen receptor (CAR T-cell) therapy can enhance the success of treatment.

Bladder cancer is a common type of cancer around the world, and the majority of patients are diagnosed with non-muscle-invasive bladder cancer (NMIBC). Although low-risk NMIBC has a good prognosis, the disease recurrence rate and development of treatment-refractory disease remain high in intermediate- to high-risk NMIBC patients. To address these challenges for the treatment of NMIBC, a novel combination therapy composed of an oncolytic adenovirus (oAd) co-expressing interleukin (IL)-12, granulocyte-macrophage colony-stimulating factor (GM-CSF), and relaxin (RLX; HY-oAd) and a clinical-stage glycogen synthase kinase (GSK)-3β inhibitor (9-ING-41; elraglusib) was investigated in the present report. Our findings demonstrate that HY-oAd and 9-ING-41 combination therapy (HY-oAd+9-ING-41) exerted superior inhibition of tumor growth compared with respective monotherapy in a syngeneic NMIBC tumor model. HY-oAd+9-ING-41 induced high-level tumor extracellular matrix (ECM) degradation and a more potent antitumor immune response than the respective monotherapy. In detail, HY-oAd+9-ING-41 induced superior accumulation of intratumoral T cells, prevention of immune cell exhaustion, and induction of tumor-specific adaptive immune response compared to either monotherapy. Collectively, these results demonstrate that the combination of HY-oAd and 9-ING-41 may be a promising approach to elicit a potent antitumor immune response against bladder cancer., Competing Interests: Author JH is an employee of the company GeneMedicine Co., Ltd. C-OY is the CEO of GeneMedicine, Co., Ltd. The remaining 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., (Copyright © 2024 Yoon, Jiao, Hong, Kim and Yun.)

Antitumor therapies based on adoptively transferred T cells or oncolytic viruses have made significant progress in recent years, but the limited efficiency of their infiltration into solid tumors makes it difficult to achieve desired antitumor effects when used alone. In this study, an oncolytic virus (rVSV-LCMVG) that is not prone to induce virus-neutralizing antibodies was designed and combined with adoptively transferred T cells. By transforming the immunosuppressive tumor microenvironment into an immunosensitive one, in B16 tumor-bearing mice, combination therapy showed superior antitumor effects than monotherapy. This occurred whether the OV was administered intratumorally or intravenously. Combination therapy significantly increased cytokine and chemokine levels within tumors and recruited CD8 + T cells to the TME to trigger antitumor immune responses. Pretreatment with adoptively transferred T cells and subsequent oncolytic virotherapy sensitizes refractory tumors by boosting T-cell recruitment, down-regulating the expression of PD-1, and restoring effector T-cell function. To offer a combination therapy with greater translational value, mRNA vaccines were introduced to induce tumor-specific T cells instead of adoptively transferred T cells. The combination of OVs and mRNA vaccine also displays a significant reduction in tumor burden and prolonged survival. This study proposed a rational combination therapy of OVs with adoptive T-cell transfer or mRNA vaccines encoding tumor-associated antigens, in terms of synergistic efficacy and mechanism., (© 2024. The Author(s).)

Oncolytic viruses (OVs) show promise as a cancer treatment by selectively replicating in tumor cells and promoting antitumor immunity. However, the current immunogenicity induced by OVs for tumor treatment is relatively weak, necessitating a thorough investigation of the mechanisms underlying its induction of antitumor immunity. Here, we show that HSV-1-based OVs (oHSVs) trigger ZBP1-mediated PANoptosis (a unique innate immune inflammatory cell death modality), resulting in augmented antitumor immune effects. Mechanistically, oHSV enhances the expression of interferon-stimulated genes, leading to the accumulation of endogenous Z-RNA and subsequent activation of ZBP1. To further enhance the antitumor potential of oHSV, we conduct a screening and identify Fusobacterium nucleatum outer membrane vesicle (Fn-OMV) that can increase the expression of PANoptosis execution proteins. The combination of Fn-OMV and oHSV demonstrates potent antitumor immunogenicity. Taken together, our study provides a deeper understanding of oHSV-induced antitumor immunity, and demonstrates a promising strategy that combines oHSV with Fn-OMV., (© 2024. The Author(s).)

Oncolytic virotherapy is a promising immunotherapy approach for cancer treatment that utilizes viruses to preferentially infect and eliminate cancer cells while stimulating the immune response. In this review, we synthesize the current literature on the molecular circuits of immune sensing and response to oncolytic virotherapy, focusing on viral DNA or RNA sensing by infected cells, cytokine and danger-associated-signal sensing by neighboring cells, and the subsequent downstream activation of immune pathways. These sequential sense-and-response mechanisms involve the triggering of molecular sensors by viruses or infected cells to activate transcription factors and related genes for a breadth of immune responses. We describe how the molecular signals induced in the tumor upon virotherapy can trigger diverse immune signaling pathways, activating both antigen-presenting-cell-based innate and T cell-based adaptive immune responses. Insights into these complex mechanisms provide valuable knowledge for enhancing oncolytic virotherapy strategies.

Onco-virotherapy is an emergent treatment for cancer based on viral vectors. The therapeutic activity is based on two different mechanisms including tumor-specific oncolysis and immunostimulatory properties. In this study, we evaluated onco-virotherapy in vitro responses on immunocompetent non-small cell lung cancer (NSCLC) patient-derived tumoroids (PDTs) and healthy organoids. PDTs are accurate tools to predict patient's clinical responses at the in vitro stage. We showed that onco-virotherapy could exert specific antitumoral effects by producing a higher number of viral particles in PDTs than in healthy organoids. In the present work, we used multiplex protein screening, based on proximity extension assay to highlight different response profiles. Our results pointed to the increase of proteins implied in T cell activation, such as IFN-γ following onco-virotherapy treatment. Based on our observation, oncolytic viruses-based therapy responders are dependent on several factors: a high PD-L1 expression, which is a biomarker of greater immune response under immunotherapies, and the number of viral particles present in tumor tissue, which is dependent to the metabolic state of tumoral cells. Herein, we highlight the use of PDTs as an alternative in vitro model to assess patient-specific responses to onco-virotherapy at the early stage of the preclinical phases., Competing Interests: HL, JD, SJ, JB and EQ were employed by Transgene S.A.. PC and AL were employed by Olink. The remaining 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., (Copyright © 2024 Lê, Deforges, Cutolo, Lamarque, Hua, Lindner, Jain, Balloul, Benkirane-Jessel and Quéméneur.)

Discovering a therapeutic that can counteract the aggressiveness of this disease's mechanism is crucial for improving survival rates for cancer patients and for better understanding the most different types of cancer. In recent years, using these viruses as an anticancer therapy has been thought to be successful. They mostly work by directly destroying cancer cells, activating the immune system to fight cancer, and expressing exogenous effector genes. For the treatment of tumors, oncolytic viruses (OVs), which can be modified to reproduce only in tumor tissues and lyse them while preserving the healthy non-neoplastic host cells and reinstating antitumor immunity which present a novel immunotherapeutic strategy. OVs can exist naturally or be created in a lab by altering existing viruses. These changes heralded the beginning of a new era of less harmful virus-based cancer therapy. We discuss three different types of oncolytic viruses that have already received regulatory approval to treat cancer as well as clinical research using oncolytic adenoviruses. The primary therapeutic applications, mechanism of action of oncolytic virus updates, future views of this therapy will be covered in this chapter., (Copyright © 2024. Published by Elsevier Inc.)

Immunotherapy failures can result from the highly suppressive tumour microenvironment that characterizes aggressive forms of cancer such as recurrent glioblastoma (rGBM) 1,2 . Here we report the results of a first-in-human phase I trial in 41 patients with rGBM who were injected with CAN-3110-an oncolytic herpes virus (oHSV) 3 . In contrast to other clinical oHSVs, CAN-3110 retains the viral neurovirulence ICP34.5 gene transcribed by a nestin promoter; nestin is overexpressed in GBM and other invasive tumours, but not in the adult brain or healthy differentiated tissue 4 . These modifications confer CAN-3110 with preferential tumour replication. No dose-limiting toxicities were encountered. Positive HSV1 serology was significantly associated with both improved survival and clearance of CAN-3110 from injected tumours. Survival after treatment, particularly in individuals seropositive for HSV1, was significantly associated with (1) changes in tumour/PBMC T cell counts and clonal diversity, (2) peripheral expansion/contraction of specific T cell clonotypes; and (3) tumour transcriptomic signatures of immune activation. These results provide human validation that intralesional oHSV treatment enhances anticancer immune responses even in immunosuppressive tumour microenvironments, particularly in individuals with cognate serology to the injected virus. This provides a biological rationale for use of this oncolytic modality in cancers that are otherwise unresponsive to immunotherapy (ClinicalTrials.gov: NCT03152318 )., (© 2023. The Author(s).)

