Improving systemic delivery of oncolytic virus by cellular carriers

Approval of OV drugs

The study of OVs can be traced back to the early 20th century. Despite the approval of related therapies in the 2000s, it was not until 2015 that OVs as viable therapeutic agents was recognized globally and solidified by the FDA approval of Amgen’s Imlygic® [talimogene laherparepvec (T-VEC)]²⁴. To date, four OV products have been granted global approval for the treatment of cancer (Table 1)^25–28. Among these OVs, T-VEC as the inaugural OVT to be approved by the FDA for intratumoral injection, therefore become a seminal advance in the field of OVT²⁸. Since the initial approval, T-VEC has been subjected to rigorous testing in numerous clinical trials. For example, a randomized phase III trial showed that treatment with T-VEC enhanced the objective response rate (ORR) in patients with unresected stage IIIB, IIIC, or IV melanoma²⁷.

Combination strategies with OVs

The efficacy of oncolytic medications in treating cancer is attributable to the intrinsic oncolysis properties. Consequently, the efficacy of monotherapy remains constrained, underscoring the pivotal role of combination therapies. The most prevalent combination strategy with OVs in clinical trials is a multimodal approach that incorporates immunotherapy, chemotherapy, and targeted therapy.

Delivery challenges in OVT

Despite the significant advances in OVT, it is important to recognize that most current methodologies for the delivery of OVs entail the localized administration of the viruses at the tumor site, which frequently constrains the efficacy and scope of treatment. A major drawback to this type of delivery is the inability to treat multifocal diseases or inaccessible tumors³⁵.

The systemic delivery of OVs remains a significant challenge due to various immune barriers and factors in the tumor microenvironment. The innate and adaptive immune systems have crucial roles in effectively protecting the host, especially in clearing circulating naked virus particles before the virus particles reach the tumor. A significant challenge is the existence of pre-existing neutralizing antibodies³⁶ and the complement system³⁷ in the host, which can restrict the efficacy of OVs. Furthermore, ineffective targeting delivery and penetration for mislocalization can negatively impact the distribution of OVs in vivo³⁸. While systemic administration of the virus allows for dissemination to occult metastases, treatment through systemic injection is limited by toxicity from high doses of OVs. The conventional intravenous administration of OVs has thus far proven ineffective in achieving sufficient viral enrichment at tumor sites³⁹.

“Trojan horse” strategy

It is widely accepted that addressing these challenges requires improving the delivery system to ultimately enhance outcomes in OVT for cancer treatment. To address these challenges, the “Trojan horse” strategy has emerged as a highly promising approach for the delivery of OVs to tumor sites⁴⁰.

This innovative method exploits the distinctive characteristics of specific cells, including stem cells, tumor cells, and genetically modified immune cells, to serve as vehicles for the delivery of OVs. Once these carrier cells reach the tumor site, the carrier cells facilitate the localized release of viruses near the cancerous cells²³. This targeted delivery system significantly enhances the concentration of therapeutic viruses at the tumor site, while concurrently minimizing potential off-target effects on healthy tissues^41,42. The “Trojan horse” strategy leverages the intrinsic migratory capabilities and tumor-tissue homing ability of these cells to ensure a more precise and effective delivery of viral therapeutics.

Conventional T cells

T cells possess a distinctive advantage in combating cancer due to direct anti-cancer effects and the capacity to facilitate recruitment of other immune response components⁴⁵. The capacity of T cells to recognize antigens presented by tumor cells significantly enhances the targeting and precision with which these cells can be directed⁴⁶.

Recently, adoptive immunotherapy utilizing extensively expanded effector T cells has demonstrated promise in the treatment of cancer patients. T cells can be easily obtained through straightforward methods, such as peripheral blood collection, followed by density gradient centrifugation to isolate mononuclear cells. These cells can then be cultured and expanded using specific cytokines, providing a convenient basis for subsequent therapeutic applications⁴⁷. This approach appears to create an optimal synergy between OVs and T cells. Qiao et al.⁴⁸ used autologous naive T cells as carriers of vesicular stomatitis virus (VSV) to purge the lymph nodes and spleen of metastatic cells. Qiao et al.⁴⁸ demonstrated that by simply co-incubating T lymphocytes with VSV, VSV-T cell isolation products can be used to target metastatic diseases. The research findings revealed that viruses adhered to the surface of T cells are released within 72–96 h. During this period T cells reach the tumor, ensuring the delivery of viruses to the tumor area. Consequently, T cell-associated viruses may readily release and infect lymph nodes.

