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Development of next-generation oncolytic viruses requires the design of vectors that are potently oncolytic, immunogenic in human tumors, and well tolerated in patients. Starting with a joint-region deleted herpes simplex virus 1 (HSV-1) to create large transgene capability, we retained a single copy of the ICP34.5 gene, introduced mutations in UL37 to inhibit retrograde axonal transport, and inserted cell-type-specific microRNA (miRNA) target cassettes in HSV-1 genes essential for replication or neurovirulence. Ten miRNA candidates highly expressed in normal tissues and with low or absent expression in malignancies were selected from a comprehensive profile of 800 miRNAs with an emphasis on protection of the nervous system. Among the genes essential for viral replication identified using a small interfering RNA (siRNA) screen, we selected ICP4, ICP27, and UL8 for miRNA attenuation where a single miRNA is sufficient to potently attenuate viral replication. Additionally, a neuron-specific miRNA target cassette was introduced to control ICP34.5 expression. This vector is resistant to type I interferon compared to ICP34.5-deleted oncolytic HSVs, and in cancer cell lines, the oncolytic activity of the modified vector is equivalent to its parental virus. In vivo, this vector potently inhibits tumor growth while being well tolerated, even at high intravenous doses, compared to parental wild-type HSV-1., Graphical Abstract, Next-generation oncolytic viral immunotherapy is designed to promote greater oncolytic potency, be highly immunogenic in human tumors, and have an improved tolerability profile among patients. This article describes the design of a highly engineered novel oncolytic HSV-1 vector, ONCR-159, demonstrates its broad therapeutic index, and confirms its potent antitumor activity.

Oncolytic viruses induce local tumor destruction and inflammation. Whether virotherapy can also overcome immunosuppression in noninfected tumor areas is under debate. To address this question, we have explored immunologic effects of oncolytic herpes simplex viruses (oHSVs) in a genetically engineered mouse model of isocitrate dehydrogenase (IDH) wild-type glioblastoma, the most common and most malignant primary brain tumor in adults. Our model recapitulates the genomics, the diffuse infiltrative growth pattern, and the extensive macrophage-dominant immunosuppression of human glioblastoma. Infection with an oHSV that was armed with a UL16-binding protein 3 (ULBP3) expression cassette inhibited distant tumor growth in the absence of viral spreading (abscopal effect) and yielded accumulation of activated macrophages and T cells. There was also abscopal synergism of oHSV(ULBP3) with anti–programmed cell death 1 (anti–PD-1) against distant, uninfected tumor areas; albeit consistent with clinical trials in patients with glioblastoma, monotherapy with anti–PD-1 was ineffective in our model. Arming oHSV with ULBP3 led to upregulation of antigen processing and presentation gene sets in myeloid cells. The cognate ULBP3 receptor NKG2D, however, is not present on myeloid cells, suggesting a noncanonical mechanism of action of ULBP3. Overall, the myeloid-dominant, anti–PD-1–sensitive abscopal effect of oHSV(ULBP3) warrants further investigation in patients with IDH wild-type glioblastoma.

