Advancements on the synergistic application of oncolytic viruses and molecularly targeted therapies for the treatment of solid tumors (Review)

Introduction

Oncolytic viruses (OVs) represent an innovative approach in cancer therapy, characterized by their ability to selectively lyse tumor cells while sparing adjacent normal tissues. They also potentiate the anti-tumor immune response through the release of tumor-associated antigens and the activation of inflammatory pathways within the tumor microenvironment (TME) (1). Preclinical toxicology studies have demonstrated that OVs exhibit low toxicity and high tolerability (2). To date, five OVs have successfully undergone clinical translation (3), including the oncolytic adenovirus H101, talimogene laherparepvec (T-VEC), Rigvir, G47Δ and nadofaragene firadenovec-vncg. They show significant potential in clinical applications (4-8). However, in solid tumors, factors such as abnormal lymphatic structures, high vascular permeability, a dense extracellular matrix and the requirement for OVs to traverse the endothelial layer to reach target cells collectively reduce the penetration range of OVs. Additionally, interactions between OVs and antigen-presenting cells can trigger innate immune responses and antiviral immunity, increasing the likelihood of clearance by the host's immune system (9). Another key obstacle is the heterogeneity of tumors, which leads to incomplete responses to specific monotherapies (10). These limitations hinder the widespread clinical translation of OV therapy.

Targeted therapy functions by selectively interacting with specific sites to inhibit enzymes and growth factor receptors essential for proliferation, inducing apoptosis in cancer cells and modulating gene expression, thereby altering protein functions in normal cells to disrupt pathways associated with carcinogenesis and tumor progression (11). However, due to genetic alterations, adaptive responses or bypass mechanisms that allow cancer cells to endure the selective pressure exerted by the treatment, resistance may be acquired, which limits the long-term efficacy and developmental potential of targeted therapies (12).

Previously research indicates that the integration of OVs with targeted therapeutics can inhibit tumor initiation and progression through various mechanisms, effectively overcoming the limitations associated with OVs and targeted drug monotherapies, thereby enhancing the overall efficacy of cancer treatment. As a result, combination therapy is gradually becoming one of the leading approaches in oncology. Earlier reviews mainly explained the basic principles and potential strategies of combining OVs with key cellular signaling pathway modulators in cancer, without categorizing the specific mechanisms (13). Certain reviews have summarized the progress of OVs in combination with various therapies, such as chemotherapy, radiotherapy and mainly novel immunotherapies like immune checkpoint inhibitors (10,14,15). This article primarily elaborates on the mechanisms and progress of OVs combined with targeted drugs, categorizing them according to different mechanisms, while further exploring how to select more rational combination approaches based on the characteristics of different tumors, such as OVs targeting tumor surface nonspecific and specific receptors and monitoring of mutated targets. By discussing the differences between human and mouse immune systems, potential combination toxicities and their solutions, as well as future strategies to address drug resistance, the article highlights key points for translating preclinical research into clinical applications. Based on the above discussions, it connects preclinical evidence with trial design elements such as patient selection criteria, recommended endpoints and overlapping toxicity safety monitoring, providing a valuable reference for the future clinical translation of OVs combined with targeted drugs.

Mechanisms of action of OVs combined with molecularly targeted therapies

The mechanism of action of OVs combined with molecularly targeted drugs is mainly related to signaling pathways, immune responses, DNA damage, apoptosis and autophagic cell death. In this chapter, a detailed explanation of the above will be provided.

Inhibition of phosphorylation at specific sites modulates downstream signaling and interferon (IFN) pathways

Inhibiting key targets in signaling pathways related to cell proliferation can enhance the replication of OVs and their oncolytic effects, thereby exerting antitumor activity. The mitogen-activated protein kinase (MAPK), Janus kinase (JAK)/signal transducer and activator of transcription (STAT) and phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathways are jointly involved in regulating cell proliferation, differentiation, survival and apoptosis, with complex crosstalk between them. MAPK kinase (MEK) is one of the key targets in the MAPK signaling pathway (16). Lee et al (17) demonstrated that inhibiting MEK phosphorylation can activate STAT3, thereby promoting oncolytic vaccinia virus (OVV) replication in doxorubicin-resistant ovarian cancer and enhancing the anti-tumor effects. STAT3 activation also promotes the replication of oncolytic herpes simplex virus (oHSV) in glioma cells (18). MEK inhibitors also suppress the antiviral response facilitated by IFN signaling through STAT1/STAT2-dependent pathways. IFNs orchestrate innate immune responses, serving as a formidable first line of defense against pathogen invasion (19). This initiates a signaling cascade through the JAK-STAT pathway (20). STAT1 and STAT2 play pivotal roles in type I and type II IFN signaling, significantly contributing to the cellular antiviral response and adaptive immunity. The primary pathways through which IFNs exert their antiviral effects include the oligoadenylate synthetase-ribonuclease L system and the RNA-dependent protein kinase (PKR) pathway, which degrade viral RNA and inhibit viral protein synthesis, respectively. Zhou et al (21) found that trametinib boosts viral replication in BRAF V600E and BRAF wild-type (wt)/KRAS mutated tumor cells by reducing STAT1 and PKR phosphorylation. Additionally, the JAK-1/2 inhibitor ruxolitinib increases vesicular stomatitis virus (VSV) replication by inhibiting the JAK/STAT signaling pathway, enhancing its sensitivity in melanoma and lung cancer (22,23). Targeting the Cysteine-rich 61 (CCN1)-AKT pathway can enhance bevacizumab's effectiveness by reducing glioma infiltration. Elevated CCN1 expression is associated with increased AKT phosphorylation in tumor cells (24). Studies have shown that CCN1 stimulates the expression of PI3K/AKT in various types of cancer cells, including glioma, breast cancer, gastric cancer and renal cancer cells (25-27). Rapid antiangiogenesis mediated by oncolytic virus (RAMBO) is an engineered oHSV designed to express angiogenesis inhibitors and is capable of suppressing the expression of CCN1 (28). A study found that the angiostatin produced by RAMBO can inhibit bevacizumab-induced CCN1 expression, reducing bevacizumab-induced glioma infiltration by blocking the CCN1-AKT signaling pathway, thereby enhancing the efficacy of bevacizumab against malignant gliomas (28).

Tyrosine kinase inhibitor (TKI) synergistically augments the antitumor efficacy of OVs by inhibiting the phosphorylation of AKT, which are activated by OVs. G47Δ-mIL12, an advanced version based on the third-generation oHSV-1 G47Δ, enhancing tumor-specific replication and safety by deleting the α47 gene and US11 promoter to prevents class I major histocompatibility complex (MHC) downregulation and inhibiting angiogenesis (4,29,30). Research conducted by indicates that the selective TKI axitinib enhances the antitumor effect of G47Δ-mIL12 in glioblastoma (GBM) by blocking the platelet-derived growth factor receptor (PDGFR) pathway, inhibiting phosphorylated ERK1/2 and reducing G47Δ-mIL12-induced AKT phosphorylation. Furthermore, this combination also promotes apoptosis and affects stem-like cell characteristics (31). TKI can also promote OV replication by inhibiting the antiviral response mediated by IFN signaling. Research by Jha et al (32) demonstrated that multi-target TKI sunitinib weakens the antiviral response by inhibiting VSV-induced activation of the IFN pathway, thereby enhancing VSV replication.