Myxoma virus (MYXV) has been shown to selectively target malignant human hematopoietic cells in vitro, while preserving normal human hematopoietic stem and progenitor cells⁴⁹. Strategies to enhance the efficacy of allogeneic hematopoietic cell transplantation (allo-HCT) encompass the suppression of T lymphocytes that instigate graft-versus-host disease (GVHD), while preserving T lymphocytes that elicit graft-versus-malignancy (GVM) effects⁵⁰. A study has shown that a strategy using a combination of MYXV with quiescent primary human T lymphocytes and subsequent transplantation into xenogeneic models avoids GVHD. Moreover, it was demonstrated that T lymphocytes effectively deliver live OVs to multiple myeloma cells, thereby enhancing GVM. Considering these findings, MYXV-T cells show promise as a potential clinical adjunct to allo-HCT⁵¹. Normal T cells can be easily prepared for cancer patients. Therefore, it should be feasible to prepare OV-T cell conjugates in vitro to achieve therapeutic effects by adoptively transferring normal lymphocytes loaded with OVs.

MDSCs

In pathologic conditions, such as cancer, myeloid cells undergo expansion and differentiate into MDSCs^52,53. MDSCs are mainly recruited by chemokines derived from tumor M-MDSCs are recruited by CCL2, CCL5, and CSF1. Polymorphonuclear neutrophils (PMNs)-MDSCs are recruited by CXC chemokines, such as CXCL1, CXCL5, CXCL8, and CXCL12⁵⁴. This recruitment mechanism highlights the preferential migration to the tumor compared to other types of immune cells⁵⁵. Thus, MDSCs have a higher potential for utilization in the delivery than other immune cell types.

Ly6C⁺ MDSCs (previously referred to as MDSCs) have been investigated as cellular carriers to deliver VSV⁵⁶. MDSCs were isolated and purified from splenocytes and single-cell suspensions from the bone marrow of tumor-bearing mice by sorting with microbeads. In addition to the role as carrier cells, the inflammatory response initiated by pathogens has been observed to induce MDSCs to transition from an M2 phenotype to an anti-tumor M1 phenotype. This finding indicates the possibility of using VSV-MDSCs to modify the immunosuppressive environment, which led to the development of a concept for utilizing MDSCs as a cell carrier to enhance the tumor-killing activity of oncolytic virus therapeutics. Indeed, it may be possible to isolate MDSCs from patients, bind VSV and MDSCs in vitro, then reintroduce the VSV-MDSCs into the patient.

However, the discrepancies between the methodologies used for the isolation of MDSCs from humans and mice restrict the potential for clinical application. The phenotypic and functional characteristics of MDSCs can vary significantly between species⁵⁷, which may impact the effectiveness in human patients. This inconsistency presents a challenge in ensuring that MDSCs are effective in the human tumor microenvironment.

Mesenchymal stem cells (MSCs)

It is a well-established fact that stem cells naturally gravitate toward tumor tissues in vivo. This phenomenon has led to the designation of stem cells as “tumor-homing cells”⁵⁸. The capacity of stem cells to migrate to tumors following systemic or remote administration has enabled development as vectors for OVs that can more effectively penetrate tumor sites and metastatic cancers while reducing systemic toxicity^59,60.

MSCs are isolated from bone marrow, adipose tissue, or umbilical cord blood and typically obtained through density gradient centrifugation and culture expansion, which makes MSCs relatively accessible for clinical applications⁶¹. Interleukin (IL)-15-carrying MYXV was utilized to infect human bone marrow-derived MSCs, which were subsequently administered to melanoma-bearing mice via intravenous injection. The results demonstrated a significant regression of lesions and improvement in survival rates⁶².

