Recent advances in oncolytic virotherapy: insights from clinical trials and combination treatment strategies.

Introduction

Cancer is among the leading causes of mortality across the world. There were almost 20 million new cases of cancer and 10 million deaths related to cancer in 2022. According to the demographic projections, the annual number of new cases of cancer will reach 35 million by 2050 [ 1 ]. Lung cancer was the most commonly diagnosed cancer worldwide in 2022 in males, followed by prostate cancer. As well, the most diagnosed cancer in females was breast cancer, followed by lung cancer and cervical cancer. In both sexes, the most common causes of cancer death were lung cancer and breast cancer, respectively [ 2 ]. Despite the advancements in cancer diagnosis and treatment options in recent years, cancer is still a major public health concern. The prognosis of cancer patients has significantly improved as a result of therapeutic measures, including immunotherapy, radiation therapy, and chemotherapy. Nevertheless, these therapies still have significant drawbacks. These are severe side effects from radiation and chemotherapy, drug resistance of tumor cells, and low effectiveness of immunotherapy in severe and severely immunosuppressed illnesses [ 3 , 4 ]. Beyond these traditional treatment cancer therapies with their limitations, the oncolytic viruses (OVs) have emerged as a new frontier in cancer therapy. This concept was first proposed in the 1900s. Afterward, various studies with different types of viruses were conducted [ 5 ].

OVs have the innate ability to infect and specifically lyse cancer cells while leaving normal cells intact. OVs can be chosen from native viral species, or they may be genetically modified. Genetically engineered OVs are created by either inserting transgenes into the viral genome to boost antitumor immunity or removing certain pathogenic genes to weaken the virus. Thus, genetic modification helps to minimize pathogenicity, boost immunogenicity, improve tumor selectivity, and raise replication competence [ 6 , 7 ]. Furthermore, by combining OVs with other therapies (especially with immunotherapy), the efficacy of OVs can be improved. Following successful outcomes from several clinical trials, the OVs attracted a lot of interest, and until now, there have been several approved OVs. In 2004, Rigvir (derived from the ECHO-7, a member of the Picornaviridae family) was approved in Latvia for the treatment of melanoma [ 8 ]. Due to the limited data regarding clinical efficacy, this non-genetically modified virus couldn’t achieve extensive use. In 2005, another OV was approved in China, namely Oncorine (H101), for treating head and neck cancer. Unlike Rigvir, H101 is a genetically modified OV. H101 is an adenovirus with a deleted E1B 55 kDa gene. By deleting this gene, H101 can only replicate in cancer cells where p53 is already non-functional [ 9 ]. As well, combining H101 with chemotherapy agents revealed better outcomes [ 10 ]. Talimogene laherparepvec (T-VEC) is the only FDA-approved OVs that was approved in 2015 for treating melanoma. T-VEC is a genetically engineered herpes simplex virus type 1 (HSV-1). T-VEC has a deletion in the ICP34.5 and ICP47 genes, and it is also designed to express human granulocyte-macrophage colony-stimulating factor (GM-CSF) [ 11 ]. Teserpaturev (G47∆) is another genetically modified HSV-1, which was approved in Japan in 2021 for treating malignant glioma. G47∆ has a deletion in the ICP34.5 and ICP47 genes, and the ICP6 has been inactivated [ 12 ].

Recently, a growing number of pre-clinical and clinical studies have been implemented to assess the efficacy and safety of this new player in the treatment of various types of cancer. Like other new approaches, OVs have several challenges and limitations. However, researchers intend to overcome these obstacles in different ways, such as combining OVs with other therapies or designing genetically modified OVs that are more tumor-specific and have fewer side effects [ 13 , 14 ]. Thus, this review aims to investigate the most recent developments in oncolytic virotherapy, with an emphasis on insights from clinical trial outcomes and the development of combination treatment strategies, given the growing interest in this field and the number of ongoing clinical trials.

Evolution of oncolytic virotherapy

Oncolytic virotherapy has undergone substantial conceptual and technological evolution over the past decades, transitioning from an experimental observation to a rationally designed therapeutic strategy. Early interest in OVs originated from clinical reports in the early 20th century describing spontaneous tumor regressions in patients with natural viral infections. In 1904, the first known instance of tumor regression following a viral infection was recorded. A patient with hematologic malignancies experienced a sharp drop in white blood cell count while suffering from a flu-like illness [ 15 ]. These observations led to the initial exploration of wild-type viruses, including flaviviruses, adenoviruses, and hepatitis viruses as potential anticancer agents, primarily based on their ability to selectively infect and lyse tumor cells (oncolysis) [ 16 ]. Nevertheless, broader assessment of wild-type viruses faced significant limitations due to several factors, including variable antitumor activity, inducing complications such as encephalitis, organ failure, and mortality, and immune-mediated neutralization and elimination by the host [ 17 ]. Afterward, with the emergence of genetic engineering as a new scientific field in 1990, the OV paradigm transitioned toward the application of genetically modified viral strains designed to exhibit reduced virulence in humans while enhancing their anti-cancer potency.

In 1991, the first application of genetically modified OVs in cancer therapy was reported [ 18 ]. Investigators employed a mutant strain of HSV-1 engineered with a targeted deletion of the gene encoding thymidine kinase. This genetically modified HSV-1, while exhibiting reduced neurovirulence, preserved its oncolytic activity against glioma monocultures as well as human tumor xenografts in murine models [ 18 ]. This represented an instance of the first generation of genetically engineered OVs, which were designed to enhance tumor specificity through the deletion of pathogenic viral genes. The second generation of OVs incorporated therapeutic transgenes along with additional genetic modifications aimed at improving tumor specificity and amplifying antitumor immune activity. For example, OncoVEXGM-CSF features the deletion of the HSV gene that encodes ICP47, along with the insertion of the human gene for GM-CSF. Additionally, it includes the deletion of ICP34.5, a modification well established to confer tumor-selective viral replication [ 19 ]. The third generation of OVs featured sophisticated engineering designed to circumvent immune-mediated elimination, deliver multiple therapeutic transgenes, and act synergistically with other cancer treatment modalities. For instance, third-generation oncolytic HSV-1, known as G47Δ, contains three mutations. It was derived from G207, a second-generation oncolytic HSV-1, through the additional removal of the α47 gene. This modification was intended to boost the virus’s cancer-destroying activity while maintaining the ability of infected tumor cells to provoke an immune response [ 20 ]. Genetically modified OVs have been generated using a diverse array of cytokine-encoding genes. These include various interleukins such as IL-2, IL-7, IL-12, and IL-15, and colony-stimulating factors such as GM-CSF, tumor necrosis factor-alpha (TNF-α), interferons, as well as chemokines such as CXCL11 and CCL21 [ 21 ].

These advancements have caused a major conceptual shift with the recognition that OVs exert significant immunomodulatory effects beyond direct oncolysis [ 16 ]. Viral infection of tumor cells was found to induce immunogenic cell death, leading to the release of tumor-associated antigens, danger-associated molecular patterns (DAMPs), and pro-inflammatory cytokines. This process facilitates the activation of innate and adaptive immune responses [ 22 ]. Consequently, oncolytic virotherapy is now increasingly viewed as a form of cancer immunotherapy, capable of reshaping the tumor microenvironment (TME) and promoting systemic antitumor immunity. However, employing OVs as a monotherapy is hindered by several drawbacks, such as tumor heterogeneity, immune evasion, and an immunosuppressive TME. To overcome these limitations, recently the field has shifted toward combination-based strategies, integrating OVs with other treatment modalities such as immune checkpoint inhibitors (ICIs), targeted therapies, chemotherapy, and radiotherapy for potentiating OVs [ 23 , 24 ].

Altogether, the field of oncolytic virotherapy continues to expand rapidly, with an increasing number of clinical trials currently evaluating its safety and efficacy across diverse cancer types. However, several challenges continue to hinder their widespread clinical translation, such as delivery challenges, antiviral immunity, therapy resistance, etc., which will be elaborated upon in the following sections. These limitations highlight the need for further optimization and innovation to fully realize the clinical potential of oncolytic virotherapy.

How OVs affects cancer cells?

OVs use different mechanisms to eliminate cancer cells [ 25 ]. Firstly, is direct lysis of tumor cells, which is considered the key mechanism. OVs exploit abnormal pathways in cancer cells, such as mutated RAS signaling, which inactivates the viral inhibitory pathway, allowing viral entry and replication within tumor cells [ 26 ]. For example, vesicular stomatitis virus (VSV) targets cancer cells exhibiting normal PKR activity but harboring p53 deficiencies or overexpressing Ras or Myc, thereby inducing apoptosis through caspase-dependent pathways [ 27 ]. Reovirus suppresses interferon-beta (IFN-β) production through oncogenic RAS activation, subsequently facilitating viral spread in cancer cells [ 28 ]. Overexpression of BCL-XL, an anti-apoptotic factor, further facilitates viral transcription and translation. Ultimately, the virus is released from the cell, resulting in the lysis of the cancer cell [ 26 ]. For instance, in glioblastoma cells, the overexpression of Bcl-XL inhibits apoptosis, but the VSV still induces cell lysis through alternative pathways [ 29 ]. In another way, OVs can induce immunogenic cell death (ICD) in cancer cells. ICD is a form of cell death that elicits a strong and durable immune response through the release of damage-associated molecular patterns (DAMPs) such as ATP, calreticulin (CRT), and HMGB1 from damaged cells [ 25 ]. This immunogenic cell death process leads to the attraction of dendritic cells and the activation of anti-tumor T-cells [ 25 ]. OVs like HSV-1 RH2 cause the release of ATP and HMGB1 and the translocation of CRT to the cell membrane, which leads to apoptosis and pyroptosis [ 30 ]. By destroying tumor cells, Adenovirus (Ad5/3-hTERT-E1A-hCD40L) can release tumor antigens and generate danger signals such as calreticulin, HMGB1, and ATP, which enhance immunogenicity and trigger apoptosis, contributing to immunogenic cell death. CD40L also directly induces apoptosis in cancer cells, stimulates both innate and adaptive immunity, and activates T cells [ 31 ]. Viral replication also induces the release of pathogen-associated molecular patterns (PAMPs), which synergize with DAMPs to enhance immune activation [ 25 ]. PAMPs activate pattern recognition receptors (PRRs) such as Retinoic acid–inducible gene )RIG-I(, cyclic GMP–AMP synthase )c-GAS (, and stimulator of interferon Genes (STING), thereby stimulating innate immune cells [ 32 ]. OVs induce ICD through several pathways: (i) DAMPs and PAMPs mediate inflammatory cytokines such as IL-2 and TNF-α, which activate CD8 + and CD4 + T cells, ultimately leading to the elimination of tumor cells by host immunity [ 33 ]. (ii) DAMPs resulting from oncolytic cell death induced by ORCA-010 promote the activation of dendritic cells and enhance tumor antigen presentation to T cells [ 34 ]. (iii) Viral proteins can function as ligands that engage death receptors, activating extrinsic apoptosis via caspase cascades or intrinsic apoptosis through mitochondrial pathways [ 26 ]. (iv) OVs like reoviruses induce autophagy through various pathways, including the formation of Atg5-Atg12 complexes [ 35 ]. In advanced cancer stages, an immunosuppressive TME develops, creating physical barriers and biochemical conditions that impair immune cell infiltration and function [ 36 ]. The primary mechanisms of TME-mediated resistance include recruitment of regulatory T cells (Tregs) and elevated TGF-β levels, which inhibit anti-tumor immunity [ 37 ]. OVs, by stimulating immune responses, induce inflammation within the TME and initiate new anti-tumor immunity, and render tumor cells susceptible to elimination by innate and adaptive immune mechanisms [ 38 , 39 ]. Chemokines released in response to DAMPs and PAMPs attract innate immune cells such as neutrophils, dendritic cells, and macrophages. Neutrophils secrete extracellular matrix-degrading enzymes, which remove structural barriers within tumors and disrupt tumor vasculature [ 26 ]. Angiogenesis is widely considered one of the main hallmarks of cancer progression, and cancer treatments at this stage include anti-angiogenic therapies; however, many of these treatments fail to completely stop angiogenesis in the TME [ 40 ]. Aberrant vascular endothelial growth factor (VEGF) expression in tumors suppresses anti-viral responses but also facilitates viral replication in endothelial cells, leading to anti-angiogenic effects [ 26 ]. After infection and replication, OVs reduce VEGF levels in endothelial cells, preventing angiogenesis [ 32 ]. Figure 1 illustrates the main mechanisms by which OVs exert their anti-tumor effects.