Boosting natural or adaptive anti-tumor immunity

IFN is also a key pathway in antiviral immunity, and the transcription of IFN-regulated genes induced by the JAK/STAT pathway inhibits OVs replication through multiple mechanisms (19). By inhibiting the type I IFN pathway to weaken the antiviral response of specific tumor cells, thereby enhancing the spread and oncolytic activity of OVs in tumor cells. Trastuzumab-DM1 (T-DM1), an antibody-drug conjugate, consists of the targeted therapeutic agent trastuzumab linked to the microtubule-disrupting agent and mertansine derivative DM1. This conjugate primarily exerts its microtubule-inhibiting effect in human EGFR2 (HER2)-overexpressing tumor cells through the specific targeting of trastuzumab (33). Arulanandam et al (34) found that T-DM1 targets HER2-overexpressing tumor cells to release DM1, which inhibits the type I IFN pathway and enhances the cytotoxic effect of TNF-α on neighboring cells, thereby increasing the spread and oncolytic effect of the VSVΔ51 virus in VSV-resistant cancer cells. This study did not emphasize the antitumor effect of trastuzumab itself (34). In addition, IκB kinase inhibitors BMS-345541 and TPCA-1 enhance VSV replication by inhibiting type I IFN-mediated antiviral responses, thereby improving the efficacy against glioma (35).

Targeted drugs combined with OVs enhance cytotoxic effects on tumor cells in the TME by activating immune cells, promoting cytokine release and reducing immunosuppression. Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells that expand in inflammation and tumors and are effective inhibitors of T cell-mediated immune responses (36). Lawson et al (37) found that sunitinib can alleviate immunosuppression induced by coxsackievirus, such as the accumulation of MDSCs, and stimulate the number of immune-stimulating cells, enhancing the anti-tumor immune response in mouse renal cell carcinoma and lung squamous cell carcinoma. Similarly, Moehler et al (38) found that sunitinib with H-1 parvovirus significantly boosts immune stimulation in melanoma. S100A8/9 are low-molecular-weight calcium-binding proteins, which can regulate the accumulation of MDSCs (39). Chai et al (40) confirmed that neural stem cell-delivered OV combined with S100A8/9 inhibitor paquinimod enhanced anti-GBM efficacy by increasing T-cell infiltration, shifting macrophages to a pro-inflammatory phenotype and reducing MDSCs. A recent study reported that KRAS inhibitors themselves promote innate and adaptive immunity, and when combined with OVV, they reduce immunosuppressive cells such as MDSCs and T-regulatory cells (Tregs) in the TME, synergistically enhancing antitumor effects (41).

TANK-binding kinase 1 (TBK1) inhibitors promote OVs replication and their sensitivity to resistant cells by enhancing immune cell responses. Intercellular adhesion molecule 1 (ICAM-1) is a cell surface receptor used by immune cells for adhesion and migration, and it is often upregulated during viral infections (42). TBK1 is a serine/threonine kinase, and its increased expression or abnormal activity can promote the survival and proliferation of cancer cells (43). Guo et al (44) reported that TBK1 inhibitors can promote VSVΔ51 replication by enhancing ICAM-1-mediated natural killer (NK) cell immunity and increase the sensitivity of VSVΔ51 to chemoresistant colorectal cancer cells.

MEK inhibitors enhance viral replication by decreasing TNF-α secretion and blocking its apoptotic effects. Due to early cell death from TNF-α-induced apoptosis, the replication and antitumor impact of oHSV-1 are limited (45). Yoo et al (46) discovered that the MEK inhibitor trametinib inhibits apoptosis in GBM cells following oHSV infection by reducing TNF-α levels, thereby promoting viral replication. Furthermore, oHSV aids trametinib in crossing the blood-brain barrier and prevents MAPK pathway reactivation, enhancing tumor sensitivity to MEK inhibition.

Proteasome inhibition promotes OVs replication by increasing the expression of heat shock proteins (HSPs). HSP90 has previously been shown to be important for the nuclear localization of HSV polymerase (47). In line with this, Yoo et al (48) found that in head and neck squamous cell carcinoma, ovarian cancer, malignant peripheral nerve sheath tumor and glioma, the proteasome inhibitor bortezomib increases the expression of HSP90 by inducing unfolded protein response accumulation, thereby promoting oHSV-1 replication and enhancing antitumor efficacy.

Using OVs to infect tumor cells allows targeted drugs to be released and connects their targets with immune cell receptors to enhance cell-killing effects. Tian et al (49) created OV-Cmab-CCL5, an oHSV encoding a bispecific fusion protein that combines cetuximab's IgG1 form with C-C motif chemokine ligand 5 (CCL5). This targets EGFR and CCL5 receptors to increase CCL5 levels in tumors and boost immune cell infiltration. It can also enhance the killing effect on GBM cells by promoting antibody-dependent cellular phagocytosis and antibody-dependent cellular cytotoxicity through linking Fcgamma receptors on macrophages and NK cells with EGFR on tumor cells.

Increasing DNA damage by inhibiting DNA damage repair and increasing ROS production to promote tumor cell death

Poly ADP-ribose polymerase (PARP) inhibitors (PARPi) enhance antitumor effects by blocking DNA repair induced by OVs, causing persistent DNA damage and promoting cancer cell death. The dl922-947, a second-generation oncolytic adenovirus with a 24-base pair deletion in the E1A-conserved region 2, enhances viral genome production in host cells (50). Passaro et al (51) found that DNA damage induced by dl922-947 activates the PARP repair mechanism in the DNA damage response signaling pathway, while the PARPi olaparib can restore PARP activation induced by it and exacerbate DNA damage, thereby promoting apoptosis and enhancing the anti-tumor efficacy against thyroid cancer. The same antitumor mechanism was observed in the treatment of melanoma with reovirus type 3 Dearing strain (ReoT3D) combined with talazoparib (52). Recent research reports that PARP1 has been identified as a replication restriction factor for HSV-1, and olaparib promotes viral replication by inhibiting PARP1, thereby enhancing antitumor efficacy in GBM and triple-negative breast cancer (53).

Furthermore, the combination of OVs and PARPi can induce excessive reactive oxygen species (ROS) production, leading to increased DNA damage and S-phase arrest, thereby enhancing the inhibition of tumor cell proliferation. While ROS are common in cells, excessive ROS can cause cell death (54) and activate oncogenic pathways, leading to DNA damage and genetic instability (55). PARPi can have antitumor effects by increasing ROS and ROS-induced DNA damage (56,57). The measles virus (MV) is a promising alternative virus (58), which has been proven to exert oncolytic effects on ovarian cancer through ROS-induced apoptosis (59). Zhang et al (60) discovered that the recombinant Chinese Hu191 MV (rMV-Hu191) modified by a viral reverse genetic system, works with olaparib to enhance oxidative DNA damage in pancreatic ductal adenocarcinoma cells by increasing ROS levels. This combination also significantly halts the cell cycle in the S phase and inhibits cell proliferation by blocking DNA division (60).

Inducing tumor cell apoptosis by disrupting specific cell cycle stages

In mammalian cells, p53 and cyclin-dependent kinases jointly manage the G1/S transition, linking apoptosis to cell cycle progression (61). Irinotecan is a topoisomerase I inhibitor that can induce the expression and phosphorylation of the tumor antigen p53, promoting the expression of apoptosis-regulating factors (62). Napabucasin (BBI608) is a stem cell inhibitor that can directly inhibit the transcription of STAT3-derived genes and can also indirectly inhibit other related genes and pathways (63). Babaei et al (64) found that combining ReoT3D, Irinotecan (CPT-11) and BBI608 significantly reduced the S phase of CT26 cells, arrested them in G0/G1 and G2/M phases, induced apoptosis by causing a sub-G1 fraction to form and downregulated KRAS and STAT3 mRNA.

