Cancer is a major global public health challenge. Traditional treatments such as surgery, radiotherapy, and chemotherapy often show limited efficacy, minimal improvements in survival rates, and high recurrence risks. With limited therapeutic options for solid tumors, tumor immunotherapy, which harness the body's immune system, has gained significant attention. Oncolytic viruses (OVs) selectively infect and destroy tumor cells, induce immunogenic cell death (ICD) and stimulate antitumor immune responses. However, current OVs therapies, which are predominantly administered via intratumoral injection, have numerous limitations, including the need for guidance, suboptimal viral spread, extracellular matrix barriers, and immune clearance. These challenges hinder repeated dosing effectiveness and restrict its clinical applicability. Although genetic engineering has improved the tumor selectivity and immune activation of OVs, significant delivery challenges remain. Recently, optimizing pharmaceutical formulations to enhance tumor targeting and viral accumulation has emerged as a key approach to improving OV therapy and expanding clinical applicability. This review highlights the critical role of pharmaceutical formulations in biologics and outlines recent advances in OVs formulations. Specifically, we discuss strategies aimed at enhancing tumor targeting, reducing adverse effects, and promoting antitumor immunity. These strategies significantly enhance OV therapeutic potential and inform novel delivery systems for clinical translation.

Oncolytic viruses (OVs) are a promising class of cancer therapy, exploiting their abilities to selectively infect and kill cancer cells while stimulating antitumor immune responses. The current assessment explores the changing horizons of OV immunotherapy, focusing on recent advances in technology plans to improve OV projects and combined approaches to improve curative efficacy. We discuss how OVs induce direct oncolysis and promote the release of tumor-associated antigens, leading to the activation of both innate and adaptive immunity. Special attention shall be given to programs for arm OVs to express curative genes, modify the tumor microenvironment and overcome immunosuppression. Moreover, we assess the synergies of uniting OVs with other immunotherapeutic techniques, such as immune checkpoint inhibitors and cell therapy, to improve tolerant outcomes. The present assessment provides an understanding of the relevant declaration of the OV analysis, highlighting the main obstacles and the future directions for the development of other capable and targeted cancer immunotherapy.

Infectious Clones represent a foundational technique in the field of reverse genetics, allowing for the construction and manipulation of full-length viral genomes. The main methods currently used for constructing viral infectious clones include Transformation-associated recombination (TAR), which is based on Yeast Artificial Chromosome (YAC) and Bacterial Artificial Chromosome (BAC). The YAC and BAC systems are powerful tools that enable the clones and manipulation of large DNA fragments, making them well-suited for the construction of full-length viral genomes. These methods have been successfully applied to construct infectious clones for a wide range of viruses, including coronaviruses, herpesviruses, flaviviruses and baculoviruses. The rescued recombinant viruses from these infectious clones have been widely used in various research areas, such as vaccine development, antiviral drug screening, pathogenesis and virulence studies, gene therapy and vector design. However, as different viruses possess unique biological characteristics, the challenge remains in how to rapidly obtain infectious clones for future research. In summary, this review introduced the development and applications of infectious clones, with a focus on the YAC, BAC and combined YAC-BAC technologies. We emphasize the importance of these platforms in various research areas and aim to provide deeper insights that can advance the platform and broaden its application horizons.

Oncolytic viruses (OVs) are natural or recombinant viruses that can directly lyse tumor cells without damaging normal cells. They enhance anti-tumor immunity by releasing antigens and activating inflammatory responses within the tumor microenvironment (TME). This offers a new therapeutic approach for MPE and solid tumors. This review discusses the progress of OVs administered via intrapleural and intratumoral routes, emphasizing their potential in MPE treatment and the challenges posed by the complex intrapleural environment, which affects the direct interaction between OVs, tumor cells, and immune cells. This review also discusses the regulatory barriers, safety concerns and accessibility of oncolytic virus therapy.

Oncolytic herpes simplex virus (oHSV) represents a promising therapeutic approach to treating cancers by virtue of its selective replication in and lysis of tumor cells, with stimulation of host antitumor immunity. At present, four OV drugs have been approved for the treatment of cancers worldwide, two of which are oHSV drugs that have received extensive attention, known as T-VEC and Delytact. This review discusses the history, mechanism of action, clinical development, quality control, and evaluation principles of oHSV products, including viral species and genetic modifications that have improved these products' therapeutic potential, limitations, and future directions. Integration of oHSVs with immunotherapeutic agents and conventional therapies has a promising future in the field of treatment of malignant tumors. Although much progress has been achieved, there is still much work to be done regarding the optimization of treatment protocols and the quality control of oncolytic virus drugs. The approval of various oncolytic virus therapies underlines their clinical relevance, safety, and efficacy, thereby paving the way for further research aimed at overcoming the existing limitations and enhancing patient responses.