Fig. 1 Oncolytic virus (OV) therapy mechanisms in tumor cells. Oncolytic viruses only infect and multiply in cancerous cells, leaving healthy cells unharmed. Viral replication is prevented in healthy cells, protecting them from harm. Viral replication causes direct oncolysis in tumor cells, which releases progeny viruses that can infect nearby tumor cells. This oncolysis includes apoptosis, pyroptosis, necrosis, necroptosis, autophagy, and immunogenic cell death (ICD). Additionally, pathogen-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs), tumor-associated antigens (TAAs), and neoantigens are released by tumor cell death. These molecules activate natural killer (NK) cells and dendritic cells (DCs), priming CD8⁺ T cells and inducing antitumor immunity. In the tumor microenvironment (TME), tumor-associated macrophages (TAMs) have the ability to change their phenotype from M2 (tumor-supportive) to M1 (pro-inflammatory). Vascular collapse brought on by OV-induced necrosis of uninfected tumor cells contributes to inflammatory reactions, tumor endothelial cell infection, and anti-angiogenic effects. Immune cell recruitment and activation are mediated by cytokines and chemokines released during these processes

Strategies for OV delivery: challenges and future prospects

Efficient delivery remains one of the most critical determinants of therapeutic success in oncolytic virotherapy. The ability to achieve adequate viral distribution within tumor tissues while minimizing systemic clearance and off-target effects is essential for maximizing antitumor efficacy [ 41 ]. Various delivery strategies have been developed to address these challenges, each with distinct advantages and limitations depending on tumor type, anatomical accessibility, and host immune status.

Local delivery

Local administration, particularly through intratumoral injection, represents one of the most widely employed delivery strategies in clinical settings. This method allows for the direct administration of high viral titers into the tumor, enabling concentration control, provoking a localized immune response, while avoiding unwanted systemic side effects associated with the infection [ 42 ]. Intratumoral delivery proves far more feasible for surface and easily injectable tumors (e.g., melanoma and head and neck cancer) than for deep or metastatic tumors, where operational delivery challenges pose significant limitations [ 42 ]. This method has been extensively used in numerous clinical trials [ 43 – 45 ]. The primary example is T-VEC (talimogene laherparepvec), an approved OVs, which is used for easily injectable skin tumors such as melanoma [ 11 ]. However, intratumoral delivery has some limitations. The dense composition of most solid tumors complicates intratumoral injection, as high interstitial pressure can prevent the virus from fully penetrating the tissue [ 46 ]. For example, studies have reported that extracellular matrix (ECM) components, particularly heparan sulfate and collagen, restrict the dissemination of Newcastle disease virus (NDV) within solid tumor tissues [ 47 ].

Furthermore, the complex nature of intratumoral delivery procedures entails considerable risks and expenses, thereby hindering the practical implementation of repeated dosing [ 42 ]. Most importantly, treating deeply situated tumors within tissues, as well as metastatic tumors, poses a significant challenge when using intratumoral injection. For instance, a clinical trial using VCN-01 (oncolytic adenovirus) in pancreatic cancer showed that while local injection effectively treated the primary tumor site, disease progression occurred in some patients [ 48 ]. This was attributed to either the emergence of new lesions or the continued growth of distant tumors that remained beyond the reach of direct intratumoral administration [ 48 ].

Hopefully, there are several approaches to overcome these limitations. There are strategies to enhance OV penetration through tumor ECM barriers which are divided into biochemical and physico-chemical methods. Biochemical modulation involves targeted degradation, inhibition of ECM synthesis, and structural modulation. Physicochemical disruption employs ultrasound, magnetic nanoparticles, and photodynamic therapy to degrade ECM barriers via mechanical or chemical effects [ 49 ]. For example, an oncolytic vaccinia virus engineered to express hyaluronidase (OVV-Hyal1) remodels the extracellular matrix, thereby improving its own spread as well as enhancing cancer chemotherapy and immunotherapy [ 50 ]. In another study, magnetic nanoparticles from magnetotactic bacteria were coassembled with HSV1716, allowing magnetically guided tumor targeting from circulation, shielding against neutralizing antibodies, and boosting viral replication inside tumors [ 51 ]. These strategies have been utilized for other OVs; however, these studies are limited to the preclinical stages [ 52 – 54 ]. Therefore, for deep-seated tumors, intratumoral injection necessitates interventional, image-guided, or surgical procedures that are challenging, costly, and often painful for routine clinical application, particularly when multiple injections are required. Moreover, considerable differences in treatment effectiveness are observed with this delivery method when applied to metastatic tumors versus inaccessible ones, such as those located in the brain. Consequently, this variability may serve as a potential explanation for the inconsistent clinical efficacy reported across various trials.

Systemic delivery

Like intratumoral delivery, systemic administration, most commonly via intravenous injection, is one of the most widely employed delivery strategies in clinical settings [ 45 ]. Because intravenous administration can distribute OVs to all parts of the body, it offers a rational treatment option for tumors originating in deep organs, as well as for recurrent and metastatic tumors. Intravenous injection is preferred in clinical trials over intratumoral delivery because it offers higher systemic bioavailability, avoids complex procedures, and targets distant metastases, making it a simpler route for physicians [ 55 ]. For instance, A Phase I/II trial involving patients with recurrent glioblastoma multiforme demonstrated that intravenous administration of NDV-HUJ (the HUJ strain) was well tolerated, with one patient achieving a complete tumor response [ 56 ]. According to another study, intravenous Pexa-Vec was well tolerated and concurrently activated both innate and adaptive immune cells, promoted lymphocyte infiltration into tumors, and was associated with extensive necrosis in some patients. As well, it significantly raised serum cytokine levels, including IFNα, IFNβ, TRAIL, and CXCL10 [ 57 ]. Additionally, Jirovec and colleagues proposed that a single intravenous dose of the oncolytic adenovirus TILT-123 leads to systemic tumor transduction and elicits an immune response in patients with advanced solid tumors [ 58 ]. Thus, the efficacy of systemic delivery has been determined in several investigations. However, in a Phase I trial, intravenous pelareorep (3 × 10¹⁰ TCID₅₀ for 5 consecutive days every 4 weeks) was well tolerated and defined as the maximum tolerated dose. No objective responses were seen in six melanoma patients, and viral shedding was rarely observed (only at day 5 or 15 in a few patients). One possible explanation for this outcome is that intravenous administration triggered high levels of neutralizing antibodies, which may have impeded viral replication [ 59 ]. Thus, like intratumoral delivery, this strategy also may face some challenges. One of them is a rapid clearance of the virus in the bloodstream. The viruses may be cleared by preexisting blood factors, including coagulation factors IX and X, as well as complement protein C4b-binding protein (C4BP), and neutralized by antibodies [ 60 ]. Thus, successful OVs delivery may only occur in patients without neutralizing antibodies against the virus.

Willmon et al. reported that two minutes after intravenous injection of VSV into an immunocompetent mouse, most detectable virus is cell‑associated rather than free in the serum, and within 30 min, all detectable virus is found associated with cells. In the case of oncolytic HSV-1, this problem can also be seen [ 61 , 62 ]. Several obstacles hinder the intravenous delivery of oncolytic HSV-1. First, abundant non‑tumor epithelial cells and sensory neurons displaying herpes virus receptors (nectin‑1 and HVEM) sequester the virus, reducing the amount reaching tumor sites and causing non‑specific targeting of healthy tissues [ 63 ]. Second, the WHO estimates that two‑thirds of the global population under 50 has been infected with HSV‑1, meaning that most individuals possess circulating HSV‑1‑specific antibodies capable of rapidly neutralizing intravenously administered oncolytic HSV-1 [ 63 ]. So, it can be postulated that HSV-1 may not be suitable for intravenous administration because it might be rapidly neutralized by preexisting antibodies against HSV-1. However, some studies are inconsistent with this statement [ 64 , 65 ]. Despite this limitation, which applies to certain viruses such as herpes, others can circumvent it. For instance, reovirus, when complexed with antibodies and loaded into human monocytes despite being fully neutralized, can be delivered as a functional replicative virus to tumor cells, resulting in tumor cell infection and lysis. FcγRIII antibody receptors enhance the ability of monocytes to internalize reovirus–antibody complexes [ 66 ]. Adair et al. also reported that following intravenous delivery of reovirus in patients with colorectal cancer metastatic to the liver, the virus was protected from neutralizing antibodies after systemic administration via immune cell carriage, which subsequently delivered reovirus to the tumor [ 67 ]. This may explain why reovirus has routinely been administered intravenously [ 68 , 69 ].

A further challenge confronting intravenous delivery is the requirement for higher viral doses relative to intratumoral injection. However, this increase in viral dose may raise the risk of contact with normal tissues, causing non-specific entry into healthy tissues and potentially severe toxicity and adverse effects [ 21 ]. Moreover, physiological barriers, including the blood-brain barrier, can significantly impede the distribution of the virus to target sites. However, there is evidence that shows that some OVs can bypass the blood-brain barrier. Samson and colleagues demonstrated that intravenously delivered oncolytic Reovirus was able to infect both glioma and brain metastases, thereby establishing that at OVs are capable of crossing the blood-brain barrier [ 69 ].