Nevertheless, there are significant challenges associated with this delivery approach. Stem cells have an intrinsic propensity to evolve into highly malignant tumors⁶³. There is a possibility that MSCs may contribute to tumor growth or metastasis⁶⁴. Although immortalized stem cell lines have demonstrated safety in preclinical animal models⁶⁵, the potential risk of secondary malignancies remains a significant concern that cannot be ignored.

Tumor cells

An early study indicated that tumor cells may serve as a potential delivery vector for OVT. Irradiated, non-tumorigenic human teratocarcinoma PA-1 cells were used as a carrier cell for the intraperitoneal delivery of HSV-1716 to combat epithelial ovarian cancer (EOC). Utilization of carrier cells resulted in enhanced oncolytic efficacy of HSV-1716 against EOC⁶⁶. In another study, AdE3-IAI.3B-infected human lung cancer A549 carrier cells were used and the cellular debris containing adenoviral particles was taken up by target cancer cells, thereby enhancing anti-tumor activity⁶⁷.

Nevertheless, previous studies have not provided evidence that tumor cells can be used as a systemic delivery carrier. A novel method has recently been introduced that utilizes liquid nitrogen shock treatment to eliminate the pathogenicity of tumor cells⁶⁸. Upon exposure to blood through intravenous injection, liquid nitrogen-treated tumor cells (LNTs) have been shown to provide effective protection for OVs from rapid neutralization and elimination. This approach facilitates the targeted delivery of oncolytic adenovirus type 11 (Ad11) to metastatic tumors, resulting in a notable reduction in tumor progression. This technique not only effectively mitigates the pathogenic risks associated with tumor cell carriers, but also preserves infectivity and functional activity. This innovative strategy represents a significant advance in enhancing the safety and efficacy of tumor cell-mediated OV delivery.

VSVΔM51 and vvDD-attached HER2 CAR-T cells

A previous study successfully loaded interferon-sensitive ΔM51 mutant of vesicular stomatitis virus (VSVΔM51) and “double-deleted” vaccina virus (vvDD) onto murine and human CAR-T cells without affecting CAR expression, viability, or functionality⁷³. These findings suggest that loading of these viruses is a relatively innocuous process. CAR-T cells were incubated with VSVΔM51-GFP and vvDD-GFP viruses at a multiplicity of infection (MOI) of 3 for 3 h at 37°C. Co-culturing mock-, VSVΔM51-GFP-, or vvDD-GFP-loaded CAR-T cells with target cells in vitro were used to investigate CAR-T-cell-mediated killing.

The study demonstrated that VSVΔM51-CAR-T cells augmented the restricted efficacy of CAR-T cells on A549 cells. Furthermore, a notable augmentation in killing occurred when VSV and HER2 CAR-T cells were combined to target D2F2/E2 cells. This finding suggests that combination therapy enhances anti-tumor activity. HER2 CAR-T cells demonstrated remarkable efficacy in eradicating T47D cells, whereas the OVs exhibited minimal impact on cancer cells. Therefore, the therapeutic effect was largely attributed to the HER2-CAR-T cells rather than the OVs. The vvDD-GFP-loaded CAR-T cells did not achieve the anticipated outcome; specifically, the vvDD-GFP-loaded CAR-T cells were unable to identify any advantages of using vvDD-loaded CAR-T cells over CAR-T cells alone. It was hypothesized that this phenomenon may be attributed to the inherent limitations of in vitro assays. Importantly, the study demonstrated compatibility between CAR-T cells and OVs.

VSV-attached EGFRvIII CAR-T cells

Evgin et al.⁷⁴ created virus-specific CAR-T cells. This strategy involves combining growth factor receptor variant III (EGFRvIII) CAR-T cells with oncolytic VSV⁷. The systemically delivered OVs enhance the efficacy of CAR-T cells in immune activity and prolong survival rates in mice with melanoma and intracranial glioblastomas.

VSV was loaded onto CAR-T cells in vitro and subsequently transported as hitchhikers to tumors in vivo, which was shown to enhance virus delivery to tumors and promote CAR-T cell expansion. Moreover, viral or virus-encoded natural TCR stimulation sites resulted in enhanced proliferation. CAR-directed anti-tumor function and distinct memory phenotypes were observed. In vivo expansion of dual-specificity (DS) CAR-T cells was facilitated by ex vivo pre-loading with oncolytic VSV or reovirus, thereby enabling further in vivo expansion and homing-enhanced reactivation of T cells.

