Oncolytic viruses (OVs) encoding interferon beta (IFN-β) are under clinical evaluation. IFN-β is an attractive transgene due to its antiproliferative and immunomodulatory properties; however, the systemic effects of sustained IFN-β expression remain poorly defined. Here, we demonstrate that prolonged circulating IFN-β induces dose-dependent toxicity in murine models. Persistent IFN-β exposure led to elevated transaminases, thrombocytopenia, lymphopenia, and reduced hemoglobin. Histopathologic analysis revealed sinusoidal endothelial sloughing, microvascular coagulation with microthrombi, and hepatocellular vacuolation and degeneration, along with bone marrow hemorrhage and cell death. These toxicities were absent in type I interferon receptor knockout mice. Importantly, termination of IFN-β production via inducible caspase-9 in adeno-associated vector-transduced cells reversed toxicity and normalized serum transaminases. In Balb/c mice bearing MPC-11 myeloma tumors which are highly permissive to IFN-β-expressing OVs, treatment induced rapid tumor lysis, elevated plasma IFN-β, and mortality. Importantly, co-administration of ruxolitinib, a clinically approved JAK1/2 inhibitor, dampened IFN-β signaling and rescued mice from lethal toxicity. Collectively, these findings define the pathophysiological consequences of sustained systemic IFN-β exposure and identify ruxolitinib as a potential mitigation strategy to manage IFN-β-mediated toxicity during OV treatment.

PURPOSE:VV1 is designed to induce selective oncolysis of tumor cells and amplify cellular antitumor immune responses. This open-label, phase I, multicenter clinical trial assessed the safety and tolerability of VV1 monotherapy administered intratumorally or intravenously or administered intravenously in combination with avelumab. PATIENTS AND METHODS:Patients with advanced solid tumors received intratumoral (n = 27) or intravenous VV1 monotherapy (n = 33) or intravenous VV1 plus avelumab (n = 16). Infusion durations (15-180 minutes) and viral dose (1.7 × 1010 or 1 × 1011 TCID50) were also evaluated. Study objectives included VV1 safety and tolerability, pharmacokinetics, pharmacodynamics, preliminary efficacy, and immune responses. RESULTS:Intratumoral and intravenous VV1 were well tolerated, and injection reactions were infrequent. The most common adverse events of any grade related to VV1 were cytokine release syndrome (58%), fatigue (33%), and decreased lymphocyte count (32%). Virus infection of tumors was confirmed by NIS imaging and increases in serum levels of virally encoded interferon-β (IFNβ). Histologic tumor analysis at 28 days after intratumoral VV1 administration indicated increases in tumor-infiltrating immune cells. Two durable partial responses were recorded: a patient with pheochromocytoma after one intravenous dose of VV1 and a patient with thymic cancer in the combination arm. Stable disease was observed in 17 of 49 patients (34.7%) who received intravenous VV1. CONCLUSIONS:This first-in-human study demonstrates that intratumoral or intravenous VV1 administration had an acceptable safety profile as monotherapy and intravenously in combination with avelumab. There is preliminary antitumor activity. Intratumoral VV1 plus cemiplimab is now showing promise in the neoadjuvant setting. SIGNIFICANCE:VV1 is a tumor-selective oncolytic vesicular stomatitis virus engineered to express IFNβ and the thyroidal NIS reporter gene. In this phase I study, we have demonstrated that a single administration of VV1, as a monotherapy or with an immune checkpoint inhibitor is safe, infects tumor lesions resulting in intratumoral infiltration of immune cells, and exhibits early signs of antitumor activity in patients with advanced unresectable and metastatic solid tumors.

Study population representativeness

Imaging following intratumoral (IT) and intravenous (IV) VV1. A, CT images of the thoracic region of a 57-year-old patient with thymic carcinoma following a 30-minute intravenous infusion of VV1 (1.7 × 1010 TCID50) + labeled dose/regimen of avelumab (part 3). In the opinion of the investigator, only scar tissue remained at the last assessment (August 2021). B, SPECT/CT/NIS imaging of 75-year-old male patient with adenocarcinoma (unknown primary cancer) following intratumoral (IT) administration of VV1 (3 × 108 TCID50) monotherapy (part 1). C, SPECT/CT/NIS imaging of a 62-year-old male patient with mesothelioma following IT administration of VV1 (1 × 108 TCID50) monotherapy (part 1). D, Immune cell infiltrates and PD-1 in a male patient with head and neck squamous cell (HNSCC) with SD, following IT administration of VV1 (part 1). Images are of an injected lesion. E, Immune cell infiltrates and PD-1 in a 70-year-old male patient with HNSCC with SD, following IT administration of VV1 (3 × 106 TCID50) monotherapy (part 1). Top, baseline. Middle, injected lesion. Bottom, noninjected lesion.

