An optimal promoter regulating cytokine transgene expression is crucial for safe and effective oncolytic virus immunotherapy.

In general, ensuring safety is the top priority of a new modality. Although oncolytic virus armed with an immune stimulatory transgene (OVI) showed some promise, the strategic concept of simultaneously achieving maximum effectiveness and minimizing side effects has not been fully explored. We generated a variety of survivin-responsive "conditionally replicating adenoviruses that can target and treat cancer cells with multiple factors (m-CRAs)" (Surv.m-CRAs) armed with the granulocyte-macrophage colony-stimulating factor (GM-CSF) transgene downstream of various promoters using our m-CRA platform technology. We carefully analyzed both therapeutic and adverse effects of them in the in vivo syngeneic Syrian hamster cancer models. Surprisingly, an intratumor injection of a conventional OVI, which expresses the GM-CSF gene under the constitutively and strongly active "cytomegalovirus enhancer and ß-actin promoter", provoked systemic and lethal GM-CSF circulation and shortened overall survival (OS). In contrast, a new conceptual type of OVI, which expressed GM-CSF under the cancer-predominant and mildly active E2F promoter or the moderately active "Rous sarcoma virus long terminal repeat", not only abolished lethal adverse events but also prolonged OS and systemic anti-cancer immunity. Our study revealed a novel concept that optimal expression levels of an immune stimulatory transgene regulated by a suitable upstream promoter is crucial for achieving high safety and maximal therapeutic effects simultaneously in OVI therapy. These results pave the way for successful development of the next-generation OVI and alert researchers about possible problems with ongoing clinical trials.

A Case Report of Pathologically Complete Response of a Huge Lymph Node Metastasis of Colorectal Cancer After Treatment with Intratumoral Oncolytic Virus H101 and Capecitabine.

Unresectable recurrent lymph node metastasis of colorectal cancer (CRC) is considered as an incurable disease clinically and has a very poor prognosis. Here, we report a male KRAS wild-type CRC case with a huge abdominal lymph node metastasis (12 cm in diameter) after CRC surgery. After three intratumoral injections of oncolytic virus (H101) combined with four cycles of low-dose oral capecitabine, the size of the metastatic lymph node shrank remarkably in response to the anticancer drug and a complete response (CR) was achieved with progression-free survival (PFS) of 19 months. The main adverse reaction was mild fever, which was relieved after physical cooling. The patient is in a general good condition now without any relapse of abdominal lymph node for over a year. On this basis, we propose that the combination therapy of oncolytic virus and capecitabine could be a promising clinical therapeutic strategy for unresectable recurrent lymph node metastasis in CRC patients.

Corrigendum: Innovative retargeted oncolytic herpesvirus against nectin4-positive cancers.

[This corrects the article DOI: 10.3389/fmolb.2023.1149973.].

The Oncolytic virus VT1092M and an Anti-PD-L1 antibody synergize to induce systemic antitumor immunity in a murine bilateral tumor model.

This study investigated the synergistic potential of an oncolytic herpes simplex virus armed with interleukin 12 (VT1092M) in combination with immune checkpoint inhibitors for enhancing antitumor responses. The potential of this combination treatment to induce systemic antitumor immunity was assessed using bilateral subcutaneous tumor and tumor re-challenge mouse models. The antitumor efficacy of various OV and ICI treatment combinations and the underlying mechanisms were explored through diverse analytical techniques, including flow cytometry and RNA sequencing. Using VT1092M, either alone or in combination with an anti-PD-L1 antibody, significantly reduced the sizes of both the injected and untreated abscopal tumors in a bilateral tumor mouse model. The combination therapy demonstrated superior antitumor efficacy to the other treatment conditions tested, which was accompanied by an increase in T cell numbers and CD8+T cell activation. Results from the survival and tumor re-challenge experiments showed that the combination therapy elicited long-term, tumor-specific immune responses, which were associated with tumor clearance and prolonged survival. Immune cell depletion assays identified CD8+T cells as the crucial mediators of systemic antitumor immunity during combination therapy. In conclusion, the combination of VT1092M and PD-L1 blockade emerged as a potent inducer of antitumor immune responses, surpassing the efficacy of each monotherapy. This synergistic approach holds promise for achieving robust and sustained antitumor immunity, with potential implications for preventing tumor metastasis in patients with cancer.

Absolute quantification of viral proteins from pseudotyped VSV-GP using UPLC-MRM.