Despite these challenges and limitations, various strategies have been developed to improve this mode of delivery. One effective approach involves the genetic modification of OVs. This strategy can both enhance antitumor activity and minimize systemic adverse effects, as it allows a greater proportion of the administered virus to successfully reach and infect tumor cells [ 70 ]. As an example, to achieve tumor-selective replication, many oncolytic adenoviruses employ mutations or deletions in the E1A and E1B genes ( NCT04771676 and NCT03178032 ). For instance, the 24-base-pair deletion in E1A restricts viral replication to pRB-deficient tumor cells while preventing it in normal cells. Likewise, E1B deletion improves selective targeting of tumor cells through the exploitation of dysfunctional p53 signaling, a common hallmark of malignancy [ 16 ]. Furthermore, VCN-11 is a novel oncolytic adenovirus engineered to express hyaluronidase and incorporate an albumin-binding domain on its capsid. The expression of hyaluronidase enables VCN-11 to degrade the tumor extracellular matrix, thereby promoting viral spread. Meanwhile, the albumin-binding domain creates an “Albumin Shield” that shields VCN-11 from antibody-mediated neutralization and allows for effective repeat administration [ 71 ]. Beyond modifying the OV itself, using other cells or materials offers another strategy. Three main strategies exist to improve OV delivery and help the virus to partially overcome the neutralizing environment of the bloodstream. First, cell carriers (e.g., monocytes, T cells, MSCs, and neural stem cells) naturally migrate to tumors and transport OVs, which then replicate and destroy cancer cells. Second, protein-based methods either coat the virus with an artificial protein corona to block immune attack or mutate the viral shell to avoid detection in blood. Third, nanoparticle carriers actively guide OVs to tumors using targeting molecules like antibodies, peptides, or aptamers that bind specifically to cancer cell markers [ 60 , 72 ]. An approved preliminary investigation involving four pediatric patients with front-line-refractory metastatic neuroblastoma who were administered multiple doses of autologous MSCs carrying ICOVIR-5, demonstrated excellent treatment tolerability. A complete clinical response was documented in one case, with the patient maintaining complete remission three years post-therapy. This suggests MSCs can deliver oncolytic adenoviruses to metastases with minimal toxicity and significant efficacy [ 73 ]. At present, multiple clinical studies are being conducted to evaluate and demonstrate the therapeutic effects of employing carrier systems for a range of different OVs ( NCT03896568 , NCT03072134 , and NCT02068794 ). While carrier cells offer a promising strategy to improve OV pharmacokinetics and biodistribution, the best cell type remains debated. Optimal carriers must have tumor tropism, the ability to internalize or bind virions, and sufficient viability for bloodstream delivery to tumors. Additionally, autologous cells are strongly preferred to prevent rejection, though this is somewhat controversial [ 72 ]. Draganov et al. showed that MSCs protect, amplify, and deliver vaccinia virus while suppressing anti-viral immunity to enhance oncolytic virotherapy [ 74 ]. However, this ability is limited by allogeneic barriers requiring patient-to-stem cell matching. Patient resistance to allogeneic carriers was linked to anti-stem cell IFNγ and cytotoxic responses, likely due to HLA or KIR mismatches [ 74 ].

Despite the significant advantages of intravenous delivery in oncolytic virotherapy, particularly its potential to target metastatic and inaccessible tumors, the number of clinical studies employing this administration route that have successfully progressed to phase III trials remains extremely limited. Several biological and technical barriers, including rapid viral clearance by the immune system, neutralizing antibodies, nonspecific uptake by the liver and spleen, insufficient tumor targeting, and concerns regarding systemic toxicity, continue to hinder the clinical translation of systemic oncolytic virotherapy. Consequently, only a small number of intravenously administered OVs have advanced to late-stage clinical evaluation (e.g., NCT01166542 ). Nevertheless, ongoing advances in the field, such as genetic engineering of viral platforms to enhance tumor selectivity and immune evasion, as well as the development of carrier-based delivery systems, including cell carriers and nanoparticles, may improve the safety and efficacy of intravenous OV delivery in the future and facilitate broader clinical application. Finally, systemic administration outcomes depend on the virus’s ability to avoid immune traps (e.g., neutralizing antibodies, complement system) and maintain an appropriate equilibrium between cytotoxic effects and systemic immune stimulation.

Other delivery approaches

Beyond the two primary delivery methods discussed earlier, investigators have also explored and applied various other administration routes. Delivery approaches, such as Intrapleural, intravesical, intrathecal, and intraperitoneal administration, aim to improve tumor targeting by delivering the virus directly into the vascular supply or body cavity associated with the tumor.

In the case of certain tumors localized to the peritoneum, abdomen, or pelvis, intraperitoneal injection could serve as a practical and viable method for delivering OVs [ 75 ]. Intraperitoneal injection offers faster absorption than subcutaneous but slower than intravenous due to the large peritoneal area. It is also easy to perform with minimal specialized skills [ 55 ]. In a Phase 1b trial, intraperitoneal Olvi-Vec showed promising safety, clinical activity, and immune activation in platinum-resistant/refractory ovarian cancer. Immune activation included enhanced CD8 + T-cell infiltration and tumor-specific T-cell activation in the blood. Several patients had extended progression-free survival and overall survival [ 76 ].

The intrathecal route is ideal for central nervous system tumors. Ochiai et al. demonstrated that intraspinal administration of PVS-RIPO in transgenic mice produced no clinical or histologic evidence of toxicity and led to an increase in median survival. These results support the potential utility of intrathecal PVS-RIPO in the treatment of neoplastic meningitis among patients with glioblastoma multiforme [ 77 ]. As well, intravesical delivery of OVs constitutes a novel treatment strategy for high-risk bladder cancer, as it enables direct exposure of tumor sites to high viral concentrations. A phase 1 clinical investigation demonstrated that intravesical administration of CG0070 conferred an acceptable safety profile and exhibited anti-neoplastic activity against bladder cancer [ 78 ]. Findings from another preclinical study suggest that intravesical treatment with Oncovex (GALV/CD) combined with the prodrug 5-FC enhances local tumor control within the bladder and may also have the potential to alter the natural progression of the disease [ 79 ]. Ultimately, given the diversity of available delivery strategies, the selection of the most suitable route depends on the individual tumor characteristics, particularly its type and anatomical site.

Combination of OVs with other treatment strategies, challenges and future prospects

Monotherapies, particularly at metastatic or end-stage cancer, generally yield poor survival rates due to viral platform and host factors [ 80 ]. Importantly, the mechanisms underlying OV-mediated tumor cell destruction are fundamentally distinct from those of other anticancer modalities. Therefore, OVs represent a rational adjunctive strategy for combination with most existing treatment approaches like radiotherapy and chemotherapy, cell therapy, ICIs, cancer vaccines, and targeted small molecules (See Fig. 2 ). Until now, different viruses have been utilized in these clinical trials including, Herpesvirus (Tables 1 and 2 ), Adenovirus (AdV) (Tables 3 and 4 ), Vaccinia virus (Tables 5 and 6 ), Reovirus (Table 7 ), Measles virus and Newcastle disease virus (NDV) (Table 8 ) and other viruses (Table 9 ), which will be described further.

Fig. 2 Combination of oncolytic viruses with other cancer treatment options

Combining with chemotherapy

Chemotherapy, a classical cancer therapy, interrupts cell growth using drugs like carboplatin, gemcitabine, and paclitaxel [ 81 ]. However, most chemotherapeutic agents cannot differentiate between normal and cancer cells, targeting all rapidly replicating cells. Tumors also commonly acquire resistance to chemotherapeutic agents, often leading to treatment failure [ 82 ]. There are several assumptions suggesting that combining this classical therapeutic approach with OVs may enhance treatment efficacy. Firstly, the antitumor activities of these two therapeutic modalities may exhibit complementary effects. In contrast to conventional therapeutic modalities, which are confined to regional application, OVs possess the distinct advantage of targeting metastatic lesions. Consequently, the combination of OVs with chemotherapy can achieve both local and systemic disease control. Secondly, these two treatment modalities may exert synergistic effects when combined. Chemotherapy can disrupt tumor barriers, thereby enhancing viral penetration and also help OVs evade antiviral immune responses [ 83 ]. Cyclophosphamide, an immunosuppressive chemotherapeutic agent, combined with OV and therapy, induces tumor apoptosis, modulates the innate and adaptive immune system, and its subsequent immunosuppression can potentiate viral oncolysis and antitumor efficacy [ 5 ]. On the other hand, the virus can promote cell cycle activation and increase the sensitivity of cells to chemotherapy. For example, apart from ONYX-015 replication-induced oncolysis, E1A gene expression from the virus can activate the cell cycle and enhance sensitivity to chemotherapy [ 84 ].

Numerous clinical studies regarding the combination of OVs with chemotherapy have been conducted or are currently underway. Notably, Oncorine (H101), the first commercialized OV worldwide, was developed by Shanghai Sunway Biotech from 1999 and received Chinese SFDA approval in November 2005 for nasopharyngeal carcinoma combined with chemotherapy after successful Phase III trials [ 9 ]. The success of T-VEC in gaining FDA approval as a monotherapy for melanoma has driven the shift toward combination strategies, leading to ongoing clinical trials that assess its efficacy alongside other modalities, including chemotherapy ( NCT02779855 ). A Phase I/II clinical trial evaluating carboplatin and paclitaxel chemotherapy in combination with intravenously administered oncolytic reovirus in patients with advanced malignancies revealed that the combination was well tolerated and exhibited evidence of therapeutic activity specifically in head and neck cancers [ 85 ]. Despite these promising results, certain challenges may exist, as suggested by other studies with conflicting outcomes.

Effective OV replication relies on the presence of active tumor cells. If chemotherapy is given first, most tumor cells are eradicated, leaving an unfavorable environment for OV replication, which is essential for their therapeutic action. On the other hand, administering OVs before chemotherapy also raises concerns, as the antitumor immune cells stimulated by the viruses might be destroyed by subsequent chemotherapy [ 86 ]. According to a preclinical study, Temozolomide chemotherapy may have a negative effect on HSV immunovirotherapy. Given that Temozolomide is the current standard of care for glioblastoma, the sequence and timing of these combined treatments should be taken into account when planning oncolytic HSV clinical trials that involve chemotherapy [ 87 ]. In another preclinical study, it was found that administering VG161 followed by chemotherapy (gemcitabine plus nab‑paclitaxel) produced the optimal therapeutic effect, emphasizing that the sequence of administration significantly influences treatment efficacy [ 88 ]. Additionally, Heise and colleagues reported that the efficacy of OVs was highly dependent on the sequence of administration. Treatment with ONYX-015 either before or simultaneously with chemotherapy (cisplatin) was significantly more effective than administering chemotherapy followed by ONYX-015 [ 89 ]. Therefore, this incident may explain the unfavorable outcomes observed in some studies combining OVs with chemotherapy.

A randomized Phase II study evaluating pelareorep in combination with docetaxel versus docetaxel monotherapy in patients with metastatic castration-resistant prostate cancer demonstrated a median overall survival of 19.1 months for the combination arm compared to 21.1 months for docetaxel alone. Consequently, this combination does not justify further clinical investigation ( NCT01619813 ). In a randomized Phase II investigation conducted by Jonker and colleagues, the addition of pelareorep to leucovorin/5-FU/oxaliplatin /bevacizumab in patients with metastatic colorectal cancer was associated with acceptable tolerability and an improved objective response rate. Nevertheless, progression-free survival was inferior in the combination group. They proposed that reduced dosing intensity of the standard chemotherapeutic agents is likely to have contributed to the absence of benefit from pelareorep [ 90 ]. These observations underscore the need for careful consideration when combining chemotherapy and OV therapy in clinical settings. Further clinical studies are needed to determine the effect of treatment sequencing between OV and chemotherapy.

