Oncolytic HSV Research Articles

The combination therapy of oncolytic HSV-1 armed with anti-PD-1 antibody and IL-12 enhances anti-tumor efficacy

Cancer immunotherapy is a new therapeutic strategy for cancer treatment that targets tumors by improving or restoring immune system function. Therapies targeting immune checkpoint molecules have exerted potent anti-tumor effects and prolonged the overall survival rate of patients. However, only a small number of patients benefit from the treatment. Oncolytic viruses exert anti-tumor effects by regulating the tumor microenvironment and affecting multiple steps of tumor immune circulation. In this study, we engineered two oncolytic viruses that express mouse anti-PD-1 antibody (VT1093M) or mouse IL-12 (VT1092M). We found that both oncolytic viruses showed significant anti-tumor effects in a murine CT26 colon adenocarcinoma model. Importantly, the intratumoral combined injection with VT1092M and VT1093M inhibited growth of the primary tumor, prevented growth of the contralateral untreated tumor, produced a vaccine-like response, activated antigen-specific T cell responses and prolonged the overall survival rate of mice. These results indicate that combination therapy with the engineered oncolytic virus may represent a potent immunotherapy strategy for cancer patients, especially those resistant to PD-1/PD-L1 blockade therapy.

DDRE-18. THERAPEUTIC EFFECTS OF TASQUINIMOD ON GLIOBLASTOMA

Abstract Glioblastoma is the most aggressive and most common primary malignant brain tumor in adults. This disease is still incurable despite multimodal therapies. We evaluated Tasquinimod, an oral HDAC4 inhibitor, in use with oncolytic HSV therapy. Tasquinimod is known to bind to HDAC4 and S100A9 to modulate myeloid population and angiogenesis in the tumors. This drug is being clinically evaluated in prostate and other solid tumors and multiple myeloma. However, the therapeutic effect of Tasquinimod in glioblastoma is not known. We found that Tasquinimod could change the immune-suppressive phenotype of MDSCs and polarize M2 macrophages towards M1 phenotype in ex vivo assay using mouse bone marrow cells treated with glioma conditioned media. We also tested Tasquinimod and oncolytic HSV in murine CT2A in C57/B6 mice and primary human GBM xenograft model in athymic mice. While the therapeutic effect of Tasquinimod or oHSV alone was not significant or marginal, the combo group show significantly longer survival. The study of underlying mechanisms is still ongoing. In conclusion, Tasquinimod combined with oncolytic HSV could have a better therapeutic effect on glioblastoma.

USP18 and USP20 restrict oHSV-1 replication in resistant human oral squamous carcinoma cell line SCC9 and affect the viability of SCC9 cells

In this study, we discovered that two human oral squamous carcinoma cell (OSCC) lines, SCC9 and SCC25, exhibited varied levels of permissivity to oncolytic HSV-1 T1012G replication and the differential virus yields may associate with the constitutive accumulation of two deubiquitinating enzymes USP18 and USP20 in tumor cells. USP18 and USP20 belong to the ubiquitin-specific protease family, mediating the deubiquitination of targets and promoting antiviral responses. Depletion of USP18 or USP20 in SCC9 cells increased T1012G virus yields; overexpression of USP18 or USP20 in SCC25 cells down-regulated T1012G virus replication. In addition, STING as a verified substrate of USP18 and USP20, was found to affect the virus multiplication of T1012G in SCC9 cells. STING knockdown led to an increase in T1012G virus yields in SCC9 cells. Besides, we introduced a deubiquitinating enzyme inhibitor GSK2643943A targeting USP20 and evaluated its effects on viral replication and tumor killing in vitro and in vivo. The results showed that the combination of GSK2643934A and T1012G treatment brought a profound anti-tumor efficacy in mice bearing SCC9 tumors. This report explored factors that play roles in mediating oHSV-1 replication in OSCC tumor cells, facilitating to offer potential targets to improve oHSV-1 oncolytic efficacy and develop candidates of biomarkers to predict the efficiency of oHSV-1 multiplication in tumors.

Response to FEC Chemotherapy and Oncolytic HSV-1 Is Associated with Macrophage Polarization and Increased Expression of S100A8/A9 in Triple Negative Breast Cancer.