The rapidly developing field of oncolytic virus (OV) therapy necessitates the development of new and improved analytical approaches for the characterization of the virus during production and development. Accurate monitoring and absolute quantification of viral proteins are crucial for OV product characterization and can facilitate the understanding of infection, immunogenicity, and development stages of viral replication. Targeted mass spectrometry methods like multiple reaction monitoring (MRM) offer a robust way to directly detect and quantify specific targeted proteins represented by surrogate peptides. We have leveraged the power of MRM by combining ultra-high performance liquid chromatography (UPLC) with a Sciex 6500 triple-stage quadrupole mass spectrometer to develop an assay that accurately and absolutely quantifies the structural proteins of a pseudotyped vesicular stomatitis virus (VSV) intended for use as a new biotherapeutic (designated hereafter as VSV-GP to differentiate it from native VSV). The new UPLC-MRM method provides absolute quantification with the use of heavy-labeled reference standard surrogate peptides. When added in known exact amounts to standards and samples, the reference standards normalize and account for any small perturbations during sample preparation and/or instrument performance, resulting in accurate and precise quantification. Because of the multiplexed nature of MRM, all targeted proteins are quantified at the same time. The optimized assay has been enhanced to quantify the ratios of the processed GP1 and GP2 proteins while simultaneously measuring any remaining or unprocessed form of the envelope protein GP complex (GPC; full-length GPC). IMPORTANCE: The development of oncolytic viral therapy has gained considerable momentum in recent years. Vesicular stomatitis virus glycoprotein (VSV-GP) is a new biotherapeutic emerging in the oncolytic viral therapy platform. Novel analytical assays that can accurately and precisely quantify the viral proteins are a necessity for the successful development of viral vector as a biotherapeutic. We developed an ultra-high performance liquid chromatography multiple reaction monitoring-based assay to quantify the absolute concentrations of the different structural proteins of VSV-GP. The complete processing of GP complex (GPC) is a prerequisite for the infectivity of the virus. The assay extends the potential for quantifying full-length GPC, which provides an understanding of the processing of GPC (along with the quantification of GP1 and GP2 separately). We used this assay in tracking GPC processing in HEK-293-F production cell lines infected with VSV-GP.

An Update on the Clinical Status, Challenges, and Future Directions of Oncolytic Virotherapy for Malignant Gliomas.

OPINION STATEMENT: Malignant gliomas are common central nervous system tumors that pose a significant clinical challenge due to the lack of effective treatments. Glioblastoma (GBM), a grade 4 malignant glioma, is the most prevalent primary malignant brain tumor and is associated with poor prognosis. Current clinical trials are exploring various strategies to combat GBM, with oncolytic viruses (OVs) appearing particularly promising. In addition to ongoing and recently completed clinical trials, one OV (Teserpaturev, Delytact®) received provisional approval for GBM treatment in Japan. OVs are designed to selectively target and eliminate cancer cells while promoting changes in the tumor microenvironment that can trigger and support long-lasting anti-tumor immunity. OVs offer the potential to remodel the tumor microenvironment and reverse systemic immune exhaustion. Additionally, an increasing number of OVs are armed with immunomodulatory payloads or combined with immunotherapy approaches in an effort to promote anti-tumor responses in a tumor-targeted manner. Recently completed oncolytic virotherapy trials can guide the way for future treatment individualization through patient preselection, enhancing the likelihood of achieving the highest possible clinical success. These trials also offer valuable insight into the numerous challenges inherent in malignant glioma treatment, some of which OVs can help overcome.

Safety of non-replicative and oncolytic replication-selective HSV vectors.

Herpes simplex virus type 1 (HSV-1) is a DNA virus and human pathogen used to construct promising therapeutic vectors. HSV-1 vectors fall into two classes: replication-selective oncolytic vectors for cancer therapy and defective non-replicative vectors for gene therapy. Vectors from each class can accommodate ≥30 kb of inserts, have been approved clinically, and demonstrate a relatively benign safety profile. Despite oncolytic HSV (oHSV) replication in tumors and elicited immune responses, the virus is well tolerated in cancer patients. Current non-replicative vectors elicit only limited immune responses. Seropositivity and immune responses against HSV-1 do not eliminate either the vector or infected cells, and the vectors can therefore be re-administered. In this review we highlight vectors that have been translated to the clinic and host-virus immune interactions that impact on the safety and efficacy of HSVs.

Oscillations in a Spatial Oncolytic Virus Model.