Combining with radiotherapy

For localized tumors, radiotherapy remains an essential treatment approach. It leverages ionizing radiation either to inflict direct DNA damage on cancer cells or to produce reactive oxygen species that interfere with normal cellular processes [ 91 , 92 ]. This approach has several shortcomings, including damage to healthy cells due to off-target effects, the development of radio-resistance in tumors, and its general ineffectiveness against metastatic growth [ 83 ]. The combination of OVs with radiotherapy elicits significant synergistic antitumor activity, particularly in aggressive malignancies refractory to standard therapies. Multiple hypotheses have been proposed to elucidate the mechanisms governing this synergistic relationship.

Firstly, OVs increase the sensitivity of tumors to radiation by disrupting the pathways responsible for repairing DNA damage [ 16 ]. Secondly, radiotherapy may promote the cellular uptake of viruses, enhance viral gene expression, and facilitate viral replication [ 93 ]. Thirdly, the abscopal phenomenon, defined as the regression of non‑irradiated tumor lesions mediated by systemic immune activation following local radiotherapy, is potentiated by OVs through enhanced antigen presentation and immune cell recruitment, thereby amplifying systemic immune responses [ 16 , 94 ]. And finally, ionizing radiation induces apoptotic cell death in tumors, resulting in the release of tumor-associated antigens and damage-associated molecular patterns. These released factors subsequently facilitate the replication and dissemination of OVs [ 80 ]. For instance, stereotactic body radiation combined with oncolytic vaccinia virus generates potent antitumor immunity and therapeutic effects. This combination enhances immune responses from both helper and cytotoxic T lymphocytes. Additionally, it triggers tumor cell necroptosis and modifies macrophage activity through the release of DAMPs [ 95 ].

This hypothesis has been strengthened according to the findings of several preclinical studies. Adusumilli et al. reported that combining radiotherapy with oncolytic HSV (NV1066) in lung cancer cell lines produced synergistic efficacy with reduced dosage and toxicity. This effect was linked to increased GADD34 expression, a radiation‑inducible DNA repair gene homologous to HSV ICP34.5 [ 96 ]. In another preclinical study, radiotherapy potentiated VSV‑mediated oncolysis through attenuation of the innate antiviral response, which resulted in enhanced VSV replication and the induction of an adaptive immune response characterized by elevated CD8 + lymphocyte counts and antitumor activity [ 97 ]. Pokrovska and colleagues demonstrated that radiation exposure is associated with enhanced EnAd production, as observed both in vitro and in a preliminary in vivo experiment [ 98 ]. It has been suggested that Y‑box binding protein‑1 (YB‑1) promotes oncolytic adenovirus replication [ 99 ]. Before irradiation, YB‑1 was mainly found in the cytoplasm surrounding the nucleus, with minimal nuclear presence. After irradiation, however, nuclear YB‑1 levels increased. This combination of radiotherapy increased the oncolytic effect of oncolytic adenovirus dl520, leading to enhanced viral replication, higher viral yield, and increased viral release [ 100 ]. These synergistic effects are not one‑sided; viruses can also potentiate the effects of radiotherapy.

Radiation‑resistant cell lines such as SCC25, MSKQLL2, and SCCVII, can become susceptible when HSV-1 (G207 virus) is combined with radiation [ 101 ]. As well, an oncolytic construct expressing tumor necrosis factor‑related apoptosis‑inducing ligand (TRAIL) in combination with radiotherapy sensitizes human colorectal cancer cells to radiation‑induced apoptotic cell death, as demonstrated both in vitro and in vivo [ 102 ]. These incidents have also been seen in oncolytic NDV and CV706, a prostate cancer-specific adenovirus variant [ 103 , 104 ]. Of particular interest, the expression of the human sodium iodide symporter (hNIS) gene facilitates the uptake of radioactive iodine isotopes within malignant cells [ 105 ]. This finding has given rise to the concept that insertion of this gene into the viruses could potentiate the effectiveness of radiotherapy. Moreover, this allows imaging of viral gene expression in cancer cells [ 105 ]. For example, intravenously administered NIS‑expressing oHSV‑1 infects and replicates selectively in prostate cancer xenografts, enabling them to concentrate ⁹⁹ᵐTcO₄ and radioiodine. This allows non‑invasive SPECT/CT imaging of viral spread within tumors and enhances the antitumor response [ 106 ]. Opyrchal et al. found that NIS expression concentrates radioactive iodine, enabling imaging of MV‑NIS infection via ¹²³I/⁹⁹ᵐTc uptake. Furthermore, radiovirotherapy combining MV‑NIS with subsequent ¹³¹I administration led to a marked increase in antitumor efficacy compared to the virus administered alone [ 107 ]. These findings have led to the initiation of further clinical studies in this area (see Table 8 ).

Most importantly, the efficacy of the OVs and radiotherapy combination has been at the clinical level [ 108 – 110 ]. In a Phase I clinical investigation, repeated endoscopic intratumoral administration of OBP-301 in conjunction with radiotherapy demonstrated feasibility and conferred clinical benefits to patients with esophageal cancer who were unsuitable for standard treatment regimens [ 111 ]. However, as noted in some studies, this combination therapy can be linked to adverse events [ 108 ]. As a future direction, the model and theory of Simbawa and colleagues suggest that an optimal timing and ideal dose of radiation exist to maximize treatment results. Their model administers virotherapy continuously over a set period. A bolus approach, delivering virotherapy in cycles, could be evaluated and compared against their continuous regimen [ 93 ].

Combining with ICIs

Immune checkpoint molecules, including PD-1, PD-L1, and CTLA-4, function as regulators of immune responses and are essential for the preservation of self‑tolerance, thus preventing non‑specific immune-mediated cellular damage [ 112 ]. Nevertheless, certain cancers are capable of evading immune surveillance by upregulating or stimulating these checkpoint targets to make an immunosuppressive tumor microenvironment [ 113 ]. ICIs directed against immune checkpoints, including PD-1, PD-L1, and CTLA-4, interfere with the capacity of malignant cells to circumvent the host immune response. ICIs mainly comprise PD-1/PD-L1 and CTLA-4 inhibitors. FDA‑approved examples include ipilimumab (anti‑CTLA‑4), pembrolizumab, cemiplimab, nivolumab (anti‑PD‑1), and atezolizumab, avelumab, durvalumab (anti‑PD‑L1) [ 114 ]. However, like other treatment options, ICIs have major limitations. A clinical benefit from ICI is restricted to just 20–30% of patients [ 115 ]. A well‑known cause of primary ICI resistance is low or absent PD-L1 expression on tumor cells. Secondly there are the immune‑related adverse effects occurring in some patients. The most important one, ICIs show limited efficacy against “cold” tumors with low TIL infiltration [ 92 ]. ICI efficacy depends heavily on the TME by which “hot” tumors with more immune infiltration respond better, while “cold” tumors show poor response. On the other hand, OVs possess characteristics that can overcome these limitations and enhance the antitumor effects of ICIs.

OVs can increase tumor cell expression of PD-L1 and CTLA-4, making tumors more susceptible to ICIs. For instance, in colon and ovarian cancer models, oncolytic vaccinia virus attracts effector T cells and induces PD-L1 expression on both cancer cells and immune cells within the tumor [ 116 ]. As well, reovirus selectively increases PD-L1 expression on malignant cells and renders cancer cells with low basal PD-L1 levels more sensitive to anti-PD-1/PD-L1 antibody treatment [ 117 ]. OV therapy can switch “cold” tumors to “hot” by increasing TIL accumulation. Cytokine‑armed OVs further remodel the TME into a proinflammatory microenvironment, making tumors more responsive to ICIs. Thus, combining OVs with ICIs is a promising strategy for treating diverse cancers, with many clinical trials already completed or underway.

In a phase 1b clinical trial, advanced melanoma patients receiving talimogene laherparepvec followed by pembrolizumab showed increased tumor CD8 + T cells, higher PD‑L1 expression, and elevated IFN‑γ gene expression across multiple cell subsets [ 118 ]. A Phase II clinical investigation demonstrated that, in patients with melanoma, combination therapy with talimogene laherparepvec and ipilimumab achieved a significantly higher objective response rate than ipilimumab monotherapy (39% versus 18%) [ 119 ]. However, some studies revealed no positive outcomes in the case of the OVs-ICI combination. A Phase III trial of T‑VEC plus pembrolizumab in advanced melanoma reported no significant improvement in PFS or OS over placebo‑pembrolizumab. ORR was 48.6% vs. 41.3%. Grade ≥ 3 treatment‑related adverse events occurred in 20.7% (T‑VEC plus pembrolizumab) and 19.5% (placebo‑pembrolizumab) [ 120 ]. This discrepancy may be explained by tumor heterogeneity, especially in advanced stages with highly immunosuppressive TMEs. Advanced melanoma uses immune escape mechanisms such as increased Tregs and MDSCs, which can suppress OV‑induced immune stimulation [ 121 ].

An additional critical issue concerns the sequence and timing of therapeutic administration. Nguyen and colleagues identified five timing categories: anti‑PD‑1 first then OV, concurrent, OV first then anti‑PD‑1, concurrent therapy first then anti‑PD‑1, and OV first then concurrent therapy [ 122 ]. Based on existing preclinical and clinical data, the most promising strategy appears to be OV first, followed by concurrent therapy. In this approach, initial OV treatment primes T cells and creates a hot TME, but engagement of PD‑L1 with PD‑1 on immune cells leads to T cell exhaustion. Therefore, after OV lead‑in, continuing OV together with anti‑PD‑1 is crucial: OV maintains T cell priming and a hot TME, while PD‑1 blockade counteracts T cell exhaustion [ 122 ]. OV‑ICI combination efficacy also depends on the OV type, ICI selection, and patient‑specific factors [ 16 ].

Interestingly, in some studies, researchers have used genetic engineering to enable the virus to express an immune checkpoint inhibitor molecule and cytokines. This can be seen in several oncolytic HSVs like RP2, which express GM-CSF + anti-CTLA-4–like molecule, T3011 expressing encoding IL-12 and an anti-PD-1 antibody, etc. (see Tables 1 and 2 ) . A legitimate question arises as to why the majority of studies, both completed and ongoing, have centered on HSV, and why this particular virus has achieved FDA approval (T-VEC) while others remain unapproved. One reason HSV has received so much attention is that its large genome permits stable and straightforward genetic modifications, including the insertion of transgenes. The large genome allows insertions > 30 kb or deletion of multiple virulence genes without affecting lytic replication in tumor cells [ 123 ]. Furthermore, a significant advantage is that HSV‑1 does not undergo integration into the host genome, thereby avoiding the risk of insertional mutagenesis [ 123 ]. Additional advantages of HSV include high replication efficiency (entry via fusion/endocytosis, nuclear DNA delivery, and use of host machinery for rapid replication), broad tropism (infects nearly all human tumor cells), and the ability to penetrate the blood–brain barrier (unlike most viruses, HSV can cross the BBB for CNS gene delivery) [ 124 ]. Additionally, anti‑herpes drugs are capable of blocking HSV‑1 replication during accidental infection and can manage the virus in non‑tumor cells [ 123 ]. Collectively, these factors have concentrated attention on HSV, driving extensive research that ultimately led to its regulatory approval for melanoma therapy.