Inducing autophagic cell death to stop tumor cells from using autophagy to survive and grow in nutrient-poor conditions

Autophagy is a fundamental biological process that is crucial for preventing cellular damage, maintaining cell survival during nutrient scarcity and responding to cytotoxic stimuli. The regulation of autophagy by the PI3K/AKT/mTOR signaling pathway allows tumors to exploit this process for survival benefits through the dysregulation of the pathway (65-67). Everolimus (RAD001) is a widely used mTOR inhibitor that targets mTOR complex 1 and inhibits autophagy (68). Alonso et al (69) demonstrated that the combination of RAD001 with Delta-24-RGD, an oncolytic adenovirus with a 24-base pair deletion in the E1A gene (70), induces autophagic cell death and enhances anti-glioma efficacy, although the precise underlying mechanism remains to be elucidated. The anti-angiogenic and immunosuppressive properties of RAD001 may also contribute to these outcomes. Furthermore, they revealed that Delta-24-RGD increases glioma cell sensitivity to RAD001 by circumventing G1 phase arrest, without impacting the replication of Delta-24-RGD, indicating an additional potential anti-tumor mechanism.

Summary of molecular mechanisms of different inhibitors in promoting OV replication and enhancing antitumor effects

The above content regarding composite molecular machineries involves various targeted drugs that exhibit significant differences in targets, mechanisms of action and tumor sensitivity. MAPK inhibitors primarily act on kinases within the MAPK pathway, such as BRAF, MEK and ERK. MEK inhibitors promote OVV replication by activating STAT3 and can also increase viral replication by reducing antiviral responses through the inhibition of STAT activation. TKIs synergistically enhance antitumor effects by reducing AKT phosphorylation activated by OVs, while OVs engineered to specifically express endostatin can increase tumor sensitivity to bevacizumab by inhibiting AKT. Pathway inhibitors can also suppress targets related to the IFN antiviral pathway, promoting OV replication. The presence of immunosuppressive cells in the TME is also a key reason for poor efficacy. Targeted drugs combined with OVs synergistically enhance antitumor effects by boosting innate and adaptive immunity and reducing immunosuppression. PARPi primarily target PARP enzymes responsible for repairing double-strand DNA damage, exerting antitumor effects by inducing sustained DNA damage through increased ROS and inhibition of OV-induced DNA repair. Furthermore, OVs can synergize with inhibitors related to apoptosis and autophagy pathways for antitumor effects (Fig. 1). The different characteristics of these inhibitors provide clearer guidance for selecting targeted drugs in combination therapy. Choosing the appropriate drugs based on tumors with different target mutations can markedly enhance the precision and antitumor efficacy of combination therapy. Due to the numerous molecular target mutations in tumors, a wide variety of targeted drugs have been developed and approved for use. However, the exploration of combination studies remains limited, with the combined mechanisms focusing only on major pathways such as MAPK, JAK/STAT and PI3K/AKT. The Wnt, Notch and Hippo signaling pathways, which also play important roles in multiple cancers (71-73), have not yet been reported, to the best of our knowledge. The potential interactions between OVs and the toxic side effects of the targeted drugs themselves could reduce the therapeutic window and introduce unpredictable pharmacokinetics/pharmacodynamics. Furthermore, this strategy also has some inherent limitations. Tumor heterogeneity may lead to certain tumors being insensitive to both treatments, and since OVs are mainly administered via local injection, their efficacy against distant metastatic lesions is limited. The use of certain potent apoptosis-inducing targeted drugs may prematurely eliminate OVs, which in turn could restrict viral replication.

Figure 1 Schematic depicting the main mechanism. The left panel mainly focuses on the mechanisms by which tumor cells interact with signaling pathways and DNA damage. Trametinib combined with oHSV inhibits STAT1, thereby affecting the type I interferon-mediated antiviral response pathway; Ruxolitinib promotes VSV replication by inhibiting JAK1/2; MEK inhibitors promote OVV replication by inhibiting STAT3 phosphorylation; Axitinib combined with G47Δ-mIL12 inhibits p-AKT and p-ERK to exert anti-tumor effects; RAMBO increases tumor sensitivity to bevacizumab by inhibiting CCN1 expression. Olaparib combined with rMV-Hu191 enhances DNA damage by increasing ROS levels; Olaparib combined with dl922-947 exacerbates DNA damage to exert anti-tumor effects. Bortezomib promotes oHSV-1 replication by increasing UPR accumulation and enhancing HSP90 expression. The right panel mainly describes the mechanism of action of combinations in the tumor immune microenvironment. Trametinib mitigates the reduction of oHSV induced by the exogenous phagocytosis pathway through the inhibition of TNF-α secretion by macrophages; OV-Cmab-CCL5 enhances the engagement of Fcγ receptors on the surfaces of macrophages and NK cells by EGFR tumor cells, thereby facilitating ADCP and ADCC; the combination of reovirus with sunitinib has been shown to decrease IFN-γ secretion and reduce the presence of Treg and MDSC within immunosuppressive environments. The co-administration of H-1PV with sunitinib significantly augments immune stimulation by promoting DC maturation, IL-6 release, and the activation of cytotoxic T lymphocytes. NSCs-OV combined with paquinimod enhances anti-tumor efficacy by increasing T cell infiltration and reducing MDSCs. IFN-γ, interferon γ; RAMBO, Rapid antiangiogenesis mediated by oncolytic virus; CCN1, Cysteine-rich 61; IFN-α, interferon α; IFN-β, interferon β; JAK, janus kinase; STAT, signal transducer and activator of transcription; TYK, tyrosine kinase; IRF9, interferon regulatory factor 9; VSV, vesicular stomatitis virus; MEK, mitogen-activated protein; OVV, oncolytic vaccinia virus; PI3K, phosphatidylinositol 3-kinase; PDGFR, platelet-derived growth factor receptor; ERK, extracellular regulated protein kinases; UPR, unfolded protein response; HSP90, heat-shock protein 90; HSV, herpes simplex virus; ROS, reactive oxygen species; IL-6, interleukin 6; MDSC, myeloid-derived suppressor cell; Treg, Regulatory T cell; ADCC, Antibody dependent cell-mediated cytotoxicity; ADCP, Antibody-dependent cell-mediated phagocytosis; H-1PV, H-1 parvovirus; NSC, neural stem cell; Cmab, cetuximab; CCL5, C-C motif chemokine ligand 5; TNF, tumor-necrosis factor; NK, natural killer; DC, Dendritic Cell.

Animal model studies on combining OVs with targeted drugs

Research indicates that combining OVs with targeted drugs is safer and more effective for treating solid tumors than using them separately. Some animal studies suggest that small molecule targeted drugs can hinder tumor growth by reducing angiogenesis, interstitial fluid pressure (IFP) and barriers to OVs reaching target cells. HF10 is a spontaneously occurring HSV-1 (74). Yamamura et al (75) found that erlotinib and HF10 work synergistically in a pancreatic cancer model by reducing angiogenesis and IFP, leading to a higher intratumoral virus distribution and significant tumor growth inhibition. Tan et al (76) found that combining the OV HF10 with bevacizumab improved tumor suppression compared to either treatment alone in breast cancer models. This synergy was linked to increased vascular permeability, VEGF-induced viral replication in endothelial cells, immune attacks on infected vessels, enhanced tumor hypoxia boosting viral protein synthesis and increased apoptosis. Another study reported that bevacizumab in combination with dl922-947 significantly inhibited tumor growth in tumor xenografts in thyroid anaplastic carcinoma tumors and increased the distribution of the virus in tumors (77). However, Mahller et al (78) found that erlotinib in combination with oHSV did not show enhanced antitumor effects in a xenograft mouse model of human malignant peripheral nerve sheath tumor. The distribution of OVs is strongly influenced by the TME, among which IFP is a great challenge to efficient virus distribution, and tumor angiogenesis is one of the main causes of high IFP.

The aforementioned preclinical studies have demonstrated that the combination of OVs with small molecule targeted therapies can partially overcome the barrier of the endothelial layer, thereby facilitating the distribution and replication of the virus within the tumor and enhancing its anti-tumor efficacy. Animal models have further indicated that this combination exhibits significant anti-tumor effects. However, the precise molecular mechanisms underlying these effects remain unidentified, and it is elusive whether they align with the mechanisms discussed in this article or involve alternative pathways. Given the discrepancies between the TMEs in vivo and in vitro, in vitro findings alone are insufficient to substantiate the translation of these therapies into clinical practice. Consequently, extensive preclinical research, including exploratory studies on the mechanisms involved, is necessary to advance the development of this combination therapy.