Simple SummaryWe have previously reported that a combination of clinical chemotherapies and oncolytic HSV-1 works to sensitize tumors to respond to immune checkpoint blockade. We further showed that this therapeutic platform worked via the upregulation of B cells and the concomitant control of immunosuppressive myeloid cells. In this manuscript, we sought to further dissect the mechanism of myeloid cell regulation and differentiation and to identify a therapeutically driven gene signature that is associated with the switch in the myeloid phenotype. This work not only impacts triple-negative breast cancer but all solid tumor phenotypes as we aim to better understand the underlying immunology associated with responses to immune checkpoint therapies in these typically refractory disease types.The era of immunotherapy has seen an insurgence of novel therapies driving oncologic research and the clinical management of the disease. We have previously reported that a combination of chemotherapy (FEC) and oncolytic virotherapy (oHSV-1) can be used to sensitize otherwise non-responsive tumors to immune checkpoint blockade and that tumor-infiltrating B cells are required for the efficacy of our therapeutic regimen in a murine model of triple-negative breast cancer. In the studies herein, we have performed gene expression profiling using microarray analyses and have investigated the differential gene expression between tumors treated with FEC + oHSV-1 versus untreated tumors. In this work, we uncovered a therapeutically driven switch of the myeloid phenotype and a gene signature driving increased tumor cell killing.

Single-cell RNA sequencing reveals a strong connection between Gadd45g upregulation and oncolytic HSV infection in tumor tissue

The oncolytic effect of virotherapy derives from the intrinsic capability of the applied virus in selectively infecting and killing tumor cells. Although oncolytic viruses of various constructions have been shown to efficiently infect and kill tumor cells in vitro, the efficiency of these viruses to exert the same effect on tumor cells within tumor tissues in vivo has not been extensively investigated. Here we report our studies using single-cell RNA sequencing to comprehensively analyze the gene expression profile of tumor tissues following herpes simplex virus 2-based oncolytic virotherapy. Our data revealed the extent and cell types within the tumor microenvironment that could be infected by the virus. Moreover, we observed changes in the expression of cellular genes, including antiviral genes, in response to viral infection. One notable gene found to be upregulated significantly in oncolytic virus-infected tumor cells was Gadd45g, which is desirable for optimal virus replication. These results not only help reveal the precise infection status of the oncolytic virus in vivo but also provide insight that may lead to the development of new strategies to further enhance the therapeutic efficacy of oncolytic virotherapy.

1093TiP An open-label, multicenter, phase I/II clinical trial of RP1 as a single agent and in combination with nivolumab in patients with solid tumors [IGNYTE

RP1 is an enhanced potency oncolytic HSV-1 which expresses a fusogenic glycoprotein (GALV-GP R-) and granulocyte macrophage colony stimulating factor (GM-CSF). Pre-clinical studies with RP1 demonstrated potent GALV-GP R-enhanced anti-tumor activity and immunogenic cell death. This phase I/II (Ph I/II) study (NCT03767348) was designed to evaluate the safety and efficacy of RP1 as single agent and in combination with nivolumab (nivo) in pts with advanced solid tumors, including pts whose disease failed prior anti-PD1/PD-L1 therapy.

Specificity, Safety, Efficacy of EGFRvIII-Retargeted Oncolytic HSV for Xenotransplanted Human Glioblastoma.

Glioblastoma is a lethal primary brain tumor lacking effective therapy. The secluded onset site, combined with the infiltrative properties of this tumor, require novel targeted therapies. In this scenario, the use of oncolytic viruses retargeted to glioblastoma cells and able to spread across the tumor cells represent an intriguing treatment strategy. Here, we tested the specificity, safety and efficacy of R-613, the first oncolytic HSV fully retargeted to EGFRvIII, a variant of the epidermal growth factor receptor carrying a mutation typically found in glioblastoma. An early treatment with R-613 on orthotopically transplanted EGFRvIII-expressing human glioblastoma significantly increased the median survival time of mice. In this setting, the growth of human glioblastoma xenotransplants was monitored by a secreted luciferase reporter and showed that R-613 is able to substantially delay the development of the tumor masses. When administered as late treatment to a well-established glioblastomas, R-613 appeared to be less effective. Notably the uninfected tumor cells derived from the explanted tumor masses were still susceptible to R-613 infection ex vivo, thus suggesting that multiple treatments could enhance R-613 therapeutic efficacy, making R-613 a promising oncolytic HSV candidate for glioblastoma treatment.

Abstract 1917: Immunomodulatory effects of a novel, enhanced potency gibbon ape leukaemia virus (GALV) fusogenic membrane glycoprotein-expressing herpes simplex virus platform with increased efficacy combined with anti PD-1 therapy