A limitation of this study was that the investigators did not investigate if viral infection induced an immune response to additional viral antigens in addition to the viral antigens initially studied. Another limitation was that there are significant biological differences between human and murine CAR-T cells. It is therefore evident that further models and clinical trials are required to guarantee that the significantly enhanced efficacy and durability observed in murine CAR-T cell loading translates into comparable effects in patients.

M@eOA-binding TCR-T cells

Nanoparticle (NP) approaches have been developed as drug transporters due to the capacity for passive or active targeting of tumor cells, reduction of toxicity and adverse effects, expansion of drug stability, improvement of pharmacokinetics, and enhancement of anti-tumor drug efficacy⁷⁵.

A novel treatment approach using oncolytic virus-T cell chimeras (Oncotech) has been developed that combines OVT and adoptive T cell therapy⁷⁶. Lipid extrusion techniques were used to encapsulate engineered oncolytic adenoviruses (eOAs) with the B16OVA cell membrane containing MHC-I-OVA257-264 to create M@eOA nanoparticles. Anchoring of M@eOA on OT-1 CD8⁺ T cells was then enabled by a biophysical interaction between the TCR and MHC-I-OVA257-264. Competitive release of the associated eOAs from the T cell surface is enabled by TCR-mediated binding in response to tumor-specific antigens on tumor cells. Concurrently, the construct encodes the CRISPR-Cas9 system, which can target the PD-L1 gene, thereby evading immune system attacks. The Oncotech treatment regimen entails the delivery of oncolytic adenovirus to the tumor site, where it is released and internalized by tumor cells. The combination of the oncolytic adenovirus and T cells results in a significant augmentation of anti-tumor immune responses. Oncotech promotes targeted OA delivery and tumor microenvironment remodeling.

MYXV-infected CAR-T and TCR-T cells

The potential to exploit CAR-T and TCR-T cells as MYXV-delivery carrier cells was examined by pre-infecting the T cells with MYXV ex vivo. This delivery strategy not only induces classic apoptosis in tumor cells but also triggers autosis in antigen-positive and -negative cells⁷⁷. MYXV targets tumor cells while sparing healthy tissues and has been shown to cause disease only in an European rabbit model, including extremely immunodeficient NSG mice⁷⁸. CAR-T cells were not effectively infected in vitro by direct introduction of MYXV. However, a modified protamine spin infection strategy was used to successfully transduce MYXV into CAR-T cells under non-physiologic conditions. This method of delivery required tumor antigen-mediated CAR signaling, as either truncation of CD3ζ in the CAR structure or knockout of the antigen, impaired MYXV delivery.

It is interesting to note that formulation of MSLN CAR-T^10%MYXV (90% MSLN CAR-T cells + 10% MSLN CAR-T MYXV cells) demonstrated superior tumor-killing activity, preserving CAR-T cell cytotoxicity while enhancing MYXV delivery to tumors. Bioluminescent imaging showed that intravenous administration of MSLN CAR-T MYXV-Luc⁺ cells effectively delivered MYXV to SKOV3 tumors with viral loads comparable to viral loads observed following intratumoral injection. These findings indicated that CAR-T cells deliver OVs to tumor cells at effective therapeutic concentrations. Furthermore, TRP-1 T^10%MYXV treatment was highly efficacious in a murine B16 melanoma model. Overall, these results demonstrated that tumor-specific T cells loaded with OVs exhibit enhanced efficacy in inhibiting tumor growth compared to T cells alone.