We studied the immunotherapeutic potential of CF33-hNIS-antiPDL1 oncolytic virus (OV) against gastric cancer with peritoneal metastasis (GCPM). We collected fresh malignant ascites (MA) or peritoneal washings (PW) during routine paracenteses and diagnostic laparoscopies from GC patients (n = 27). Cells were analyzed for cancer cell markers and T cells, or treated with PBS, CF33-GFP, or CF33-hNIS-antiPDL1 (MOI = 3). We analyzed infectivity, replication, cytotoxicity, CD107α upregulation of CD8 + and CD4 + T cells, CD274 (PD-L1) blockade of cancer cells by virus-encoded anti-PD-L1 scFv, and the release of growth factors and cytokines. We observed higher CD45 - /large-size cells and lower CD8 + T cell percentages in MA than PW. CD45 - /large-size cells were morphologically malignant and expressed CD274 (PD-L1), CD252 (OX40L), and EGFR. CD4 + and CD8 + T cells did not express cell surface exhaustion markers. Virus infection and replication increased cancer cell death at 15 h and 48 h. CF33-hNIS-antiPDL1 treatment produced functional anti-PD-L1 scFv, which blocked surface PD-L1 binding of live cancer cells and increased CD8 + CD107α + and CD4 + CD107α + T cell percentages while decreasing EGF, PDGF, soluble anti-PD-L1, and IL-10. CF33-OVs infect, replicate in, express functional proteins, and kill ex vivo GCPM cells with immune-activating effects. CF33-hNIS-antiPDL1 displays real potential for intraperitoneal GCPM therapy.

Oncolytic virotherapy has currently emerged as a powerful therapeutic approach in cancer treatment. Although the history of using viruses goes back to the early 20th century, the approval of talimogene laherparepvec (T-VEC) in 2015 increased interest in oncolytic viruses (OVs). OVs are multifaceted biotherapeutic agents because they replicate in and kill tumor cells and augment immune responses by releasing immunostimulatory molecules from lysed cells. Despite promising results, some limitations hinder the efficacy of oncolytic virotherapy. The delivery challenges and the upregulation of checkpoints following oncolytic virotherapy also mediate resistance to OVs by diminishing immune responses. Furthermore, the localization of receptors of viruses in the tight junctions, interferon responses, and the aberrant expression of genes involved in the cell cycle of the virus, including their infection and replication, reduce the efficacy of OVs. In this review, we present different mechanisms of resistance to OVs and strategies to overcome them.

Recognizing immune responses to oncolytic virotherapy opens the way for new combinations.

In recent years, it became apparent that cancers either associated with viral infections or aberrantly expressing endogenous retroviral elements (EREs) are more immunogenic, exhibiting an intense intra-tumor immune cell infiltration characterized by a robust cytolytic apparatus. On the other hand, epigenetic regulation of EREs is crucial to maintain steady-state conditions and cell homeostasis. In line with this, epigenetic disruptions within steady-state cells can lead to cancer development and trigger the release of EREs into the cytoplasmic compartment. As such, detection of viral molecules by intracellular innate immune sensors leads to the production of type I and type III interferons that act to induce an antiviral state, thus restraining viral replication. This knowledge has recently gained momentum due to the possibility of triggering intratumoral activation of interferon responses, which could be used as an adjuvant to elicit strong anti-tumor immune responses that ultimately lead to a cascade of cytokine production. Accordingly, several therapeutic approaches are currently being tested using this rationale to improve responses to cancer immunotherapies. In this review, we discuss the immune mechanisms operating in viral infections, show evidence that exogenous viruses and endogenous retroviruses in cancer may enhance tumor immunogenicity, dissect the epigenetic control of EREs, and point to interferon pathway activation in the tumor milieu as a promising molecular predictive marker and immunotherapy target. Finally, we briefly discuss current strategies to modulate these responses within tumor tissues, including the clinical use of innate immune receptor agonists and DNA demethylating agents., Competing Interests: 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., (Copyright © 2021 Vitiello, Ferreira, Cordeiro de Lima and Medina.)

Objective: Our objective was to assess safety and adverse events associated with intraperitoneal Olvi-Vec virotherapy in patients with platinum-resistant or refractory ovarian cancer (PRROC). Secondary objectives included objective response rate (ORR) per RECIST 1.1 and progression-free survival (PFS)., Methods: Olvi-Vec is a modified vaccinia virus that causes oncolysis and immune activation. An open-label phase 1b trial using a 3 + 3 dose escalation was conducted. Intraperitoneal Olvi-Vec was given as monotherapy in two consecutive daily doses. Translational analyses included anti-virus antibody levels, viral shedding, circulating tumor cells (CTCs) and T cells., Results: Twelve patients (median age: 69 years, range: 45-77) with median 5 prior therapies (range: 2-10) and 2 prior platinum lines (range: 1-5) were enrolled. There were three dose level cohorts: 3 × 10 9 (n = 6), 1 × 10 10 (n = 5), and 2.5 × 10 10 (n = 1) plaque forming units (PFU)/day on two consecutive days. Treatment-related adverse events (TRAEs) included G1/G2 nausea (n = 6), fever (n = 6), abdominal distention (n = 5), and abdominal pain (n = 4). There were no Grade 4 TRAEs, no dose relationship to TRAEs, and no deaths attributed to Olvi-Vec. The ORR was 9% (1/11). Stable disease (SD) was 64% (7/11), and SD ≥15 weeks was 46% (5/11). Median PFS was 15.7 weeks (95%CI: 5.7-34.5), including extended PFS in four patients (23.2, 34.5, 59.4+ and 70.8 weeks). Three patients had extended overall survival (deceased 33.6 months, and alive with disease at 54 and 59 months). CTCs diminished in 6/8 (75%) baseline-positive patients. Immune activation was demonstrated from virus-enhanced tumor infiltration of CD8+ T-cells and activation of tumor-specific T-cells in peripheral blood., Conclusions: Oncolytic viral therapy with intraperitoneal Olvi-Vec showed promising safety, clinical activities, and immune activation in patients with PRROC, warranting further clinical investigation., Competing Interests: Declaration of Competing Interest Dr. Robert W. Holloway serves as Chair, Clinical Advisory Board on Gynecologic Cancers to Genelux. None of the other authors have any potential financial or commercial conflict of interest associated with this research manuscript., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Although the recent treatment in melanoma through the use of anti-PD-1 immunotherapy is successful, the efficacy of this approach remains to be improved. Here, we explore the feasibility of combination strategy with the armed oncolytic adenovirus ZD55-IL-24 and PD-1 blockade. We find that combination therapy with localized ZD55-IL-24 and systemic PD-1 blockade leads to synergistic inhibition of both local and distant established tumors in B16-bearing immunocompetent mouse model. Our further mechanism investigation reveals that synergistic therapeutic effect is associated with marked promotion of tumor immune infiltration and recognition in both local and distant tumors as well as spleens. PD-1 blockade has no obvious effect on promotion of tumor immune infiltration and recognition. Localized therapy with ZD55-IL-24, however, can help PD-1 blockade to overcome the limitation of relatively low tumor immune infiltration and recognition. This study provides a rationale for investigation of such combination therapy in the clinic., (© 2021. The Author(s).)