Although oncolytic viruses have shown clinical efficacy for local treatment of cancerous tumors, the ability to induce immune-mediated regression to visceral lesions is not robust. Immune checkpoint modulation has been efficacious in a fraction of cancers associated with an inflamed microenvironment but is associated with toxicity due to nonspecific T-cell activation. Therefore, combining these two strategies has the exciting potential to be an effective cancer therapy. Oncolytic virus can be genetically modified to decrease pathogenicity to normal cells and to increase their lytic potential and ability to stimulate antitumor immunity, thus improving the risk-benefit ratio for cancer patients. Oncorus oHSV (ONCR) are novel oncolytic herpes simplex virus type-1 vectors developed for the treatment of solid tumors. ONCR utilizes a unique conditional-lethal strategy in which tissue-specific miRs binding sites are inserted into early genes essential for viral replication and in the neurovirulence gene ICP34.5. This strategy curtails viral replication in normal cells, including neurons, and limits the expression of g34.5 to tumor cells, thus promoting potent viral replication in presence of type I interferon. Fifteen immunostimulatory transgenes and combination thereof were systematically evaluated in dual flank syngeneic mouse tumor models to test both for oncolysis and abscopal efficacy on the contralateral noninjected tumor. Among the selected transgenes, IL-12 was found to elicit the most potent efficacy on the ipsilateral and contralateral tumors. We demonstrated that the replication of ONCR and the expression of IL-12 was limited to the injected tumors. By contrast, induction of systemic immunity as assessed by IFNg production in contralateral tumors and in plasma was observed. ONCR expressing IL-12 elicited efficacy in multiple subcutaneous and metastatic models. Additional payloads designed to stimulate antigen presentation, recruitment of immune cells and inhibition of tumor immune suppression were identified as further enhancing IL-12 activity. Data supporting the selection of a suite of payloads cloned into a single ONCR vector will be presented. ONCR represents a new class of oncolytic virus that promote antitumor responses through a multiprong mechanism of action dependent on selective tumor cell killing, the induction of systemic antitumor immunity and reversion of immune suppression. Citation Format: Lorena Lerner, Edward M. Kennedy, Terry Farkaly, Allison Colthart, Caity Montagna, Prajna Behera, Judith Jacques, Peter Grzesik, Jennifer Lee, Agnieszka Denslow, Jacqueline Gursha, Brian Haines, Michael Ball, Daniel Wambua, Cecilia Kwong, Lingxin Kong, Michael Paglia, Laura Viggiano Salta, Lorenz Ponce, Caroline Webb, Mitchell Finer, Christophe Quéva. microRNA attenuated oHSV-1 armed with multiple immunomodulatory payloads mediates robust and selective antitumor immune response in preclinical tumor models [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B23.

Glioblastoma is a deadly disease with median survival ranging below one year. T-cell-based immunotherapy approaches failed to prolong survival of glioblastoma patients in two phase III clinical trials, thus reflecting the immunologically inert features of glioblastoma. Pre-clinical efficacy of oncolytic virus therapy in combination with immune checkpoint inhibition was demonstrated in syngeneic glioblastoma models. However, given the extensive spreading of glioblastoma cells throughout the brain, we hypothesized that efficacious immunotherapy approaches in patients will need to elicit abscopal responses, i.e. the secondary targeting of tumor cells at sites distant from treated lesions. We addressed this issue in an immune competent RCAS/tv-a-based genetic glioblastoma model that recapitulates the genotype, gene expression, histology and immune phenotype of human glioblastomas. Spatial distance of virus-treated and untreated glioblastoma lesions was modeled by generating two tumors in both hemispheres of N/tv-a;Ink4a/Arf(-/-);PTEN(fl/fl);Luc(LSL/LSL)mice. One tumor was generated by transduction with RCAS-Pdgfb and RCAS-shPTEN. A second, contralateral tumor was generated with RCAS-Pdgfb and RCAS-Cre, thus enabling additional monitoring of tumor growth through luciferase activity. We infected luciferase-negative tumor lesions with a miR-124-attenuated oncolytic herpes simplex virus 1 (oHSV) armed with the NKG2D ligand ULBP3. oHSV-ULBP3 infection slowed the growth of distant, non-infected glioblastoma lesions and prolonged survival. oHSV-ULBP3 led to influx and activation of T-cells in both, treated and untreated glioblastoma lesions. Massive influx of monocytes yielded an increase of mostly PD1+ macrophages in both tumors. Neutrophils were also increased ubiquitously, whereas NK-cells were suppressed. Treatment with an anti-PD1 antibody alone had no effect, but combination with oHSV-ULBP3 counteracted macrophage and neutrophil influx and enhanced abscopal anti-tumor responses. We conclude that oHSV-ULBP3 in combination with anti-PD-1 is a feasible approach to induce abscopal T-cell-mediated immune responses in glioblastoma. Unraveling the role of myeloid components of the tumor microenvironment may be critical to overcome resistance to such treatment.

