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Oncolytic viral therapy utilizes a tumor-selective replicating virus which preferentially infects and destroys cancer cells and triggers antitumor immunity. The Western Reserve strain of vaccinia virus (VV) is the most virulent strain of VV in animal models and has been engineered for tumor selectivity through two targeted gene deletions (vvDD). We performed the first-in-human phase 1, intratumoral dose escalation clinical trial of vvDD in 16 patients with advanced solid tumors. In addition to safety, we evaluated signs of vvDD replication and spread to distant tumors, pharmacokinetics and pharmacodynamics, clinical and immune responses to vvDD. Dose escalation proceeded without dose-limiting toxicities to a maximum feasible dose of 3 × 109 pfu. vvDD replication in tumors was reproducible. vvDD genomes and/or infectious particles were recovered from injected (n = 5 patients) and noninjected (n = 2 patients) tumors. At the two highest doses, vvDD genomes were detected acutely in blood in all patients while delayed re-emergence of vvDD genomes in blood was detected in two patients. Fifteen of 16 patients exhibited late symptoms, consistent with ongoing vvDD replication. In summary, intratumoral injection of the oncolytic vaccinia vvDD was well-tolerated in patients and resulted in selective infection of injected and noninjected tumors and antitumor activity.

\"Dr. Stephen Hawking was the most renowned scientist since Einstein, known both for his groundbreaking work in physics and cosmology and for his mischievous sense of humor. He educated millions of readers about the origins of the universe and the nature of black holes, and inspired millions more by defying a terrifying early prognosis of ALS, which originally gave him only two years to live. In later life he could communicate only by using a few facial muscles, but he continued to advance his field and serve as a revered voice on social and humanitarian issues. Hawking not only unraveled some of the universe's greatest mysteries but also believed science plays a critical role in fixing problems here on Earth. Now he turns his attention to the most urgent issues facing us. Will humanity survive? Should we colonize space? Does God exist? These are just a few of the questions Hawking addresses in this wide-ranging, passionately argued final book from one of the greatest minds in history. Featuring a foreword by Eddie Redmayne, who won an Oscar playing Stephen Hawking, an introduction by Nobel Laureate Kip Thorne, and an afterword from Hawking's daughter, Lucy.\" -- (Source of summary not specified)

Serum lactate dehydrogenase (LDH) is a prognostic factor for patients with stage IV melanoma. To gain insights into the biology underlying this prognostic factor, we analyzed total serum LDH, serum LDH isoenzymes, and serum lactate in up to 49 patients with metastatic melanoma. Our data demonstrate that high serum LDH is associated with a significant increase in LDH isoenzymes 3 and 4, and a decrease in LDH isoenzymes 1 and 2. Since LDH isoenzymes play a role in both glycolysis and oxidative phosphorylation (OXPHOS), we subsequently determined using tissue microarray (TMA) analysis that the levels of proteins associated with mitochondrial function, lactate metabolism, and regulators of glycolysis were all elevated in advanced melanomas compared with nevic melanocytes. To investigate whether in advanced melanoma, the glycolysis and OXPHOS pathways might be linked, we determined expression of the monocarboxylate transporters (MCT) 1 and 4. Analysis of a nevus-to-melanoma progression TMA revealed that MCT4, and to a lesser extend MCT1, were elevated with progression to advanced melanoma. Further analysis of human melanoma specimens using the Seahorse XF24 extracellular flux analyzer indicated that metastatic melanoma tumors derived a large fraction of energy from OXPHOS. Taken together, these findings suggest that in stage IV melanomas with normal serum LDH, glycolysis and OXPHOS may provide metabolic symbiosis within the same tumor, whereas in stage IV melanomas with high serum LDH glycolysis is the principle source of energy.

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.

The present study offers novel insights into the molecular circuitry of accelerated in vivo tumor growth by Notch2 knockdown in triple-negative breast cancer (TNBC) cells. Therapeutic vulnerability of Notch2-altered growth to a small molecule (withaferin A, WA) is also demonstrated. MDA-MB-231 and SUM159 cells were used for the xenograft studies. A variety of technologies were deployed to elucidate the mechanisms underlying tumor growth augmentation by Notch2 knockdown and its reversal by WA, including Fluorescence Molecular Tomography for measurement of tumor angiogenesis in live mice, Seahorse Flux analyzer for ex vivo measurement of tumor metabolism, proteomics, and Luminex-based cytokine profiling. Stable knockdown of Notch2 resulted in accelerated in vivo tumor growth in both cells reflected by tumor volume and/or latency. For example, the wet tumor weight from mice bearing Notch2 knockdown MDA-MB-231 cells was about 7.1-fold higher compared with control ( P  < 0.0001). Accelerated tumor growth by Notch2 knockdown was highly sensitive to inhibition by a promising steroidal lactone (WA) derived from a medicinal plant. Molecular underpinnings for tumor growth intensification by Notch2 knockdown included compensatory increase in Notch1 activation, increased cellular proliferation and/or angiogenesis, and increased plasma or tumor levels of growth stimulatory cytokines. WA administration reversed many of these effects providing explanation for its remarkable anti-cancer efficacy. Notch2 functions as a tumor growth suppressor in TNBC and WA offers a novel therapeutic strategy for restoring this function.

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 micoenvironment.