Although the immune organs of humans and mice are highly conserved in their macroscopic anatomical structure, there are significant differences in the composition, distribution and expression of key immune molecules in their immune cells (Table I) (79). Due to significant immune differences between the TME of mouse tumor models and human spontaneous tumors, their clinical application value is limited. The discovery and application of immunodeficient mice provide a crucial breakthrough for reconstructing the human immune system in vivo, thereby accurately simulating the human TME. Their development has undergone continuous iterative upgrades, evolving from simple T-cell deficiencies (such as nude mice) to severe combined immunodeficiency models with multiple functional deficiencies in immune cells such as T, B and NK cells (79). Most of the research data in this study are derived from this type of model. However, immunodeficient mice completely lose the ability to initiate adaptive immune responses, displaying severe defects in the innate immune system. They are not suitable for studying the human immune system itself or the diseases it participates in, but this is the primary requirement for constructing humanized mouse models from immunodeficient mice (80). Humanized mice refer to immunodeficient mice co-implanted with human tumors and immune components, which can realistically assess the mechanisms of immune system activation and efficacy in preclinical studies of OVs in combination with targeted drugs, providing more optimized combination treatment strategies for clinical trial design.

Table I Comparison of the differences between the human and mouse immune systems.

Table I

Comparison of the differences between the human and mouse immune systems.

Item	Human immune system	Mouse immune system
Neutrophil proportion, %	40-65	4-6
Lymphocyte proportion, %	15-40	60-80
IgG classification	IgG1, IgG2a, IgG2b, IgG3	IgG1, IgG2, IgG3, IgG4
Splenic T cells	Scattered in GCs	Clustered distribution
Epidermal immune cells	αβT	γδT
NK cell inhibitory receptor	KIR	Ly49
NKG2D binding ligands	MHC-I, UL16	H-60, Rae1β
Anti-parasitic infection	Th2 cells activate eosinophils and B cells to secrete IgE	Th1 cells produce IFN-γ

[i] Th1, type 1 T-helper; Ig, immunoglobulin; NK, natural killer; NKG2D, NK group 2 member D; GC, germinal center; KIR, killer-cell immunoglobulin-like receptors; MHC, major histocompatibility complex; Th2, type 2 T-helper; IFN, interferon.

Clinical studies of OVs with targeted drugs

Certain OVs have the ability to preferentially infect certain types of tumor cells before any modifications. This natural tropism primarily depends on the specific binding between viral surface proteins and specific receptors on the host cell surface. Many tumor cells overexpress certain receptors required for viral invasion. For example, the coxsackievirus and adenovirus receptor (CAR) is expressed at low levels in normal tissues but is often overexpressed in various malignancies, allowing coxsackievirus and adenovirus to preferentially target and infect these tumors such as laryngeal cancer, thyroid cancer, lung cancer and female reproductive system tumors (81,82). CD46 is mainly present on the surface of prostate cancer and bladder cancer and can be recognized by adenoviruses and measles viruses (83-86). The expression of integrins in tumor cells is usually abnormal, allowing various viruses such as adenovirus, coxsackievirus and reovirus to enter cells through integrin-associated ligands (87-91). Table II shows the natural affinity of many viruses for nonspecific receptors on the surface of certain tumor cells (Table II). It can serve directly as a therapeutic option or as a framework for viral modification. In addition to common receptors, the surface receptors of different types of tumors also have their specific characteristics. OVs Ankara-5T4, constructed to target the specific receptors 5T4 for colorectal cancer, have shown higher specificity and efficacy (92,93). In addition, colorectal cancer is associated with gut microbiota dysbiosis, but there are no reports on the genetic modification of OVs in relation to this connection (94,95). R-405, designed to target the prostate cancer-specific receptor prostate-specific membrane antigen, also demonstrates strong antitumor effects (96,97). The development and research of OVs targeting different tumor-specific receptors are shown in the table (Table III). Therefore, we can enable OVs to acquire the ability to bind new receptors and further enhance tumor targeting by inserting antibody fragments or peptides that specifically bind to tumor-associated antigens.

Table II Currently common types of OVs, their nonspecific receptors and the expression of these receptors in major solid tumors.

Table II

Currently common types of OVs, their nonspecific receptors and the expression of these receptors in major solid tumors.

OV type	Nonspecific receptor	Tumor expression	(Refs.)
Adenovirus	CAR	Elevated expression in laryngeal cancer, thyroid cancer, lung cancer and female reproductive system tumors; decreased expression in kidney cancer and prostate cancer	(81,82)
	CD46	Prostate cancer and bladder cancer	(83-85)
	Integrin	The expression of integrins in tumor cells is usually abnormal	(87-89)
	Desmoglein 2	Upregulated in melanoma, malignant skin cancer, lung cancer, colon adenocarcinoma, primary liver cancer and cervical cancer; downregulated in gastric cancer, malignant ovarian tumors and pancreatic cancer	(169-178)
	Sialic acid	Widely overexpressed in cancer	(179,180)
Herpes virus	Herpesvirus entry mediator	Lung cancer, gastric cancer, breast cancer, esophageal squamous cell carcinoma	(181-186)
	Nectin-1	Bladder urothelial carcinoma, colorectal cancer	(187-189)
Vaccinia virus	Heparan sulfate proteoglycan	Bladder cancer, breast cancer, gallbladder cancer	(190-194)
Reovirus	Integrin	Same as adenovirus	(90)
	Junctional adhesion molecule A	Esophageal squamous cell carcinoma, glioma, stomach cancer, breast cancer	(195-199)
Measles virus	CD46	Same as adenovirus	(86)
	Signaling lymphocytic activation molecule	Hepatocellular carcinoma, head and neck squamous cell carcinoma	(200,201)
Poliovirus	CD155	Pancreatic cancer, breast cancer, osteosarcoma, colorectal cancer	(202-206)
Vesicular stomatitis virus	Low-density lipoprotein receptor	Breast cancer, renal cell carcinoma, ovarian cancer	(207-210)
Coxsackie virus	CAR	Same as adenovirus	(81,82)
	Integrin	Same as adenovirus	(91)
	ICAM-1/CD54	Liver cancer, pancreatic cancer, ovarian cancer, prostate cancer	(211-215)
	DAF/CD55	Colorectal cancer, breast cancer, malignant glioma, medullary thyroid carcinoma	(216-220)
	Scavenger receptor class B member 2	Breast cancer, hepatocellular carcinoma, glioma	(221-223)

[i] OV, oncolytic virus; ICAM-1, intercellular adhesion molecule 1, CAR, coxsackievirus and adenovirus receptor; DAF, decay-accelerating factor.

Table III Tumor specificity and corresponding OVs of the world's 10 most ommon cancers.

Table III

Tumor specificity and corresponding OVs of the world's 10 most ommon cancers.