Abstract Oncolytic viral immunotherapies are attractive because they are self-amplifying, can kill tumors through multiple, immunologically visible mechanisms and have the ability to promote anti-tumor immune responses. RP1 is a novel, enhanced potency oncolytic HSV that has been genetically engineered to express human GM-CSF and the gibbon ape leukemia virus fusogenic membrane glycoprotein with the R sequence deleted (GALV-GP R-), providing constitutive fusion activity. GALV-GP R- binds pit-1 receptors on human and rat, but not mouse, cells to mediate cell-cell fusion. RP1 potently kills human tumor-derived cells, including a panel of melanoma and head and neck cell lines, as demonstrated here. In vivo, using a human melanoma model in nude mice, higher titres (2-3 log) of virus were retrieved from tumors treated with the virus expressing GALV-GP R- (i.e. RP1) versus an equivalent non-GALV-GP R- expressing virus. No virus was retrieved from uninjected tumors. In addition to enhanced direct effects on injected tumors, contralateral uninjected tumors were significantly smaller in groups treated with RP1 versus the GALV-GP R- non-expressing virus despite restriction of virus replication to the injected tumor. This effect was seen in nude mice, i.e. without an adaptive immune system, suggesting contribution of innate (e.g. NK cell-mediated) immunity to the contralateral effects observed. In the immunocompetent mouse 4434 melanoma model, RP1 also reduced tumor burden in both injected and uninjected tumors, again with absolute restriction of virus replication to injected tumors. Animals treated with RP1 had splenomegaly and a population of tumor-infiltrating PD-L1+ cells within injected tumors at one week post-treatment, albeit without a contribution of fusion mediated by GALV-GP R- in this model. FACS-based analysis identified these PD-L1+ cells as predominantly CD45+/CD11b(med/low)/LY6G+ neutrophils. Co-treatment with RP1 and an anti-mouse PD-1 antibody increased anti-tumor effects and the levels of CD3+ cells in uninjected tumors. Data from detailed RNAseq transcriptional analyses from injected and uninjected tumors will also be presented. Citation Format: Victoria Roulstone, Joan Kyula, Suzanne Thomas, Linta Kuncheria, Praveen K. Bommareddy, Henry Smith, Harriet Whittock, Robert S. Coffin, Kevin Harrington. Immunomodulatory effects of a novel, enhanced potency gibbon ape leukaemia virus (GALV) fusogenic membrane glycoprotein-expressing herpes simplex virus platform with increased efficacy combined with anti PD-1 therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1917.

Abstract LB180: Clinical biomarker studies with two fusion-enhanced versions of oncolytic HSV (RP1 and RP2) alone and in combination with nivolumab in cancer patients indicate potent immune activation

Abstract Introduction:RP1 and RP2 are novel, enhanced potency oncolytic versions of HSV1 engineered to express human GM-CSF and the gibbon ape leukemia virus membrane R- glycoprotein (GALV-GP R-), providing constitutive fusion activity and increased immunogenic cell death. RP2 further expresses an anti-CTLA-4 antibody-like molecule. Murine versions of RP1 and RP2 exhibited synergy in combination with anti-mouse-PD-1 leading to enhanced regression of both injected and un-injected tumors in mice (Thomas et al JITC 2019). RP1 and RP2 are currently being evaluated in clinical trials in a range of solid tumors alone and combined with anti-PD1 therapy, where deep and durable responses have been demonstrated (SITC 2020). Here we present biomarker data from the Phase 1/2 clinical trial of RP1 alone and combined with nivolumab (NCT03767348) and from the Phase 1 portion of the clinical trial with RP2 alone (NCT04336241). Methods: In the Phase 1/2 studies tumor biopsies and peripheral blood mononuclear samples (PBMCs) were collected at screening and at D43 for biomarker analysis, after combination therapy with nivolumab for RP1 and following single agent treatment for RP2. Immunohistochemistry (IHC) was performed for CD8 (SP57 clone, Ventana) and for PD-L1 (28-8 clone, pharmDx assay). Gene expression was analysed using NanoString to assess effects on a range of genes. The tumor inflammation signature score (TIS) was also calculated. Results:Preliminary Phase 1/2 biomarker data from paired tumor biopsies include the following: Immunohistochemistry for CD8 and PD-L1 (n=30) indicated robust and increased infiltration of CD8+ T cells and PD-L1 expression, both after combined treatment with RP1 and nivolumab and after single agent RP2 across different tumor types, and including reversal of T cell exclusion following prior combined treatment with ipilimumab and nivolumab in melanoma. Gene expression analysis (n=15) demonstrated a significant increase in the expression levels of genes associated with innate and adaptive immune activation and genes previously reported to be associated with responsiveness to anti-PD1 therapy, particularly CD8, CXCL9, CD27 and TIGIT, as well as consistently increased TIS. Conclusion:Consistent with the pre-clinical data, preliminary clinical biomarker data indicate substantial increase in CD8 T cell infiltration and PD-L1 expression, as well as increased TIS score in the majority of patients treated with RP2 alone or RP1 and nivolumab combination. Particularly marked effects were seen in some patients with clinical responses which occurred independent of both baseline PD-L1 and prior anti-PD1 therapy status, which suggests potential broad utility of the RP1/2 treatment approach in igniting an anti-tumor immune response. Tumor mutation burden analysis and T cell receptor sequencing are currently underway and further updates of the dataset will be presented. Citation Format: Kevin J. Harrington, Francesca Aroldi, Joseph J. Sacco, Mohammed M. Milhem, Brendan D. Curti, Ari M. Vanderwalde, Scott Baum, Adel Samson, Anna C. Pavlick, Jason A. Chesney, Jiaxin Niu, Terence D. Rhodes, Tawnya L. Bowles, Robert Conry, Anna Olsson-Brown, Douglas E. Laux, Pablo Nenclares, Lavita Menezes, Alex Deterding, Victoria Roulstone, Joan Kyula, Suzanne Thomas, Praveen K. Bommareddy, Selda Samakoglu, Andrea Pirzkall, Robert S. Coffin, Mark R. Middleton. Clinical biomarker studies with two fusion-enhanced versions of oncolytic HSV (RP1 and RP2) alone and in combination with nivolumab in cancer patients indicate potent immune activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB180.