BackgroundOncolytic viruses are an underappreciated immunotherapy capable of inflaming the tumor microenvironment (TME), vaccinating a patient against their own tumor, and delivering gene therapy to the TME. However, apart from the oncolytic HSV T-vec, these therapies have not seen widespread use, due in part to incomplete understanding of their immunologic mechanisms of action. We sought to determine features of oncolytic vaccinia virus (VV) response and resistance using subclones of the HPV+ head and neck cancer model MEER rendered sensitive or resistant to VV.MethodsA VV sensitive MEER tumor resisting treatment was serially passaged in mice and treated with VV until a stably resistant line was generated (Fig1). Sensitive or resistant MEER tumors were implanted, treated with a single intratumoral dose of VV, and harvested 4–7 days later for cytometric analysis. A genetically encoded TGFβ inhibitor was recombined into oncolytic VV (VV-TGFβi).ResultsWe used serial in vivo passaging to generate a VV-resistant MEER line (MEERvvR) from one sensitive to VV (MEERvvS, figure 1) and compared their immune infiltrate. While VV promoted acute cytokine production and cytotoxicity in conventional T cells, the major determining factor between sensitivity and resistance was the phenotype of Treg cells. At baseline, Treg cells in MEERvvS had lower Nrp1 expression and higher IFNγ-STAT1 signaling compared to MEERvvR, indicative of Treg 'fragility'. VV treatment induced MEERvvS Treg cells to become immunostimulatory and produce IFNγ (figure 2). RNAseq revealed MEERvvR produced more TGFβ than MEERvvS cells, suggesting these tumors directly stabilize Treg cells. To determine if MEERvvR could be sensitized to VV, we engineered oncolytic vaccinia to produce a genetically-encoded TGFβ inhibitor which binds TGFβRII, preventing TGFβ1-3 binding (VV-TGFβi). When MEERvvR were treated with VV-TGFβi, elite responses were restored, with commensurate increase in survival (figure 3) associated with increased STAT1 signaling in Treg cells.ConclusionsResistance to oncolytic vaccinia is controlled by Treg cell phenotype; tumors harboring more fragile Treg cells respond exquisitely to VV. An oncolytic vaccinia engineered to produce a novel TGFβi could remodel the TME to be less supportive of Tregs, rendering resistant tumors sensitive to VV. Our data highlight the importance of Treg cell status in resistance to oncolytic virus therapy and suggest TGFβ can be effectively targeted through an inhibitor encoded within the virus. Importantly, this TME directed production of the TGFβi carries no toxicity previously associated with systemic TGFβ inhibition, suggesting a viral approach to TGFβ inhibition can be an effective strategy support broader immunotherapy response.Abstract 743 Figure 1Strategy used to generate a vaccinia resistant MEER (MEERvvR) from vaccinia sensitive MEER (MEERvvS)Abstract 743 Figure 2IFNγ production in Treg cells in MEERvvS and MEERvvR after treatment with PBS or control vaccinia (VV-Ctrl)Abstract 743 Figure 3Survival of VV-resistant MEER treated with PBS, control vaccinia (VV-Ctrl), or vaccinia engineered to deliver a potent inhibitor of TGFβ (VV-TGFβi)

Since previous work using a nonreplicating adenovirus-expressing mouse interferon-β (Ad.mIFNβ) showed promising preclinical activity, we postulated that a vector-expressing IFNβ at high levels that could also replicate would be even more beneficial. Accordingly a replication competent, recombinant vaccinia viral vector-expressing mIFNβ (VV.mIFNβ) was tested. VV.mIFNβ-induced antitumor responses in two syngeneic mouse flank models of lung cancer. Although VV.mIFNβ had equivalent in vivo efficacy in both murine tumor models, the mechanisms of tumor killing were completely different. In LKRM2 tumors, viral replication was minimal and the tumor killing mechanism was due to activation of immune responses through induction of a local inflammatory response and production of antitumor CD8 T-cells. In contrast, in TC-1 tumors, the vector replicated well, induced an innate immune response, but antitumor activity was primarily due to a direct oncolytic effect. However, the VV.mIFNβ vector was able to augment the efficacy of an antitumor vaccine in the TC-1 tumor model in association with increased numbers of infiltrating CD8 T-cells. These data show the complex relationships between oncolytic viruses and the immune system which, if understood and harnessed correctly, could potentially be used to enhance the efficacy of immunotherapy.

The development of effective cancer therapy will require the simultaneous targeting of multiple steps in tumor development. We have previously described an antiangiogenic gene therapy vector, Ad Flk1-Fc, which expresses a soluble VEGF receptor capable of inhibiting tumor angiogenesis and growth. We have also described an oncolytic virus, dl922/947, whose replication and subsequent cytotoxicity are restricted to cancer cells with a loss of the G1–S cell cycle checkpoint. Here we have optimized methods for combining these therapies, yielding significantly greater anti-tumor effects than the respective monotherapies. In cultured tumor lines, co-infection with both Ad Flk1-Fc and dl922/947 allowed replication and repackaging of the replication-deficient Ad Flk1-Fc and enhanced soluble VEGF receptor expression. Similar repackaging and increased gene expression were demonstrated in vivo using bioluminescence imaging studies. Finally, coadministration of these therapeutic viral therapies in vivo produced significantly enhanced anti-tumor effects in colon HCT 116 and prostate PC-3 xenografts in mice. This increased therapeutic benefit correlated with replication of Ad Flk1-Fc viral genomes, increased intratumoral levels of Flk1-Fc protein, and decreased microvessel density, consistent with enhanced antiangiogenic activity.