Tumor type	Tumor-specific receptor	Genetically engineered OV targeting tumor-specific receptor	(Refs.)
Colorectal cancer	5T4	Ankara-5T4	(92,93)
	Abnormal gut microbiota	-	(94,95)
	CEA	rV	(224,225)
Prostate cancer	PSMA	R-405	(96,97)
	PSCA	-
	TMPRSS2	-
	STEAP1	-
Stomach cancer	CLDN18.2	OV-BiTE	(226,227)
Liver cancer	AFP	Ha2bm-d19	(228)
	ASGPR	-	(229)
	GPC3	-	(230)
Thyroid cancer	TSHR	-	(231)
	RET
Bladder cancer	PD-L1	-	(232)
	FGFR	-	(233)
Melanoma	BRAF V600E/K	-	(234)
	GD2	-	(235)
	CSPG4/MCSP	Coxsackievirus B3	(236)

[i] OV, oncolytic virus; CEA, carcinoembryonic antigen; PSMA, prostate-specific membrane antigen; PSCA, prostate stem cell antigen; TMPRSS2, transmembrane serine protease 2; STEAP1, six-transmembrane epithelial antigen of prostate 1; CLDN18.2, claudin18.2; AFP, alpha-fetoprotein; ASGPR, asialoglycoprotein receptor; GPC3, glypican-3; TSHR, thyrotropin receptor; PD-L1, programmed cell death ligand 1; FGFR, fibroblast growth factor receptor; CSPG4/MCSP, chondroitin sulfate proteoglycan 4.

Over the past two decades, clinical research has progressively investigated the integration of OVs with targeted therapies, yielding promising outcomes in certain instances. A meta-analysis encompassing 12 studies evaluated the objective response rate, survival rate and incidence of adverse reactions in cancer patients receiving a combination of OVs and conventional therapy compared to conventional therapy alone. The findings revealed that the combination therapy was significantly associated with an increased objective response rate (ORR) (P=0.04) and a higher incidence of grade ≥3 adverse reactions (P=0.02) and grade ≥3 neutropenia (P=0.01), although no significant difference in survival was observed (98). Nonetheless, further investigation is warranted. In a phase I trial conducted by Hirooka et al (99), patients with histologically confirmed locally advanced pancreatic cancer without distant metastasis and unresectable tumors received one cycle of erlotinib and gemcitabine, followed by intratumoral injection of HF10 under endoscopic ultrasound guidance. The primary endpoint was safety assessment and the secondary endpoint was efficacy evaluation. The study results showed that among the 10 subjects, 2 experienced serious adverse events unrelated to HF10, indicating that the doses used in this trial were safe and effective. Among the 9 subjects who completed all four HF10 injections, the overall response included 3 partial responses, 4 patients with stable disease and 2 cases with progressive disease, with a median progression-free survival (PFS) of 6.3 months and a median overall survival (OS) of 15.5 months, and 2 subjects were able to undergo surgical resection. The results suggest the safety and efficacy of this therapeutic approach (100). However, the study only compared the efficacy between different HF10 titers and did not set up a control group or erlotinib monotherapy group, gemcitabine monotherapy group or erlotinib plus gemcitabine to further demonstrate the superiority of HF10 injection after treatment with erlotinib and gemcitabine.

Sorafenib in combination with JX-594 has been studied in hepatocellular carcinoma (HCC). JX-594 (Pexa-Vec) was constructed by inserting human granulocyte-monocyte colony-stimulating factor and lacZ into the thymidine kinase gene region of the Wyeth strain vaccinia virus (101). After infecting tumor cells, it can stimulate toll like receptors, activate dendritic cells (DCs), increase NK, cytotoxic T-lymphocyte and white blood cell infiltration, and promote the secretion of a variety of cytokines, such as IFN, IL-1, IL-6 and IL-12 (101). Heo et al (102) discovered that administering JX-594 before sorafenib significantly inhibited tumor growth compared to control and other treatment sequences in mouse models. Encouraged by these findings, they tested this sequence in patients with advanced HCC and found it to be well-tolerated, leading to rapid and significant tumor necrosis, with a 50-100% increase in tumor necrosis for patients who did not achieve durable objective tumor remission with JX-594 alone completed JX-594 treatment, and JX-594 was injected directly into the tumors under imaging guidance (102). However, only 3 patients were evaluated in this study and are not representative of the entire HCC population, and did not set evaluation endpoints or provide statistical analysis results, resulting in poor generalizability of the results and reduced reliability, potentially leading to misleading conclusions. Rare adverse events could also not be detected. In a randomized, multicenter phase IIb trial, intravenous injection of Pexa-Vec in patients with advanced HCC who have progressed after sorafenib treatment or are intolerant to sorafenib, OS is primarily being evaluated, with secondary endpoints including time to progression (TTP), ORR, disease control rate (DCR), time to symptomatic progression, safety, tolerability and quality of life. The study results showed that Pexa-Vec was generally well-tolerated, but there were no significant differences in the evaluated endpoints, which may be due to a high dropout rate preventing accurate assessment of the outcomes (103). In a phase Ⅲ, randomized, open-label study, for patients with advanced HCC who have not received systematic treatment, Pexa-Vec was first administered via intratumoral injection, followed by sorafenib, to evaluate OS compared to sorafenib alone. Secondary objectives included TTP, PFS, ORR and DCR, as well as assessing and comparing the safety of the two treatment groups. The study results showed that compared to sorafenib alone, treatment with sorafenib following Pexa-Vec did not show a statistically significant benefit and was less effective, with a higher incidence of serious adverse events (104). The study indicates that the dosing sequence of JX-594 and sorafenib may impact their effectiveness. Investigating the mechanisms behind their interaction could advance the use of OVs with targeted drugs in clinical research. Table IV summarizes the study subjects, treatment methods, endpoints, results and limitations of the aforementioned clinical studies on OVs combined with targeted drugs and/or other chemotherapeutic agents, as well as those have not reported results. There are currently relatively few clinical trials of OVs combined with targeted drugs, suggesting that there may be obstacles to clinical translation and highlighting the importance of preclinical research in clinical trials.

Table IV Completed or ongoing clinical trials of oncolytic viruses with targeted and other drugs.

Table IV

Completed or ongoing clinical trials of oncolytic viruses with targeted and other drugs.

Type of virus	Object	Groups	Primary endpoint	Key secondary endpoints	Results	Limitations	Clinical trial phase	Clinical trial registration no.
Oncolytic herpes simplex virus	Unresectable locally advanced pancreatic cancer	HF10 + Erlotinib + gemcitabine	Safety (n=10)	Efficacy (n=9)	No AEs related to HF10; PR (n=3), SD (n=4), PD (n=2)	-	Ⅰ	UMIN000010150
	Late-stage microsatellite stable and mismatch repair proficient colorectal cancer	RP2/RP3 + Bevacizumab + Atezolizumab	ORR	OS, PFS, DOR, DoCB	-	-	II (incomplete)	NCT05733611
	Locally advanced unresectable or metastatic HCC	RP2 + Bevacizumab + Atezolizumab	ORR	Safety, DOR	-	-	II (incomplete)	NCT05733598
Oncolytic vaccinia virus	Advanced HCC not previously treated with sorafenib	JX-594 + Sorafenib	-	-	Significant tumor necrosis (tumor density quantification)	The small number of subjects cannot represent the entire HCC population, no control group	II	NCT00554372
	Sorafenib-failed HCC	Pexa-vec + BSC BSC	OS	QoL, TTP, safety	No statistical difference at the endpoints	High rates of drop-out and inability to assess	IIb	NCT01387555
	Advanced HCC not previously treated with systemic therapy	Pexa-vec + Sorafenib Sorafenib	OS	TTP, PFS, ORR, DCR	No significant difference at the endpoints	Patient samples not collected after treatment to assess whether there is benefit from Pexa-vec	III	NCT02562755
	Platinum-resistant/refractory ovarian cancer	Olvi-Vec + Bevacizumab + Platinum	PFS	DOR, ORR, OS	-	-	III (incomplete)	NCT05281471
Natural alphavirus M1	Advanced HCC	M1-c6v1 + Camrelizumab + Apatinib	Safety, tolerance	ORR, OS, PFS	ORR was 70%, OS was 15.4 months and PFS was 8.9 months	-	I	NCT04665362
Oncolytic adenovirus	Advanced HCC	H101 + Sorafenib	ORR	Changes in AFP serum levels, DCR, PFS	-	-	IV	NCT05113290
	Colorectal cancer liver metastasis	BioTTT001 + Regorafenib, Toripalimab	Safety, tolerance	OS, PFS, ORR, plasma adenovirus copies	-	-	I	NCT06283134
Reovirus	KRAS-mutant metastatic colorectal cancer	REOLYSIN + Bevacizumab + FOLFIRI	MTD	ORR, PFS, OS	-	-	I	NCT01274624

[i] HCC, hepatocellular carcinoma; PR, partial response; SD, stable disease; PD, progressive disease; ORR, objective response rate; OS, overall survival; DCR, disease control rate; PFS, progression-free survival; DOR, duration of response; DoCB, duration of clinical benefit; BSC, best supportive care; RR, relative risk; TTP, time to progression; DCR, disease control rate; QoL, quality of life; MTD, maximum tolerated dose.