In addition to inducing apoptosis and pyroptosis in tumor cells, tumor cell autosis induced by MSLN CAR-T^10%MYXV cells, a form of tumor cell death that is distinct from any known T cell killing pathway, was observed. Furthermore, during autosis, tumor cells near the target cells are also destroyed through “bystander killing,” which inhibits antigen escape. CAR-T cell-derived IFNγ-AKT signaling synergizes with MYXV-induced M-T5-SKP-1-VPS34 signaling to induce tumor autosis by inhibiting SKP-1 and AKT signaling. Collectively, the strategy specifically combines MYXV with tumor-specific T cells to demonstrate that T^10%MYXV ACT induces autosis and adaptive immunity to overcome treatment resistance in solid tumors.

oHSV-infected B7-H3 CAR-T cells

HSV-1-infected B7-H3 CAR-T cells were used as carriers in a recent study to deliver HSV systemically to solid tumors while maintaining anti-tumor functionality of CAR-T cells for a defined period. In orthotopic mouse models of glioblastoma multiforme (GBM) with immunocompromised and immunocompetent backgrounds, B7-H3 CAR-T cells effectively delivered oHSV to tumor lesions, enhancing T cell infiltration and significantly prolonging mouse survival⁷⁹. B7-H3 CAR-T cells could be infected with the HSV-1^dko-GFP virus during co-culturing of B7-H3 CAR-T cells with HSV-1^dko-GFP-infected U251 or A375 tumor cells. The researchers observed that oHSV-infected B7-H3 CAR-T exhibited the ability to deliver oHSV into orthotopic intracranial tumors whether hCAR T or mCAR T. Moreover, notable tumor regression was observed in cases in which a combination of B7-H3 CAR-T and oHSV-administered intratumoral (IT) injection, as well as instances in which oHSV-infected B7-H3 CAR-T was used alone.

It is worth noting that modified oHSV consisting of three mutations was used, including deletions in the antigen processing inhibition gene (ICP47) and two copies of the neurovirulent factor (ICP34.5), which has been improved in terms of safety and efficacy.

Potential adverse events of tumor-specific T cell delivery strategy

The utilization of cells as delivery vehicles for OVs may introduce potential adverse effects. The toxicity profiles of different types of OVs display considerable variation. It has been demonstrated that herpes simplex virus (HSV) can have a detrimental impact on the nervous system with the potential to precipitate conditions, such as encephalitis. To mitigate the toxic side effects associated with wild-type viruses, genetic engineering can be used to optimize wild-type virus characteristics, thereby reducing the pathogenicity. An alternative approach is to select non-pathogenic viruses. For example, MYXV is not a pathogen in humans and other non-leporid animals.

Another factor to consider is the potential for adverse effects associated with cellular therapies, including cytokine release syndrome (CRS), neurologic toxicity, tumor lysis syndrome, cytopenia, and infections⁸⁰. Tumor cells may alter antigen expression or upregulate inhibitory pathways to evade T-cell attack, leading to off-target effects^81,82. The immunosuppressive factors present within the tumor microenvironment serve to inhibit T cell activity, thereby reducing the capacity to eradicate tumors and increase the likelihood of immune-related adverse events.

Currently, there is an insufficient quantity of empirical data to validate the occurrence of additional side effects resulting from OV delivery by tumor-specific T cells. However, given the discrepancies in in vitro preparation techniques, the use of surface attachment may facilitate easier detachment during in vivo delivery, which could potentially result in adverse effects. In contrast, the infection-based approach, whereby the virus is released only after tumor-specific T cells have recognized the antigen, has the notable advantage of significantly reducing the incidence of side effects. In conclusion, while the utilization of tumor-specific T cells as OV carriers is a promising avenue of research, the potential adverse effects and challenges require careful consideration and further investigation to ensure the safety and efficacy of this therapeutic approach.

