Padma Sampath

Chemokine Expression From Oncolytic Vaccinia Virus Enhances Vaccine Therapies of Cancer

Tumor vaccines can induce robust immune responses targeting tumor antigens in the clinic, but antitumor effects have been disappointing. One reason for this is ineffective tumor infiltration of the cytotoxic T lymphocytes (CTLs) produced. Oncolytic viruses are capable of selectively replicating within tumor tissue and can induce a strong immune response. We therefore sought to determine whether these therapies could be rationally combined such that modulation of the tumor microenvironment by the viral therapy could help direct beneficial CTLs induced by the vaccine. As such, we examined the effects of expressing chemokines from oncolytic vaccinia virus, including CCL5 (RANTES), whose receptors are expressed on CTLs induced by different vaccines, including type-1-polarized dendritic cells (DC1). vvCCL5, an oncolytic vaccinia virus expressing CCL5, induced chemotaxis of lymphocyte populations in vitro and in vivo, and displayed improved safety in vivo. Interestingly, enhanced therapeutic benefits with vvCCL5 in vivo correlated with increased persistence of the viral agent exclusively within the tumor. When tumor-bearing mice were both vaccinated with DC1 and treated with vvCCL5 a further significant enhancement in tumor response was achieved which correlated with increased levels of tumor infiltrating lymphocytes. This approach therefore represents a novel means of combining biological therapies for cancer treatment.

Defining Effective Combinations of Immune Checkpoint Blockade and Oncolytic Virotherapy

Purpose: Recent data from randomized clinical trials with oncolytic viral therapies and with cancer immunotherapies have finally recapitulated the promise these platforms demonstrated in preclinical 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 antitumor 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. Clin Cancer Res; 21(24); 5543–51. ©2015 AACR. See related commentary by Slaney and Darcy, p. 5417

Targeting Localized Immune Suppression Within the Tumor Through Repeat Cycles of Immune Cell-oncolytic Virus Combination Therapy

A major limitation to the use of immunotherapy in the treatment of cancer has been the localized immune suppressive environment within the tumor. Although there is evidence that tumor-selective (oncolytic) viruses may help to overcome this immune suppression, a primary limitation to their use has been limited systemic delivery potential, especially in the face of antiviral immunity. We recently demonstrated that tumor-trafficking immune cells can efficiently deliver oncolytic viral therapies to their tumor targets. These cells act as both a therapeutic agent and also a carrier vehicle for the oncolytic virus. Here, we demonstrate that such delivery is also possible in the face of pre-existing antiviral immunity, so overcoming the limited systemic delivery of naked, cell-free virus. It was also found that treatment of previously immunized mice or repeat treatments leading to immunization resulted in a switch from a primarily oncolytic to an immunotherapeutic mechanism of action. Furthermore, repeat cycles of treatment with combination immune cell-viral therapy resulted in increased tumor infiltration of effector T-cells and a general reduction in the levels of known immune suppressive lymphocyte populations. This therefore represents a novel and effective means to overcome localized immune suppression within the tumor microenvironment.

Modulation of NKG2D-ligand cell surface expression enhances immune cell therapy of cancer.

A variety of immune cell therapies proposed for use in the treatment of cancer, including both autologous cells (Lymphokine Activated Killer, Cytokine Induced Killer) or cell lines (TALL-104, NK-92), rely on recognition of NKG2D ligands on malignant cells for targeting. These ligands, such as MICA and MICB in humans are stress response ligands and are commonly, but not ubiquitously expressed within tumors. Several tumor escape mechanisms have been reported, including ligand downregulation and internalization, or proteolytic cleavage and shedding of their exposed portions (releasing soluble MICA and MICB; sMICA, sMICB). Therefore, an ability to prescreen patients for the level of tumor cell surface expression and shedding of these ligands would prevent needless treatment of patients that are unable to respond, whereas targeted pretreatment of patients to increase surface expression and/or block shedding would enhance the subsequent effectiveness of these therapies. Here, we report that serum tests of sMICA and sMICB in conjunction with tumor measurements might be used to determine rates of shedding from a tumor and that treatment with a selected combination of histone deacetylase inhibitors (to upregulate cell surface MICA/B in some tumors), and metalloproteinase inhibitors (to block MICA/B shedding in others) can be incorporated to regulate cell surface MICA/B levels before immune cell therapy, significantly enhancing their effectiveness (either used alone or as carrier vehicles for oncolytic viruses). Ultimately prescreening patients undergoing such immune cell therapies might be used to personalize cancer treatment regimens based on the NKG2D-ligand status of the tumor.

Alternate mechanisms of initial pattern recognition drive differential immune responses to related poxviruses.

Vaccinia immunization was pivotal to successful smallpox eradication. However, the early immune responses that distinguish poxvirus immunization from pathogenic infection remain unknown. To address this, we developed a strategy to map the activation of key signaling networks in vivo and applied this approach to define and compare the earliest signaling events elicited by immunizing (vaccinia) and lethal (ectromelia) poxvirus infections in mice. Vaccinia induced rapid TLR2-dependent responses, leading to IL-6 production, which then initiated STAT3 signaling in dendritic and T cells. In contrast, ectromelia did not induce TLR2 activation, and profound mouse strain-dependent responses were observed. In resistant C57BL/6 mice, the STAT1 and STAT3 pathways were rapidly activated, whereas in susceptible BALB/c mice, IL-6-dependent STAT3 activation did not occur. These data link early immune signaling events to infection outcome and suggest that activation of different pattern-recognition receptors early after infection may be important in determining vaccine efficacy.

Crosstalk Between Immune Cell and Oncolytic Vaccinia Therapy Enhances Tumor Trafficking and Antitumor Effects

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.

Regulating Cytokine Function Enhances Safety and Activity of Genetic Cancer Therapies

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.

Oncolytic vaccinia virus demonstrates antiangiogenic effects mediated by targeting of VEGF

Oncolytic vaccinia virus has been shown to induce a profound, rapid and tumor‐specific vascular collapse in both preclinical models and 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 revascularization after viral therapy was dramatically delayed and did not occur until after viral clearance. This indicated that oncolytic vaccinia may possess a previously undescribed antiangiogenic 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 antiangiogenic 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 antiangiogenic 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.

Fluoromodule-based reporter/probes designed for in vivo fluorescence imaging

Optical imaging of whole, living animals has proven to be a powerful tool in multiple areas of preclinical research and has allowed noninvasive monitoring of immune responses, tumor and pathogen growth, and treatment responses in longitudinal studies. However, fluorescence-based studies in animals are challenging because tissue absorbs and autofluoresces strongly in the visible light spectrum. These optical properties drive development and use of fluorescent labels that absorb and emit at longer wavelengths. Here, we present a far-red absorbing fluoromodule-based reporter/probe system and show that this system can be used for imaging in living mice. The probe we developed is a fluorogenic dye called SC1 that is dark in solution but highly fluorescent when bound to its cognate reporter, Mars1. The reporter/probe complex, or fluoromodule, produced peak emission near 730 nm. Mars1 was able to bind a variety of structurally similar probes that differ in color and membrane permeability. We demonstrated that a tool kit of multiple probes can be used to label extracellular and intracellular reporter-tagged receptor pools with 2 colors. Imaging studies may benefit from this far-red excited reporter/probe system, which features tight coupling between probe fluorescence and reporter binding and offers the option of using an expandable family of fluorogenic probes with a single reporter gene.

Manipulating TLR Signaling Increases the Anti-tumor T Cell Response Induced by Viral Cancer Therapies

SUMMARY The 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.