In some rare case studies, OVs combined with molecularly targeted drugs can alleviate the patient's disease and improve the prognosis. CF33-hNIS-anti-PD-L1 (CHECKvacc) is a novel chimeric orthopoxvirus encoding a single-stranded variable fragment of human sodium iodide symporter and anti-programmed cell death ligand 1, with potent anticancer activity in triple-negative breast cancer xenografts (105). A pathology report reported that a patient with metastatic triple-negative breast cancer progressed after multiple lines of therapy with extensive skin metastases, received intratumoral injection of CHECKvacc followed by sequential treatment with trastuzumab deruxtecan, with a clinical complete response lasting 7 months and a DFS of 10 months, The tumor marker CA15-3 was significantly reduced (106). The patient's decision to cease therapy after multiple treatments makes it unclear whether remission is solely due to the combination therapy, requiring more research. A pathology report highlighted a patient with advanced poorly differentiated rectal adenocarcinoma and liver metastases who achieved complete remission and stable disease for 7 years following FOLFOX-4 chemotherapy, bevacizumab and Rigvir (107). However, since most pathological reports are retrospective, issues of data completeness and consistency cannot be verified, and they also have disadvantages such as high selectivity, poor population representativeness and a high risk of bias, and the results do not represent the safety and efficacy of this combination in the overall population, but can be linked to the existing literature as a way to share lessons learned to help identify new trends and potential uses.

Table V summarizes the classification, genetic modification, immunostimulatory properties and current clinical status of OVs involved in combination therapies discussed in this article. oHSV is the most genetically modified OV, and most applications of modified OVs remain at the preclinical research stage and have not yet progressed to clinical trials.

Table V Comparison of the characteristics of OVs.

Table V

Comparison of the characteristics of OVs.

Type of virus		Genetic modification	Immune microenvironment	Clinical trial phase	Clinical trial registration no.
Oncolytic vaccinia virus	JX-594	Insertion mutations (hGM-CSF)	TLRs stimulation, DCs activation, NK, CTL, WBC expansion, IFN, IL-1, IL-6 and IL-12 secretion	Ⅲ	NCT02562755
	CF33-Hnis-anti-PD-L1	Insertion mutations (hNIS and PD-L1)	IFN-γ, IL-1β, IL-6, IL-10, TNF-α and TGF-β1 secretion	I	NCT05081492
Oncolytic HSV	G207	γ34.5 deletion, ICP insertion mutation	Specific CTL responses	II	NCT00028158
	G47Δ	γ34.5 deletion, ICP insertion mutation, α47 deletion	Recovery of MHC expression in HSV-1-infected human cells, antiangiogenic	II	UMIN000015995
	G47Δ-mIL12	mIL-12	Recovery of MHC expression in HSV-1-infected human cells, antiangiogenic, IFN-γ secretion, Th1 differentiation, immunosuppression improvement	-	-
	RAMBO	Angiotin	Downregulation of CCN1 expression	-	-
	OV-Cmab-CCL5	IgG1 form of cetuximab and CCL5	Enhances migration and activation of NK cells, macrophages and T cells	-	-
	HF10	Gene deletions, insertional mutations, frameshift mutations	CD8 T-cell infiltration, IL-2, IFN-γ and TNF-α secretion	I	NCT01017185
Reovirus	ReoT3D	-	Promotes DC maturation, IFN-α, IFN-γ and TNF-α secretion, NK cell activation	-	-
Parvovirus	H-1PV	-	Increases phagocytosis, maturation and cross-presentation of DCs	II	NCT02653313
Oncolytic adenovirus	Delta-24-RGD	24bp E1A deletion	Stimulates Th1 type immunity, high expression of OX40L on DCs and macrophages, increase in NK and T cells, IFN-γ secretion	II	NCT01582516
	dl922-947	24bp E1A deletion	Decreased secretion of IL-8 and CCL5, vascular damage and reduced macrophage infiltration	-	-
VSV	VSVΔ51	M protein mutations	-	-	-
MV	rMV-Hu191	Defective MTase region of MV L protein	Pyroptosis, apoptosis	-	-

[i] OV, oncolytic virus; TLRs, Toll-like receptors; CTL, cytotoxic T lymphocyte; Th1, type 1 T-helper cell; CCL5, C-C motif chemokine ligand 5; NK, natural killer; DC, dendritic cell; IFN, interferon; MV, measles virus; VSV, vesicular stomatitis virus; CCN1, Cysteine-rich 61; HSV, herpes simplex virus; hGM-CSF, human granulocyte-monocyte colony-stimulating factor; WBC, white blood cell; IL, interleukin; TNF, tumor-necrosis factor; TGF, transforming growth factor; hNIS, human sodium-iodide symporter; PD-L1, programmed cell death ligand 1; MHC, major histocompatibility complex; RAMBO, rapid antiangiogenesis mediated by oncolytic virus; Ig, immunoglobulin; ReoT3D, reovirus type 3 Dearing strain; H-1PV, H-1 parvovirus; OX40L, OX40 ligand; Cmab, cetuximab.

Discussion

Malignant tumors constitute the primary cause of mortality worldwide, with their incidence continuously rising (108). Therapeutic strategies, encompassing surgery, chemotherapy, radiotherapy, immunotherapy and targeted therapy, have progressed from monotherapy to combination therapy to address tumor drug resistance (109,110). OVs have emerged as an innovative approach to cancer treatment, distinguished by their tumor-targeting capabilities, which minimize harm to normal cells and reduce the occurrence of side effects. Clinical trials have not reported any fatalities or severe adverse events attributable to OV therapy, highlighting its favorable safety profile. However, the inherent limitations of OVs may affect their therapeutic efficacy. Targeted drugs can activate the phosphorylation of relevant targets to promote viral replication, and inhibit pathway activation and antiviral responses caused by OVs, thereby increasing OV replication, distribution and oncolytic effects. This enhancement may extend to other targets involved in tumorigenesis or immune microenvironment regulation. The relationship between MEK and STAT3 leads to the inhibition of MEK promoting STAT3 activation, thereby enhancing viral replication. Existing studies have reported a negative correlation between ERK and STAT3 (111-113). This negative correlation has been confirmed as a potential mechanism underlying the development of drug resistance, suggesting that a similar mechanism may also exist. Targeted drugs can also synergize with OVs to enhance immunity, exacerbate DNA damage and promote tumor cell apoptosis, exerting a strong antitumor effect. The diversity in the types and mechanisms of action of targeted drugs, coupled with the variety of OVs and the potential for their genetic modification, provides numerous opportunities. These opportunities include the elimination of factors that adversely affect their anti-tumor effects, as well as the potential for synergistic and cumulative effects. Such interactions underscore the promising prospects for the combined use of OVs and targeted drugs. In addition to the simultaneous administration of these therapeutic agents, researchers have also developed genetically modified OVs that incorporate molecularly targeted drugs. This innovative strategy seeks to leverage the anti-tumor properties of both the targeted drugs and the OVs. However, extensive genetic modifications limit the replication and efficacy of the virus. Therefore, revisiting the fundamental principles of virology and basic immunology is crucial for rationally designing more effective oncolytic virus constructs (114). Multiple preclinical studies have confirmed that OVs combined with targeted drugs can enhance anti-tumor immune responses through multiple mechanisms, and initial results have been achieved in clinical trial progress. To this end, in the present study, preclinical evidence was connected with trial design elements, including patient selection, recommended endpoints, and overlapping toxicity safety monitoring, to construct a clinical translation pathway (Fig. 2). Based on mechanisms of action, potential combination strategies and priority tumor types can be suggested, which in turn provides criteria for patient selection, while the combined efficacy observed in animal models offers guidance on drug dosage and administration sequence. Continuous safety monitoring during these explorations may help prevent serious adverse events caused by potential toxicities. How to further optimize OVs and targeted drugs based on their own characteristics and limitations to advance clinical trials more effectively is a key focus for the future.