Combining with cell therapies

Cellular immunotherapy entails the administration of immune cells with antitumor activity to patients, thereby eliciting an anti‑neoplastic response. The principal modalities of adoptive cellular immunotherapy encompass chimeric antigen receptor (CAR) T cell therapy, natural killer (NK) cell therapy, T cell receptor (TCR) T cell therapy, and tumor‑infiltrating lymphocyte (TIL) therapy [ 125 ]. CAR T‑cell therapy has recently become a promising cancer treatment, especially for blood cancers. FDA‑approved CAR T products are available for acute lymphoblastic leukemia, B‑cell lymphoma, and mantle cell lymphoma [ 14 ]. Although CAR‑T cell therapy has shown promise in clinical studies for hematological cancers, its further application is limited by certain barriers, such as poor tumor penetration, low persistence of the engineered T cells, and a lack of response in “cold” tumors [ 126 ]. To circumvent these limitations, researchers have proposed combining cell therapy with OVs, and clinical trials on this topic are now ongoing. OVs can provide synergistic and complementary effects when combined with cell therapy in various ways.

Firstly, OVs can facilitate the migration and infiltration of T cells into the tumor microenvironment. To achieve this, OVs have been armed with chemokines such as CXCL11 that potently enhance T‑cell tumor infiltration. In a preclinical model, oAd‑CXCL11 (oncolytic adenovirus armed with CXCL11) increased tumor infiltration of CD8 + T cells, NK cells, and M1 macrophages, while reducing MDSCs, Tregs, and M2 macrophages in the TME [ 127 ]. Secondly, OVs have the capacity to mitigate T‑cell exhaustion and prolong T‑cell persistence within the immunosuppressive TME. For this purpose, OVs have been engineered to express cytokines critical for T‑cell survival and proliferation. In vitro, oAD‑IL7 (IL‑7‑loaded oncolytic adenovirus) combined with B7H3‑CAR‑T increased T cell proliferation and decreased apoptosis [ 128 ]. An ex vivo study also demonstrated that oAD‑IL7 enhanced the proliferation and persistence of tumor‑infiltrating B7H3‑CAR‑T cells [ 128 ]. Moreover, OVs armed with IL‑2 generated TILs with fewer exhausted PD‑1 hi Tim‑3 + CD8 + T cells and fewer Tregs [ 129 ]. Third, OVs can address a key limitation of CAR‑T and TCR‑T therapies in solid tumors, which is antigen loss or heterogeneity. For this purpose, OVs can be used as carriers for bispecific T‑cell engagers (BiTEs), which allow T cells to recognize and kill tumor cells expressing a variety of antigens, thereby overcoming antigen escape [ 14 , 130 ]. By engineering an oncolytic adenovirus to carry an EGFR‑directed bispecific T‑cell engager (OAd‑BiTE), researchers achieved marked enhancements in CAR‑T cell function, including greater activation, more robust proliferation, elevated cytokine secretion, and stronger tumor‑killing activity, all while preserving a favorable safety profile in a laboratory setting [ 131 ]. The BiTEs secreted from infected cells proved capable of redirecting CAR‑T cells toward EGFR‑expressing tumors irrespective of FR‑α expression, thereby overcoming the problem of antigen heterogeneity [ 131 ]. Another promising avenue is the combination of cell therapy with OVs that carry multiple therapeutic payloads (multi‑armed OVs). Using an oncolytic adenovirus equipped with BiTE, a cytokine, and a checkpoint inhibitor, Porter et al. enhanced CAR T cell function. The strategy allows simultaneous targeting of two tumor antigens through both CAR and native TCR engagement, leading to markedly better tumor control and increased survival [ 132 ]. A phase I clinical trial is currently underway for RGV004, an engineered oncolytic vaccinia virus that carries a bispecific CD19/CD3 antibody, aimed at treating patients with refractory or relapsed B‑cell lymphoma ( NCT04887025 ).

While the integration of OVs with adoptive cell therapy offers a promising strategy for solid tumors by optimizing the tumor microenvironment and enhancing the recruitment and functionality of adoptive cells, CAR‑T cell therapy is not without significant safety concerns, notably immune effector cell‑associated neurotoxicity syndrome and cytokine release syndrome [ 5 , 24 ]. Therefore, any combination regimen involving CAR‑T cells and OVs must prioritize safety assessment and rational design, and additional clinical investigations are urgently required.

Combining with targeted small-molecule

Targeted therapy has emerged as a dominant paradigm in oncological treatment, distinguished by its capacity for precision and enhanced efficacy. This therapeutic approach selectively modulates distinct biological processes inherent to malignant cells, thereby restraining their proliferation and dissemination [ 86 ]. Targeted drugs combined with OVs can be grouped by function, such as angiogenesis inhibitors, anti‑cancer antibodies, small molecule inhibitors, etc [ 133 ]. However, mutations in therapeutic target genes may either increase drug sensitivity or, more often, lead to resistance [ 80 ]. Thus, combining targeted therapy with oncolytic virotherapy can enhance treatment precision and effectiveness. Administering ruxolitinib, a JAK‑1/2 inhibitor, to mice before oncolytic HSV treatment led to reduced expression of interferon‑stimulated genes (ISGs). This suppression made tumors more vulnerable to oncolytic HSV infection. In addition, ruxolitinib pretreatment enhanced viral replication and modified the immune response triggered by oncolytic HSV [ 134 ]. Both JAK1/2 inhibitor experiments and in vivo Jak2 knockout studies in B16‑F10 melanoma models showed that JAK/STAT pathway inhibition significantly enhanced susceptibility to VSV‑Δ51 [ 135 ]. Moreover, in vitro experiments demonstrated that oHSV acts synergistically with the PI3K inhibitor BKM120 to kill prostate cancer sphere-forming cells. This combination also proved highly effective in vivo, where it significantly slowed tumor growth and, in some instances, produced complete tumor regression [ 136 ]. Animals receiving both 3TSR (an anti‑angiogenic peptide) and oncolytic NDV experienced the greatest decreases in primary tumor size, ascites, and secondary lesions, with half of the mice completely free of peritoneal metastases. This combination also improved immune cell infiltration into the primary tumor center [ 137 ]. In summary, the integration of targeted therapies with OVs represents a promising strategy to overcome treatment resistance and enhance anti-tumor efficacy across multiple cancer models. It is even possible to combine viruses with cancer vaccines, an approach that will require additional clinical studies to establish their safety and therapeutic potential [ 138 ].

Table 1 HSV in clinical trials as a monotherapy Agent Genetic modification Cancer type Phase Administration Status Ref M032 HSV-1 with γ134.5-deletion + expressing IL-12 Glioma I Intratumoral Not yet recruiting NCT07076498 Glioma I Intratumoral Active, not recruiting NCT02062827 RP1 HSV-1 expressing GALV-GP-R and GM-CSF Melanoma I Intradermal (local injection at tumor biopsy site) Recruiting NCT06216938 Advanced cutaneous malignancies Ib/II Intratumoral Recruiting NCT04349436 Cutaneous squamous cell carcinoma I/II Intratumoral Recruiting NCT05858229 RP2 HSV-1 with codon-optimized GM-CSF + fusogenic GALV-GP-R⁻ + anti-CTLA-4–like molecule Oral precancer disease II Intralesional Recruiting NCT06623110 C134 HSV-1 with deletion of γ134.5 and insertion of HCMV IRS-1 gene Glioma Ib Intratumoral Enrolling by invitation NCT06193174 Glioma I Intratumoral Active, not recruiting NCT03657576 MVR-C5252 Genetically engineered HSV-1 expressing human IL-12 and anti-PD-1 Fab fragment Glioma I Intratumoral Recruiting NCT06126744 Glioblastoma I Intratumoral Not yet recruiting NCT05095441 R130 Modified HSV-1 containing the gene coding for anti-CD3 scFv/CD86/PD1/HSV2-US11 Bone and soft tissue tumors I Intratumoral Recruiting NCT06171282 Advanced solid tumors I Intratumoral or intraperitoneal Recruiting NCT05961111 Advanced solid tumors I Intratumoral or intraperitoneal Unknown status NCT05886075 Advanced solid tumors I Intratumoral or intraperitoneal Recruiting NCT05860374 Relapsed/refractory bone and soft tissue tumors I Intratumoral Recruiting NCT05851456 Relapsed/refractory head and neck cancer I Intratumoral Recruiting NCT05830240 Relapsed/refractory cervical and endometrial cancer I Intratumoral or Intraperitoneal Recruiting NCT05812677 Relapsed/refractory ovarian cancer. I Intratumoral or Intraperitoneal Unknown status NCT05801783 T3011 HSV-1 encoding IL-12 and an anti-PD-1 antibody Breast cancer, esophagus cancer, lung cancer, melanoma skin cancer, Malignant head and neck, sarcoma I/IIa Intratumoral Unknown status NCT05602792 Advanced solid tumors I/IIa Intravenous Unknown status NCT05598268 Non-muscle-Invasive bladder cancer I Intravesical instillation Recruiting NCT06427291 BS006 HSV-2 (HG52 strain) with deletion of ICP34.5 and ICP47, expressing a bispecific T-cell engager (anti-CD3 and anti-PD-L1). Recurrent cervical cancer I Intratumoral Unknown status NCT05393440 OH2 Genetically engineered HSV-II to express human GM-CSF Melanoma III Intratumoral Recruiting NCT05868707 Central nervous system tumors I/II Intracavitary Recruiting NCT05235074 Bladder cancer Ib/II Intravesical instillation Recruiting NCT05232136 Solid tumors I Intratumoral Not yet recruiting NCT05954091 Liver cancer I Transcatheter Intraarterial infusion Recruiting NCT05698459 HSV1716 HSV-I with deleted in the RL1 gene which encodes the protein ICP34.5 Malignant pleural mesothelioma I/IIa Intrapleural Completed NCT01721018 Refractory Non-Central Nervous System Solid Tumors I Intravenous and intratumoral Completed NCT00931931 HF10 HSV-1 which lacks UL43, UL49.5, UL55, UL56 and overexpression UL53 and UL54 Head and neck cancer or solid tumors with cutaneous and/or superficial lesions I Intratumoral Completed NCT01017185 Solid tumors I Intratumoral Completed NCT02428036 G207 HSV-1 with γ134.5 deletion and UL39 inactivating mutant Malignant glioma Ib/II Intracerebral Completed NCT00028158 BS006 HSV-2 expressing a PD- L1/CD3 bispecific antibody-like molecule Advanced/metastatic solid tumors I Intratumoral Suspended NCT05938296 Cervical cancer I Intratumoral Unknown status NCT05393440 OrienX010 HSV-1 with deletions/inactivation of ICP34.5, ICP47, and ICP6; expressing human GM-CSF (hCMV IE promoter ( Melanoma Ic Intratumoral Unknown status NCT03048253 T-VEC HSV-1 with ICP34.5 and ICP47 deletion and expressing GM-CSF Peritoneal surface malignancies I Intraperitoneal Completed NCT03663712 Non-melanoma skin cancer I Intralesional Completed NCT03458117 Kaposi sarcoma II Intralesional Unknown status NCT04065152 VG161 HSV-1 expressing IL-12, IL-15, IL-15Rα and a PD-1–PD-L1-blocking fusion protein Primary liver cancer I Intratumoral Recruiting NCT04806464 Advanced intrahepatic cholangiocarcinoma II Intratumoral Recruiting NCT06746480 Solid Tumors I Intratumoral Unknown status NCT04758897 Advanced Bone and Soft Tissue Sarcoma II Intratumoral Recruiting NCT06126510 VG2025 HSV-1 with IL-12, IL-15, IL-15R⍺ under CXCR4 promoter Solid tumors I Recruiting NCT05477849 These data are extracted from the https://clinicaltrials.gov/