ATRX (alpha-thalassemia/mental retardation, X-linked), a chromatin remodeler, is one of the most commonly mutated genes in human cancer. The ATRX protein functions as a histone chaperone, facilitating the proper folding and assembly of histone proteins into nucleosome cores. Investigations into its molecular mechanisms have significantly advanced our understanding of its roles in diseases associated with chromosomal instability and defective DNA repair. In this comprehensive review, we delineate ATRX's critical function in maintaining heterochromatin integrity and genomic stability under physiological conditions. We further explore the pathogenesis of ATRX-deficient tumors and ATRX syndrome, systematically evaluate current therapeutic strategies for these conditions, and propose novel perspectives on potential targeted therapies for ATRX-mutated malignancies. This review provides useful resource for regarding the etiology and treatment of ATRX deficiency-related diseases.

Breast cancer is the most prevalent malignant tumor among women, with triple-negative breast cancer (TNBC) being one of the most aggressive forms due to its high invasiveness and metastatic potential. Traditional treatments such as endocrine therapy and anti-HER2-targeted therapy are largely ineffective for TNBC, and while chemotherapy shows some promise, resistance remains a significant hurdle. Recently, there has been increasing interest in biological therapies, especially oncolytic viruses (OVs). OVs promote anti-tumor effects by selectively killing tumor cells and stimulating immune responses, and have achieved notable breakthroughs in breast cancer treatment. OVs have demonstrated effectiveness comparable to surgery, radiotherapy, or chemotherapy in selected cancers, but data are sparse in the context of TNBC. This review provides an overview of recent progress in the application of OVs as a tool for precision TNBC treatment.

KD01, a third-generation conditionally replicating adenovirus serotype 5 developed by our team, has approved by the China Center for Drug Evaluation (CDE) for Phase I clinical trials (NCT06552598). However, 60% seroprevalence of anti-Ad5 neutralizing antibodies is a major hurdle for Ad5-based oncolytic viruses. To address this issue, we developed oAd5/35-HF, a fourth-generation oncolytic adenovirus vector designed to enhance infection efficiency and evade pre-existing neutralizing antibodies (NABs). To achieve this, we introduced targeted capsid modifications, replacing hexon hypervariable regions (HVRs) 1 and 5 with those from adenovirus serotype 35 (Ad35), along with alterations to the fiber region. These combined modifications significantly improved infection efficiency, maintained high viral titers, and enabled the virus to resist NABs. This is the first report of replacing both the Ad5 hexon HVRs and fiber regions with those from Ad35 in an oncolytic adenovirus, resulting in potent antitumor activity across multiple cancer types, even in the presence of high NAB levels. The oAd5/35-HF backbone provides a versatile platform for developing new chimera oncolytic adenovirus and adenovirus vector-based vaccine.

The pleiotropic roles of tumor-associated macrophages (TAMs) render them attractive targets in antitumor drug development. CD47/SIRPα (signal regulatory protein alpha) and CD24/Siglec-10 (sialic acid-binding immunoglobulin-like lectin 10) signaling pathways have been found to suppress macrophage phagocytosis of malignant cells. Systemic blockade of CD47/SIRPα has shown severe side effects. Intratumoral delivery of a CD47 inhibitor by oncolytic viruses (OVs) may circumvent this hurdle.

To identify the characteristics of recombinant adenovirus (AdV)-SIRPα/Siglec-10, we conducted CCK8 assay, quantitative PCR, western blot, competitive binding assay, in vitro cytotoxicity assay, ELISA and phagocytosis assay. We investigated the antitumor immune responses of AdV-SIRPα/Siglec-10 using flow cytometry, various tumor-bearing mouse models, humanized tumor-bearing mouse models, immune cell depletion, RNA sequencing, and in vitro T cell activation assay.

Here, we developed a novel AdV encoding a fusion protein composed of the extracellular domains of murine or human SIRPα and Siglec-10 (SIRPα/Siglec-10), termed AdV-mSS or AdV-huSS. The SIRPα/Siglec-10 was effectively secreted by cells infected with AdV-mSS and functioned as a dual blocker of CD47 and CD24, thereby significantly enhancing macrophage phagocytosis. In a series of tumor models, including subcutaneous and ascitic H22 hepatocellular carcinoma (HCC), subcutaneous Hepa1-6 HCC, MC38 colorectal carcinoma, and Lewis lung carcinoma, AdV-mSS treatment markedly enhanced antitumor efficacy. Mechanistically, AdV-mSS reprogrammed TAMs toward an antitumor phenotype and enhanced the expression of major histocompatibility complex (MHC)-I/II, promoting CD8+T cell proliferation and activation. Depletion of either macrophages or CD8+T cells abrogated the antitumor efficacy of AdV-mSS. Similarly, in a humanized LM3 HCC mouse model, AdV-huSS significantly inhibited tumor growth and prolonged survival.

Dual SIRPα/Siglec-10 inhibitor delivered intratumorally by AdV not only reinvigorated the TAM-CD8+T cell axis but also potentially reduced the risk of off-target effects. Further investigation of AdV-huSS in patients with cancer is warranted in the near future.