Figure 2 Clinical translation roadmap. In preclinical studies, in vitro experiments provide guidance on tumor type choice for patient selection in clinical trial design, and further help set appropriate recommended endpoints according to different stages of the trial. Mechanistic exploration suggests possible combination strategies, and the combined efficacy in animal models provides references for drug dosage and administration sequence. Potential toxicities in these three areas indicate the need for safety monitoring to avoid adverse events.

Future development direction: OVs and molecularly targeted drugs combined with precision strategy and OV delivery optimization

Overactivation of signaling pathways is a cause of cancer pathogenesis, being involved in various tumors and playing a critical role in guiding clinical treatment choices and classification (115). Different gene mutation statuses, such as EGFR and KRAS mutations in lung adenocarcinoma, show significant differences in biological behavior and clinicopathological features, leading to completely different treatment options (116). In breast cancer, classification is based on the expression of estrogen receptor, progesterone receptor and HER2 (Luminal A, Luminal B, HER2-positive, triple-negative), which directly determines subsequent treatment strategies (117). Once the key signaling pathways driving tumor growth are identified, targeted drugs can be developed against critical nodes in these pathways. These pathways can also serve as predictive markers for evaluating treatment efficacy and prognosis. For instance, phosphatase and tensin homolog protein loss leading to overactivation of the PI3K-AKT pathway in prostate cancer generally indicates poor prognosis (118). Single-cell RNA sequencing suggests that the C2 insulin-like growth factor 2 tumor subtype is associated with poor prognosis in high-grade serous ovarian cancer, providing a promising target for future therapies (119). Additionally, signaling pathway targets can be used as monitoring indicators for studying resistance mechanisms and adjusting treatment plans; analyzing pathway changes through re-biopsy can reveal resistance mechanisms and guide subsequent treatment. Approximately half of the resistance to first-generation EGFR inhibitors is due to the emergence of the secondary T790M mutation. To address this, the third-generation EGFR inhibitor Osimertinib was developed, which effectively overcomes T790M resistance and has become the standard treatment option after resistance develops (120). Through time-of-flight cytometry, it was found that AXL inhibition can induce JAK1-STAT3 signaling to compensate for the loss of AXL, enhancing the potential for distant metastasis in lung cancer (121). AXL is a member of the TAM receptor family, typically overexpressed in cancers, and has become a potential target for malignant tumors (122). The use of sequencing technology to predict targeted drug targets has formed a set of multi-omics integration strategies. By sequencing the genome, transcriptome and by other omics, key genes related to diseases can be systematically identified, and their potential as drug targets can be evaluated. Advances in targeted drug development have led to new drugs addressing various mutations, enhancing the effectiveness and overcoming the limitations of older treatments. Consequently, precise combination strategies based on molecular typing can significantly enhance anti-tumor effects and reduce drug resistance.

As the first line of defense against the invasion of external pathogenic microorganisms, the antiviral response pathway, in addition to the IFN pathway, The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) and retinoic-acid-inducible gene-I (RIG-I)/mitochondrial antiviral signaling protein (MAVS), also play key roles in antiviral innate immunity, belonging to the cellular stress response pathway. The release of viral DNA and tumor cell nuclear DNA can activate the cGAS-STING pathway, subsequently triggering a series of signaling cascades, thereby inducing the production of type I IFNs and initiating adaptive immune responses (123). However, in various cancers, interruption or loss of the cGAS-STING pathway has been observed, which prevents OVs from effectively activating this pathway, resulting in a failure to initiate immune responses (124-126). The development of various STING agonists has shown enhanced stability and efficacy in anti-tumor applications (127-129). Sibal et al (130) found that the STING activator 2'3'-cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) combined with the attenuated HSV-1 strain C-REV could induce a more sustained anti-tumor immune response, but they did not conduct an in-depth analysis of how 2'3'-cGAMP affects the intracellular process dynamics related to viral replication mechanisms (130). The RIG-I/MAVS pathway primarily recognizes viral RNA in the cytoplasm. RIG-I has high affinity for double-stranded (ds)RNA with three phosphates on the 5' end. After binding, it also activates the transcription and expression of type I IFNs and various pro-inflammatory cytokines (131). It was reported that HSV-1 infection induces mitochondrial damage and release of mitochondrial DNA, which can trigger cGAS/STING/interferon regulatory factor 3 and RIG-I-MAVS signaling pathways, suggesting that the RIG-I-MAVS pathway can recognize more than just viral RNA (132). Current research mainly focuses on OVs transforming 'cold' tumors into 'hot' tumors and promoting immune cell infiltration and tumor killing by activating the cGAS-STING and RIG-I/MAVS pathway, generating a strong antitumor immune response, and tumors lacking cGAS or STING expression are more easily infected, thereby enhancing oncolytic activity (133-137). However, if this pathway, which is central to antiviral immunity, is excessively or prematurely activated, it may lead to viral clearance, thereby weakening the direct oncolytic effect of the virus. Precisely regulating the balance between viral replication and immune activation is a major challenge for studying the combined use of OVs and related pathway activators. Furthermore, there are no reports in the literature on whether specific inhibitors exist that can temporarily modulate innate antiviral pathways to allow OV spread without causing systemic immunosuppression, which may become a direction for future research.

When OVs enter the body, their ability to induce a strong anti-tumor immune response is also influenced by another key factor. Previous research has indicated that OVs are swiftly neutralized by antibodies upon entering the body, thereby significantly diminishing their therapeutic efficacy (138,139). To overcome this challenge, researchers have proposed employing vector delivery systems to shield OVs from complement and neutralizing antibodies, thereby enhancing tumor targeting. Polymer coatings (140) and cell-derived nanovesicles (141) has been identified as potential vectors for delivering OVs. Another promising approach involves cell-based drug delivery systems, which provide advantages such as extended drug circulation, improved efficacy, controlled drug release and reduced immunogenicity and cytotoxicity (142). Furthermore, chimeric antigen receptor T-cells have been reported as effective carriers for OVs, offering significant advantages in OV therapy against distant tumors (143). However, the immunosuppressive mechanisms present within the TME may restrict the effectiveness of T cells as OV vectors to tumors that are not classified as immune deserts (144). By contrast, mesenchymal stem cells have been extensively investigated as vectors for various OVs, including oncolytic adenovirus, oncolytic HSV, oncolytic MV and oncolytic reovirus (145). This is attributed to their advantageous properties, such as tumor homing, intrinsic anti-cancer capabilities, protection of OVs from neutralizing antibodies and the ability to deliver viruses to tumor sites via trojan horse strategies (146-148). The development and refinement of targeted delivery systems for OVs substantially mitigate the risk of clearance by the host immune system, thereby addressing some of the challenges associated with combining OVs with molecular targeted therapies and enhancing their anti-tumor efficacy. However, in the development of targeted delivery systems, we must consider not only the immunogenicity of the OVs themselves, but also evaluate whether the delivery system could synergize with the OVs to trigger potential risks such as cytokine storms. Therefore, it is necessary to make a rational selection and modification of the carrier. In addition, efficiently and stably assembling OVs with the delivery system while maintaining the activity of both, and achieving large-scale production at a relatively low cost, remains a significant challenge.