Viruses have been widely used to treat cancer for many years and they achieved tremendous success in clinical trials with outstanding results, which has led to the foundation of companies that develop recombinant viruses for a better tumor treatment. Even though there has been a great progress in the field of viral tumor immunotherapy, until now only one virus, the oncolytic virus talimogene laherparepvec (TVEC), a genetically modified herpes simplex virus type 1 (T-VEC), has been approved by the FDA for cancer treatment. Although oncolytic viruses showed progress in certain cancer types and patient populations but they have yet shown limited efficacy when it comes to solid tumors. Only recently it was demonstrated that the immune stimulatory aspect of oncolytic viruses can strongly contribute to their anti-tumoral activity. One specific example in this context are arenaviruses, which have been shown to be non-cytopathic in nature lead to the massive immune activation within the tumor resulting in strong anti-tumoral activity. This strong immune activation might be also linked to their noncytopathic features, as their immune stimulatory potential is not self-limiting as is the case for oncolytic viruses due to their fast eradication by anti-viral immune effects. Because of this strong immune activation, arenaviruses appear superior to oncolytic viruses when it comes to potent and long-lasting anti-tumor effects in a broad variety of tumor types. Currently one of the most promising therapeutics which has turned to be very much beneficial for the treatment of different cancer types is represented by antibodies targeting checkpoint inhibitors such as PD-1/PD-L-1. In this review, we will summarize anti-tumoral effects of arenaviruses, and will discuss their potential to be combined with checkpoint inhibitors for a more efficient tumor treatment, which further emphasizes that arenavirus therapy as a viroimmunotherapy can be an efficient tool for the better clearance of tumors., Competing Interests: The authors declare that no conflict of interests exists., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

Immunotherapy has revolutionised cancer treatment through restoration of host antitumour immune response. Immune checkpoint inhibitors (ICIs) confer durable responses in only a subset of patients. Mechanisms of ICI resistance to improve durable response rates and overall survival are an area of intense clinical research. Robust clinical development is ongoing to evaluate novel combination therapies to overcome ICI resistance, including targeting immunoregulatory pathways in the tumour microenvironment. Intratumoural (IT) immunotherapies such as toll-like receptor agonists, stimulator of interferon-induced gene agonists, retinoic-inducible gene I-like receptor agonists and oncolytic viruses may represent potential combination treatment options to overcome ICI resistance. Use of IT immunotherapies in combination with ICIs may alter the tumour microenvironment to address resistance mechanisms and improve antitumour response. Optimisation of IT immunotherapy clinical trials will elucidate resistance mechanisms, facilitate clinical trial design, define pharmacodynamic predictors that identify patients who may most benefit and inform clinical development of combination immunotherapy regimens. Here we provide an overview of IT immunotherapy principles, mechanisms of action, categories of IT immunotherapeutics, emerging data, clinical development strategies, response assessment, dose and schedule determination, clinical trial design and translational study design., Competing Interests: Conflict of interest statement J.Y. is an employee of Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA. A.K. is an employee of Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA, and has stock ownership interests in Merck & Co., Inc., Kenilworth, NJ, USA. J.B. reports grants and others from Acerta, Celgene, Genentech, Kite/Gilead and Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA. R.H.I.A is an employee of Seven and Eight Biopharmaceuticals Inc and reports personal fees from Aduro, Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA, Moderna Therapeutics, Novartis, Oncosec, Pfizer, Pulse Biosciences and Takara. S.H.-L. reports no conflicts of interest. J.J.L. reports scientific advisory board from 7 Hills, Actym, Alphamab Oncology, Arch Oncology, Kanaph, Mavu, Onc.AI, Pyxis, Spring Bank and Tempest; consultancy from AbbVie, Algios, Array, Bayer, Bristol Myers Squibb, CStone, Eisai, EMD Serono, Janssen, Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA, Mersana, Novartis, PTx, RefleXion, Regeneron, Rubius, Silicon, Tesaro and Xilios; research support (to his institution for clinical trials unless noted) from AbbVie, Agios (IIT), Array (IIT), Astellas, Bristol Myers Squibb, Corvus, EMD Serono, Immatics, Incyte, Kadmon, MacroGenics, Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA, Spring Bank, Tizona and Xencor; travel from Bristol Myers Squibb, EMD Serono, Janssen, Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA, Mersana and Pyxis; patents (both provisional): Serial #15/612,657 (Cancer Immunotherapy), PCT/US18/36052 (Microbiome Biomarkers for Anti-PD-1/PD-L1 Responsiveness: Diagnostic, Prognostic and Therapeutic Uses Thereof). A.D. reports consulting fees from Apexigen, Idera and Nektar; and institutional research funding from Apexigen, Idera Pharmaceuticals, Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA, and Nektar Therapeutics. A.M. reports no conflict of interest. A.S. is an employee and stockholder of Two River, Inc. Z.A.C is an employee of Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA, and has stock ownership interests in Merck & Co., Inc., Kenilworth, NJ, USA, and in Bristol Myers Squibb. F.S.H. reports grants, personal fees, and others from Bristol Myers Squibb; personal fees from Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA, and from EMD Serono; grants, personal fees and others from Novartis; personal fees from Surface, Compass Therapeutics, Apricity, Aduro, Sanofi, Pionyr, Torque, Rheos, Kairos, Bicara, Eisai, Idera, Takeda; and others from Pieris Pharmaceutical and from Corner Therapeutics. In addition, Dr. Hodi has the following patents: ‘Methods for Treating MICA-Related Disorders’ (#20100111973) with royalties paid, ‘Tumor Antigens and Uses Thereof’ (#7250291) issued, ‘Angiopoietin-2 Biomarkers Predictive of Anti-Immune Checkpoint Response’ (#20170248603) pending, ‘Compositions and Methods for Identification, Assessment, Prevention, and Treatment of Melanoma Using PD-L1 Isoforms’ (#20160340407) pending, ‘Therapeutic Peptides’ (#20160046716) pending, a patent Therapeutic Peptides (#20140004112) pending, a patent Therapeutic Peptides (#20170022275) pending, ‘Therapeutic Peptides’ (#20170008962) pending, ‘Therapeutic Peptides’ issued (#9402905), ‘Methods of Using Pembrolizumab and Trebananib’ pending, ‘Vaccine Compositions and Methods for Restoring NKG2D Pathway Function Against Cancers’ issued (#10279021), ‘Antibodies That Bind to MHC Class I Polypeptide-Related Sequence A’ issued (#10106611) and ‘Únti-Galectin Antibody Biomarkers Predictive of Anti-Immune Checkpoint and Anti-Angiogenesis Responses’ pending (#20170343552)., (Copyright © 2021 Merck Sharp & Dohme Corp. and the Author(s). Published by Elsevier Ltd.. All rights reserved.)

Oncolytic viruses as cancer vaccines modulate the tumor microenvironment and act synergistically with immune checkpoint inhibitors to overcome resistance. Taking advantage of the loading capacity for exogenous genes, we generated a recombinant herpes simplex virus type 1 (HSV-1), HSV-aPD-1, carrying a full-length humanized anti-PD-1 monoclonal antibody (anti-PD-1 mAb) that replicates and expresses anti-PD-1 mAbs in tumor cells in vitro and in vivo. Its anti-tumor effect was assessed in human PD-1 knock-in mice by analyzing tumor inhibition, cell populations and RNA expression in tumors, and serum cytokine levels. Enhanced anti-tumor immune responses and T-cell infiltration were induced by HSV-aPD-1 compared with unloaded virus or anti-PD-1 therapy in both MC38 and B16-F10 models, resulting in improved treatment efficacy in the latter. Moreover, compared with unloaded HSV-1 or HSV-1 loaded with GM-CSF/IL-2 combined with anti-PD-1 mAbs, HSV-aPD-1 displayed similar therapeutic control of tumor growth. Finally, tumor RNAseq analysis in the B16-F10 model showed upregulated IFN pathway and antigen processing and presentation genes, and downregulated angiogenesis and cell adhesion genes, which all contribute to tumor inhibition. These findings indicate the clinical potential of HSV-aPD-1 as monotherapy or combination therapy, especially in tumors resistant to immune checkpoint inhibitors., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Oncolytic herpes simplex virus-1 is capable of lysing tumor cells while alerting the immune system. CD47, in collaboration with SIRPα, represents an important immune checkpoint to inhibit phagocytosis by innate immune cells. Here we show locoregional control of glioblastoma by an oncolytic herpes virus expressing a full-length anti(α)-human CD47 IgG1 or IgG4 antibody. The antibodies secreted by the virus-infected glioblastoma cells block the CD47 'don't eat me' signal irrespective of the subclass; however, αCD47-IgG1 has a stronger tumor killing effect than αCD47-IgG4 due to additional antibody-dependent cellular phagocytosis by macrophages and antibody-dependent cellular cytotoxicity by NK cells. Intracranially injected αCD47-IgG1-producing virus continuously releases the respective antibody in the tumor microenvironment but not into systemic circulation; additionally, αCD47-IgG1-producing virus also improves the survival of tumor-bearing mice better than control oncolytic herpes virus combined with topical αCD47-IgG1. Results from immunocompetent mouse tumor models further confirm that macrophages, and to a lesser extent NK cells, mediate the anti-tumor cytotoxicity of antibody-producing oncolytic herpesviruses. Collectively, oncolytic herpes simplex virus-1 encoding full-length antibodies could improve immune-virotherapy for glioblastoma., (© 2021. The Author(s).)