Abstract We have developed engager proteins that reversibly block VSV-G, directing VSV-G-pseudotyped lentiviral vectors (LVV) into resting CD3-positive T cells. Following selective binding of the engager-modified LVV particles to CD3, they are internalized into endosomes, where the engager is detached by the low pH and the absence of calcium ions, releasing unmodified LVV. Free LVV particles then fuse with the endosomal membrane and efficiently deliver their genetic cargo to the cytoplasm. These engager proteins are stable and easily produced in high yields, making them suitable for practical use. Reversible bi-specific engagers that bind to the homotrimeric VSV-G protein and redirect it to a targeted receptor were initially developed by genetically fusing a cell-targeting polypeptide to the VSV-G binding domains CR2 and CR3 of the LDL receptor (Israel Patent application No. 296547, 2022. Inventors: M. Rubinstein, G. Schreiber, D. Gataulin, Weizmann Institute). When pre-incubated with VSV and VSV-G-LVV, these engagers mediated selective vector entry into mammalian cells via the targeted receptor while preventing non-specific entry through LDLR. The monomeric engagers blocked binding of VSV-G to LDLR with an ED50 of 3 nM. Trimeric engager versions exhibited an ED50 of 20 pM. To increase the binding avidity of the engager to VSV-G, we engineered and produced trimeric engagers consisting of CD3-specific scFv fused through a trimerizing peptide to VSV-G binding domains of LDLR. When co-incubated with LVV, the engagers bound stably to LVV, preventing transduction through LDLR or its other family members, while mediating efficient transduction via the targeted CD3 receptor. CD3-directed engager-modified LVVs efficiently transduced and activated primary human T cells in PBMC cultures, exhibiting stable CD3 targeting even after prolonged storage at room temperature and freeze-thaw cycling. CD3-engager-modified LVV particles encoding an anti-BCMA CAR were administered intravenously to (human) PBMC-engrafted NSG-DKO mice bearing established BCMA-positive human myeloma xenografts. Rapid and complete tumor regressions were observed in all animals treated with the engager-modified LVVs. In summary, we have generated trimerizing bifunctional engagers that redirect the attachment and entry of VSV-G-pseudotyped lentiviral vectors into resting human T cells. Engager-modified LVVs encoding a CAR transgene were therapeutically effective when administered intravenously to tumor-bearing animals. Clinical translation is planned. Citation Format: Gopal Naik Nenavath, Nandakumar Packiriswamy, Diana Gataulin, Md Sharif Hasan, Tatenda Kadungure, Harshitha Anantharama, Pragati Jain, Kyle Gromer, Karina Krotova, Miriam Eisenstein, Gideon Schreiber, Patrycja Lech, Luis Blancas Mejia, Colin Caine, Thipparat Suwanmanee, Luke Breigenzer, Darren Phung, Tim Carey, Hamid Salimi, Emma Buck, Christian Kinney, Luke Russell, Kah-Whye Peng, Stephen J Russell, Menachem Rubinstein. High-avidity reversible engager proteins efficiently redirect the entry of intravenously delivered lentiviral vectors, simplifying the direct in vivo delivery of CAR-T cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 273.

Vesicular stomatitis virus (VSV) is a promising anticancer agent but rapidly loses its infectivity in human serum. Here, we demonstrate that low-density and very low-density lipoproteins (LDLs and VLDLs) compete with VSV-G-displaying vectors for occupancy of cellular LDL receptors (LDLRs). Infectivity of VSV-G-displaying vectors was reduced two- to three-logs in the presence of heat-inactivated human serum but not if the serum was depleted of lipoproteins. The inhibitory effect was replicated in serum-free media supplemented with physiological concentrations of purified lipoprotein particles that contain the LDLR-binding protein Apo B-100, and the degree of inhibition correlated with the concentration of LDL or VLDL. When VSV-G was retargeted to the epidermal growth factor receptor, competitive inhibition by human serum or by LDL/VLDL was no longer observed. Similar results were obtained when VSV-G was retargeted to other receptors. Our findings demonstrate that serum LDL and VLDL competitively block the entry of VSV-G-displaying viral particles, but this barrier for in vivo delivery can be circumvented through a display of a receptor-targeting ligand on VSV-G.