Lidocaine, a local anesthetic, has been shown to modulate immune responses. This study examines its effects on cytokine production in peripheral blood mononuclear cells (PBMCs) from healthy donors and tumor-infiltrating immune cells (TIICs) from gastric cancer patients. PBMCs from healthy donors and TIICs from gastric cancer patients were treated with lidocaine. Cytokine production was assessed using flow cytometry and cytokine assays, with a focus on IFN-γ, IL-12, IL-10, TGF-β, and IL-35 levels. Cytotoxicity against primary gastric cancer cells (PGCCs) was also evaluated. Lidocaine inhibited IFN-γ production in CD8+ PBMCs and IL-12 in CD14+ PBMCs while increasing anti-inflammatory cytokines (IL-10, TGF-β, IL-35) in CD4+CD25+ and CD14+ cells. In TIICs, lidocaine enhanced IFN-γ and IL-12 production in CD8+ and CD14+ cells while reducing IL-10, TGF-β, and IL-35 levels, promoting an M1-like phenotype in macrophages. Mechanistically, lidocaine enhanced IFN-γ production in sorted CD8+ TIICs through G-protein-coupled receptor (GPCR) signaling and increased IL-12 production in sorted CD14+ TIICs via the toll-like receptor 4 (TLR4) signaling pathway. Lidocaine also increased IFN-γ production and cytotoxicity in CD8+ TIICs via NF-κB activation. Importantly, lidocaine did not affect the viability of PBMCs, TIICs, or PGCCs at concentrations up to 1.5 mM. Lidocaine reprogrammed the tumor immune microenvironment, enhancing anti-tumor immune responses, suggesting its potential to modulate immune functions in gastric cancer.

The delivery of biomolecules to target cells has been a longstanding challenge in biotechnology. DNA viruses naturally evolved the ability to deliver genetic material to cells and modulate cellular processes. As such, they inherently possess requisite characteristics that have led to their extensive study, engineering, and development as biotechnological tools. Here, we overview the application of DNA viruses to biotechnology, with specific implications in basic research, health, biomanufacturing, and agriculture. For each application, we review how an increasing understanding of virology and technological methods to genetically manipulate DNA viruses has enabled advances in these fields. Additionally, we highlight the remaining challenges to unlocking the full biotechnological potential of DNA viral technologies. Finally, we discuss the importance of balancing continued technological progress with ethical and biosafety considerations.

Skin cancer, including melanoma and non-melanoma types, presents a significant and growing global health challenge due to its increasing incidence and mortality rates. While conventional treatments such as surgical excision, immunotherapy, and targeted therapies are well-established, microorganism-based approaches represent an innovative and promising alternative. These therapies employ live, genetically engineered, or commensal bacteria, viral vectors, or bacterial components to achieve various therapeutic mechanisms, including tumor targeting, immune system modulation, vascular disruption, competitive exclusion, drug delivery, and direct oncolysis. Despite their potential, these approaches require further investigation to address safety concerns, optimize treatment protocols, and gain a comprehensive understanding of their long-term outcomes.

Cancer continues to be a major global health burden, with high morbidity and mortality. Building on the success of immune checkpoint inhibitors and adoptive cellular therapy, cancer vaccines have garnered significant interest, but their clinical success remains modest. Benefiting from advancements in technology, many meticulously designed cancer vaccines have shown promise, warranting further investigations to reach their full potential. Cancer vaccines hold unique benefits, particularly for patients resistant to other therapies, and they offer the ability to initiate broad and durable T cell responses. In this review, we highlight the antigen selection for cancer vaccines, introduce the immune responses induced by vaccines, and propose strategies to enhance vaccine immunogenicity. Furthermore, we summarize key features and notable clinical advances of various vaccine platforms. Lastly, we delve into the mechanisms of tumor resistance and explore the potential benefits of combining cancer vaccines with standard treatments and other immunomodulatory approaches to improve vaccine efficacy.

Ovarian cancer (OC) remains the most lethal gynecologic malignancy with limited effective treatment options. Oncolytic viruses (OVs) have emerged as a promising therapeutic approach for cancer treatment, capable of selectively infecting and lysing cancer cells while stimulating anti-tumor immune responses. Preclinical studies have demonstrated significant tumor regression and prolonged survival in OC models using various OVs, such as herpes simplex. Early-phase clinical trials have shown a favorable safety profile, though the impact on patient survival has been modest. Current research focuses on combining OVs with other treatments like immune checkpoint inhibitors to enhance their efficacy. We provide a comprehensive overview of the current understanding and future directions for utilizing OVs in the management of OC.

Oncolytic virus (OV) mediated immunotherapy is one of the recent techniques used to treat higher grade cancers where conventional therapies like chemotherapy, radiation fail. OVs as a therapeutic tool show high efficacy and fewer side effects than conventional methods as supported by multiple preclinical and clinical studies since they are engineered to target tumours. In this chapter, we discuss the modifications in viruses to make them oncolytic, types of strains commonly administered, mechanisms employed by viruses to specifically target and eradicate malignancy and progress achieved as reported in case studies (preclinical and clinical trials). OVs also face some unique challenges with respect to the malignancy being treated and the varied pathogen exposure of the patients, which is also highlighted here. Since pathogen exposure varies according to population dynamics worldwide, chances of generating a non-specific recall response to an OV cannot be negated. Lastly, the future perspectives and ongoing practises of combination therapies are discussed as they provide a leading edge over monotherapies in terms of tumour clearance, blocking metastasis and enhancing patient survival. Efforts undertaken to overcome current challenges are also highlighted.