Talimogene laherparepvec (T-VEC) is a herpes simplex virus type 1-based intralesional oncolytic immunotherapy approved for the treatment of unresectable melanoma. The present, ongoing study aimed to estimate the treatment effect of neoadjuvant T-VEC on recurrence-free survival (RFS) of patients with advanced resectable melanoma. An open-label, phase 2 trial (NCT02211131) was conducted in 150 patients with resectable stage IIIB-IVM1a melanoma who were randomized to receive T-VEC followed by surgery (arm 1, n = 76) or surgery alone (arm 2, n = 74). The primary endpoint was a 2-year RFS in the intention-to-treat population. Secondary and exploratory endpoints included overall survival (OS), pathological complete response (pCR), safety and biomarker analyses. The 2-year RFS was 29.5% in arm 1 and 16.5% in arm 2 (overall hazard ratio (HR) = 0.75, 80% confidence interval (CI) = 0.58-0.96). The 2-year OS was 88.9% for arm 1 and 77.4% for arm 2 (overall HR = 0.49, 80% CI = 0.30-0.79). The RFS and OS differences between arms persisted at 3 years. In arm 1, 17.1% achieved a pCR. Increased CD8 + density correlated with clinical outcomes in an exploratory analysis. Arm 1 adverse events were consistent with previous reports for T-VEC. The present study met its primary endpoint and estimated a 25% reduction in the risk of disease recurrence for neoadjuvant T-VEC plus surgery versus upfront surgery for patients with resectable stage IIIB-IVM1a melanoma., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Oncolytic adenovirus therapy is gaining importance as a novel treatment option for the management of various cancers. Different concepts of modification within the adenovirus vector have been identified that define the mode of action against and the interaction with the tumour. Adenoviral vectors allow for genetic manipulations that restrict tumour specificity and also the expression of specific transgenes in order to support the anti-tumour effect. Additionally, replication of the virus and reinfection of neighbouring tumour cells amplify the therapeutic effect. Another important aspect in oncolytic adenovirus therapy is the virus induced cell death which is a process that activates the immune system against the tumour. This review describes which elements in adenovirus vectors have been identified for modification not only to utilize oncolytic adenovirus vectors into conditionally replicating adenoviruses (CRAds) that allow replication specifically in tumour cells but also to confer specific characteristics to these viruses. These advances in development resulted in clinical trials that are summarized based on the conceptual design.

We report on the efficacy of the non-attenuated HER2-retargeted oHSV named R-337 against the immunologically hot CT26-HER2 tumor, and an insight into the basis of the immune protection. Preliminarily, we conducted an RNA immune profiling and immune cell content characterization of CT26-HER2 tumor in comparison to the immunologically cold LLC1-HER2 tumor. CT26-HER2 tumor was implanted into HER2-transgenic BALB/c mice. Hallmarks of R-337 effects were the protection from primary tumor, long-term adaptive vaccination directed to both HER2 and CT26-wt cell neoantigens. The latter effect differentiated R-337 from OncoVEXGM-CSF. As to the basis of the immune protection, R-337 orchestrated several changes to the tumor immune profile, which cumulatively reversed the immunosuppression typical of this tumor (graphical abstract). Thus, Ido1 (inhibitor of T cell anticancer immunity) levels and T regulatory cell infiltration were decreased; Cd40 and Cd27 co-immunostimulatory markers were increased; the IFNγ cascade was activated. Of note was the dampening of IFN-I response, which we attribute to the fact that R-337 is fully equipped with genes that contrast the host innate response. The IFN-I shut-down likely favored viral replication and the expression of the mIL-12 payload, which, in turn, boosted the antitumor response. The results call for a characterization of tumor immune markers to employ oncolytic herpesviruses more precisely.

Functional tumor-specific cytotoxic T cells elicited by therapeutic cancer vaccination in combination with oncolytic viruses offer opportunities to address resistance to checkpoint blockade therapy. Two cancer vaccines, the self-adjuvanting protein vaccine KISIMA, and the recombinant oncolytic vesicular stomatitis virus pseudotyped with LCMV-GP expressing tumor-associated antigens, termed VSV-GP-TAA, both show promise as a single agent. Here we find that, when given in a heterologous prime-boost regimen with an optimized schedule and route of administration, combining KISIMA and VSV-GP-TAA vaccinations induces better cancer immunity than individually. Using several mouse tumor models with varying degrees of susceptibility for viral replication, we find that priming with KISIMA-TAA followed by VSV-GP-TAA boost causes profound changes in the tumor microenvironment, and induces a large pool of poly-functional and persistent antigen-specific cytotoxic T cells in the periphery. Combining this heterologous vaccination with checkpoint blockade further improves therapeutic efficacy with long-term survival in the spectrum. Overall, heterologous vaccination with KISIMA and VSV-GP-TAA could sensitize non-inflamed tumors to checkpoint blockade therapy., (© 2021. The Author(s).)

Background: Despite the success of immune checkpoint inhibitors against PD-L1 in the clinic, only a fraction of patients benefit from such therapy. A theoretical strategy to increase efficacy would be to arm such antibodies with Fc-mediated effector mechanisms. However, these effector mechanisms are inhibited or reduced due to toxicity issues since PD-L1 is not confined to the tumor and also expressed on healthy cells. To increase efficacy while minimizing toxicity, we designed an oncolytic adenovirus that secretes a cross-hybrid Fc-fusion peptide against PD-L1 able to elicit effector mechanisms of an IgG1 and also IgA1 consequently activating neutrophils, a population neglected by IgG1, in order to combine multiple effector mechanisms., Methods: The cross-hybrid Fc-fusion peptide comprises of an Fc with the constant domains of an IgA1 and IgG1 which is connected to a PD-1 ectodomain via a GGGS linker and was cloned into an oncolytic adenovirus. We demonstrated that the oncolytic adenovirus was able to secrete the cross-hybrid Fc-fusion peptide able to bind to PD-L1 and activate multiple immune components enhancing tumor cytotoxicity in various cancer cell lines, in vivo and ex vivo renal-cell carcinoma patient-derived organoids., Results: Using various techniques to measure cytotoxicity, the cross-hybrid Fc-fusion peptide expressed by the oncolytic adenovirus was shown to activate Fc-effector mechanisms of an IgA1 (neutrophil activation) as well as of an IgG1 (natural killer and complement activation). The activation of multiple effector mechanism simultaneously led to significantly increased tumor killing compared with FDA-approved PD-L1 checkpoint inhibitor (Atezolizumab), IgG1-PDL1 and IgA-PDL1 in various in vitro cell lines, in vivo models and ex vivo renal cell carcinoma organoids. Moreover, in vivo data demonstrated that Ad-Cab did not require CD8+ T cells, unlike conventional checkpoint inhibitors, since it was able to activate other effector populations., Conclusion: Arming PD-L1 checkpoint inhibitors with Fc-effector mechanisms of both an IgA1 and an IgG1 can increase efficacy while maintaining safety by limiting expression to the tumor using oncolytic adenovirus. The increase in tumor killing is mostly attributed to the activation of multiple effector populations rather than activating a single effector population leading to significantly higher tumor killing., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Talimogene laherparepvec (T-VEC) is a genetically modified herpes simplex type 1 virus and known as an effective oncolytic immunotherapy for injectable cutaneous, subcutaneous and nodal melanoma lesions in stage IIIB-IVM1a patients. This study set out to identify prognostic factors for achieving a complete response that can be used to optimize patient selection for T-VEC monotherapy., Methods: Patients with stage IIIB-IVM1a melanoma, treated with T-VEC at the Netherlands Cancer Institute between 2016-12 and 2020-01 with a follow-up time > 6 months, were included. Data were collected on baseline characteristics, responses and adverse events (AEs). Uni- and multivariable analyses were conducted, and a prediction model was developed to identify prognostic factors associated with CR., Results: A total of 93 patients were included with a median age of 69 years, median follow-up time was 16.6 months. As best response, 58 patients (62%) had a CR, and the overall response rate was 79%. The durable response rate (objective response lasting > 6 months) was 51%. Grade 1-2 AEs occurred in almost every patient. Tumor size, type of metastases, prior treatment with systemic therapy and stage (8Th AJCC) were independent prognostic factors for achieving CR. The prediction model includes the predictors tumor size, type of metastases and number of lesions., Conclusions: This study shows that intralesional T-VEC monotherapy is able to achieve high complete and durable responses. The prediction model shows that use of T-VEC in patients with less tumor burden is associated with better outcomes, suggesting use earlier in the course of the disease., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