Abstract CT018: Phase I immunovirotherapy trial of oncolytic HSV-1 G207 alone or combined with radiation in pediatric high-grade glioma

Abstract Introduction: Pediatric high-grade gliomas (pHGGs) are routinely fatal with a median overall survival (OS) at recurrence of 5.6 months (mos). A safe, effective immunotherapy for pHGG has eluded investigators. Oncolytic HSV-1 G207 contains mutations that prevent a productive infection of normal cells but permit replication in tumor cells. In addition to direct tumor cell lysis, G207 activates innate/adaptive immune cells and promotes cross-presentation of tumor antigens to generate an anti-tumor immune response. A 5 Gy radiation dose increases viral replication and spread. We evaluated the safety and efficacy of G207 alone and combined with radiation in children with progressive supratentorial HGG (NCT02457845). Methods: We employed a 3 + 3 design with 4 cohorts. Children 3-18 years old with biopsy-confirmed HGG underwent stereotactic placement of up to four intratumoral catheters. The next day, we administered 107 or 108 plaque-forming units (pfu) of G207 by controlled rate infusion over 6 hours. Within 24 hours of G207, patients in dose level 3 and 4 received 5 Gy to the gross tumor volume. The primary objective was safety/tolerability. We assessed secondary objectives of virus shedding in blood, saliva and conjunctiva by PCR, response by MRI and evaluation of matched pre- and post-G207 tissue for tumor-infiltrating lymphocytes (TILs), and seroconversion by immunofluorescence assay. Results: 12 patients (age range 7-18) with progressive, IDH wild-type pHGG received G207. At screening, 10 patients had tumors with a bi-perpendicular sum ≥ 5.5 cm, 3 had multi-focal disease, 8 had failed ≥ 2 prior treatment regimens, and 4 had failed ≥ 3 regimens. 3-4 catheters (44 total) were placed safely throughout the cerebrum and resulted in no neurologic sequelae. G207 alone or with radiation was safe and tolerable in all patients with no dose-limiting toxicities, attributable grade 3 or 4 toxicities/serious adverse events, or evidence of virus shedding. 11 participants had radiographic, neuropathologic, and/or clinical responses. Median OS was 12.2 mos (95% CI 8.0, 16.4). Thus far, 36% of patients have lived >18 mos, the median OS for newly diagnosed pHGG. Compared to patients who seroconverted post-G207 (n=3), patients with baseline HSV-1 antibodies (n=3) had a shorter median survival: 5.1 mos (3.0, 7.2) vs 18.3 mos (9.2, 27.4). G207 significantly increased CD4+ and CD8+ TILs. Conclusions: G207 alone and combined with radiation was tolerable and safe with evidence of responses in children with pHGG. The promising median OS (12.2 mos) compares favorably with historical data (5.6 mos). Baseline HSV-1 seropositivity and seroconversion are potential biomarkers of treatment response that require further investigation. Importantly, G207 converted ‘cold' tumors to ‘hot' with a dramatic increase in TILs. A multi-institutional Phase II clinical trial of G207 in pHGG is forthcoming (NCT04482933). Citation Format: Gregory K. Friedman, James M. Johnston, Asim K. Bag, Joshua D. Bernstock, Rong Li, Inmaculada Aban, Kara Kachurak, Li Nan, Kyung-Don Kang, Stacie Totsch, Charles Schlappi, Allison M. Martin, Devang Pastakia, Sameer Farouk Sait, Yasmin Khakoo, Matthias A. Karajannis, Karina Woodling, Joshua D. Palmer, Diana S. Osorio, Jeffrey Leonard, Mohamed S. Abdelbaki, Avi Madan-Swain, T. Prescott Atkinson, Richard J. Whitley, John B. Fiveash, James M. Markert, G. Yancey Gillespie. Phase I immunovirotherapy trial of oncolytic HSV-1 G207 alone or combined with radiation in pediatric high-grade glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT018.