Recent advances in oncology research have highlighted the promising synergy between low-dose radiation therapy (LDRT) and immunotherapies, with growing evidence highlighting the unique benefits of the combination. LDRT has emerged as a potent tool for stimulating the immune system, triggering systemic antitumor effects by remodeling the tumor microenvironment. Notably, LDRT demonstrates remarkable efficacy even in challenging metastatic sites such as the liver (uveal) and brain (cutaneous), particularly in advanced melanoma stages. The increasing interest in utilizing LDRT for secondary metastatic sites of uveal, mucosal, or cutaneous melanomas underscores its potential efficacy in combination with various immunotherapies. This comprehensive review traverses the journey from laboratory research to clinical applications, elucidating LDRT's immunomodulatory role on the tumor immune microenvironment (TIME) and systemic immune responses. We meticulously examine the preclinical evidence and ongoing clinical trials, throwing light on the promising prospects of LDRT as a complementary therapy in melanoma treatment. Furthermore, we explore the challenges associated with LDRT's integration into combination therapies, addressing crucial factors such as optimal dosage, fractionation, treatment frequency, and synergy with other pharmacological agents. Considering its low toxicity profile, LDRT presents a compelling case for application across multiple lesions, augmenting the antitumor immune response in poly-metastatic disease scenarios. The convergence of LDRT with other disciplines holds immense potential for developing novel radiotherapy-combined modalities, paving the way for more effective and personalized treatment strategies in melanoma and beyond. Moreover, the dose-related toxicities of immunotherapies may be reduced by synergistic amplification of antitumor efficacy with LDRT.

Oncolytic viruses (OVs) are promising immunotherapeutics to treat immunologically cold tumors. However, research on the mechanism of action of OVs in humans and clinically relevant biomarkers is still sparse. To induce strong T-cell responses against solid tumors, TILT-123 (Ad5/3-E2F-d24-hTNFa-IRES-hIL2, igrelimogene litadenorepvec) was developed. TILT-123 encodes two transgenes: tumor necrosis alpha (TNFa) and interleukin-2 (IL-2). TUNIMO (NCT04695327) was a phase I clinical trial using TILT-123 in patients with advanced solid tumors aiming to assess the safety, efficacy, and immunological effects of TILT-123. Research presented in this study evaluated the immunological effects of TILT-123 in the TUNIMO trial by using biological samples collected from the patients during the study, with an objective to leverage the findings to develop possible biomarkers of response and gain insights into possible synergistic combination treatments.

20 patients with advanced solid tumors were treated with TILT-123. Response to therapy was assessed with contrast-enhanced CT and fluorodeoxyglucose positron emission tomography, along with overall survival (OS) calculation. Biological samples from patients were collected in the form of blood and tumor biopsies. Collected samples were analyzed with immunohistochemistry, transcriptomics, proteomics, and flow cytometry.

TILT-123 induced cyclical decreases in blood lymphocyte count, and more substantial blood lymphocyte count correlated with better radiographical response and longer OS. Lymphocyte count findings were confirmed with external control dataset of 96 patients. More substantial lymphocyte count change was linked to stronger immune activation in plasma proteome after intravenous TILT-123 and the presence of TILT-123 mRNA in tumors. Regarding other assays. tumor biopsies profiled showed increased amounts of CD8+ T cells, CD4+ T cells and NK cells after intravenous TILT-123, but not after intratumoral TILT-123. Transcriptional differences were seen in tumors after intravenous therapy and intratumoral therapy, with patients benefitting therapy showing stronger downregulation of immune activation at all time points.

TILT-123 therapy induced accumulation of effector lymphocytes in tumors. Peripheral lymphocyte count decrease is a promising biomarker for assessing oncolytic adenovirus therapy response.

Cancer remains a major global health challenge. Immunotherapy has revolutionized the management of cancer, yet only a limited number of patients respond to such treatments. This is largely attributed to the immunosuppressive tumor microenvironment, which diminishes the effectiveness of immunotherapy. Recent studies have underscored the potential of naturally derived caerin 1 peptides, particularly caerin 1.1 and caerin 1.9, which exhibit strong antitumor effects and enhance the efficacy of immunotherapies in animal models. This review encapsulates the current research aimed at augmenting the effectiveness of immunotherapy, focusing on the role of caerin 1.1 and caerin 1.9 in boosting immunotherapeutic outcomes, elucidating possible mechanisms, and discussing their limitations and challenges.