Oncolytic viruses (OVs) are an emerging class of anti-cancer agents that replicate selectively within malignant cells and generate potent immune responses. Their potential efficacy has been shown in clinical trials, with talimogene laherparepvec (T-VEC or IMLYGIC ®) now approved both in the United States and Europe. In healthy individuals, NK cells provide effective surveillance against cancer and viral infections. In oncolytic viral therapy, NK cells may render OV ineffective by rapid elimination of the propagating virus but could also improve therapeutic efficacy by preferential killing of OV-infected malignant cells. Existing evidence suggests that the overall effect of NK cells against OV is context dependent. In the past decade, the understanding of cancer and OV biology has improved significantly, which helped refine this class of treatments in early-phase clinical trials. In this review, we summarised different strategies that have been evaluated to modulate NK activities for improving OV therapeutic benefits. Further development of OVs will require a systematic approach to overcome the challenges of the production and delivery of complex gene and cell-based therapies in clinical settings.

Clinical immunotherapy approaches are lacking efficacy in the treatment of glioblastoma (GBM). In this study, we sought to reverse local and systemic GBM-induced immunosuppression using the Helicobacter pylori neutrophil-activating protein (NAP), a potent TLR2 agonist, as an immunostimulatory transgene expressed in an oncolytic measles virus (MV) platform, retargeted to allow viral entry through the urokinase-type plasminogen activator receptor (uPAR). While single-agent murine anti-PD1 treatment or repeat in situ immunization with MV-s-NAP-uPA provided modest survival benefit in MV-resistant syngeneic GBM models, the combination treatment led to synergy with a cure rate of 80% in mice bearing intracranial GL261 tumors and 72% in mice with CT-2A tumors. Combination NAP-immunovirotherapy induced massive influx of lymphoid cells in mouse brain, with CD8+ T cell predominance; therapeutic efficacy was CD8+ T cell dependent. Inhibition of the IFN response pathway using the JAK1/JAK2 inhibitor ruxolitinib decreased PD-L1 expression on myeloid-derived suppressor cells in the brain and further potentiated the therapeutic effect of MV-s-NAP-uPA and anti-PD1. Our findings support the notion that MV strains armed with bacterial immunostimulatory antigens represent an effective strategy to overcome the limited efficacy of immune checkpoint inhibitor-based therapies in GBM, creating a promising translational strategy for this lethal brain tumor.

Oncolytic virus can selectively recognize cancer cells, target tumors, and stimulate an oncolytic and immune response. Recombinant armed oncolytic vaccinia virus has emerged as an attractive tool in oncolytic virotherapy because it has tumor-specific cytotoxicity and serves as a vector to express immune genes. A novel thymidine kinase (TK) gene-deleted oncolytic vaccinia virus (named ΔTK-Armed-VACV) armed with anti-human-programed cell death-1 protein (PD-1) antibody and anti-human-tumor necrosis factor receptor superfamily, member 9 (4-1BB) antibody genes was constructed based on Western Reserve in our previous study. The present study evaluated the ability of this virus for cancer-targeted therapy both in vitro and in vivo. A complete morphological structure of ΔTK-Armed-VACV was verified using transmission electron microscopy. The antibody was co-expressed with the replication of ΔTK-Armed-VACV in vitro assessed by Western blot analysis, enzyme-linked immunosorbent assay, and quantitative real-time polymerase chain reaction. The 3-(4,5-dimethylthiazol-2-yl)-5-(3-rboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt assay showed that the ΔTK-Armed-VACV exhibited significant tumor-specific cytotoxicity in vitro. The ΔTK-Armed-VACV inhibited the tumor growth in a 4T1 or A549 tumor-bearing mouse model. ELISpot assay showed that ΔTK-Armed-VACV-treated mice induced the expression of interferon-gamma, and lactate dehydrogenase-dependent cytotoxicity assay revealed that the ΔTK-Armed-VACV treatment activated tumor-specific cytotoxic T lymphocytes. The results indicated that oncolytic VACV with Western Reserve-mediated anti-human-PD-1 and anti-human-4-1BB antibody co-expression exerted a significant antitumor effect, indicating that the combination of oncolytic virotherapy and immunotherapy by the oncolytic VACV expressing one or more immune checkpoint genes might have satisfactory clinical expectations., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Oncolytic viruses (OV) have shown excellent safety and efficacy in preclinical and clinical studies. Influenza A virus (IAV) is considered a promising oncolytic virus. In this report, we generated a recombinant influenza virus expressing an immune checkpoint blockade agent targeting CTLA4. Using reverse genetics, a recombinant influenza virus, termed rFlu-CTLA4, encoding the heavy chain of a CTLA4 antibody on the PB1 segment and the light chain of the CTLA4 antibody on the PA segment was produced. RFlu-CTLA4 could replicate to high titers, and antibodies were produced in the allantoic fluid of infected eggs. Furthermore, the selective cytotoxicity of the virus was higher in various hepatocellular carcinoma cancer cell lines than in the normal cell line L02 in vitro, as indicated by MTS assays. More importantly, in a subcutaneous H22 mouse hepatocarcinoma model, intratumoral injections of rFlu-CTLA4 inhibited the growth of treated tumors and increased the overall survival of mice compared with injections of the PR8 virus. Taken together, these results warrant further exploration of this novel recombinant influenza virus for its potential use as a single or combination agent for cancer immunotherapy., (© 2021 International Federation for Cell Biology.)

Cancer immunotherapy targeting immune checkpoint inhibitors shows efficacy in several human cancers, but "cold tumors" that lack immune cells are typically unresponsive. Among the potential therapeutic approaches that could "heat" or promote lymphocyte infiltration of cold tumors, oncolytic viruses have attracted interest for their lytic and immunogenic mechanisms of action. In this article, we review the use of oncolytic adenoviruses in cancer immunotherapy, with a particular focus on preclinical and clinical data of oncolytic adenovirus-triggered immune responses against tumor antigens. We also discuss parameters to consider in clinical trial design and the combination of oncolytic adenoviruses with conventional treatments or other immunotherapies., (©2021 American Association for Cancer Research.)

Although the oncolytic parvovirus H-1PV has entered clinical trials, predicting therapeutic success remains challenging. We investigated whether the antiviral state in tumor cells determines the parvoviral oncolytic efficacy. The interferon/interferon-stimulated genes (IFN/ISG)-circuit and its major configurator, human endogenous retroviruses (HERVs), were evaluated using qRT-PCR, ELISA, Western blot, and RNA-Seq techniques. In pancreatic cancer cell lines, H-1PV caused a late global shutdown of innate immunity, whereby the concomitant inhibition of HERVs and IFN/ISGs was co-regulatory rather than causative. The growth-inhibitory IC50 doses correlated with the power of suppression but not with absolute ISG levels. Moreover, H-1PV was not sensitive to exogenous IFN despite upregulated antiviral ISGs. Such resistance questioned the biological necessity of the oncotropic ISG-shutdown, which instead might represent a surrogate marker for personalized oncolytic efficacy. The disabled antiviral homeostasis may modify the activity of other viruses, as demonstrated by the reemergence of endogenous AluY-retrotransposons. This way of suppression may compromise the interferogenicity of drugs having gemcitabine-like mechanisms of action. This shortcoming in immunogenic cell death induction is however amendable by immune cells which release IFN in response to H-1PV.