Virotherapy treatment is a new and promising target therapy that selectively attacks cancer cells without harming normal cells. Mathematical models of oncolytic viruses have shown predator-prey like oscillatory patterns as result of an underlying Hopf bifurcation. In a spatial context, these oscillations can lead to different spatio-temporal phenomena such as hollow-ring patterns, target patterns, and dispersed patterns. In this paper we continue the systematic analysis of these spatial oscillations and discuss their relevance in the clinical context. We consider a bifurcation analysis of a spatially explicit reaction-diffusion model to find the above mentioned spatio-temporal virus infection patterns. The desired pattern for tumor eradication is the hollow ring pattern and we find exact conditions for its occurrence. Moreover, we derive the minimal speed of travelling invasion waves for the cancer and for the oncolytic virus. Our numerical simulations in 2-D reveal complex spatial interactions of the virus infection and a new phenomenon of a periodic peak splitting. An effect that we cannot explain with our current methods.

Hypothesis: AdAPT-001 and pseudoprogression - when seeing is not necessarily believing.

The purpose of this commentary is to highlight the high occurrence of clinical pseudoprogression and delayed responses that have been observed to date with the locally injected oncolytic adenovirus, AdAPT-001, currently in a Phase 1/2 clinical trial (NCT04673942) for the treatment of treatment-refractory tumors. Not surprisingly, these have led to confusion about response assessment and whether to continue patients on treatment. AdAPT-001 carries a transforming growth factor (TGF)-beta trap (TGF-ß), which sequesters TGF-ß, a cytokine that potently regulates inflammation, fibrosis, and immunosuppression in cancer. Pseudoprogression (PsP) or progression prior to response or stabilization, has been widely recognized with radiotherapy for primary brain tumors and immune checkpoint inhibitors (ICIs). PsP has also been described and documented in the context of oncolytic virotherapy but perhaps to a lesser extent. However, repeated intratumoral injections with these immunostimulatory agents may induce a more intense immune response and release more antigenic epitopes than with ICIs, for example, which are strictly T-cell directed rather than also tumor-directed like AdAPT-001.

Current and future immunotherapeutic approaches in pancreatic cancer treatment.

Pancreatic cancer is a major cause of cancer-related death, but despondently, the outlook and prognosis for this resistant type of tumor have remained grim for a long time. Currently, it is extremely challenging to prevent or detect it early enough for effective treatment because patients rarely exhibit symptoms and there are no reliable indicators for detection. Most patients have advanced or spreading cancer that is difficult to treat, and treatments like chemotherapy and radiotherapy can only slightly prolong their life by a few months. Immunotherapy has revolutionized the treatment of pancreatic cancer, yet its effectiveness is limited by the tumor's immunosuppressive and hard-to-reach microenvironment. First, this article explains the immunosuppressive microenvironment of pancreatic cancer and highlights a wide range of immunotherapy options, including therapies involving oncolytic viruses, modified T cells (T-cell receptor [TCR]-engineered and chimeric antigen receptor [CAR] T-cell therapy), CAR natural killer cell therapy, cytokine-induced killer cells, immune checkpoint inhibitors, immunomodulators, cancer vaccines, and strategies targeting myeloid cells in the context of contemporary knowledge and future trends. Lastly, it discusses the main challenges ahead of pancreatic cancer immunotherapy.

Emerging delivery strategy for oncolytic virotherapy.

Oncolytic virotherapy represents a promising approach in cancer immunotherapy. The primary delivery method for oncolytic viruses (OVs) is intratumoral injection, which apparently limits their clinical application. For patients with advanced cancer with disseminated metastasis, systemic administration is considered the optimal approach. However, the direct delivery of naked viruses through intravenous injection presents challenges, including rapid clearance by the immune system, inadequate accumulation in tumors, and significant side effects. Consequently, the development of drug delivery strategies has led to the emergence of various bio-materials serving as viral vectors, thereby improving the anti-tumor efficacy of oncolytic virotherapy. This review provides an overview of innovative strategies for delivering OVs, with a focus on nanoparticle-based or cell-based delivery systems. Recent pre-clinical and clinical studies are examined to highlight the enhanced efficacy of systemic delivery using these novel platforms. In addition, prevalent challenges in current research are briefly discussed, and potential solutions are proposed.

A modified HSV-1 oncolytic virus reconciles antiviral and antitumor immunity via promoting IFNß expression and inhibiting PKR.