Recently, we demonstrated that the oncolytic Coxsackievirus B3 (CVB3) strain PD-H can be efficiently adapted to resistant colorectal cancer cells through dose-dependent passaging in colorectal cancer cells. However, the method is time-consuming, which limits its clinical applicability. Here, we investigated whether the manufacturing time of the adapted virus can be reduced by replacing the dose-based passaging with volume-based passaging. For this purpose, the murine colorectal carcinoma cell line MC38, resistant to PD-H-induced lysis, was initially infected with PD-H at 0.1 multiplicity of infection (MOI). For subsequent passages, 15-30 µL of a 1:10 dilution of the cell culture supernatant was transferred to fresh MC38 cells early after virus-induced cell lysis became visible. By virus passage 10, complete cell lysis of MC38 cells was achieved. Sequencing of the passage 10 virus (P-10) revealed two nucleotide substitutions in the 5' UTR and six amino acid changes in the viral polyprotein compared to the PD-H founder. P-10, however, consisted of a heterogeneous virus population. Therefore, the detected mutations were introduced into the cDNA of PD-H, from which the recombinant virus PD-MC38 was generated. PD-MC38 exhibited significantly enhanced replication and lytic activity in MC38 cells compared to PD-H, whereas its oncolytic activity in other colorectal cancer cell lines was comparable to or even lower than that of PD-H. These findings demonstrate that volume-based passaging is suitable to generate tumor cell-specific adapted PD-H. Moreover, compared to the dose-dependent passaging, volume-based passaging significantly reduced the time required to generate the adapted virus.

Oncolytic adenoviruses (OAds) are the most clinically tested viral vectors for solid tumors. However, most clinically tested "Armed" OAds show limited antitumor effects in patients with various solid tumors even with increased dosages and multiple injections. We developed a binary oncolytic/helper-dependent adenovirus system (CAdVEC), in which tumors are coinfected with an OAd and a non-replicating helper-dependent Ad (HDAd). We recently demonstrated that a single low-dose CAdVEC expressing interleukin-12, programmed death-ligand 1 blocker, and HSV thymidine kinase safety switch (CAdTrio) induces significant antitumor effects in patients, including complete response. Similar to previous OAd studies, all patients primarily amplified Ad-specific T cells after treatment however, CAdVEC was still able to induce clinical responses even given at a 100-fold lower dose.

To address the mechanisms of CAdTrio-mediated antitumor effect in patients, we analyzed patients' samples using Enzyme-linked immunosorbent spot (ELISpot) to measure T-cell specificity and quantitative polymerase chain reaction (qPCR) to measure CAdVEC viral genome copies at tumor sites. We then evaluated potential mechanisms of CAdVEC efficacy in vitro using live-cell imaging. Based on those results, we developed a new CAdVEC additionally expressing a T-cell engager molecule targeting CD44v6 to redirect tumor-infiltrating irrelevant T cells against cancer stem cell populations (CAdTetra) for further improvement of local CAdVEC treatment. We tested its efficacy against different cancer types both in vitro and in vivo including Ad pre-immunized humanized mice.

We found that HDAd-infected cells escape Ad-specific T-cell recognition with enhanced tumor-specific T-cell activity through immunomodulatory transgenes. Since CAdVEC treatment initially amplified Ad-specific T cells in patients, we re-direct these virus-specific T cells to target tumor cells by additionally expressing CD44v6.BiTE from CAdTetra. CAdTetra significantly controlled tumor growth, repolarizing local and systemic responses against cancer cells in both immunologically "hot" and "cold" tumor models and also induced immunologic memory against rechallenged tumors.

Our results indicate that CAdTetra effectively induces adaptive T-cell responses against cancer cells by using tumor-infiltrating irrelevant T cells.

Oncolytic viruses have emerged as a highly promising modality for cancer treatment due to their ability to replicate specifically within tumors, carry therapeutic genes, and modulate the immunosuppressive tumor microenvironment through various mechanisms. Additionally, they show potential synergy with immune checkpoint inhibitors. A study report indicates that from 2000 to 2020, 49.5% of oncolytic viruses were administered intratumorally and 35% intravenously during clinical trials. However, both administration methods face significant challenges, particularly with intravenous delivery, which encounters issues such as non-specific tissue uptake, neutralizing antibody responses, and antiviral effects mediated by various immune cells. Despite extensive research into the antiviral roles of CD8+ T cells and NK cells in oncolytic virus therapy, neutrophils-constituting approximately 50% to 70% of human peripheral blood leukocytes-have received relatively little attention. Neutrophils are the most abundant leukocyte subset in peripheral circulation, known for their phagocytic activity. Beyond their traditional roles in bacterial and fungal infections, emerging literature suggests that neutrophils also play a critical role in the body's antiviral responses. Given the gaps in understanding the role of neutrophils in oncolytic virus therapy, this article reviews current literature on this topic. It aims to provide a theoretical foundation for developing oncolytic virus-based cancer therapies and enhancing their anti-tumor efficacy in future clinical treatments.