Oncolytic viruses (OV) have shown clinical efficacy for treatment of malignancies when administered intratumorally. Attempts to use OV for systemic treatment of cancer have shown very limited success due to the presence of neutralizing antibodies. Development of an intravenously delivered oncolytic viral therapy that maintains its potency and effectiveness in the presence of neutralizing antibodies is greatly needed for patients with visceral lesions that are metastatic and difficult to inject. Therefore, the OV field is moving toward the design of vector platforms that can be used for systemic therapy to take full advantage of the oncolytic activity and the induction of anti-tumor immunity. Oncorus is developing a new strategy for systemic delivery of OV genomes by using tumor targeted nanoparticles, designed to be immunologically inert systemically, but can initiate a local robust intratumoral oncolytic virus infection. The Nanoparticle Encapsulated Polynucleotide (NEP) platform is designed for systemic delivery of replication competent OV genomes encoded by a plasmid DNA loaded into tumor-homing nanoparticles. To initiate viral replication intratumorally from a plasmid DNA we engineered a mammalian mRNA cassette that codes for an infectious +sense picornaviral RNA. This RNA is liberated from the larger capped and polyadenylated mammalian transcript by combination of ribozymes and siRNA cleavage, enabling rapid expression of viral proteins and viral replication. We selected Seneca Valley Virus (SVV) as our first candidate virus for the NEP platform. SVV is a potent oncolytic virus in neuroendocrine tumors previously evaluated in clinical trial after IV administration and well tolerated in the patients. The robust initiation of SVV replication from the pDNA expression construct results in cellular lysis in permissive cells when transfected in vitro. In vivo, intratumoral injection of a lipid based formulated SVV plasmid resulted in robust viral replication and dramatic tumor growth inhibition in multiple tumor models. This viral construct has also been modified to express immunomodulatory payloads to enhance the adaptive immune response by recruitment of immune effector cells. Finally, we have enabled miRNA attenuation of his construct to add a genetic tissue selectivity switch for safety, and we have shown that both miR-1 and miR-122 effectively and specifically attenuate viral replication. To enable ONCR-NEP therapy for intravenously administration, we have begun developing a lipid nanoparticle formulation with an active tumor targeting moiety. We have obtained lipid nanoparticle formulations with favorable physical and functional characteristics that are effective both in vitro and in vivo. We show here for the first time the development of a synthetic virus, a breakthrough therapeutic technology that is highly effective in vivo. Citation Format: Edward M. Kennedy, Jennifer S. Lee, Judy Jacques, Terry Farkaly, Prajna Behara, Peter Grzesik, Brian B. Haines, Agnieszka Denslow, Jacqueline Gursha, Lingxin Kong, Mitchell Finer, Christophe Quéva, Lorena Lerner. Development of ONCR-NEP, a lipid nanoparticle delivered oncolytic virus capable of robust in situ amplification resulting in tumor lysis and regression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4773.

Glioblastoma multiforme (GBM) is a highly aggressive brain tumor associated with poor prognosis and resistance to current therapies. ONCR-1 is a novel oncolytic herpes simplex virus type-1 vector in development for the treatment of GBM. ONCR-1 utilizes a unique conditional-lethal strategy in which miR124 binding sites are inserted into the ICP4 locus to prevent viral replication in neuronal cells while preserving one copy of the γ34.5 gene and enabling potent cytotoxicity in tumor cells. ONCR-1 is armed with a matrix metalloproteinase 9 (MMP9) transgene to facilitate viral spread in the tumor extracellular matrix. Further, ONCR-1 expresses a UL16 binding protein 3 (ULBP3) transgene to activate NK and CD8+ T cells via the NKG2D receptor and promote antitumor immune responses. In human and murine tumor cells in vitro, ONCR-1 evoked potent cytotoxicity. ONCR-1 increased proteolytic MMP in cell supernatants and ULBP3 cell surface expression with no effect on related NKG2D ligands. In mice bearing subcutaneous or orthotopic human U251 GBM tumors, ONCR-1 administered intratumorally (3x105-3x106 PFU QDx1) inhibited tumor growth and prolonged survival. Ex vivo analysis of immune cells in subcutaneous tumors showed significant increases in virally evoked NKG2D+ NK cells with ONCR-1 treatment. In mice bearing dual flank subcutaneous syngeneic A20 tumors, ONCR-1 significantly inhibited tumor growth in injected tumors (1x105-3x105 PFU, Q2Dx3) and evoked potent systemic antitumor immune responses as evidenced by significant inhibition of distal noninjected left flank tumors. IFNγ production from isolated splenocytes cultured in the absence and presence of A20 tumor cells was significantly augmented in ONCR-1-treated animals, providing further evidence of an enhanced antitumor immune response. Moreover, in mice bearing subcutaneous A20 tumors in which ONCR-1 evoked complete tumor regression, complete protection from rechallenge with A20 tumor cells was observed. Depletion of NK cells or CD8+ T cells in mice resulted in inhibition of ONCR-1-mediated antitumor effects in both injected and noninjected murine A20 tumors, supporting an immune mechanism of action. Our preclinical studies demonstrate that ONCR-1 evokes tumor cell killing by direct oncolysis and by enhancing antitumor immune responses. ONCR-1 represents a novel clinical candidate for the treatment of GBM. Citation Format: Alexandra Hicks, Paola Grandi, Michael Paglia, Jingzang Miu, Cecilia Kwong, Jacqueline Gursha, Michael Ball, Weiguo Yao, Daniel Wambua, Terry Farkaly, Kyle Grant, Laura Viggiano Salta, Lorenz Ponce, Joseph Glorioso, Christophe Queva, Mitchell Finer. ONCR-1, a novel herpes simplex virus expressing MMP9 and ULBP3 transgenes, evokes potent oncolysis and development of antitumor immune responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4698.