Type I interferon (IFN-I) is a potent immune modulator intricately involved in regulating tumor immunity. Meanwhile, the integrity of the IFN-I signaling pathway is essential for radiotherapy, chemotherapy, targeted therapy, and immunotherapy. However, the clinical application of IFN-I remains challenging due to its non-specific cytotoxicity and limited half-life. To overcome these limitations, we developed a gene delivery platform, CRISPR-V, enabling the rapid creation of novel HSV-1 oncolytic viruses. Utilizing this platform, we created an oncolytic virus, OVH-IFNß, in which the IFNß gene was incorporated into the HSV-1 genome. However, exogenous IFNß expression significantly inhibited OVH-IFNß replication. Through transcriptome data analyses, we identified several ISG genes inhibiting OVH-IFNß replication. By gene knockout and functional studies of the downstream effectors, we confirmed the prominent antiviral activities of protein kinase R (PKR). To balance the antitumor and antiviral immunity of IFNß, we developed a novel HSV-1 oncolytic virus, OVH-IFNß-iPKR, which can express IFNß while inhibiting PKR, leading to a potent antitumor immunity while reducing the antiviral capacity of IFNß. OVH-IFNß-iPKR shows a strong ability to induce immunogenic cell death and activate tumor-specific CD8+ T cells, leading to de novo immune responses and providing a novel strategy for tumor immunotherapy.

Revealing the therapeutic properties of gut microbiota: transforming cancer immunotherapy from basic to clinical approaches.

The immune system plays a pivotal role in the battle against cancer, serving as a formidable guardian in the ongoing fight against malignant cells. To combat these malignant cells, immunotherapy has emerged as a prevalent approach leveraging antibodies and peptides such as anti-PD-1, anti-PD-L1, and anti-CTLA-4 to inhibit immune checkpoints and activate T lymphocytes. The optimization of gut microbiota plays a significant role in modulating the defense system in the body. This study explores the potential of certain gut-resident bacteria to amplify the impact of immunotherapy. Contemporary antibiotic treatments, which can impair gut flora, may diminish the efficacy of immune checkpoint blockers. Conversely, probiotics or fecal microbiota transplantation can help re-establish intestinal microflora equilibrium. Additionally, the gut microbiome has been implicated in various strategies to counteract immune resistance, thereby enhancing the success of cancer immunotherapy. This paper also acknowledges cutting-edge technologies such as nanotechnology, CAR-T therapy, ACT therapy, and oncolytic viruses in modulating gut microbiota. Thus, an exhaustive review of literature was performed to uncover the elusive link that could potentiate the gut microbiome's role in augmenting the success of cancer immunotherapy.

Pharmacokinetic enhancement of oncolytic virus M1 by inhibiting JAK‒STAT pathway.

Oncolytic viruses (OVs), a group of replication-competent viruses that can selectively infect and kill cancer cells while leaving healthy cells intact, are emerging as promising living anticancer agents. Unlike traditional drugs composed of non-replicating compounds or biomolecules, the replicative nature of viruses confer unique pharmacokinetic properties that require further studies. Despite some pharmacokinetics studies of OVs, mechanistic insights into the connection between OV pharmacokinetics and antitumor efficacy remain vague. Here, we characterized the pharmacokinetic profile of oncolytic virus M1 (OVM) in immunocompetent mouse tumor models and identified the JAK‒STAT pathway as a key modulator of OVM pharmacokinetics. By suppressing the JAK‒STAT pathway, early OVM pharmacokinetics are ameliorated, leading to enhanced tumor-specific viral accumulation, increased AUC and Cmax, and improved antitumor efficacy. Rather than compromising antitumor immunity after JAK‒STAT inhibition, the improved pharmacokinetics of OVM promotes T cell recruitment and activation in the tumor microenvironment, providing an optimal opportunity for the therapeutic outcome of immune checkpoint blockade, such as anti-PD-L1. Taken together, this study advances our understanding of the pharmacokinetic-pharmacodynamic relationship in OV therapy.

Open-label, phase II study of talimogene laherparepvec plus pembrolizumab for the treatment of advanced melanoma that progressed on prior anti-PD-1 therapy: MASTERKEY-115.