Interest in virus-based therapeutics for the treatment of genetic and oncolytic diseases has created a demand for high-yield, low-cost virus-manufacturing processes. However, traditional analytical methods of assessing infectious virus titer require multiple processing steps and manual counting, limiting sample throughput, and increasing human error. This bottleneck severely limits the development of new manufacturing unit operations to drive down costs. In this work, we utilize an Incucyte Live-Cell Analysis System to develop a high-throughput infectious titer assay for adenovirus expressing a GFP-transgene. Although previous studies have demonstrated live-cell imaging's potential for use with other viruses, they provide little guidance regarding the selection of the viewing and analysis parameters. To fill this gap, we develop an algorithmic approach to identify the optimum viewing and analysis parameters and create a statistical workflow for quantifying infectious adenovirus in a sample dilution series in a standard 24-well microplate. The developed assay is comparable to Hexon staining, the gold-standard for adenovirus infectious titer, with a Pearson correlation coefficient of 0.9. Finally, the developed algorithmic approach and statistical workflow were applied to create an assay for adenovirus titer using a 96-well microplate, allowing five times more samples to be quantified compared to the standard 24-well plate. While this assay uses a GFP-insert that precludes its use in a clinical environment, the key learnings surrounding the careful use of viewing and analysis parameters, and the statistical workflow are widely applicable to implementing life-cell imaging for dilution-series-based assays. Moreover, this method directly enables the fast and accurate evaluation of virus samples in a preclinical environment.

Gene therapies hold significant promise for treating previously incurable diseases. A number of gene therapies have already been approved for clinical use. Currently, gene therapies are mostly limited to the use of adeno-associated viruses and the herpes virus. Viral vectors, particularly those derived from human viruses, play a critical role in this therapeutic approach due to their ability to efficiently deliver genetic material to target cells. Despite their advantages, such as stable gene expression and efficient transduction, viral vectors face numerous limitations that hinder their broad application. These limitations include small cloning capacities, immune and inflammatory responses, and risks of insertional mutagenesis. This review explores the current landscape of viral vectors used in gene therapy, discussing the different types of DNA- and RNA-based viral vectors, their characteristics, limitations, and current medical and potential clinical applications. The review also highlights strategies to overcome existing challenges, including optimizing vector design, improving safety profiles, and enhancing transgene expression both using molecular techniques and nanotechnologies, as well as by approved drug formulations.

Oncolytic viruses are either naturally occurring or genetically engineered viruses that can activate immune cells and selectively replicate in and destroy cancer cells without damaging healthy tissues. Oncolytic virus therapy (OVT) represents an emerging treatment approach for cancer. In this review, we outline the properties of oncolytic viruses and then offer an overview of the immune cells and tumor microenvironment (TME) across various OVTs. A thorough understanding of the immunological mechanisms involved in OVTs could lead to the identification of novel and more effective therapeutic targets for cancer treatment.

SARS-CoV-2, a highly contagious coronavirus, is responsible for the global pandemic of COVID-19 in 2019. Currently, it remains uncertain whether SARS-CoV-2 possesses oncogenic or oncolytic potential in influencing tumor progression. Therefore, it is important to evaluate the clinical and functional role of SARS-CoV-2 on tumor progression.

Here, we integrated bioinformatic analysis of COVID-19 RNA-seq data from the GEO database and performed functional studies to explore the regulatory role of SARS-CoV-2 in solid tumor progression, including lung, colon, kidney and liver cancer.

Our results demonstrate that infection with SARS-CoV-2 is associated with a decreased expression of genes associated with cancer proliferation and metastasis in lung tissues from patients diagnosed with COVID-19. Several cancer proliferation or metastasis related genes were frequently downregulated in SARS-CoV-2 infected intestinal organoids and human colon carcinoma cells. In vivo and in vitro studies revealed that SARS-CoV-2 nucleocapsid (N) protein inhibits colon and kidney tumor growth and metastasis through the N-terminal (NTD) and the C-terminal domain (CTD). The molecular mechanism indicates that the N protein of SARS-CoV-2 interacts with YBX1, resulting in the recruitment of PKM mRNA into stress granules mediated by G3BP1. This process ultimately destabilizes PKM expression and suppresses glycolysis.

Our study reveals a new function of SARS-CoV-2 nucleocapsid protein on tumor progression.

A limitation of approved oncolytic viruses is their requirement for intratumoral (i.t.) injection. TILT-123 (igrelimogene litadenorepvec, Ad5/3-E2F-D24-hTNFα-IRES-hIL-2) is a chimeric oncolytic adenovirus suitable for intravenous (i.v.) delivery due to its capsid modification and dual selectivity devices. It is armed with tumor necrosis alpha and interleukin-2 for promoting T-cell activation and lymphocyte trafficking to tumors, thereby enhancing the antitumor immune response. Here, we present the findings after a single i.v. administration of TILT-123 in three phase I dose escalation clinical trials.

Patients with advanced solid tumors initially received a single i.v. dose of TILT-123 ranging from 3 × 109 to 4 × 1012 viral particles (VP). Blood was collected at baseline, 1, 16, and 192 h (7 days) post-treatment for bioavailability and serum analysis. Tumor biopsies were collected prior to treatment and 7 days post-treatment for analysis of viral presence and immunological effects. Patients did not receive any other cancer therapies during this period.