Abstract 1309: HSV1 oncolytic therapy for breast cancer meningeal metastases

Abstract Background: Meningeal metastasis is a fatal complication of breast cancer which results when cancer cells seed in the subarachnoid space. Subsequent tumor growth in the leptomeninges presents severe neurological complications of the cranial nerves, cerebrum and spinal cord limiting life expectancy to less than 4 months. Currently there is no cure. Aggressive multimodal therapies such as radiation, systemic and intra-cerebrospinal fluid (CSF) chemotherapy are ineffective. Chemotherapy is cleared rapidly from the CSF while doses deemed therapeutic are highly toxic. Oncolytic Herpes Simplex Virus type 1 (OHSV1) was selected as a potential therapeutic in this regard. Destruction of cancer cells by lytic virus replication is under clinical investigation where multiple virus replication cycles amplify the injected dose. We present OHSV1 therapeutics in a murine model of meningeal metastases where disease progression resembles that in the clinic. Methods: Meningeal metastases were induced in nude Balb/c mice by stereotaxic injection of MDA-MB-231 breast cancer cells into the right lateral ventricle of the brain. OHSV1 (1x10^8 pfu) was injected into the lateral ventricle on either day 9, 12 or 16 to target the early, intermediate and late disease phases of tumor growth in the model. Virus replication kinetics, tumor response, neurological and physical changes, and survival were studied for each treatment group. Virus replication was imaged with 18F-FHBG-PET and Fluc bioluminescence while tumor response to oncolytic HSV1 was imaged with Gd-MRI and Rluc-bioluminescence. The brains were studied ex vivo to confirm in vivo scans, virus with LacZ and antibody to viral TK, and titer with plaque assay. Results: A remarkable reduction metastatic growth to OHSV1 was evident in the early and intermediate groups where tumor growth was inhibited in some mice while existing tumors regressed in others. Tumor load in the base of the brain and spinal cord seen in control mice was markedly reduced in the two groups highlighted by minimal contrast uptake on Gd-MRI. As such the onset of neurological symptoms (bradykinesia, ataxia and paralysis) was delayed accompanied by weight gain. Inhibition of tumor growth in the late treatment group was relatively modest. Virus replication was evident as waves over the course of treatment in the three groups where the peaks represented virions released at the completion of a replication cycle. In vitro analyses correlated with in vivo observations. Early and intermediate OHSV1 therapy extended survival 12 and 7 days beyond the control group (day 21), while late OHSV1 therapy extended survival beyond 4 days. Conclusion: OHSV1 is therapeutic against life threatening disease progression when administered at early and intermediate stage disease with potential to target late stage disease. This treatment holds promise for breast cancer meningeal metastases that can be translated to the clinic. Citation Format: Darshini Kuruppu, Deepak Bhere, Khalid Shah, Anna-Liisa Brownell, Umar Mahmood, Kenneth K. Tanabe. HSV1 oncolytic therapy for breast cancer meningeal metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1309.

The Effect of Herpes Simplex Virus-Type-1 (HSV-1) Oncolytic Immunotherapy on the Tumor Microenvironment.

The development of cancer causes disruption of anti-tumor immunity required for surveillance and elimination of tumor cells. Immunotherapeutic strategies aim for the restoration or establishment of these anti-tumor immune responses. Cancer immunotherapies include immune checkpoint inhibitors (ICIs), adoptive cellular therapy (ACT), cancer vaccines, and oncolytic virotherapy (OVT). The clinical success of some of these immunotherapeutic modalities, including herpes simplex virus type-1 derived OVT, resulted in Food and Drug Administration (FDA) approval for use in treatment of human cancers. However, a significant proportion of patients do not respond or benefit equally from these immunotherapies. The creation of an immunosuppressive tumor microenvironment (TME) represents an important barrier preventing success of many immunotherapeutic approaches. Mechanisms of immunosuppression in the TME are a major area of current research. In this review, we discuss how oncolytic HSV affects the tumor microenvironment to promote anti-tumor immune responses. Where possible we focus on oncolytic HSV strains for which clinical data is available, and discuss how these viruses alter the vasculature, extracellular matrix and immune responses in the tumor microenvironment.

EPCT-22 SAFETY AND EFFICACY OF INTRAVENTRICULAR IMMUNOVIROTHERAPY WITH ONCOLYTIC HSV-1 G207 FOR TREATMENT OF LEPTOMENINGEAL DISEASE