Background: Newcastle disease virus (NDV) is an oncolytic virus with excellent selectivity against cancer cells, both in vitro and in vivo. Unfortunately, prolonged in vitro NDV infection results in the development of persistent infection in the cancer cells which are then able to resist NDV-mediated oncolysis. However, the mechanism of persistency of infection remains poorly understood., Methods: In this study, we established persistently NDV-infected EJ28 bladder cancer cells, designated as EJ28P. Global transcriptomic analysis was subsequently carried out by microarray analysis. Differentially expressed genes (DEGs) between EJ28 and EJ28P cells identified by the edgeR program were further analysed by Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA) analyses. In addition, the microarray data were validated by RT-qPCR., Results: Persistently NDV-infected EJ28 bladder cancer cells were successfully established and confirmed by flow cytometry. Microarray analysis identified a total of 368 genes as differentially expressed in EJ28P cells when compared to the non-infected EJ28 cells. GSEA revealed that the Wnt/β-catenin and KRAS signalling pathways were upregulated while the TGF-β signalling pathway was downregulated. Findings from this study suggest that the upregulation of genes that are associated with cell growth, pro-survival, and anti-apoptosis may explain the survivability of EJ28P cells and the development of persistent infection of NDV., Conclusions: This study provides insights into the transcriptomic changes that occur and the specific signalling pathways that are potentially involved in the development and maintenance of NDV persistency of infection in bladder cancer cells. These findings warrant further investigation and is crucial towards the development of effective NDV oncolytic therapy against cancer.

By conferring systemic protection and durable benefits, cancer immunotherapies are emerging as long-term solutions for cancer treatment. One such approach that is currently undergoing clinical testing is a therapeutic anti-cancer vaccine that uses two different viruses expressing the same tumor antigen to prime and boost anti-tumor immunity. By providing the additional advantage of directly killing cancer cells, oncolytic viruses (OVs) constitute ideal platforms for such treatment strategy. However, given that the targeted tumor antigen is encoded into the viral genomes, its production requires robust infection and therefore, the vaccination efficiency partially depends on the unpredictable and highly variable intrinsic sensitivity of each tumor to OV infection. In this study, we demonstrate that anti-cancer vaccination using OVs (Adenovirus (Ad), Maraba virus (MRB), Vesicular stomatitis virus (VSV) and Vaccinia virus (VV)) co-administered with antigenic peptides is as efficient as antigen-engineered OVs and does not depend on viral replication. Our strategy is particularly attractive for personalized anti-cancer vaccines targeting patient-specific mutations. We suggest that the use of OVs as adjuvant platforms for therapeutic anti-cancer vaccination warrants testing for cancer treatment.

Colorectal cancer is the third most common neoplasm in the world and the third leading cause of cancer-related deaths in the USA. A safer and more effective therapeutic intervention against this malignant carcinoma is called for given the limitations and toxicities associated with the currently available treatment modalities. Immuno-oncolytic or oncolytic virotherapy, the use of viruses to selectively or preferentially kill cancer cells, has emerged as a potential anticancer treatment modality. Oncolytic viruses act as double-edged swords against the tumors through the direct cytolysis of cancer cells and the induction of antitumor immunity. A number of such viruses have been tested against colorectal cancer, in both preclinical and clinical settings, and many have produced promising results. Oncolytic virotherapy has also shown synergistic antitumor efficacy in combination with conventional treatment regimens. In this review, we describe the status of this therapeutic approach against colorectal cancer at both preclinical and clinical levels. Successes with and the challenges of using oncolytic viruses, both as monotherapy and in combination therapy, are also highlighted.

Introduction : Despite diverse treatment modalities and novel therapies, many cancers and patients are not effectively treated. Cancer immunotherapy has recently achieved breakthrough status yet is not effective in all cancer types or patients and can generate serious adverse effects. Oncolytic viruses (OVs) are a promising new therapeutic modality that harnesses virus biology and host interactions to treat cancer. OVs, genetically engineered or natural, preferentially replicate in and kill cancer cells, sparing normal cells/tissues, and mediating anti-tumor immunity. Areas covered : This review focuses on OVs as cancer therapeutic agents from a historical perspective, especially strategies to boost their immunotherapeutic activities. OVs offer a multifaceted platform, whose activities are modulated based on the parental virus and genetic alterations. In addition to direct viral effects, many OVs can be armed with therapeutic transgenes to also act as gene therapy vectors, and/or combined with other drugs or therapies. Expert opinion : OVs are an amazingly versatile and malleable class of cancer therapies. They tend to target cellular and host physiology as opposed to specific genetic alterations, which potentially enables broad responsiveness. The biological complexity of OVs have hindered their translation; however, the recent approval of talimogene laherparepvec (T-Vec) has invigorated the field.

Background: While programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) antagonists have improved the prognosis for many patients with melanoma, around 60% fail therapy. PVSRIPO is a non-neurovirulent rhinovirus:poliovirus chimera that facilitates an antitumor immune response following cell entry via the poliovirus receptor CD155, which is expressed on tumor and antigen-presenting cells. Preclinical studies show that oncolytic virus plus anti-PD-1 therapy leads to a greater antitumor response than either agent alone, warranting clinical investigation., Methods: An open-label phase I trial of intratumoral PVSRIPO in patients with unresectable melanoma (American Joint Committee on Cancer V.7 stage IIIB, IIIC, or IV) was performed. Eligible patients had disease progression on anti-PD-1 and V-raf murine sarcoma viral oncogene homolog B (BRAF)/mitogen activated protein kinase kinase (MEK) inhibitors (if BRAF mutant). The primary objective was to characterize the safety and tolerability of PVSRIPO. Twelve patients in four cohorts received a total of 1, 2 or 3 injections of PVSRIPO monotherapy, with 21 days between injections., Results: PVSRIPO injections were well tolerated with no serious adverse events (SAEs) or dose-limiting toxicities (DLTs) reported. All adverse events (AEs) were grade (G) 1 or G2 (G1 pruritus most common at 58%); all but two PVSRIPO-treatment related AEs were localized to the injected or adjacent lesions (n=1 G1 hot flash, n=1 G1 fatigue). Four out of 12 patients (33%) achieved an objective response per immune-related response criteria (two observations, 4 weeks apart), including 4/6 (67%) who received three injections. In the four patients with in-transit disease, a pathological complete response (pCR) was observed in two (50%) patients. Following study completion, 11/12 patients (92%) reinitiated immune checkpoint inhibitor-based therapy, and 6/12 patients (50%) remained without progression at a median follow-up time of 18 months., Conclusion: Intratumoral PVSRIPO was well tolerated. Despite the limited number of PVSRIPO treatments relative to the overall lesion burden (67% patients>5 lesions), intratumoral PVSRIPO showed promising antitumor activity, with pCR in injected as well as non-injected lesions in select patients., Trial Registration Number: NCT03712358., Competing Interests: Competing interests: SKN owns intellectual property related to this research, which has been licensed to Istari Oncology, Inc. DDB and MG have financial interest in Istari Oncology, Inc. Duke University (Licensor of PVSRIPO) has a financial interest in Istari Oncology, Inc., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Cancer virotherapy is a paradigm-shifting treatment modality based on virus-mediated oncolysis and subsequent antitumor immune responses. Clinical trials of currently available virotherapies showed that robust antitumor immunity characterizes the remarkable and long-term responses observed in a subset of patients. These data suggest that future therapies should incorporate strategies to maximize the immunotherapeutic potential of oncolytic viruses. In this review, we highlight the recent evidence that the antiviral immunity of the patients may limit the immunotherapeutic potential of oncolytic viruses and summarize the most relevant approaches to strategically redirect the immune response away from the viruses and toward tumors to heighten the clinical impact of viro-immunotherapy platforms., Competing Interests: Competing interests: FL, JF and CG-M are reporting ownership interest (including patents). CG-M and JF are consultants and shareholders of DNATrix., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: OH2 is a genetically engineered oncolytic herpes simplex virus type 2 designed to selectively amplify in tumor cells and express granulocyte-macrophage colony-stimulating factor to enhance antitumor immune responses. We investigated the safety, tolerability and antitumor activity of OH2 as single agent or in combination with HX008, an anti-programmed cell death protein 1 antibody, in patients with advanced solid tumors., Methods: In this multicenter, phase I/II trial, we enrolled patients with standard treatment-refractory advanced solid tumors who have injectable lesions. In phase I, patients received intratumoral injection of OH2 at escalating doses (10 6 , 10 7 and 10 8 CCID50/mL) as single agent or with fixed-dose HX008. The recommended doses were then expanded in phase II. Primary endpoints were safety and tolerability defined by the maximum-tolerated dose and dose-limiting toxicities (DLTs) in phase I, and antitumor activity assessed per Response Evaluation Criteria in Solid Tumors (RECIST version 1.1) and immune-RECIST in phase II., Results: Between April 17, 2019 and September 22, 2020, 54 patients with metastatic cancers were enrolled. Forty patients were treated with single agent OH2, and 14 with OH2 plus HX008. No DLTs were reported with single agent OH2 in phase I. Four patients, having metastatic mismatch repair-proficient rectal cancer or metastatic esophageal cancer, achieved immune-partial response, with two from the single agent cohort and two from the combination cohort. The duration of response were 11.25+ and 14.03+ months for the two responders treated with single agent OH2, and 1.38+ and 2.56+ months for the two responders in the combination cohort. The most common treatment-related adverse event (TRAE) with single agent OH2 was fever (n=18, 45.0%). All TRAEs were of grade 1-2, except one case of grade 3 fever in the 10 8 CCID50/mL group. No treatment-related serious AEs occurred. Single agent OH2 induced alterations in the tumor microenvironment, with clear increases in CD3 + and CD8 + cell density and programmed death-ligand 1 expression in the patients' post-treatment biopsies relative to baseline., Conclusions: Intratumoral injection of OH2 was well-tolerated, and demonstrated durable antitumor activity in patients with metastatic esophageal and rectal cancer. Further clinical development of OH2 as single agent or with immune checkpoint inhibitors in selected tumor types is warranted., Competing Interests: Competing interests: BL is the founder of Binhui Biopharmaceutical Co., Ltd. XG is a salaried employee of Binhui Biopharmaceutical Co., Ltd., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