VSV-IFNβ-NIS (VV1) is a Vesicular Stomatitis Virus (VSV) that encodes the interferon beta (IFNβ) and the iodide symporter (NIS) gene. The IFNβ gene confers tumor selectivity and enhances host antitumor immunity while the NIS gene acts as an imaging biomarker, allowing noninvasive imaging of viral infection sites using PET/SPECT. Multiple myeloma (MM) is a cancer of plasma cells and poses a challenge for treatment due to its biological heterogeneity. As MM usually relapses and is refractory, current therapies face the issue of drug resistance and patients may have to turn to novel therapies. Therefore, we set out to understand the activity of VSV which could be a new approach for patients with relapsed refractory MM (RRMM). Our study utilized two unique bortezomib resistant Vk*MYC transplantable models, Vk12598 and Vk12653, with distinct genomic features. Upon re-transplantation of splenocytes from these mice, flow cytometry revealed that Vk12598 and Vk12653 mice had high MM populations in the spleen and bone marrow. To assess VV1 efficacy in tumor bearing mice, we performed a VV1 dose escalation study, showing efficacy in delaying MM development and prolonging survival in both mice models, with Vk12598 being the more responsive model. Later studies also showed that VV1 induces tumor remission in the Vk12598 model when treatment was initiated at low tumor burden (gamma/albumin ratio ∼0.2). IFNβ levels increased significantly post 24-72h of VV1 treatment, indicating targeted infection and replication of the virus in MM cells. However, high doses of VV1 led to toxicity, as observed from early mortality and weight loss. To determine the sites of MM metastasis and VV1 infection, timepoint harvests were carried out. Hematoxylin and eosin and immunostainings showed the onset of MM of Vk*MYC in the bone marrow and spleen first, then later in the liver. Immunofluorescent staining confirmed that the higher the tumor burden observed, the more VV1 was present in the tissues. To enhance VV1 efficacy, we performed in vivo combination therapies of VV1 with anti-PD-1, an inhibitor of PD-1 which is overexpressed on MM patient T cells; and bortezomib, which inhibits NF-κB which in turn prevents IFN pathway protein transcription, potentially enhancing the replication of VV1 virus. In Vk12598 mice, VV1 combined with both drugs induced tumor regression and doubled the median survival time compared to treatment with VV1 only or VV1 coupled with either of the drugs individually. Addition of VV1 to either anti-PD-1 or bortezomib alone also increased survival time significantly. Studies are still ongoing to understand the mechanisms of VSV and its combination treatments in Vk*MYC models. This translational study will allow us to gain the knowledge needed to refine VSV treatments and provide novel therapy combinations to improve MM patient survival. Yi Ying Ma, Lianwen Zhang, Rebecca A. Nace, Marta Chesi, Stephen J. Russell, Kah Whye Peng. Harnessing oncolytic virus VSV and combination therapy to advance treatments for relapsed refractory multiple myeloma using Vk*MYC models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 940.

Herpes simplex virus (HSV)-based oncolytic virotherapy has demonstrated promising antitumor effects across various cancer types. However, its application remains limited in scope, and expanding its use to additional cancers poses ongoing challenges. Recently, two other human herpesviruses—varicella-zoster virus (VZV) and cytomegalovirus (CMV)—have emerged as potential platforms for oncolytic virotherapy. In this review, we describe the potential tumor cross-reactivity of the T cell and natural killer (NK) cell responses that are activated and amplified by VZV and CMV, highlighting clinical observations and experimental findings that support the feasibility of redirecting and harnessing these virus-driven immune responses for effective tumor control. We also summarize recent progress in developing oncolytic VZV and CMV vectors, including advances in virus engineering, production, and delivery strategies. This review offers critical insights and highlights key challenges in establishing VZV- and CMV-based cancer viroimmunotherapy platforms.