Leptomeningeal metastatic disease (LMD) occurs in 30–50% of newly diagnosed and recurrent pediatric malignant cerebellar tumors and 20–45% of malignant supratentorial tumors. Radiation and chemotherapy often cause substantial long-term neurotoxicity and outcomes remain poor for patients with LMD. At recurrence, LMD is generally minimally responsive to conventional therapies. Immunovirotherapy with engineered oncolytic HSV-1 G207 has emerged as a promising treatment for children with high-grade brain tumors. G207 infects and kills tumor cells while sparing normal cells and stimulates a robust anti-tumor immune response. Intratumoral G207 inoculation demonstrated safety and preliminary efficacy in a pediatric Phase 1 trial in recurrent/progressive high-grade glioma (NCT02457845), and a Phase 2 trial (NCT04482933) is forthcoming. Additionally, a Phase 1 trial of intratumoral G207 in recurrent/progressive malignant pediatric cerebellar tumors is ongoing (NCT03911388). While intratumoral inoculation delivers G207 directly to a primary tumor, it requires neurosurgical procedures thereby limiting repeat doses. Thus, we sought to establish the safety and efficacy of intraventricular G207. Utilizing an immunocompetent, HSV-sensitive murine strain, we determined that a standard 1x107 plaque-forming units (PFU) dose of G207 resulted in damage to the ependymal lining. However, interferon induction with an intraventricular low-dose (1x104 PFU) of G207 or polyinosinic-polycytidylic acid (poly I:C), a toll-like receptor 3 agonist, three days prior to standard treatment dose protected the ependymal lining. This approach enabled safe delivery of multiple subsequent doses. Importantly, with these protective measures, G207 significantly prolonged survival in pediatric patient-derived xenograft models and an immunocompetent murine LMD model of group 3 medulloblastoma, the most aggressive and fatal subtype. Collectively, these data indicate that toxicity from intraventricular G207 can be safely mitigated prior to a therapeutic dose, and that intraventricular G207 effectively targets group 3 medulloblastoma including LMD. These findings provide support for clinical translation of intraventricular G207.

A phase 1, open-label, dose escalation study of the safety and tolerability of T3011 in advanced cutaneous or subcutaneous malignancies.

2526 Background: T3011 is a genetically modified, next-generation oncolytic HSV-1 with 2 exogenous genes encoding the active heterodimer human interleukin 12 (IL-12) and the Fab fragment of an anti-human PD-1 antibody. Locally produced IL-12 induces the synthesis of interferon-gamma (IFN-γ) production, enhancing cytolytic activity of natural killer cells and cytotoxic T lymphocytes. The anti PD-1 antibody blocks checkpoint inhibition of T effector cells. Extensive preclinical studies demonstrate that T3011 (and murine equivalent T3855) has potent antitumor activities. Methods: This phase 1 multicenter, open-label, dose escalation study evaluates the safety of intratumoral (IT) T3011 given once every other week (Q2W) in patients (pts) with advanced cutaneous or subcutaneous malignancies. The primary objective is to determine the Recommended Phase 2 Dose of T3011 based on the overall safety, pharmacokinetic and pharmacodynamic profile. Eligible pts are ≥ 18 years, have cutaneous or subcutaneous advanced cancer that has progressed on standard treatment and at least 1 measurable tumor lesion (≥ 10 mm) suitable for T3011 IT injection. Part 1 of the study uses a 3+3 design to evaluate the safety and tolerability of T3011 monotherapy in 4 escalating doses (1 × 106, 1 × 107, 5 × 107, and 1 × 108 PFU/mL). Up to 4 mL of T3011 may be injected based on tumor size. Total enrollment will be determined by toxicities observed. Results: As of Feb. 14, 8 pts have received IT T3011 (Q2W): 3 in Cohort 1 (1 × 106), 3 in Cohort 2 (1 × 107), and 2 in Cohort 3 (5.0 × 107 PFU/ml). Maximum doses per pt was 11. Enrollment continues in Cohort 3. T3011 was well tolerated with no ≥ Grade 3 treatment-related adverse events (AEs), no DLTs or treatment-related SAEs reported to date. Common AEs were pain at injection site, leukopenia, anemia, hypocalcemia, nausea, fever, headache, dermatitis, and diaphoresis. Viral shedding was analyzed in blood, urine and saliva at various times during the study. No Viral DNA was detected in blood or urine samples (first 3 pts analyzed) to date. Biopsy samples taken from injected tumors from 2 melanoma pts (Cohort 1) revealed significant reduction of viable tumor cells after 4 injections (Week 9) compared with baseline. In particular, one post-treatment biopsy contained 45% tumor necrosis area with dramatic increases of CD8 + and NKT cells. CD3+ and CD4+ cells as well as PD-1 expression were increased in post-treatment biopsies of both pts. Conclusions: T3011 IT injection was well tolerated at the first 2 dose levels. Post treatment biopsies from 2 pts (Cohort 1) demonstrated significantly reduced tumor cell viability as well as increased lymphocyte infiltration indicating on-target anti-tumor activities of T3011. To date, 5 out of 6 evaluable pts had SD as best response and 6 enrolled pts remain on study. Dose escalation is continuing. Clinical trial information: NCT04370587.

ONCR-177, an Oncolytic HSV-1 Designed to Potently Activate Systemic Antitumor Immunity.

