Oncolytic virotherapy (OVT) has received significant attention in recent years, especially since the approval of talimogene Laherparepvec (T-VEC) in 2015 by the Food and Drug administration (FDA). Mechanistic studies of oncolytic viruses (OVs) have revealed that most, if not all, OVs induce direct oncolysis and stimulate innate and adaptive anti-tumour immunity. With the advancement of tumour modelling, allowing characterisation of the effects of tumour microenvironment (TME) components and identification of the cellular mechanisms required for cell death (both direct oncolysis and anti-tumour immune responses), it is clear that a “one size fits all” approach is not applicable to all OVs, or indeed the same OV across different tumour types and disease locations. This article will provide an unbiased review of oncolytic reovirus (clinically formulated as pelareorep), including the molecular and cellular requirements for reovirus oncolysis and anti-tumour immunity, reports of pre-clinical efficacy and its overall clinical trajectory. Moreover, as it is now abundantly clear that the true potential of all OVs, including reovirus, will only be reached upon the development of synergistic combination strategies, reovirus combination therapeutics will be discussed, including the limitations and challenges that remain to harness the full potential of this promising therapeutic agent.

Background The oncolytic virus, coxsackievirus A21 (CVA21), has shown promise as a single agent in several clinical trials and is now being tested in combination with immune checkpoint blockade. Combination therapies offer the best chance of disease control; however, the design of successful combination strategies requires a deeper understanding of the mechanisms underpinning CVA21 efficacy, in particular, the role of CVA21 anti-tumor immunity. Therefore, this study aimed to examine the ability of CVA21 to induce human anti-tumor immunity, and identify the cellular mechanism responsible. Methods This study utilized peripheral blood mononuclear cells from i) healthy donors, ii) Acute Myeloid Leukemia (AML) patients, and iii) patients taking part in the STORM clinical trial, who received intravenous CVA21; patients receiving intravenous CVA21 were consented separately in accordance with local institutional ethics review and approval. Collectively, these blood samples were used to characterize the development of innate and adaptive anti-tumor immune responses following CVA21 treatment. Results An Initial characterization of peripheral blood mononuclear cells, collected from cancer patients following intravenous infusion of CVA21, confirmed that CVA21 activated immune effector cells in patients. Next, using hematological disease models which were sensitive (Multiple Myeloma; MM) or resistant (AML) to CVA21-direct oncolysis, we demonstrated that CVA21 stimulated potent anti-tumor immune responses, including: 1) cytokine-mediated bystander killing; 2) enhanced natural killer cell-mediated cellular cytotoxicity; and 3) priming of tumor-specific cytotoxic T lymphocytes, with specificity towards known tumor-associated antigens. Importantly, immune-mediated killing of both MM and AML, despite AML cells being resistant to CVA21-direct oncolysis, was observed. Upon further examination of the cellular mechanisms responsible for CVA21-induced anti-tumor immunity we have identified the importance of type I IFN for NK cell activation, and demonstrated that both ICAM-1 and plasmacytoid dendritic cells were key mediators of this response. Conclusion This work supports the development of CVA21 as an immunotherapeutic agent for the treatment of both AML and MM. Additionally, the data presented provides an important insight into the mechanisms of CVA21-mediated immunotherapy to aid the development of clinical biomarkers to predict response and rationalize future drug combinations. Electronic supplementary material The online version of this article (10.1186/s40425-019-0632-y) contains supplementary material, which is available to authorized users.

BackgroundMultiple 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.MethodsThis 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.ResultsUsing 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.ConclusionThese 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.

Introduction: The aim of this study was to examine the contribution of antitumor immunity for the efficacy of oncolytic reovirus therapy against multiple myeloma (MM). Oncolytic viruses (OV) have two main mechanisms of action; direct lytic killing and potentiation of antitumor immunity. The direct oncolytic potential of reovirus in MM has previously been demonstrated both in vitro and in vivo, but the importance of an enhanced immunologic antitumor response remains underinvestigated. Reovirus-induced antitumor immunity has been demonstrated in other types of cancer such as melanoma and chronic leukemia, and is of major importance for the efficacy of OV treatment. Thus, it was hypothesized that reovirus-induced antimyeloma immunity would contribute significantly to the efficacy of reovirus treatment for MM. Experimental Procedures: C57BL/KaLwRij mice were used in the 5TGM1 model system to establish MM in vivo. This model closely resembles human MM with induction of osteolytic bone disease and secretion of paraprotein. C57BL/KaLwRij mice have a fully functional immune system, comparable to C57BL/6 mice, and to our knowledge, this is the first immunocompetent model of MM for the study of reovirus efficacy. After establishment of MM in the bone by intravenous injection of bone-homing 5TGM1 cells, mice were treated with repeated injections of reovirus or PBS. Upon sacrifice, direct cytotoxicity and immune activation was examined using flow cytometry. All animal experiments were performed under an appropriate project license following approval by a local ethical review committee. In vivo findings were translated into human in vitro studies, using MM cell lines, healthy donor (HD) blood, and MM patient samples. Reovirus-induced Natural Killer (NK) cell activation and degranulation was examined using flow cytometry and priming of myeloma-specific T cells was performed using long-term priming cultures. Results: Tumor burden was reduced by reovirus treatment both in the bone marrow (BM) and spleen of tumor-bearing mice. The immune cell populations, including NK cells, CD4+ T cells, and CD8+ T cells, were restored to levels of tumor-naïve mice in the BM. NK cells were activated in the BM following reovirus treatment, which indicates the onset of an innate immune response. In the spleen, an increase in CD4+ T cells, in combination with CD8+ activation, was indicative of an early adaptive immune response. These results translate into human in vitro findings, using both HD and MM patient samples, with activation of NK cells in response to reovirus treatment and subsequent enhancement of NK cell degranulation and killing of MM target cells. Encouragingly, reovirus-activated NK cells were able to kill OPM2 cells, which are resistant to direct lytic killing. Preliminary human in vitro studies suggest that reovirus treatment can prime antimyeloma CD8+ T cells for the induction of a long-term protective response. Conclusions: Introducing a viral agent into the body requires a delicate immunologic balance to avoid neutralizing the virus by an antiviral response and simultaneously allowing the enhancement of antitumor immunity. The importance of enhanced antitumor immunity for OV therapy efficacy is becoming more widely recognized and antiviral immunity can in some circumstances contribute to tumor eradication. The findings in this study indicate that the antitumor immune response is also important in the MM setting, with activation of both innate and adaptive immune responses resulting in enhanced killing of MM cells, in particular those resistant to direct lytic killing. This suggests that both arms of OV therapy could play a role for MM eradication, including any minimal residual disease. Future work will explore whether the antimyeloma response can be further enhanced by combinatorial treatments, including current standard of care treatments. Citation Format: Louise M. E. Müller, Christopher Parrish, Gemma Migneco, Gina B. Scott, Matthew Holmes, Alan A. Melcher, Michelle A. Lawson, Gordon Cook, Fiona Errington-Mais. Evaluating the contribution of anti-myeloma immunity for the efficacy of oncolytic reovirus therapy [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 18.

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