In our clinical trials of oncolytic vesicular stomatitis virus expressing interferon beta (VSV-IFNβ), several patients achieved initial responses followed by aggressive relapse. We show here that VSV-IFNβ-escape tumors predictably express a point-mutated CSDE1 P5S form of the RNA-binding Cold Shock Domain-containing E1 protein, which promotes escape as an inhibitor of VSV replication by disrupting viral transcription. Given time, VSV-IFNβ evolves a compensatory mutation in the P/M Inter-Genic Region which rescues replication in CSDE1 P5S cells. These data show that CSDE1 is a major cellular co-factor for VSV replication. However, CSDE1 P5S also generates a neo-epitope recognized by non-tolerized T cells. We exploit this predictable neo-antigenesis to drive, and trap, tumors into an escape phenotype, which can be ambushed by vaccination against CSDE1 P5S , preventing tumor escape. Combining frontline therapy with escape-targeting immunotherapy will be applicable across multiple therapies which drive tumor mutation/evolution and simultaneously generate novel, targetable immunopeptidomes associated with acquired treatment resistance.

Colorectal cancer is an aggressive malignancy for which there are limited treatment options. Oncolytic vaccinia virus is being developed as a novel strategy for cancer therapy. Arming vaccinia virus with immunostimulatory cytokines can enhance the tumor cell-specific replication and antitumor efficacy. Interleukin-24 (IL-24) is an important immune mediator, as well as a broad-spectrum tumor suppressor. We constructed a targeted vaccinia virus of Guang9 strain harboring IL-24 (VG9-IL-24) to evaluate its antitumor effects. In vitro, VG9-IL-24 induced an increased number of apoptotic cells and blocked colorectal cancer cells in the G₂/M phase of the cell cycle. VG9-IL-24 induced apoptosis in colorectal cancer cells via multiple apoptotic signaling pathways. In vivo, VG9-IL-24 significantly inhibited the tumor growth and prolonged the survival both in human and murine colorectal cancer models. In addition, VG9-IL-24 stimulated multiple antitumor immune responses and direct bystander antitumor activity. Our results indicate that VG9-IL-24 can inhibit the growth of colorectal cancer tumor by inducing oncolysis and apoptosis as well as stimulating the antitumor immune effects. These findings indicate that VG9-IL-24 may exert a potential therapeutic strategy for combating colorectal cancer.

Talimogene laherparepvec (T-VEC) is a genetically modified herpes simplex 1 virus (HSV-1) approved for cancer therapy. We investigate its effect on the clinical, histological, single-cell transcriptomic, and immune repertoire level using repeated fine-needle aspirates (FNAs) of injected and noninjected lesions in primary cutaneous B cell lymphoma (pCBCL). Thirteen patients received intralesional T-VEC, 11 of which demonstrate tumor response in the injected lesions. Using single-cell sequencing of the FNAs, we identify the malignant population and separate three pCBCL subtypes. Twenty-four hours after the injection, we detect HSV-1 T-VEC transcripts in malignant and nonmalignant cells of the injected lesion but not of the noninjected lesion. Oncolytic virotherapy results in a rapid eradication of malignant cells. It also leads to interferon pathway activation and early influx of natural killer cells, monocytes, and dendritic cells. These events are followed by enrichment in cytotoxic T cells and a decrease of regulatory T cells in injected and noninjected lesions., Competing Interests: Declaration of interests The project was sponsored by University of Zurich, Switzerland, partially supported by a research grant provided by Amgen. R.D. has intermittent, project focused consulting and/or advisory relationships with Novartis, Merck Sharp & Dhome, Bristol-Myers Squibb (BMS), Roche, Amgen, Takeda, Pierre Fabre, Sun Pharma, Sanofi, Catalym, Second Genome, Regeneron, and Alligator outside the submitted work. Research in C.B.’s lab was supported by the Swiss National Science Foundation, ETH domain Personalized Health and Related Technologies (PHRT-510). E.G. was supported by the Promedica Stiftung (1406/M and 1412/M), the Swiss Cancer Research Foundation (KFS-4243-08-2017), and the Clinical Research Priority Program of the University of Zurich outside the submitted work. E.R. has intermittent, project focused consulting relationships with Amgen, BMS, and Sanofi outside the submitted work. Other authors have no interests to declare., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Programmed death-ligand 1 (PD-L1) is an important immune checkpoint protein that can be regarded as a pan-cancer antigen expressed by multiple different cell types within the tumor. While antagonizing PD-L1 is well known to relieve PD-1/PD-L1-mediated T cell suppression, here we have combined this approach with an immunotherapy strategy to target T cell cytotoxicity directly toward PD-L1-expressing cells. We developed a bi-specific T cell engager (BiTE) crosslinking PD-L1 and CD3ε and demonstrated targeted cytotoxicity using a clinically relevant patient-derived ascites model. This approach represents an immunological 'volte-face' whereby a tumor immunological defense mechanism can be instantly transformed into an Achilles' heel for targeted immunotherapy., Methods: The PD-L1 targeting BiTE comprises an anti-PD-L1 single-chain variable fragment (scFv) or nanobody (NB) domain and an anti-CD3 scFv domain in a tandem repeat. The ability to activate T cell cytotoxicity toward PD-L1-expressing cells was established using human carcinoma cells and PD-L1-expressing human ('M2') macrophages in the presence of autologous T cells. Furthermore, we armed oncolytic herpes simplex virus-1 (oHSV-1) with PD-L1 BiTE and demonstrated successful delivery and targeted cytotoxicity in unpurified cultures of malignant ascites derived from different cancer patients., Results: PD-L1 BiTE crosslinks PD-L1-positive cells and CD3ε on T cells in a 'pseudo-synapse' and triggers T cell activation and release of proinflammatory cytokines such as interferon-gamma (IFN-γ), interferon gamma-induced protein 10 (IP-10) and tumour necrosis factor-α (TNF-α). Activation of endogenous T cells within ascites samples led to significant lysis of tumor cells and M2-like macrophages (CD11b+CD64+ and CD206+/CD163+). The survival of CD3+ T cells (which can also express PD-L1) was unaffected. Intriguingly, ascites fluid that appeared particularly immunosuppressive led to higher expression of PD-L1 on tumor cells, resulting in improved BiTE-mediated T cell activation., Conclusions: The study reveals that PD-L1 BiTE is an effective immunotherapeutic approach to kill PD-L1-positive tumor cells and macrophages while leaving T cells unharmed. This approach activates endogenous T cells within malignant ascites, generates a proinflammatory response and eliminates cells promoting tumor progression. Using an oncolytic virus for local expression of PD-L1 BiTE also prevents 'on-target off-tumor' systemic toxicities and harnesses immunosuppressive protumor conditions to augment immunotherapy in immunologically 'cold' clinical cancers., Competing Interests: Competing interests: None., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ.)