ONCR-177 is an engineered recombinant oncolytic herpes simplex virus (HSV) with complementary safety mechanisms, including tissue-specific miRNA attenuation and mutant UL37 to inhibit replication, neuropathic activity, and latency in normal cells. ONCR-177 is armed with five transgenes for IL12, FLT3LG (extracellular domain), CCL4, and antagonists to immune checkpoints PD-1 and CTLA-4. In vitro assays demonstrated that targeted miRNAs could efficiently suppress ONCR-177 replication and transgene expression, as could the HSV-1 standard-of-care therapy acyclovir. Although ONCR-177 was oncolytic across a panel of human cancer cell lines, including in the presence of type I IFN, replication was suppressed in human pluripotent stem cell-derived neurons, cardiomyocytes, and hepatocytes. Dendritic cells activated with ONCR-177 tumor lysates efficiently stimulated tumor antigen-specific CD8+ T-cell responses. In vivo, biodistribution analyses suggested that viral copy number and transgene expression peaked approximately 24 to 72 hours after injection and remained primarily within the injected tumor. Intratumoral administration of ONCR-177 mouse surrogate virus, mONCR-171, was efficacious across a panel of syngeneic bilateral mouse tumor models, resulting in partial or complete tumor regressions that translated into significant survival benefits and to the elicitation of a protective memory response. Antitumor effects correlated with local and distant intratumoral infiltration of several immune effector cell types, consistent with the proposed functions of the transgenes. The addition of systemic anti-PD-1 augmented the efficacy of mONCR-171, particularly for abscopal tumors. Based in part upon these preclinical results, ONCR-177 is being evaluated in patients with metastatic cancer (ONCR-177-101, NCT04348916).

Safety and interim survival data after intracranial administration of M032, a genetically engineered oncolytic HSV-1 expressing IL-12, in pet dogs with sporadic gliomas.

The diagnosis of glioma remains disheartening in the clinical realm. While a multitude of studies and trials have shown promise, improvements in overall survival have been disappointing. Modeling these tumors in the laboratory setting has become increasingly challenging, given their complex in situ behavior and interactions for therapeutic evasion. Dogs, particularly brachycephalic breeds, are known to spontaneously develop gliomas that resemble human gliomas both clinically and pathophysiologically, making canines with sporadic tumors promising candidates for study. Typically, survival among these dogs is approximately 2 months with palliation alone. The authors have completed the first stage of a unique phase I dose-escalating canine clinical trial in which the safety and tolerability of M032, a nonneurovirulent oncolytic herpes simplex virus-1 vector genetically engineered to express interleukin-12, are being studied in pet dogs with gliomas undergoing maximum safe tumor resection and inoculation of the cavity with the viral infusate. Twenty-five canine patients were enrolled between January 2018 and August 2020. One patient was electively withdrawn from the trial by its owner, and 3 did not receive the virus. For the 21 dogs that remained, 13 had high-grade gliomas, 5 had low-grade gliomas, and 3 were undetermined. According to histopathological analysis, 62% of the tumors were oligodendrogliomas. At the time of this report, the median overall survival from the date of treatment was 151 days (± 78 days). No significant adverse events attributable to M032 or dose-limiting toxicities have been observed to date. In this largest study of oncolytic viral therapy for canine brain tumors to date, treatment with M032 did not cause harm and the combination of surgery and oncolytic viral therapy may have contributed to prolonged survival in pet dogs with spontaneous gliomas. Forthcoming in-depth radiographic, immunohistochemical, and genetic analyses will afford a more advanced understanding of how this treatment impacts these tumors and the immune system. Our goal is to utilize these findings bitranslationally to inform human studies and refine therapies that will improve outcomes in both humans and pet dogs with gliomas.

HSV-1 Oncolytic Viruses from Bench to Bedside: An Overview of Current Clinical Trials.

Simple SummaryOncolytic Herpes simplex virus-1 (HSV-1) offers the dual potential of both lytic tumor-specific cell killing and inducing anti-tumor immune responses. The HSV-1 genome can be altered to enhance both components and this may be applicable for the treatment of a broad range of cancers. Several engineered oncolytic viruses based on the HSV-1 backbone are currently under investigation in various clinical trials, both as single agents and in combination with various immunomodulatory drugs.Herpes simplex virus 1 (HSV-1) provides a genetic chassis for several oncolytic viruses (OVs) currently in clinical trials. Oncolytic HSV1 (oHSV) have been engineered to reduce neurovirulence and enhance anti-tumor lytic activity and immunogenicity to make them attractive candidates in a range of oncology indications. Successful clinical data resulted in the FDA-approval of the oHSV talimogene laherparepvec (T-Vec) in 2015, and several other variants are currently undergoing clinical assessment and may expand the landscape of future oncologic therapy options. This review offers a detailed overview of the latest results from clinical trials as well as an outlook on newly developed HSV-1 oncolytic variants with improved tumor selectivity, replication, and immunostimulatory capacity and related clinical studies.