BACKGROUND: Treatment options for immunotherapy-refractory melanoma are an unmet need. The MASTERKEY-115 phase II, open-label, multicenter trial evaluated talimogene laherparepvec (T-VEC) plus pembrolizumab in advanced melanoma that progressed on prior programmed cell death protein-1 (PD-1) inhibitors. METHODS: Cohorts 1 and 2 comprised patients (unresectable/metastatic melanoma) who had primary or acquired resistance, respectively, and disease progression within 12 weeks of their last anti-PD-1 dose. Cohorts 3 and 4 comprised patients (resectable disease) who underwent complete surgery, received adjuvant anti-PD-1, and experienced recurrence. Cohort 3 were disease-free for < 6 months and cohort 4 for ≥ 6 months after starting the adjuvant anti-PD-1 therapy and before confirmed recurrence. The primary endpoint was objective response rate (ORR) per RECIST v1.1. Secondary endpoints included complete response rate (CRR), disease control rate (DCR) and progression-free survival (PFS) per RECIST v1.1 and irRC-RECIST, and safety. RESULTS: Of the 72 enrolled patients, 71 were treated. The ORR (95% CI) was 0%, 6.7% (0.2-32.0), 40.0% (16.3-67.7), and 46.7% (21.3-73.4) in cohorts 1-4, respectively; iORR was 3.8% (0.1-19.6), 6.7% (0.2-32.0), 53.3% (26.6-78.7), and 46.7% (21.3-73.4). iCRR was 0%, 0%, 13.3%, and 13.3%. Median iPFS (months) was 5.5, 8.2, not estimable [NE], and NE for cohorts 1-4, respectively; iDCR was 50.0%, 40.0%, 73.3%, and 86.7%. Treatment-related adverse events (TRAEs), grade ≥ 3 TRAEs, serious AEs, and fatal AEs occurred in 54 (76.1%), 9 (12.7%), 24 (33.8%), and 10 (14.1%) patients, respectively. CONCLUSION: T-VEC-pembrolizumab demonstrated antitumor activity and tolerability in PD-1-refractory melanoma, specifically in patients with disease recurrence on or after adjuvant anti-PD-1. TRIAL REGISTRATION: ClinicalTrials.gov identifier - NCT04068181.

Combination of FOLFOXIRI Drugs with Oncolytic Coxsackie B3 Virus PD-H Synergistically Induces Oncolysis in the Refractory Colorectal Cancer Cell Line Colo320.

FOLFOXIRI chemotherapy is a first-line therapy for advanced or metastatic colorectal cancer (CRC), yet its therapeutic efficacy remains limited. Immunostimulatory therapies like oncolytic viruses can complement chemotherapies by fostering the infiltration of the tumor by immune cells and enhancing drug cytotoxicity. In this study, we explored the effect of combining the FOLFOXIRI chemotherapeutic agents with the oncolytic coxsackievirus B3 (CVB3) PD-H in the CRC cell line Colo320. Additionally, we examined the impact of the drugs on the expression of microRNAs (miRs), which could be used to increase the safety of oncolytic CVB3 containing corresponding miR target sites (miR-TS). The measurement of cytotoxic activity using the Chou-Talalay combination index approach revealed that PD-H synergistically enhanced the cytotoxic activity of oxaliplatin (OX), 5-fluorouracil (5-FU) and SN-38. PD-H replication was not affected by OX and SN-38 but inhibited by high concentrations of 5-FU. MiR expression levels were not or only slightly elevated by the drugs or with drug/PD-H combinations on Colo320 cells. Moreover, the drug treatment did not increase the mutation rate of the miR-TS inserted into the PD-H genome. The results demonstrate that the combination of FOLFOXIRI drugs and PD-H may be a promising approach to enhance the therapeutic effect of FOLFOXIRI therapy in CRC.

Current Progress in Vaccines against Merkel Cell Carcinoma: A Narrative Review and Update.

Merkel cell carcinoma is a rare, aggressive skin cancer that mainly occurs in elderly and immunocompromised patients. Due to the success of immune checkpoint inhibition in MCC, the importance of immunotherapy and vaccines in MCC has increased in recent years. In this article, we aim to present the current progress and perspectives in the development of vaccines for this disease. Here, we summarize and discuss the current literature and ongoing clinical trials investigating vaccines against MCC. We identified 10 articles through a PubMed search investigating a vaccine against MCC. From the international clinical trial database Clinical.Trials.gov, we identified nine studies on vaccines for the management of MCC, of which seven are actively recruiting. Most of the identified studies investigating a vaccine against MCC are preclinical or phase 1/2 trials. The vaccine principles mainly included DNA- and (synthetic) peptide-based vaccines, but RNA-based vaccines, oncolytic viruses, and the combination of vaccines and immunotherapy are also under investigation for the treatment of MCC. Although the management of MCC is changing, when compared to times before the approval of immune checkpoint inhibitors, it will still take some time before the first MCC vaccine is ready for approval.