Across all three trials (TUNIMO, TUNINTIL, and PROTA), 52 total patients were treated with i.v. TILT-123. Overall, TILT-123 was found to be well-tolerated, with no dose-limiting toxicities observed. Post-treatment tumor biopsies showed expression of viral genes, presence of TILT-123 adenovirus proteins or DNA, and changes in immune cell infiltration from baseline. Increased virus dose did not lead to increased virus detection in tumors. Median overall survival was longer in patients with confirmed presence of TILT-123 in post-treatment biopsies (280 versus 190 days, p = 0.0405).

TILT-123 demonstrated safety and significant intratumoral immunomodulation following a single i.v. administration, warranting further investigation.

TUNIMO-NCT04695327. Registered 4 January 2021, https://clinicaltrials.gov/study/NCT04695327 . TUNINTIL-NCT04217473. Registered 19 December 2019, https://clinicaltrials.gov/study/NCT04217473 . PROTA-NCT05271318. Registered 4 February 2022, https://clinicaltrials.gov/study/NCT05271318 .

Dendritic cells (DCs) are crucial in cancer immunity, because they activate cytotoxic T cells by presenting tumor antigens. Recently, oncolytic virus therapy has been recognized as a systemic immune stimulator. We previously developed a telomerase-specific oncolytic adenovirus (OBP-301) and a p53-armed OBP-301 (OBP-702), demonstrating that these viruses strongly activate systemic antitumor immunity. However, their effects on DCs remained unclear. In the present study, the aim was to elucidate the mechanisms of DC activation by OBP-702, focusing particularly on tumor-derived exosomes. Exosomes (Exo53, Exo301, or Exo702) were isolated from conditioned media of human or murine pancreatic cancer cell lines (Panc-1, MiaPaCa-2, and PAN02) after treatment with Ad-p53, OBP-301, or OBP-702. Exo702 derived from Panc-1 and MiaPaCa-2 cells significantly upregulated CD86, CD80, CD83 (markers of DC maturation), and IFN-γ in DCs in vitro. Similarly, Exo702 derived from PAN02 cells upregulated CD86 and IFN-γ in bone marrow-derived DCs in a bilateral PAN02 subcutaneous tumor model. This DC maturation was inhibited by GW4869, an inhibitor of exosome release, and anti-CD63, an antibody targeting the exosome marker. Intratumoral injection of OBP-702 into PAN02 subcutaneous tumors significantly increased the presence of mature DCs and CD8-positive T cells in draining lymph nodes, leading to long-lasting antitumor effects through the durable activation of systemic antitumor immunity. In conclusion, tumor-derived exosomes play a significant role in DC maturation following OBP-702 treatment and are critical for the systemic activation of antitumor immunity, leading to the abscopal effect.

Pancreatic cancer is one of the deadliest cancers globally, with limited success from existing therapies, including chemotherapies and immunotherapies like checkpoint inhibitors for patients with advanced pancreatic ductal adenocarcinoma (PDAC). A promising new approach is the use of oncolytic viruses (OV), a form of immunotherapy that has been demonstrated clinical effectiveness in various cancers. Here we investigated the potential of the oncolytic coxsackievirus B3 strain (CVB3) PD-H as a new treatment for pancreatic cancer. In vitro, PD-H exhibited robust replication, as measured by plaque assays, and potent lytic activity, as assessed by XTT assays, in most pancreatic tumor cell lines, outperforming two other coxsackievirus strains tested, H3N-375/1TS and CVA21. Thus, H3N-375/1TS showed efficient replication and lytic efficiency in distinctly fewer tumor cell lines, while most tumor cells were resistant to CVA21. The oncolytic efficiency of the three OV largely correlated with mRNA expression levels of viral receptors and their ability to induce apoptosis, as measured by cleaved caspase 3/7 activity in the tumor cells. In a syngeneic mouse model with subcutaneous pancreatic tumors, intratumoral administration of PD-H significantly inhibited tumor growth but did not completely stop tumor progression. Importantly, no virus-related side effects were observed. Although pancreatic tumors respond to PD-H treatment, its therapeutic efficacy is limited. Combining PD-H with other treatments, such as those aiming at reducing the desmoplastic stroma which impedes viral infection and spread within the tumor, may enhance its efficacy.

Although impactful scientific advancements have recently been made in cancer therapy, there remains an opportunity for future improvements. Immunotherapy is perhaps one of the most cutting-edge categories of therapies demonstrating potential in the clinical setting. Genetically engineered T cells express chimeric antigen receptors (CARs), which can detect signals expressed by the molecules present on the surface of cancer cells, also called tumor-associated antigens (TAAs). Their effectiveness has been extensively demonstrated in hematological cancers; therefore, these results can establish the groundwork for their applications on a wide range of requirements. However, the application of CAR-T cell technology for solid tumors has several challenges, such as the existence of an immune-suppressing tumor microenvironment and/or inadequate tumor infiltration. Consequently, combining therapies such as CAR-T cell technology with other approaches has been proposed. The effectiveness of combining CAR-T cell with oncolytic virus therapy, with either genetically altered or naturally occurring viruses, to target tumor cells is currently under investigation, with several clinical trials being conducted. This narrative review summarizes the current advancements, opportunities, benefits, and limitations in using each therapy alone and their combination. The use of oncolytic viruses offers an opportunity to address the existing challenges of CAR-T cell therapy, which appear in the process of trying to overcome solid tumors, through the combination of their strengths. Additionally, utilizing oncolytic viruses allows researchers to modify the virus, thus enabling the targeted delivery of specific therapeutic agents within the tumor environment. This, in turn, can potentially enhance the cytotoxic effect and therapeutic potential of CAR-T cell technology on solid malignancies, with impactful results in the clinical setting.