Table 2 HSV in clinical trials as a combination therapy Agent Genetic modification Combination Cancer type Phase Administration Status Ref T-VEC HSV-1 with ICP34.5 and ICP47 deletion and expressing GM-CSF Anti-PD1 Monoclonal Antibody (pembrolizumab) Sarcoma II Intralesional Active, not recruiting NCT03069378 Anti-PD1 Monoclonal Antibody (pembrolizumab) Melanoma II Intralesional Recruiting NCT03842943 Paclitaxel chemotherapy Breast cancer I/II Intratumoral Active, not recruiting NCT02779855 Radiotherapy Melanoma, merkel cell carcinoma, or other solid tumors II Intralesional Completed NCT02819843 Pembrolizumab Solid tumors Ib/II Intralesional Completed NCT02509507 RP1 HSV-1 expressing GALV-GP-R and GM-CSF Anti-PD-1 monoclonal antibody (nivolumab) Melanoma, non-melanoma skin cancer, and non-small cell lung cancer II Intratumoral Active, not recruiting NCT03767348 Cemiplimab Cutaneous Squamous Cell Carcinoma I/II Intratumoral Active, not recruiting NCT04050436 Nivolumab Melanoma III Recruiting NCT06264180 RP2 HSV-1 with codon-optimized GM-CSF + fusogenic GALV-GP-R⁻ + anti-CTLA-4–like molecule Nivolumab and ipilimumab Melanoma II/III Recruiting NCT06581406 Atezolizumab, bevacizumab and durvalumab Hepatocellular carcinoma and biliary tract cancer II Intratumoral Recruiting NCT05733598 PD-1 blocking antibody (nivolumab) Solid tumors I Intratumoral Recruiting NCT04336241 RP2/RP3 HSV-1 that express exogenous genes (RP2: GM-CSF, GALV, and anti-CTLA-4; RP3: GALV, and anti-CTLA-4 hCD40L, and h4-1BBL) anti-PD-L1 therapy (atezolizumab) and anti-VEGF therapy (bevacizumab) Colorectal cancer II Intratumoral Active, not recruiting NCT05733611 RP3 HSV-1 that expresses exogenous genes (anti-CTLA-4 antibody, CD40 ligand and h4-1BBL) anti-PD1therapy (Nivolumab) Advanced solid tumors I Intratumoral Active, not recruiting NCT04735978 T3011 HSV-1 encoding IL-12 and an anti-PD-1 antibody Toraplizumab and Regorafenib Liver Metastases from Colorectal Cancer I Hepatic Artery Infusion Not yet recruiting NCT06283303 Pembrolizumab Solid Tumors I/IIa Intratumoral Recruiting NCT04370587 ON-01 Engineered HSV-1 expressing yeast cytosine deaminase Flucytosine Glioma I/II Intratumoral Completed NCT06562621 OH2 Genetically engineered HSV-II to express human GM-CSF HX008 (anti-PD-1 antibody ( Melanoma Ib Injection (route not specified) Recruiting NCT04616443 HX008 (anti-PD-1 antibody ( Gastrointestinal cancers, head and neck cancers, soft tissue sarcomas I/II Intratumorally Recruiting NCT03866525 Keytruda (an anti-PD-1 antibody) Melanoma I Injection (route not specified) Recruiting NCT04386967 G207 HSV-1 with γ134.5 deletion and UL39 inactivating mutant Radiation Recurrent or refractory cerebellar brain tumor I Infusion via catheters Recruiting NCT03911388 Radiation Recurrent supratentorial brain tumors I Intratumoral infusion via catheters Completed NCT02457845 rQNestin34.5v.2 HSV-1 that retains expression of the ICP34.5 gene under glioma-selective transcriptional regulation Cyclophosphamide Glioma I Intratumoral Recruiting NCT03152318 JNJ-87,704,916 NA Cetrelimab Advanced solid tumors I Intratumoral Recruiting NCT06311578 KB707 HSV-1 engineered to express IL-12 PD-1 immune checkpoint inhibitor (Pembrolizumab), Chemotherapy and Docetaxel Advanced solid tumor malignancies affecting the lungs I/II Nebulization Recruiting NCT06228326 PD-1 and LAG-3 immune checkpoint inhibitors ( Opdualag) and PD-1 immune checkpoint inhibitor (Pembrolizumab) locally advanced or metastatic solid tumor I/II Intratumoral Recruiting NCT05970497 VG161 Recombinant Human IL12/15-PDL1B Oncolytic HSV-1 Nivolumab Hepatocellular carcinoma or intrahepatic cholangiocarcinoma II Intratumoral Recruiting NCT05223816 VG2025 Nivolumab Solid tumors I Intratumoral Recruiting NCT05266612 These data are extracted from the https://clinicaltrials.gov/

Table 3 AdV in clinical trials as a monotherapy Agent Genetic modification Cancer type Phase Administration Status Ref Ad-TD-nsIL12 Recombinant adenovirus genetically engineered to express human non-secreted interleukin-12 (nsIL-12) Glioma I/II Intratumoral Enrolling by invitation NCT07126990 Human nsIL12 Oncolytic Adenovirus (BioTTT001) Genetically engineered adenovirus to express human nsIL-12 Glioma Ib/II Intratumoral Enrolling by invitation NCT06763965 Solid tumors I Intratumoral Enrolling by invitation NCT06215846 NRG103 NA Glioblastoma I Intratumoral )via an Ommaya reservoir( Recruiting NCT06757153 VT-101 NA Bladder cancer I Intravesical instillation Recruiting NCT06632964 TS-2021 Oncolytic adenovirus that carried Ki67 promoter, TGF-β2 5’UTR, and IL-15 Glioma I Intratumoral Recruiting NCT06585527 KD01 Human adenovirus type 5 engineered with a deletion in the E3 region, in which the ADP gene is replaced by the pro-apoptotic tBID gene, and a 27-base pair deletion in the E1A region Cervical malignancies I Intratumoral Recruiting NCT06552598 YSCH-01 Recombinant adenovirus encoding L-IFN Solid tumors I Intratumoral Unknown status NCT05180851 Oncorine (H101) E1B 55KD and partial E3 deleted Refractory malignant ascites II Intraperitoneal Completed NCT04771676 DNX-2440 Oncolytic adenovirus expressing human OX40 ligand. Colorectal Liver Metastasis I Intratumoral Suspended NCT04714983 DNX-2401 A 24 bp deletion in the E1A gene and RGD peptide insertion into the fiber knob Glioma I Intratumoral Completed NCT03178032 Glioma I Intraarterial and intratumoral Recruiting NCT03896568 TILT-123 Oncolytic adenovirus expressing human TNF-α and IL-2 Solid tumors I Recruiting NCT04695327 Melanoma I Completed NCT04217473 SynOV1.1 E1B/partial E3 deletion + synthetic sensory switch Solid tumors I Intratumoral Unknown status NCT04612504 ORCA-010 Contains the E1A-Δ24 + fiber-RGD mutation + the T1 mutation Prostate cancer I/IIa Intratumoral Unknown status NCT04097002 OBP-301 Ad5 based adenoviral construct that incorporates a human telomerase reverse transcriptase gene (hTERT) promoter Melanoma II Intratumoral orIntralesional Unknown status NCT03190824 CG0070 Oncolytic adenovirus with a E2F promotor and expresses GMCSF Non-muscle invasive bladder cancer II Intravesical Completed NCT02365818 These data are extracted from the https://clinicaltrials.gov/

Table 4 AdV in clinical trials as a combination therapy Agent Genetic modification Combination Cancer type Phase Administration Status Ref Human nsIL12 Oncolytic Adenovirus (BioTTT001) Genetically engineered adenovirus to express human nsIL-12 Neoadjuvant* Breast cancer I Intratumoral Withdrawn NCT07150767 SOX and Toripalimab Gastric cancer II Intraperitoneal Not yet recruiting NCT06283121 Toraplizumab and Regorafenib Colorectal cancer I Hepatic artery infusion Not yet recruiting NCT06283134 TILT-123 TNFα and IL-2 coding oncolytic adenovirus Pembrolizumab (a monoclonal antibody binding PD-1) Non-small cell lung cancer I NA Active, not recruiting NCT06125197 Pembrolizumab or Pembrolizumab and Pegylated Liposomal Doxorubicin Ovarian Cancer I/Ib Intratumoral or intraperitoneal Recruiting NCT05271318 Avelumab )anti-PDL1 antibody) Solid tumors (SCCHN and melanoma) I Active, not recruiting NCT05222932 chemotherapy (cyclophosphamide + fludarabine) and adoptive TIL therapy. Melanoma I Recruiting NCT06961786 H101 Knockout of e1B-55KD; partial deletion of E3 region PD-1 inhibitor Pleural mesothelioma * 2 Intratumoral or intrapleural Recruiting NCT06031636 Tislelizumab + Lenvatinib Biliary tract cancer II Intratumoral Recruiting NCT05823987 Tislelizumab + Lenvatinib Hepatocellular carcinoma I Intratumoral Recruiting NCT05675462 Camrelizumab (PD-1 inhibitor) Non-muscle-invasive bladder cancer II Intravesical Unknown status NCT05564897 Camrelizumab (anti-PD-1 antibody) Cervical cancer II Intratumoral Unknown status NCT05234905 HAIC of FOLFOX Intrahepatic cholangiocarcinoma IV Intratumoral Recruiting NCT05124002 Sorafenib Hepatocellular carcinoma IV Intratumora Unknown status Recombinant Human Adenovirus Type 5 Tremelimumab (PD-1 monoclonal antibody) Melanoma I Intratumoral Unknown status NCT05928962 MEM-288 Oncolytic adenovirus vector encoding transgenes for human IFNβ and a recombinant chimeric form of CD40-ligand (MEM40) Nivolumab (anti-PD1 monoclonal antibody) or docetaxel Solid tumors I Intratumoral Recruiting NCT05076760 AdAPT-001 Carries a TGF-ß trap that neutralizes the immunosuppressive and profibrotic cytokine namely TGF-ß Immune checkpoint inhibitor Sarcoma and refractory solid tumors II Intratumoral Recruiting NCT04673942 Enadenotucirev Live and replicating chimeric Ad11p/3 oncolytic adenovirus Radiotherapy and/or capecitabine. Rectal cancer I Intravenous Completed NCT03916510 Nivolumab Epithelial tumors I Intravenous Completed NCT02636036 NG-350 A Oncolytic adenoviral vector which expresses a full-length agonist anti-CD40 antibody Check point inhibitor Epithelial tumors Ia Intravenous Completed NCT03852511 CAdVEC HER2-Specific Autologous CAR T Cells HER2 Positive Solid Tumors I Intratumoral Recruiting NCT03740256 LOAd703 Oncolytic adenovirus (Ad5/35) engineered to express the immunostimulatory transgenes TMZ-CD40L and 4-1BBL Chemotherapy or using gemcitabine immune-conditioning Pancreatic, biliary, colorectal or ovarian I/II Intratumoral Completed NCT03225989 Atezolizumab (anti-PD-L1 antibody) Melanoma I/II Intratumoral Completed NCT04123470 Chemotherapy and anti-PD-L1 antibody Pancreatic cancer I/IIa Intratumoral Active, not recruiting NCT02705196 NSC-CRAd-Survivin-pk7 Neural stem cells loaded with the virus Radiation and chemotherapy Glioma I Intratumoral * 3 Completed NCT03072134 ADV/HSV-tk Adenovirus-mediated expression of HSV thymidine kinase Valacyclovir, Radiation and Pembrolizumab Negative breast cancerand metastatic non-small cell lung cancer II Intratumoral Completed NCT03004183 ONCOS-102 Engineered oncolytic adenovirus expressing GM-CSF Cyclophosphamide and Pembrolizumab Melanoma I Intratumoral Completed NCT03003676 VCN-01 Genetically modified human adenovirus encoding human PH20 hyaluronidase Gemcitabine and Abraxane Solid tumors I Intravenous Completed NCT02045602 DNX-2401 24 bp deletion in the E1A gene and an RGD peptide insertion into the fiber knob Pembrolizumab Glioblastoma or gliosarcoma II Intratumoral Completed NCT02798406 *Albumin-bound paclitaxel, trastuzumab, pertuzumab, carboplatin, toripalimab, docetaxel, doxorubicin, cyclophosphamide *2 Observational *3 Delivered via neural stem cells These data are extracted from the https://clinicaltrials.gov/