Identification of restrictive molecules involved in oncolytic virotherapy using genome-wide CRISPR screening.

Oncolytic viruses (OVs) offer a novel approach to treat solid tumors; however, their efficacy is frequently suboptimal due to various limiting factors. To address this challenge, we engineered an OV containing targets for neuron-specific microRNA-124 and Granulocyte-macrophage colony-stimulating factor (GM-CSF), significantly enhancing its neuronal safety while minimally compromising its replication capacity. Moreover, we identified PARP1 as an HSV-1 replication restriction factor using genome-wide CRISPR screening. In models of glioblastoma (GBM) and triple-negative breast cancer (TNBC), we showed that the combination of OV and a PARP inhibitor (PARPi) exhibited superior efficacy compared to either monotherapy. Additionally, single-cell RNA sequencing (scRNA-seq) revealed that this combination therapy sensitized TNBC to immune checkpoint blockade, and the incorporation of an immune checkpoint inhibitor (ICI) further increased the survival rate of tumor-bearing mice. The combination of PARPi and ICI synergistically enhanced the ability of OV to establish durable tumor-specific immune responses. Our study effectively overcomes the inherent limitations of OV therapy, providing valuable insights for the clinical treatment of TNBC, GBM, and other malignancies.

Oncolytic virus V937 in combination with PD-1 blockade therapy to target immunologically quiescent liver and colorectal cancer.

V937 is an investigational, genetically unmodified Kuykendall strain of coxsackievirus A21, which has been evaluated in the clinic for advanced solid tumor malignancies. V937 specifically infects and lyses tumor cells that overexpress intercellular adhesion molecule-1 (ICAM-1). Intratumoral V937 as a monotherapy and in combination with anti-PD-1 antibody pembrolizumab has shown clinical response in patients with metastatic melanoma, which overexpresses ICAM-1. Here, we investigate in preclinical studies the potential bidirectional cross-talk between hepatocellular carcinomas (HCC) or colorectal carcinomas (CRC) and immune cells when treated with V937 alone or in combination with pembrolizumab. We show that while V937 treatment of tumor cell lines or organoids or peripheral blood mononuclear cells (PBMCs) alone induced a minimal immunological response, V937 treatment of non-contact co-cultures of tumor cell lines or CRC organoids with PBMCs led to robust production of proinflammatory cytokines and immune cell activation. In addition, both recombinant interferon-gamma and pembrolizumab increased ICAM-1 on tumor cell lines or organoids and, in turn, amplified V937-mediated oncolysis and immunogenicity. These findings provide critical mechanistic insights on the cross-talk between V937-mediated oncolysis and immune responses, demonstrating the therapeutic potential of V937 in combination with PD-1 blockade to treat immunologically quiescent cancers.

Enhancing antitumor efficacy of oncolytic virus M1 via albendazole-sustained CD8+ T cell activation.

The immune response plays a crucial role in the functionality of oncolytic viruses. In this study, Albendazole, an antihelminthic drug known to modulate the immune checkpoint PD-L1, was combined with the oncolytic virus M1 (OVM1) to treat mice with either prostate cancer (RM-1) or glioma (GL261) tumors. This combination therapy enhanced anti-tumor effects in immunocompetent mice, but not in immunodeficient ones, without increasing OVM1 replication. Instead, it led to an increase in the number of CD8+ T cells within the tumor, downregulated the expression of PD1 on CD8+ T cells, and upregulated activation markers such as Ki67, CD44, and CD69 and the secretion of cytotoxic factors including interferon (IFN)-γ, granzyme B, and tumor necrosis factor (TNF)-α. Consistently, it enhanced the in vitro tumor-killing activity of lymphocytes from tumor-draining lymph nodes or spleens. The synergistic effect of Albendazole on OVM1 was abolished by depleting CD8+ T cells, suggesting a CD8+ T cell-dependent mechanism. In addition, Albendazole and OVM1 therapy increased CTLA4 expression in the spleen, and the addition of CTLA4 antibodies further enhanced the anti-tumor efficacy in vivo. In summary, Albendazole can act synergistically with oncolytic viruses via CD8+ T cell activation, and the Albendazole/OVM1 combination can overcome resistance to CTLA4-based immune checkpoint blockade therapy.