Integrity of the Antiviral STING-mediated DNA Sensing in Tumor Cells Is Required to Sustain the Immunotherapeutic Efficacy of Herpes Simplex Oncolytic Virus.

Simple SummaryOncolytic viruses are emerging immunotherapeutics in cancer treatments. The conflicting role of innate immunity in the antitumor activity of oncolytic viruses is still a matter of debate. The STING-dependent DNA sensing axis is considered detrimental for viral replication and cancer cell clearance. Accordingly, we observed that STING loss in tumor cells was associated with improved lytic potential by a herpes-based oncolytic virus. However, STING-knockout cancer cells infected with the oncolytic virus showed impaired immunogenicity, as immunogenic cell death was improperly triggered. In agreement with these observations, STING-knockout tumors raised in a murine syngeneic model were more resistant to a combined treatment of the oncolytic virus with PD-1 blockade. The present study demonstrates the antitumor benefit of antiviral immunity and sheds lights on the mechanisms of immune resistance to oncovirotherapy exerted by STING-loss in tumor cells. The dichotomic contribution of cancer cell lysis and tumor immunogenicity is considered essential for effective oncovirotherapy, suggesting that the innate antiviral immune response is a hurdle for efficacy of oncolytic viruses. However, emerging evidence is resizing this view. By sensing cytosolic DNA, the cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) axis can both counteract viral spread and contribute to the elicitation of adaptive immunity via type I interferon responses. In this paper, we analyzed the tumor-resident function of Sting-mediated DNA sensing in a combined approach of oncovirotherapy and PD-1 immune checkpoint blockade, in an immunocompetent murine model. While supporting increased lytic potential by oncolytic HER2-retargeted HSV-1 in vitro and in vivo, Sting-knockout tumors showed molecular signatures of an immunosuppressive tumor microenvironment. These signatures were correspondingly associated with ineffectiveness of the combination therapy in a model of established tumors. Results suggest that the impairment in antiviral response of Sting-knockout tumors, while favoring viral replication, is not able to elicit an adequate immunotherapeutic effect, due to lack of immunogenic cell death and the inability of Sting-knockout cancer cells to promote anti-tumor adaptive immune responses. Accordingly, we propose that antiviral, tumor-resident Sting provides fundamental contributions to immunotherapeutic efficacy of oncolytic viruses.

Herpes Simplex Virus Oncolytic Immunovirotherapy: The Blossoming Branch of Multimodal Therapy.

Oncolytic viruses are smart therapeutics against cancer due to their potential to replicate and produce the needed therapeutic dose in the tumor, and to their ability to self-exhaust upon tumor clearance. Oncolytic virotherapy strategies based on the herpes simplex virus are reaching their thirties, and a wide variety of approaches has been envisioned and tested in many different models, and on a range of tumor targets. This huge effort has culminated in the primacy of an oncolytic HSV (oHSV) being the first oncolytic virus to be approved by the FDA and EMA for clinical use, for the treatment of advanced melanoma. The path has just been opened; many more cancer types with poor prognosis await effective and innovative therapies, and oHSVs could provide a promising solution, especially as combination therapies and immunovirotherapies. In this review, we analyze the most recent advances in this field, and try to envision the future ahead of oHSVs.

Antitumor effects of IL-12 and GM-CSF co-expressed in an engineered oncolytic HSV-1.

Oncolytic viruses selectively replicate and destroy cancer cells while sparing normal cells, prompting their recognition as promising antitumor agents. Herpes simplex virus (HSV) is suitable as an anticancer agent, given its considerable therapeutic gene capacity and excellent safety profile in clinical trials. Interleukin (IL)-12 induces a Th1-type immune response that mediates interferon (IFN)-γ release from natural killer(NK), CD4+ and CD8+ T cells. Granulocyte-macrophage colony-stimulating factor (GM-CSF) induces the generation of antigen-presenting cells and promotes dendritic cell differentiation. We established a novel oncolytic HSV-1 (∆6/GM/IL12) co-expressing IL-12 and GM-CSF and tested its effects against a B16-F10 murine melanoma model. ∆6/GM/IL12 administration diminished tumor growth and prolonged survival compared to treatment with ∆6/GM or ∆6/IL12 expressing each individual cytokine. Flow cytometry and histological analysis showed increased activation of CD4+ and CD8+ T cells in ∆6/GM/IL12-treated mice. Enzyme-linked immunosorbent spot assay showed an increase in the phenotypically characterized IFN-γ-producing cell population in ∆6/GM/IL12-treated mice. Moreover, ∆6/GM/IL12 induced a B16-F10-specific cytotoxic immune response that enhanced IFN-γ production by CD3+CD8+ T cells. Therefore, IL-12 and GM-CSF from an engineered oncolytic HSV have a synergistic effect, boosting the immune response to increase their antitumor effects.