Chimeric antigen receptor (CAR) T cells have had limited success against solid tumors. Here, we used an oncolytic foamy virus (oFV) to display a model CAR target antigen (CD19) on tumors in combination with anti-CD19 CAR T cells. We generated oFV-Δbel2 and oFV-bel2 vectors to test the efficiency and stability of viral/CD19 spread. While both viruses conferred equal CAR T killing in vitro, the oFV-Δbel2 virus acquired G-to-A mutations, whereas oFV-bel2 virus had genome deletions. In subcutaneous tumor models in vivo, CAR T cells led to a significant decrease in oFV-specific bioluminescence, confirming clearance of oFV-infected tumor cells. However, the most effective therapy was with high-dose oFV in the absence of CAR T cells, indicating that CAR T clearance of oFV was detrimental. Moreover, in tumors that escaped CAR T cell treatment, resurgent virus contained deletions within the oFV-CD19 transgene, allowing the virus to escape CAR T elimination. Therefore, oFV represents a slow smoldering type of oncolytic virus, whose chronic spread through tumors generates anti-tumor therapy, which is abolished by CAR T therapy. These results suggest that further development of this oncolytic platform, with additional immunotherapeutic arming, may allow for an effective combination of chronic oncolysis.

TPS657 Background: Poorly differentiated neuroendocrine carcinoma (NEC) is an aggressive malignancy comprising both pulmonary and extrapulmonary primary sites. NEC includes both small cell lung cancer (SCLC) and large cell neuroendocrine carcinoma (LCNEC), as well as other neuroendocrine carcinomas arising from any primary organ. A substantial portion (~40%) of NEC arises from gastrointestinal primary sites. The optimal systemic therapy beyond first line platinum and etoposide is not established. There is a critical need to improve upon the median survival in the second line, as most patients do not survive more than 6 months. The efficacy of single agent immune checkpoint inhibitors (ICIs) in NEC has been disappointing. One possible explanation for this is that the tumor microenvironment in NEC is non-inflamed. VSV-IFNβ-NIS is a vesicular stomatitis virus (VSV)-based oncolytic virus being tested in multiple early phase clinical trials. Preliminary studies of immune responses in patients receiving VSV-IFNβ-NIS therapy suggest some patients develop T cell responses to viral antigens and known tumor antigens. We hypothesize that VSV-IFNβ-NIS therapy may convert a non-inflamed or immune-excluded phenotype in NEC to a highly inflamed phenotype that sensitizes the tumor to ICIs. Methods: This is a phase 1-2 safety run-in study designed to determine the safety of VSV-IFNβ-NIS in combination with a single agent ICI, pembrolizumab, followed by dose expansion in patients with refractory non-small cell lung cancer (NSCLC) or NEC. The safety run-in portion of this study has been completed, and we are presently testing the recommended phase 2 dose (RP2D) of VSV-IFNβ-NIS in an expansion cohort of patients with SCLC or NEC of any primary site. Patients must have previously progressed on at least one line of systemic therapy. Prior treatment with checkpoint inhibitors is permitted. Patients are treated one time with the RP2D of 1.0x10^11 TCID50 VSV-IFNβ-NIS on day 1, followed by pembrolizumab on day 8 and then pembrolizumab every 21 days until progression, up to 2 years. The primary objective is to estimate the response rate by RECIST 1.1. Secondary objectives include estimation of disease-control rate, duration or response, progression-free survival, overall survival, and safety signals. The NEC expansion cohort will seek to enroll 10 patients. If at least one objective response is observed, and safety is confirmed, the regimen will be considered for future study. Clinical trial information: NCT03647163 .

BackgroundThe varicella-zoster virus (VZV), belonging to the group of human α-herpesviruses, has yet to be developed as a platform for oncolytic virotherapy, despite indications from clinical case reports suggesting a potential association between VZV infection and cancer remission.MethodsHere, we constructed oncolytic VZV candidates based on the vaccine strain vOka and the laboratory strain Ellen. These newly engineered viruses were subsequently assessed for their oncolytic properties in the human MeWo melanoma xenograft model and the mouse B16-F10-nectin1 melanoma syngeneic model.ResultsIn the MeWo xenograft model, both vOka and Ellen exhibited potent antitumor efficacy. However, it was observed that introducing a hyperfusogenic mutation into glycoprotein B led to a reduction in VZV’s effectiveness. Notably, the deletion of ORF8 (encodes viral deoxyuridine triphosphatase) attenuated the replication of VZV both in vitro and in vivo, but it did not compromise VZV’s oncolytic potency. We further armed the VZV Ellen-ΔORF8 vector with a tet-off controlled mouse single-chain IL12 (scIL12) gene cassette. This augmented virus was validated for its oncolytic activity and triggered systemic antitumor immune responses in the immunocompetent B16-F10-nectin1 model.ConclusionsThese findings highlight the potential of using Ellen-ΔORF8-tet-off-scIL12 as a novel VZV-based oncolytic virotherapy.