Advantages of cellular carriers over other delivery strategies

One of the primary advantages of cellular carriers is the intrinsic tumor-homing capability, particularly because these carriers can be engineered to target tumor-associated antigens, as exemplified by CAR T cell therapy. This finding enables cellular carriers to specifically target and accumulate at tumor sites, providing a significant advantage in the treatment of metastatic cancers^83,84. The quantity of cellular carriers that successfully reach the tumor site exceeds 10% of the injected dose⁵⁹. In contrast, only 0.7% of the injected NP dose ultimately reaches the tumor site⁸⁵. Moreover, the MOI for viral infection in vitro is higher in cells than in NPs^73,77,86, further indicating that cellular delivery strategies have higher delivery efficiency at the tumor site compared to NPs. This ability improves therapeutic efficacy while minimizing off-target effects on healthy tissues. Non-cellular carriers, such as NPs and liposomes, lack the intrinsic homing capabilities of cellular carriers. Non-cellular carriers are often distributed non-specifically throughout the body and are rapidly cleared from circulation by the mononuclear phagocyte system (MPS)⁸⁷. A significant portion of these carriers is taken up by the liver and other reticuloendothelial organs, which limits carrier capacity to effectively reach metastatic tumors and contributes to increased liver toxicity¹¹. Additionally, cellular carriers can persist in the body for prolonged periods, thereby facilitating sustained immune modulation over time. Additionally, non-cellular delivery systems typically exhibit a shorter duration of effect and necessitate repeated administration to maintain or prolong immune responses. Augmented dosing can exacerbate the risk of adverse effects^88,89.

Furthermore, using tumor-specific T cells as delivery carriers not only engages and eliminates tumor cells but also works synergistically to modulate the tumor microenvironment, overcoming immune suppression and enhancing the overall anti-tumor response through combined mechanisms of action⁹⁰. Cellular carriers release cytokines and chemokines that recruit more immune cells, boosting the immune response. As an example, T and stem cells are capable of releasing a range of cytokines and chemokines, including IL-2, tumor necrosis factor-alpha (TNF-α), and chemokine ligand 2 (CCL2)⁹¹. These cytokines and chemokines serve to attract further immune cells to the tumor microenvironment. This recruitment fosters a more potent immune response by creating a localized inflammatory environment conducive to anti-tumor activity.

Cell carriers, particularly immune and stem cells, are inherently capable of traversing biological barriers, such as the blood-brain barrier (BBB) or the dense stroma of tumors. Traversing biological barriers renders cell carriers particularly useful for the delivery of pharmaceutical agents to areas that are otherwise inaccessible⁹². For example, a recent study⁷⁹ successfully demonstrated that the intravenous injection of B7-H3 CAR-T HSV-Luci cells facilitates the systemic delivery of HSV-Luci to intracranial tumors. While the BBB and complex tumor microenvironment present significant challenges for immune cell infiltration and OV delivery in brain tumors, the successful delivery of OVs by CAR-T cells to GBM suggests that tumors without such stringent barriers may permit more straightforward delivery of OVs by cell carriers. Non-cellular carriers may have more difficulty overcoming biological barriers, especially the BBB. While liposomes and nanoparticles can be engineered for improved penetration, the ability to cross such barriers is still more limited compared to some cell types.

Limitations of cellular carriers vs. other delivery strategies

Despite these advantages, a significant challenge in utilizing cellular carriers is identification of the optimal combination of OVs and carrier cells. Some OVs have intrinsic toxicity, especially when coupled with carrier cells through attachment methods, leading to premature release that not only prevents OVs from reaching the intended tumor site but also causes systemic toxicity. Interestingly, when utilizing T cells to deliver MYXV, significant replication and release only occur upon T cell activation, indicating that the cellular delivery strategy enables MYXV release specifically at the tumor site^51,77. However, this mechanism is not universally applicable. Therefore, it is essential to explore and determine the most effective combinations of OVs and cellular carriers to achieve the desired therapeutic outcomes.

The preparation of cellular carriers can be complex and labor-intensive, involving isolation, activation, and genetic modification processes that require specialized techniques. The variability inherent to these processes can result in inconsistencies in therapeutic outcomes. For examples, discrepancies in the efficacy of genetic modification or the activation status of the cells can result in disparate degrees of tumor targeting and efficacy when the carriers are infused into the patient. The intricate manufacturing process can result in inconsistencies between batches, which can impede the transition of research findings into clinical applications. In contrast, non-cellular delivery strategies, such as NPs, offer more straightforward manufacturing processes and allow for easier customization of the delivery system, which facilitates greater scalability and consistency, offering a clear advantage in terms of cost-effectiveness and large-scale production.