Effective treatment of advanced stage cancer with oncolytic viruses requires potent cancer cell-specific oncolytic activity. We modified oncolytic HSV (oHSV) to utilize a novel conditional-lethal strategy that retains all viral genes to maintain potency of the oncolytic virus, yet block replication in normal cells. In addition, we armed the virus with extracellular matrix decreasing enzyme to promote viral spread and immunomodulatory payloads to increase recruitment and effector function of immune cells. To enhance the selectivity of viral replication to tumor cells, Oncorus engineered oHSVs containing microRNA (miRNA)-binding cassettes (miR-T) in essential genes required for viral replication. The first Oncorus clinical candidate, ONCR-1, under development for the treatment recurrent glioblastoma multiform (GBM), has been attenuated for replication in neurons, the most trophic cell type for HSV. Neuronal attenuation was achieved by inserting binding sites for a highly specific neuronal miRNA, miR-124, into the 3′UTR of the essential ICP4 gene. We demonstrate that these engineered miR-124 response elements can selectively inhibit the expression of ICP4 and VP5 as well as the expression of ONCR-1 payloads. This attenuation strategy eliminates viral replication in miR-124 expressing cells without compromising oncolytic activity in cancer cells, thereby providing a therapeutic window. We extended the design oHSV attenuation by profiling of miRNAs expression in panels of solid tumors and non-diseased tissue counterparts in other potentially high-risk tissues, prioritized to indications amenable to local or systemic administration. We leveraged our comprehensive normal and malignant tissue dataset to identify numerous highly expressed unique non-cancerous tissue-specific candidate miRNAs. Insertion of miR-T cassettes into HSV-1 genes collaborated to stall the early steps in the viral replication cycle and further increased the depth and breadth of oHSV attenuation in non-disease tissue relative to the single miR-T cassette in ICP4. We found that miR-122, a highly expressed miRNA in normal hepatocytes, is low or absent in many carcinoma cells. Insertion of miR-122 cassette into the 3′UTR of an immediate early gene, ICP27, inhibited completely viral replication in miR-122 expressing cells. oHSV with dual miR-124 and miR-122 was potently attenuated cells expressing one or both miRs while displaying a robust oncolytic activity in malignant cells. We have shown that insertion of miR-T cassettes in essential viral genes effectively attenuates oHSV replication in normal human cells, and most importantly, robust oncolysis was retained in cancer cells. This work extends the miR-T paradigm to multiple viral genes with diverse miRNA targets, thus expanding the protection of normal tissues and facilitating the application of oHSV to the treatment of multiple malignancies. Citation Format: Edward M. Kennedy, Terry Farkaly, Caitlin Goshert, Allison Colthart, Prajna Behera, Peter Grzesik, Kyle Grant, Michael Paglia, Paola Grandi, Christophe Quéva, Mitchell Finer, Lorena Lerner. microRNA control of an oHSV vector allows for robust oncolysis and selective control of viral replication in normal tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5929.