Background: Talimogene laherparepvec (T-VEC), an oncolytic virus, was designed to selectively replicate in and lyse tumor cells, releasing tumor-derived antigen to stimulate a tumor-specific immune response., Methods: In this phase II study in patients with unresectable stage IIIB-IV melanoma, we evaluated non-injected lesions to establish whether baseline or change in intratumoral CD8 + T-cell density (determined using immunohistochemistry) correlated with T-VEC clinical response., Results: Of 112 enrolled patients, 111 received ≥1 dose of T-VEC. After a median follow-up of 108.0 weeks, objective/complete response rates were 28%/14% in the overall population and 32%/18% in patients with stage IIIB-IVM1a disease. No unexpected toxicity occurred. Baseline and week 6 change from baseline CD8 + T-cell density results were available for 91 and 65 patients, respectively. Neither baseline nor change in CD8 + T-cell density correlated with objective response rate, changes in tumor burden, duration of response or durable response rate. However, a 2.4-fold median increase in CD8 + T-cell density in non-injected lesions from baseline to week 6 was observed. In exploratory analyses, multiparameter immunofluorescence showed that after treatment there was an increase in the proportion of infiltrating CD8 + T-cells expressing granzyme B and checkpoint markers (programmed death-1, programmed death-ligand 1 (PD-L1) and cytotoxic T-lymphocyte antigen-4) in non-injected lesions, together with an increase in helper T-cells. Consistent with T-cell infiltrate, we observed an increase in the adaptive resistance marker PD-L1 in non-injected lesions., Conclusions: This study indicates that T-VEC induces systemic immune activity and alters the tumor microenvironment in a way that will likely enhance the effects of other immunotherapy agents in combination therapy., Trial Registration Number: NCT02366195., Competing Interests: Competing interests: JM has acted as a consultant/advisor for and has received honoraria from Almirall, Amgen, Bristol-Myers Squibb, GlaxoSmithKline, Merck Sharp & Dohme, Leo Pharma, Novartis, Pierre Fabre and Roche. IS has acted as a consultant and served on a speaker’s bureau for Biocad, Bristol-Myers Squibb, Merck Sharp & Dohme, Novartis and Roche. DS has received research support from Bristol-Myers Squibb and Novartis; has acted as a consultant for and received royalties from Amgen, Array, AstraZeneca, Bristol-Myers Squibb, Merck Sharp & Dohme, Novartis, Pierre Fabre, and Roche; and has acted as a consultant for Immunocore, Incyte, LeoPharma, Merck Serono, Pfizer, Philogen, and Regeneron. RG has received research support from Johnson & Johnson, Novartis, Merck Serono, Amgen and Pfizer; has received honoraria for advice and/or lectures from Almirall Hermal, Amgen, Bayer, Bristol-Myers Squibb, Merck Serono, Merck Sharp & Dohme, Novartis, Pierre Fabre, Roche, Sanofi/Regeneron, SUN Pharma and 4SC. J-JG has acted as a consultant/in an advisory role for and has received honoraria from Bristol-Myers Squibb, GlaxoSmithKline, Merck Sharp & Dohme, Merck-Pfizer, Pierre Fabre, and Roche. JJS has received research support from AstraZeneca, Bristol-Myers Squibb and Immunocore; has acted as a consultant/in an advisory role for Immunocore, Delcath, Merck Sharp & Dohme, Amgen and Pierre Fabre; and served on a speaker’s bureau for Bristol-Myers Squibb. KSG and AA are employees and stock holders of Amgen. KL is an employee of Amgen. CAP was previously an employee of Amgen. HG has acted as a consultant/in an advisory role for Bristol-Myers Squibb, Merck Sharp & Dohme, Novartis, Amgen, Roche and Pierre Fabre., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Osteosarcoma is the most common malignant solid tumor that affects bones, however, survival rates of patients with relapsed osteosarcoma have not improved in the last 30 years. Oncolytic virotherapy, which uses viruses designed to selectively replicate in cancer cells, has emerged as a promising treatment for solid tumors. Our group uses mesenchymal stem cells (MSCs) to transport oncolytic adenoviruses (OAds) to the tumor site, a therapeutic strategy called Celyvir. This treatment has been already applied in human patients, canine patients and different mouse models. In parallel, previous results have probed that administration of granulocyte-colony stimulating factor (G-CSF) increased immune infiltration in tumors. We then hypothesized that the mobilization of immune cells by G-CSF may increase the antitumor efficacy of Celyvir treatment by increasing the immune infiltration into the tumors., Methods: In this study, we use a murine version of Celyvir consisting in murine MSCs carrying the murine OAd dlE102-here called OAd-MSCs-in an immunocompetent model of osteosarcoma. We tested the antitumoral efficacy of the combination of OAd-MSCs plus G-CSF., Results: Our results show that treatment with OAd-MSCs or the union of OAd-MSCs with G-CSF (Combination) significantly reduced tumor growth of osteosarcoma in vivo. Moreover, treated tumors presented higher tumor infiltration of immune cells-especially tumor-infiltrating lymphocytes-and reduced T cell exhaustion, which seems to be enhanced in tumors treated with the Combination. The comparison of our results to those obtained from a cohort of pediatric osteosarcoma patients showed that the virotherapy induces immunological changes similar to those observed in patients with good prognosis., Conclusions: The results open the possibility of using cellular virotherapy for the treatment of bone cancers. Indeed, its combination with G-CSF may be considered for the improvement of the therapy., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Multiple myeloma (MM) remains an incurable disease and oncolytic viruses offer a well-tolerated addition to the therapeutic arsenal. Oncolytic reovirus has progressed to phase I clinical trials and its direct lytic potential has been extensively studied. However, to date, the role for reovirus-induced immunotherapy against MM, and the impact of the bone marrow (BM) niche, have not been reported., Methods: This study used human peripheral blood mononuclear cells from healthy donors and in vitro co-culture of MM cells and BM stromal cells to recapitulate the resistant BM niche. Additionally, the 5TGM1-Kalw/RijHSD immunocompetent in vivo model was used to examine reovirus efficacy and characterize reovirus-induced immune responses in the BM and spleen following intravenous administration. Collectively, these in vitro and in vivo models were used to characterize the development of innate and adaptive antimyeloma immunity following reovirus treatment., Results: Using the 5TGM1-Kalw/RijHSD immunocompetent in vivo model we have demonstrated that reovirus reduces both MM tumor burden and myeloma-induced bone disease. Furthermore, detailed immune characterization revealed that reovirus: (i) increased natural killer (NK) cell and CD8 + T cell numbers; (ii) activated NK cells and CD8 + T cells and (iii) upregulated effector-memory CD8 + T cells. Moreover, increased effector-memory CD8 + T cells correlated with decreased tumor burden. Next, we explored the potential for reovirus-induced immunotherapy using human co-culture models to mimic the myeloma-supportive BM niche. MM cells co-cultured with BM stromal cells displayed resistance to reovirus-induced oncolysis and bystander cytokine-killing but remained susceptible to killing by reovirus-activated NK cells and MM-specific cytotoxic T lymphocytes., Conclusion: These data highlight the importance of reovirus-induced immunotherapy for targeting MM cells within the BM niche and suggest that combination with agents which boost antitumor immune responses should be a priority., Competing Interests: Competing interests: MC is affiliated with Oncolytics Biotec Inc., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ.)