Additionally, a key limitation of cellular carriers is the potential for immune responses, particularly with allogeneic cells, which can lead to immune rejection⁹³. Even autologous cells, when genetically modified to carry therapeutic agents like OVs, may trigger immune responses. In contrast, non-cellular carriers, typically composed of stable synthetic materials, exhibit lower immunogenicity. However, non-cellular carriers still pose risks, especially when made from synthetic polymers or metals, which can provoke immune responses and compromise therapeutic efficacy⁹⁴.

In conclusion, while cellular and other delivery strategies each have distinctive benefits and limitations, cellular carriers have the potential to significantly enhance targeted cancer therapies over other strategies due to intrinsic tumor-homing capabilities, immune modulation, and the ability to overcome biological barriers.

Preclinical research

In the future, preclinical research on the delivery of OVs using cellular carriers will facilitate the exploration of various innovative strategies for the combination of modified OVs with engineered tumor-specific cells. This approach has the potential to facilitate more efficacious targeted therapy and enhance both efficacy and safety.

The design can incorporate engineered T cell strategies that are capable of recognizing tumor-associated antigens and sensing specific microenvironmental changes, such as hypoxia, an acidic pH, or metabolite concentrations. Upon detecting tumor signals, these T cells can automatically activate genes that initiate the replication of OVs. OVs can be genetically modified to stably replicate in hypoxic conditions, thereby enhancing survival and replication in tumor tissues. This combination allows for the realization of synergistic effects within the hypoxic tumor microenvironment. Developing dynamic release systems that intelligently adjust OVs release based on tumor metabolic states or microenvironment changes ensures effective delivery at the most critical moments. It may also be worth considering the development of a dual-cell carrier strategy, such as the combination of tumor-specific T cells with macrophages, which may offer advantages in removing immunosuppressive elements within the tumor microenvironment. This strategy, in turn, may facilitate a more conducive setting for T cell anti-tumor activity.

Furthermore, the formation of multifunctional delivery systems is possible through the combination of NPs or polymer carriers with engineered cellular carriers. As an example, engineered T cells can be utilized to carry modified NPs that target the tumor and release OVs upon reaching the site, thereby enhancing treatment efficacy. Such delivery systems can be designed to trigger the release of OVs based on specific features of the tumor microenvironment, including pH changes and enzyme activity, which improve targeting and control. In conclusion, the integration of these strategies will facilitate more innovative and diverse research on cellular carrier delivery of OVs, ultimately leading to the development of more personalized therapeutic options for patients.

Clinical research

The current challenge for cellular carrier strategies in the delivery of OVs in clinical research is to translate preclinical research into safe clinical trials. Two clinical trials are currently underway (NCT03896568 and NCT02068794), both of which use MSCs as delivery carriers. To optimize the clinical use of cellular carriers for OV delivery, a multifaceted approach is essential. The utilization of advances in genetic engineering has the potential to markedly enhance the specificity and efficacy of these carriers, thereby enabling the carriers to more effectively target tumor-associated antigens. Similarly, it is important to determine the most efficacious combinations of OVs and carrier cells to enhance therapeutic efficacy and minimize systemic toxicity. Additionally, systematic modifications to infection parameters, including viral concentrations and incubation times in vitro, enhance viral load and therapeutic outcomes. Moreover, the combination of cellular carriers with other therapeutic modalities, including ICIs, may result in a synergistic effect that enhances overall anti-tumor responses.

It is also imperative to implement comprehensive monitoring protocols for adverse events to guarantee the safety of patients throughout the treatment process. The implementation of these strategies will facilitate refinement of the clinical application of cellular carriers, thereby enhancing the effectiveness and safety of OV therapies for cancer patients.

Despite the theoretical advantages of cell-based therapies, practical application in clinical settings is constrained by significant limitations. The successful translation of these therapies into clinical practice is contingent upon overcoming of numerous technological, economic, and legal challenges. The production of cell-based therapeutic products requires a significant investment in research and development, as well as a thorough assessment of cost-effectiveness for large-scale manufacturing. In addition, ensuring the stability and consistency of manufacturing processes represents a critical challenge that directly influences the reliability and efficacy of treatments. Thus, addressing these complexities requires interdisciplinary collaboration, innovative approaches, and sustained long-term research and development efforts.