Table 5 Vaccinia virus in clinical trials as a monotherapy Agent Genetic modification Cancer type Phase Administration Status Ref OTS-412 Vaccinia virus-tk gene (thymidine kinase) has been replaced with an HSV-tk Healthy volunteer I Systemic Injection Completed NCT07302945 hV01 hV01 is a recombinant vaccinia virus with deletions of the viral thymidine kinase and viral growth factor genes and insertion of the human IL-21 gene Advanced solid tumors NA Intratumoral Recruiting NCT07185243 Pancreatic cancer II Intratumoral Recruiting NCT07006077 Advanced solid tumors I Intratumoral Active, not recruiting NCT05914376 GO306 - Solid tumors I Intratumoral or intracavitary Recruiting NCT07128914 GC001 - Gliomas I Intratumoral Recruiting NCT06660056 GC001 - Solid tumors I Intratumoral Recruiting NCT06508307 vvDD-hIL2-2-RG-1 Recombinant Western Reserve strain vaccinia virus (vvDD) with double deletion of thymidine kinase and vaccinia growth factor genes, engineered to express membrane-bound human IL-2 for localized T-cell activation in the tumor microenvironment Metastatic Gastrointestinal and Peritoneal Tumors I Intratumoral Recruiting NCT07001592 IDOV-Immune - Solid tumors I Intravenous Recruiting NCT06910657 RGV004 Genetically-engineered vaccinia virus (encoding CD19/CD3 bispecific antibody) B-cell Lymphoma I Intratumoral Unknown status NCT04887025 GL-ONC1 Genetically-engineered vaccinia virus (encoding Renilla luciferase-Aequorea green fluorescent protein fusion, β-galactosidase, and β-glucuronidase) Peritoneal carcinomatosis I/II Intraperitoneal Completed NCT01443260 Solid tumors I Intravenous Completed NCT00794131 Pexa-Vec (JX-594) Thymidine kinase-deactivated vaccinia virus + transgenes encoding GM-CSF and beta-galactosidase Solid tumors I Intratumoral Completed NCT01169584 Hepatic carcinoma I Transdermal Completed NCT00629759 Solid tumors I Intravenous Completed NCT00625456 Hepatocellular carcinoma II Intratumoral Completed NCT00554372 Melanoma I/II Intratumoral Completed NCT00429312 IDOV-SAFETM - Advanced solid tumors I Intravenous Recruiting NCT06346041 IDOV-SAFETM - Advanced solid tumors I Intravenous Recruiting NCT06718946 These data are extracted from the https://clinicaltrials.gov/

Table 6 Vaccinia virus in clinical trials as a combination therapy Agent Genetic modification Combination Cancer type Phase Administration Status Ref OTS-412 Vaccinia virus-tk gene (thymidine kinase) has been replaced with an HSV-tk Hydroxyurea alone and Hydroxyurea/Atezolizumab Solid tumor I Intratumoral Not yet recruiting NCT07190833 Hydroxyurea alone and Hydroxyurea/Atezolizumab Solid tumor I Intratumoral Not yet recruiting NCT07170592 Olvi-Vec (Olvimulogene nanivacirepvec, GL-ONC1 and laboratory name GLV-1h68) Replacing the TK, hemagglutinin, and F145L genes with three expression cassettes encoding the β-galactosidase, β-glucuronidase, and RLuc-GFP fusion proteins, respectively Chemotherapy + Physician’s choice of ICI Non-small-cell lung cancer II Intravenous Recruiting NCT06463665 Chemotherapy and Bevacizumab Ovarian cancer III Intraperitoneal Recruiting NCT05281471 GL-ONC1 + chemotherapy ± bevacizumab Ovarian cancer Ib/II Intraperitoneal Completed NCT02759588 Radiation therapy and cisplatin Head and neck cancer I Intravenous Completed NCT01584284 KM1 NA Chemotherapy Ovarian Cancer I Intraperitoneal Recruiting NCT05684731 T601 TK-RR deletion + Expression of FCU1 gene T601 alone and T601 + 5-FC Solid tumor I/IIa Unknown status NCT04226066 ASP9801 Encoding IL-7 and IL-12 ASP9801 alone and ASP9801 + Pembrolizumab Solid tumor I Intratumoral Completed NCT03954067 Pexa-Vec (JX-594) Thymidine kinase-deactivated vaccinia virus + transgenes encoding GM-CSF and beta-galactosidase Cemiplimab (monoclonal antibody to PD-1) Renal cell carcinoma Ib/IIa Intratumoral Active, not recruiting NCT03294083 Durvalumab and Tremelimumab Colorectal cancer I/II Intravenous Completed NCT03206073 Sorafenib Hepatocellular carcinoma III Intratumoral Completed NCT02562755 Irinotecan Colorectal carcinoma I/IIa Intravenous Completed NCT01394939 Supportive care Liver cancer II Intravenous Completed NCT01387555 TG6002 5-flucytosine Glioblastoma I/II Intravenous Unknown status NCT03294486 IDOV-SAFETM Toripalimab and fruquintinib (targeted cancer treatments) Advanced solid tumors I Intravenous Recruiting NCT06380309 These data are extracted from the https://clinicaltrials.gov/

Table 7 Reovirus in clinical trials as a monotherapy and combination therapy Agent Genetic modification Combination Cancer type Phase Administration Status Ref Pelareorep (REOLYSIN) non-engineered / naturally occurring oncolytic virus - Gliomas I/II Intratumoral Completed NCT00528684 - Bone and soft tissue sarcomas metastatic to the lung II Intravenous Completed NCT00503295 Retifanlimab (INCMGA00012) (Anti-PD-1) Triple negative breast cancer II Intravenous Active, not recruiting NCT04445844 Pelareorep + paclitaxel, or Pelareorep + paclitaxel + avelumab (anti-PD-L1) Metastatic breast cancer II Intravenous Active, not recruiting NCT04215146 Carfilzomib + dexamethasone + nivolumab + Pelareorep Multiple myeloma I Intravenous Completed NCT03605719 Chemotherapy + Pembrolizumab + Pelareorep Pancreatic adenocarcinoma I Intravenous Completed NCT02620423 Gemcitabine Pancreatic adenocarcinoma II Intravenous Completed NCT00998322 Irinotecan/Fluorouracil/Leucovorin (FOLFIRI) + Bevacizumab + Pelareorep Metastatic colorectal cancer I Intravenous Completed NCT01274624 Chemotherapy Pancreatic cancer II Intravenous Completed NCT01280058 Paclitaxel alone and paclitaxel + Pelareorep Ovarian epithelial, fallopian tube, or primary peritoneal cancer II Intravenous Completed NCT01199263

Table 8 MV and NDV in clinical trials as a monotherapy and combination therapy Agent Genetic modification Combination Cancer type Phase Administration Status Ref MV-s-NAP Oncolytic Measles Virus Encoding H. pylori NAP - Breast cancer I Intratumoral Recruiting NCT04521764 MV-NIS Genetically engineered to express neurofibromatosis type 1 (NIS) - Urothelial carcinoma I Intravesical Completed NCT03171493 - Peripheral nerve sheath tumor I Intratumoral Completed NCT02700230 - Myeloma II Intravenous Completed NCT02192775 - Ovarian, primary peritoneal or fallopian tube cancer I/II Intraperitoneal* Active, not recruiting NCT02068794 - Breast Cancer I Intratumoral Completed NCT01846091 - Pleural Mesothelioma I Intrapleural Completed NCT01503177 MV-CEA Carcinoembryonic Antigen-expressing measles virus - Glioblastoma Multiforme I Resection cavity administration and intratumoral Completed NCT00390299 MV-CEA and MV-NIS Recombinant carcinoembryonic antigen (CEA)-expressing measles virus (MV-CEA) and oncolytic measles virus encoding thyroidal sodium iodide symporter (MV-NIS) - Ovarian Epithelial Cancer or Primary Peritoneal Cancer I Intraperitoneal Completed NCT00408590 MV-NIS Genetically engineered to produce sodium iodine symporter (NIS) MV-NIS alone and MV-NIS + Cyclophosphamide Multiple Myeloma I/II Intravenous Completed NCT00450814 MEDI5395 Expressing GM-CSF Durvalumab Advanced Solid Tumors I Intravenous Completed NCT03889275 These data are extracted from the https://clinicaltrials.gov/ * MV-NIS infected mesenchymal stem cells which given thorough intraperitoneal route

Predictive biomarkers and patient selection for oncolytic virotherapy

Clinical responses show considerable variability, with some patients achieving substantial tumor reduction and longer survival, while others derive no meaningful benefit despite receiving the same OV platform [ 139 ]. One possible explanation for this variability is that patients receiving these therapies have often undergone multiple prior conventional cancer treatments, leading to alterations in tumor cell characteristics compared with their original state. These changes may contribute to marked differences in therapeutic effectiveness among patients [ 140 ]. This highlights the need for more precise patient selection strategies. A key approach to improving patient selection is the use of predictive biomarkers, which help identify individuals who are more likely to benefit from specific cancer therapies and thereby support more personalized treatment planning.

