Purpose Recent data from randomized clinical trials with oncolytic viral therapies and with cancer immunotherapies have finally recapitulated the promise these platforms demonstrated in pre-clinical models. Perhaps the greatest advance with oncolytic virotherapy has been the appreciation of the importance of activation of the immune response in therapeutic activity. Meanwhile, the understanding that blockade of immune checkpoints (with antibodies that block the binding of PD1 to PDL1 or CTLA4 to B7-2) is critical for an effective anti-tumor immune response has revitalized the field of immunotherapy. The combination of immune activation using an oncolytic virus and blockade of immune checkpoints is therefore a logical next step. Experimental Design Here we explore such combinations and demonstrate their potential to produce enhanced responses in mouse tumor models. Different combinations and regimens were explored in immunocompetent mouse models of renal and colorectal cancer. Bioluminescence imaging and immune assays were used to determine the mechanisms mediating synergistic or antagonistic combinations. Results Interaction between immune checkpoint inhibitors and oncolytic virotherapy was found to be complex, with correct selection of viral strain, antibody and timing of the combination being critical for synergistic effects. Indeed, some combinations produced antagonistic effects and loss of therapeutic activity. A period of oncolytic viral replication and directed targeting of the immune response against the tumor were required for the most beneficial effects, with CD8+ and NK, but not CD4+ cells mediating the effects. Conclusions These considerations will be critical in the design of the inevitable clinical translation of these combination approaches.

SUMMARY Immunotherapies are highly promising cancer treatments, but understanding the factors mediating their resistance remains critical. Successes in randomized clinical testing have supported the growing appreciation that oncolytic virotherapies primarily act as immunotherapies. Here we identified prostaglandin E2 (PGE2) in the tumor as a key mediator of resistance to immunotherapies, including oncolytic vaccinia virotherapy. Elevated levels of PGE2 coupled to suppressive chemokine profiles and high levels of granulocytic myeloid derived suppressor cells (MDSC) resulted in loss of immunotherapeutic potential. Viral vectors engineered to target PGE2 were capable of overcoming localized immunosuppression leading to profound changes in the tumor’s immune status. This allowed the viral vectors to raise robust anti-tumor adaptive immune responses and sensitized established and previously resistant tumors to immunotherapies.

Multiple studies have associated the transcription factor IRF1 with tumor-suppressive activities. Here, we report an opposite tumor cell-intrinsic function of IRF1 in promoting tumor growth. IRF1-deficient tumor cells showed reduced tumor growth in MC38 and CT26 colon carcinoma and B16 melanoma mouse models. This reduction in tumor growth was dependent on host CD8 þ T cells. Detailed profiling of tumor-infiltrating leukocytes did not show changes in the various T-cell and myeloid cell populations. However, CD8 þ T cells that had infiltrated IRF1-deficieint tumors in vivo exhibited enhanced cytotoxicity. IRF1-deficient tumor cells lost the ability to upregulate PD-L1 expression in vitro and in vivo and were more susceptible to T-cell-mediated killing. Induced expression of PD-L1 in IRF1-deficient tumor cells restored tumor growth. These results indicate differential activity of IRF1 in tumor escape.

Oncolytic vaccinia virus has been shown to induce a profound, rapid and tumor-specific vascular collapse in both preclinical models and in clinical studies, however a complete examination of the kinetics and levels of collapse and revascularization has not been described previously. Contrast-enhanced ultrasound was used to follow tumor perfusion levels in mouse tumor models at times after vaccinia therapy. It was observed that re-vascularization after viral therapy was dramatically delayed and did not occur until after viral clearance. This indicated that oncolytic vaccinia may posses a previously undescribed anti-angiogenic potential that might synergize with the reported anti-vascular effects. Despite a rapid loss of perfusion and widespread hypoxia within the tumor it was observed that VEGF levels in the tumor were suppressed throughout the period of active viral infection. Although tumor vasculature could eventually reform after the viral therapy was cleared in mouse models, anti-tumor effects could be significantly enhanced through additional combination with anti-VEGF therapies. This was initially examined using a gene therapy approach (Ad-Flk1-Fc) to target VEGF directly, demonstrating that the timing of application of the anti-angiogenic therapy was critical. However, it is also known that oncolytic vaccinia sensitizes tumors to tyrosine kinase inhibitors (TKI) in the clinic through an unknown mechanism. It is possible this phenomenon may be mediated through the anti-angiogenic effects of the TKIs. This was modeled in mouse tumors using sunitinib in combination with oncolytic vaccinia. It was observed that prevention of angiogenesis mediated by oncolytic vaccinia can be utilized to enhance the TKI therapy.

The combination of an oncolytic virus, that directly destroys tumor cells and mediates an acute immune response, with an immune cell therapy, capable of further enlisting and enhancing the host immune response, has the potential to create a potent therapeutic effect. We have previously developed several strategies for optimizing the delivery of oncolytic vaccinia virus vectors to their tumor targets, including the use of immune cell-based carrier vehicles and the incorporation of mutations that increase production of the enveloped form of vaccinia (extracellular enveloped viral (EEV)) that is better adapted to spread within a host. Here, we initially combine these approaches to create a novel therapeutic, consisting of an immune cell (cytokine-induced killer, CIK) preloaded with an oncolytic virus that is EEV enhanced. This resulted in direct interaction between the viral and immune cell components with each assisting the other in directing the therapy to the tumor and so enhancing the antitumor effects. This effect could be further improved through CCL5 expression from the virus. The resulting multicomponent therapy displays the ability for synergistic crosstalk between components, so significantly enhancing tumor trafficking and antitumor effects.

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Genetic therapies, including transfected immune cells and viral vectors, continue to show clinical responses as systemically deliverable and targeted therapeutics, with the first such approaches having been approved for cancer treatment. The majority of these employ cytokine transgenes. However, expression of cytokines early after systemic delivery can result in increased toxicity and nonspecific induction of the immune response. In addition, premature immune-mediated clearance of the therapy may result, especially for viral-based approaches. Here, it was initially verified that cytokine (interleukin (IL)2) or chemokine (CCL5) expression from a systemically delivered oncolytic virus resulted in reduced oncolytic activity and suboptimal immune activation, while IL2 also resulted in increased toxicity. However, all these limitations could be overcome through incorporation of exogenous regulation of cytokine or chemokine transgene function through fusion of a small and externally controllable destabilizing domain to the protein of interest. Regulation allowed an initial phase without cytokine function, permitting enhanced delivery and oncolytic activity before activation of cytokine function and a subsequent phase of enhanced and tumor-targeted immunotherapeutic activity. As a result of this exogenous regulation of cytokine function, both oncolytic and immune-mediated mechanisms of action were optimized, greatly enhancing therapeutic activity, while toxicity was significantly reduced.

SUMMARYThe immune response plays a key role in enhancing the therapeutic activity of oncolytic virotherapies. However, to date, investigators have relied on inherent interactions between the virus and the immune system, often coupled to the expression of a single cytokine transgene. Recently, the importance of TLR activation in mediating adaptive immunity has been demonstrated. We therefore sought to influence the type and level of immune response raised after oncolytic vaccinia therapy through manipulation of TLR signaling. Vaccinia naturally activates TLR2, associated with an antibody response, whereas a CTL response is associated with TLR3-TRIF-signaling pathways. We manipulated TLR signaling by vaccinia through deglycosylation of the viral particle to block TLR2 activation and expression of a TRIF transgene. The resulting vector displayed greatly reduced production of anti-viral neutralizing antibody as well as an increased anti-tumor CTL response. Delivery in both naive and pre-treated mice was enhanced and immunotherapeutic activity dramatically improved.