Core challenges in clinical translation

The integration of OVs with molecularly targeted therapies constitutes a promising domain for further exploration. Current research predominantly focuses on preclinical trials, with relatively few studies advancing to clinical trials. This limited progression is likely due to the fact that many viruses do not successfully advance beyond phase I trials (149), which are primarily designed to assess safety and determine appropriate dosing parameters. To date, only 13 viruses have exhibited adequate safety profiles and preliminary clinical success to warrant progression to phase II trials, which are intended to assess clinical efficacy (149). Current methodologies for drug combination exhibit inherent limitations, such as the issue of cumulative toxicity, necessitating further clinical trials to ascertain their safety and efficacy. It is crucial to evaluate the rationale behind these combination strategies and to optimize the use of both the shared and unique properties of the drugs, especially in cases where combinations are ineffective (114). Given that tumor cells frequently develop resistance to monotherapies, employing a combination of diverse therapeutic approaches may partially alleviate this resistance (150-152). However, the potential development of new drug resistance following combination therapy, and the subsequent challenges it may pose to tumor eradication, warrant further rigorous investigation and validation.

The dosing sequence guides the transition from preclinical studies to clinical trials

The efficacy of JX-594 in combination with sorafenib in HCC mentioned in the aforementioned clinical study above is affected by the order of administration, and priority administration of JX-594 may be superior to sorafenib. The underlying mechanisms may be related to the following factors. Immune 'cold tumors' may be more dependent on OV to preferentially initiate immunity (133-135). As a result, preferential injection of OVs can induce immunogenic death of tumor cells, release tumor-associated antigens, promote DC maturation, T-cell infiltration and cytokine secretion, transform 'cold tumors' into 'hot tumors' and provide a more favorable immune environment for subsequent targeted drugs to exert anti-tumor effects (136,153). Some targeted drugs may inhibit OV replication, so prioritizing OV treatment ensures sufficient viral spread and prevents targeted drugs from weakening its oncolytic effect. When OVs are combined with certain antiangiogenic drugs, which induced reduction in blood supply to tumors may limit delivery of OV (154,155). Conversely, targeted drug pretreatment can promote OV replication by downregulating the IFN signaling pathway, a tumor cell defense mechanism (21,34). The use of immunomodulatory drugs to remove immunosuppressive cells such as Tregs and relieve immunosuppression may improve the efficacy of immunotherapy for OV (37,40,41). IFP is one of the key factors hindering the intratumoral distribution of OVs, and the prior use of chemotherapy drugs such as cyclophosphamide can reduce tumor IFP, further improve the intratumoral distribution of OV, and enhance its oncolytic effect (156). Considering the above factors, based on the dynamic changes of tumor characteristics, viral characteristics, drug mechanism and TME, adjusting the order and dose of drugs can enhance the anti-tumor effect.

Potential toxicity and safety management of combinations

OVs and targeted drugs each have their own toxic effects, and when used in combination, careful consideration should be given to whether potential additive toxicity could cause serious adverse events. To date, the combination of therapies mentioned in this study has not yet undergone clinical translation, and the unique toxicities that may arise warrant attention. Protein kinase inhibitors (PKIs) inhibit downstream signaling by targeting specific oncogenic kinases and are one of the research focuses in cancer therapy. However, most analyzed PKI product characteristic summaries and European public assessment reports indicated severe hepatotoxicity (157). Furthermore, the hepatotoxicity caused by OVs themselves also needs to be taken seriously when combined with targeted therapy. Therefore, before treatment, patients at high risk of hepatotoxicity should be identified through relevant biomarkers. In addition, the nonspecific receptor CAR targeted by OVs is also present in normal liver cells (158), and the virus can be genetically engineered to avoid being taken up by the liver or infecting liver cells.

Sunitinib, which is the most involved targeted drug in mechanistic studies, shows a higher prevalence of reduced left ventricular ejection fraction and bone marrow suppression (159). OVs therapy is generally not considered to have significant bone marrow suppression characteristics, and the incidence and severity of hematologic toxicity are usually low. However, certain wild-type OVs, such as oHSV-1, when administered locally to lesions, do not cause viremia, but in rare cases, intravenous administration can lead to a strong systemic immune response due to large-scale viral replication, which may indirectly affect bone marrow function and result in transient cytopenia (160). In addition, bone marrow suppression is also the most common side effect of PARPi (161). Therefore, when considering the combination of sunitinib or PARPi with OVs, the hematological toxicity caused by both should be of concern. The complete blood count should be monitored regularly, and when grade ≥3 toxicity occurs, it is usually necessary to suspend treatment. Once recovery to grade ≤1 is achieved, the dose needs to be reduced upon resuming to avoid irreversible consequences.

Delta-24-RGD is commonly used for intratumoral injection in the treatment of recurrent gliomas, with neurological symptoms being the most common adverse events, such as headaches, speech disorders and cerebral edema (70). ONC201 has shown significant efficacy against certain high-grade gliomas in clinical trials, but it can also lead to adverse events related to the nervous system (162). The therapeutic effect of their combined treatment on tumors is undeniable, but the potential overlapping side effects on the central nervous system should not be overlooked either. Furthermore, distinguishing whether these neurological symptoms are caused by treatment-related inflammation, which may indicate the effectiveness of the treatment, or by the progression of the tumor itself, is crucial in clinical practice and is also a challenge in management. Certain wild-type viruses naturally have neurotropism, and by genetically modifying these viruses, their ability to infect and damage normal nerve cells can be significantly weakened (163-165).

At the same time, the size and location of the tumor, previous treatment history such as radiotherapy that can damage the blood-brain barrier, and pre-existing neurological deficits may all amplify the neurotoxicity of combined therapy (166). Bevacizumab combined with RAMBO can enhance the efficacy against malignant glioma through the CCN1-AKT pathway, but its dual anti-angiogenic effect could significantly increase its impact on the vascular system, excessively suppressing angiogenesis in both the tumor and surrounding normal tissues, leading to tissue ischemia and necrosis (167,168). Therefore, when conducting further clinical research, the optimal order, dosage and interval for administering the two drugs should be determined to balance efficacy and toxicity. In addition, the new genes carried by the genetically engineered recombinant oncolytic viruses may lead to additional side effects. Based on studies of relevant OVs and the toxicity of targeted drugs, future clinical development requires cautious dose exploration and safety management.

In conclusion, the present study collected literature on the mechanisms and clinical research of OVs combined with targeted drugs over the past two decades. There may be potential selection bias, while literature on this research area is relatively sparse and outdated. Additionally, the weighting between preclinical and clinical evidence is uneven and quantitative synthesis of efficacy across studies is limited.

Availability of data and materials

Not applicable.

Authors' contributions

SB and TS conceived the study and wrote the manuscript, and conducted the literature search/selection and data extraction. YT and QW revised and edited the manuscript. All authors have read and approved the final manuscript. Data authentication is not applicable.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Acknowledgements

Not applicable.

Funding

This research was funded by the Guangxi Science and Technology Program (grant nos. 2025GXNSFDA069034 and AD25069077), the National Natural Science Foundation of China (grant nos. 82260484, 81860459, 82360587 and 82560548), the Key Laboratory of Early Prevention and Treatment for Regional High-Frequency Tumors at Guangxi Medical University, Ministry of Education (grant nos. GKE-ZZ2023021 and GKE-ZZ202407) and the Key Projects of Guangxi Natural Science Foundation (grant no. 2024GXNSFDA010022). Additionally, support was provided by the '139' Plan for Cultivating High-Level and Key Talents in Guangxi Medicine, China (grant no. 201903036).

References