Several candidate biomarkers have been investigated in different studies. A systematic review and meta-analysis indicated that several biomarkers are associated with response to OV therapy [ 139 ]. For example, high tumor mutational burden, loss-of-function mutations in interferon signaling pathways, baseline CD8⁺ T-cell infiltration, post-treatment upregulation of IFN-γ and PD-L1, and favorable gut microbiome profiles were linked to improved outcomes. In contrast, intact antiviral signaling, immune-excluded tumor microenvironments, and myeloid cell dominance were associated with therapeutic resistance. Moreover, OV treatment, especially when combined with checkpoint inhibitors, is consistently associated with an increase in CD8 + T cells, along with increased PD-L1 expression and IFN-γ gene expression in the tumor microenvironment [ 139 ]. Evidence from a study conducted by Hao et al. in 2023 suggests that infection with EV-A71 can markedly influence gene expression patterns in glioma [ 141 ]. They found that polypyrimidine tract binding protein 1 (PTBP1) overexpression in low-grade glioma has been associated with advanced tumor stage and poorer clinical outcomes, and it also correlates with immune cell infiltration, supporting its potential role as a prognostic biomarker [ 141 ]. A study by Park et al. identified BAP1 as a potential predictive biomarker for response to JX-594 therapy and explored its underlying mechanisms. Their findings showed that JX-594 inhibits the growth of BAP1-deficient tumors via IRF7-dependent induction of IFN-β, suggesting that reactivation of this pathway may represent a promising therapeutic strategy in renal cell carcinoma [ 142 ]. Another study found that a decrease in peripheral lymphocyte levels predicts imaging response and survival in advanced solid tumor patients treated with TILT-123, suggesting a simple and practical biomarker, although further validation is needed [ 143 ]. As well, there is also evidence that defects in interferon signaling pathways may serve as potential biomarkers of sensitivity to OV therapy [ 144 ]. For instance, Nguyen et al. reported that mutations in the IFNγ–JAK–STAT signaling pathway, which are associated with resistance to immune checkpoint inhibitors in melanoma, can simultaneously enhance tumor sensitivity to OV therapy [ 135 ]. Another study investigated oncolytic virotherapy in pancreatic ductal adenocarcinoma using patient-derived tumor cultures treated with five OVs, including Ad5-hTERT adenovirus, T-VEC, MV-NIS, reovirus jin-3, and H-1 protoparvovirus (H-1PV), to assess therapeutic response and identify predictive biomarkers [ 145 ]. Overall, viral entry receptor expression was not strongly associated with sensitivity, except for LGALS1, which correlated with H-1PV-induced cell killing. Instead, the response was mainly influenced by immune, metabolic, and proliferative pathways, with interferon-stimulated genes linked to resistance to MV-NIS and low cGAS expression associated with better response, while cGAS inhibition improved viral efficacy. The authors proposed that, instead of relying on single biomarkers, combining ex vivo sensitivity testing with transcriptomic analysis may provide a more effective approach for patient stratification [ 145 ]. Interestingly, the research team established a transcriptomic biomarker framework, termed ViroPredict 1.0, aimed at pinpointing individuals most responsive to VG161 therapy [ 146 ]. By analyzing baseline tumor RNA‑sequencing profiles, they applied a least absolute shrinkage and selection operator (LASSO)–based Cox regression model, which revealed five genes (SEZ6, AKR1C1, LILRA5, NCKAP5, and SETD9) that correlated with treatment outcomes. The derived risk score effectively classified participants into high‑ and low‑response cohorts, demonstrating statistically significant disparities in both progression‑free and overall survival rates [ 146 ].

However, despite these promising findings regarding identifying predictive biomarkers for patient selection in oncolytic virotherapy, there is no universally validated biomarker. This can be attributed to the fact that the majority of clinical trials conducted thus far have failed to incorporate stratification based on biomarkers, leading to a generalized approach that may lessen the effectiveness of treatments. As the landscape shifts towards precision oncology, there is a critical necessity to integrate diverse biomarker information covering genomic, immunological, and microbiome aspects into the framework for designing and analyzing OV trials. This need is particularly pronounced with the growing adoption of combination therapies, such as the concurrent use of OVs and ICIs, which demands a more profound understanding of the biomarkers that can predict potential synergies, resistance mechanisms, or adverse effects [ 139 ]. As a future perspective, artificial intelligence can be used for oncolytic viral therapy predictions. Uthamacumaran et al. suggested an AI-driven hybrid ecological framework for anticipating the dynamics associated with oncolytic viral therapy. This hybrid model synergizes Generalized Lotka–Volterra equations with a set of AI-driven optimization methods such as reinforcement learning, genetic algorithms, differential evolution, and random forest for feature importance analysis to accurately model oncolytic viral therapy dynamics, optimize parameters, and pinpoint key genes that predict intricate tumor–immune interactions [ 147 ]. This model offers real-time predictions of oncolytic viral therapy responses and simultaneously identifies critical biomarkers to improve therapeutic effectiveness. Nonetheless, these computationally inferred biomarkers require rigorous experimental and clinical validation before therapeutic implementation [ 147 ]. Furthermore, according to the recent study, extracellular vesicles carrying viral DNA may also be employed as biomarkers to monitor and predict responses in oncolytic adenovirus therapy [ 148 ]. This strategy offers a promising framework for personalized oncolytic virotherapy monitoring and holds potential for adaptation to other virus-based therapies in future.

Conclusions and future perspective

Oncolytic virotherapy has advanced quickly, with its main objective now being to harness and boost virus-induced anti-tumor immune responses rather than just directly lysing tumor cells. The therapeutic potential of OVs has been demonstrated by clinical approvals (e.g., T-VEC and other products) and multiple phase I–III trials across a variety of viral backbones (HSV-1, adenovirus, vaccinia, VSV, and others), especially when used to overcome resistance to conventional modalities. Immunostimulatory transgenes (GM-CSF, IL-12, TNFα), enhanced biosafety and tumor selectivity through genetic engineering, and early evidence of durable responses when OVs are rationally combined with immune checkpoint inhibitors, cell therapies, radiotherapy, or chemotherapy are some of the major advancements.

Despite encouraging outcomes, significant obstacles still exist. Preexisting antiviral immunity, quick neutralization, poor tumor penetration in fibrotic or poorly vascularized lesions, and off-target inflammatory risks are the main obstacles to systemic delivery. Single-agent efficacy is further complicated by tumor heterogeneity, underscoring the necessity of predictive biomarkers for patient stratification [ 149 , 150 ]. As well, it should be mentioned that there is inconsistent clinical efficacy of OVs across trials (e.g., reovirus studies), which can be attributed to several factors [ 16 , 151 ]. These factors are: tumor heterogeneity, virus type, delivery routes, rapid immune clearance, insufficient tumor specificity, limited spread within the tumor, and the immunosuppressive TME.

Looking forward, next-generation strategies should prioritize precision: engineered tropism for metastatic tumors, controllable gene expression, matrix-modifying payloads to improve penetration, and delivery innovations (cellular carriers, nanoparticles, hydrogels, cell robots) [ 152 – 154 ]. Moreover, extracellular vesicles (EVs) are emerging as effective carriers for targeted delivery of therapeutic agents into the tumor microenvironment. They can also improve the tumor-specific delivery of OVs, shielding them from immune clearance after systemic administration. Moreover, EVs can facilitate the spread of OVs to metastatic sites and enhance anti-tumor immune responses [ 155 ]. Integrating OVs with CAR-T/CAR-NK platforms, checkpoint blockade, and personalized biomarker-driven regimens offers the most promise to convert cold tumors into immunologically hot tumors. Realizing this potential will demand interdisciplinary collaboration, advanced modeling for individualized dosing, and large, biomarker-enabled clinical trials to define safe, effective combination paradigms and translate preclinical promise into durable patient benefit.

Table 9 Other oncolytic viruses in clinical trials as a combination therapy Agent Genetic modification Combination Cancer type Phase Administration Status Ref Gebasaxturev (coxsackievirus A21 or V937 or CAVATAK) Genetically unmodified Pembrolizumab + Vibostolimab, and Pembrolizumab + Gebasaxturev and Pembrolizumab alone Melanoma I/II Intravenous Completed NCT04303169 Pembrolizumab Melanoma I Intratumoral Completed NCT02565992 Ipilimumab Melanoma metastatic to the liver I Intravenous Completed NCT03408587 Pembrolizumab Non-small cell lung cancer I/II Intravenous Unknown status NCT02824965 None Melanoma II Intratumoral Completed NCT01227551 Melanoma I Intratumoral Completed NCT00438009 PVSRIPO Genetically unmodified None Breast Cancer I Intratumoral Completed NCT03564782 Malignant Glioma Ib Intratumoral Completed NCT03043391 Glioblastoma I Intratumoral Completed NCT01491893 Glioblastoma II Intratumoral Recruiting NCT06177964 VSV-hIFNβ-NIS Recombinant VSV carrying the human sodium iodide symporter and Interferon-beta genes Ipilimumab and cemiplimab Peripheral T-cell Lymphoma I Intravenous Recruiting NCT06508463 With or Without Cyclophosphamide and Combinations of Ipilimumab, Nivolumab, and Cemiplimab Multiple myeloma, acute myeloid leukemia or lymphoma I Intravenous Recruiting NCT03017820 Alone and in combination with ruxolitinib Endometrial Cancer I Intravenous Active, not recruiting NCT03120624 Parvovirus H-1 (ParvOryx) Genetically unmodified None Glioblastoma multiforme I/IIa Intratumoral or intravenous Completed NCT01301430 MG1-MAGEA3 MG1 Maraba Expressing MAGE-A3 With Adenovirus Vaccine Expressing MAGE-A3 (Ad-MAGEA3), in Combination With Pembrolizumab Non-Small Cell Lung Cancer Patients I/II Intravenous Completed NCT02879760 Maraba/MAGE-A3 (MG1MA3) Maraba virus modified to express tumour antigen MAGE-A3 With and Without Adenovirus vaccine, With Transgenic MAGE-A3 Insertion (AdMA3) Solid Tumors I/II Intravenous Active, not recruiting NCT02285816 These data are extracted from the https://clinicaltrials.gov/

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Acknowledgements

Not applicable.

Author contributions

O.SA Writing original draft, review and editing, supervision, Investigating. M.Z and B.M, N.A and T.S Review and editing, Investigating. B.M Designing figures, Review and editing. All authors accepted the final version of the manuscript.

Funding

Not applicable.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Using of language models

During the preparation of this work the authors used language models in order to enhancing the writing quality and minimize unintentional plagiarism. After using language models, the authors reviewed and edited the content as needed and takes full responsibility for the content of the published article.

Abbreviations

Oncolytic viruses

CSF-Granulocyte-macrophage colony-stimulating factor

2-Interleukin-2

α-Tumor Necrosis Factor alpha

Protein kinase receptor

1-Herpes simplex virus type 1

2-Herpes simplex virus type 2

Vesicular stomatitis virus

Newcastle disease virus

Adenovirus

β-Interferon-beta

Immunogenic cell death

Damage-associated molecular patterns

Pathogen-associated molecular patterns

Pattern recognition receptors

Tumor microenvironment

Vascular Endothelial Growth Factor

Neutrophil-activating protein

Thymidine Kinase

VEC-Talimogene laherparepvec

Oncorine

L1-Programmed Death-Ligand 1

Extracellular vesicles

Regulatory T cells

Tumor-associated antigens

Natural killer

Dendritic cells

Tumor-associated macrophages

5-Fluorouracil

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