Interleukin-12 (IL-12) is considered to be a promising cytokine for enhancing an antitumor immune response; however, recombinant IL-12 has shown significant toxicity and limited efficacy in early clinical trials. Recently, many strategies for delivering IL-12 to tumor tissues have been developed, such as modifying IL-12, utilizing viral vectors, non-viral vectors, and cellular vectors. Previous studies have found that the fusion of IL-12 with extracellular matrix proteins, collagen, and immune factors is a way to enhance its therapeutic potential. In addition, studies have demonstrated that viral vectors are a good platform, and a variety of viruses such as oncolytic viruses, adenoviruses, and poxviruses have been used to deliver IL-12-with testing previously conducted in various cancer models. The local expression of IL-12 in tumors based on viral delivery avoids systemic toxicity while inducing effective antitumor immunity and acting synergistically with other therapies without compromising safety. In addition, lipid nanoparticles are currently considered to be the most mature drug delivery system. Moreover, cells are also considered to be drug carriers because they can effectively deliver therapeutic substances to tumors. In this article, we will systematically discuss the anti-tumor effects of IL-12 on its own or in combination with other therapies based on different delivery strategies.

Urinary tumors pose a significant health threat because of their high prevalence and recurrence rates. Despite the availability of various treatment options, many patients poorly respond to traditional therapies, highlighting the urgent need for alternative approaches. Oncolytic viruses are promising therapeutic agents. These viruses exploit the unique characteristics of cancer cells to specifically target and destroy them, thereby triggering potent antitumor immune responses. This review delves into recent advancements and future prospects of oncolytic viruses, focusing on their application in renal, bladder, and prostate cancers. By discussing practical implications and the potential of different viruses, including the cowpox virus, adenovirus, measles virus, coxsackievirus, and reovirus, we pave the way for further exploration and refinement of this exciting field.

Oncolytic virotherapy is a promising approach in cancer immunotherapy. We have previously described a recombinant hybrid oncolytic virus (OV), VSV-NDV, which has a favorable safety profile and therapeutic immunogenicity, leading to direct oncolysis, abscopal effects, and prolonged survival in syngeneic in vivo tumor models. While OVs are known to mediate systemic anti-tumor immune responses, the detailed characterization of local and systemic immune responses to fusogenic oncolytic virotherapy remains unexplored.

We analyzed immune cell compartments in the spleen, blood, tumor-draining lymph nodes (TDLNs), and tumors over the course of VSV-NDV therapy in a bilateral syngeneic melanoma mouse model. Our results revealed significant local infiltration and activation of T lymphocytes in tumors and globally in the blood and spleen. Notably, in vivo CD8+ T cell depletion led to complete abrogation of the tumor response, highlighting the crucial role of T cells in promoting the therapeutic effects of oncolytic VSV-NDV. In vitro co-culture experiments enabled the interrogation of human immune cell responses to VSV-NDV-mediated oncolysis. Human peripheral blood mononuclear cells (PBMCs) were efficiently stimulated by exposure to VSV-NDV-infected cancer cells, which recapitulates the in vivo murine findings.

Taken together, these data characterize a broad anti-tumor immune cell response to oncolytic VSV-NDV therapy and suggest that CD8+ T cells play a decisive role in therapeutic outcome, which supports the further development of this chimeric vector as a multimechanistic immunotherapy for solid cancers.

Cancer is one of the leading causes of death worldwide. Traditional cancer treatments include surgery, radiotherapy, and chemotherapy, as well as combinations of these treatments. Despite significant advances in these fields, the search for innovative ways to treat malignant tumors, including the application of oncolytic viruses, remains relevant. One such virus is the vesicular stomatitis virus (VSV), which possess a number of useful oncolytic properties. However, VSV-based drugs are still in their infancy and are yet to be approved for clinical use. This review discusses the mechanisms of oncogenesis, the antiviral response of tumor and normal cells, and markers of tumor cell resistance to VSV virotherapy. In addition, it examines methods for producing and arming recombinant VSV and provides examples of clinical trials. The data presented will allow better assessment of the prospects of using VSV as an oncolytic.

We model interactions between cancer cells and viruses during oncolytic viral therapy. One of our primary goals is to identify parameter regions that yield treatment failure or success. We show that the tumor size under therapy at a particular time is less than the size without therapy. Our analysis demonstrates two thresholds for the horizontal transmission rate: a "failure threshold" below which treatment fails, and a "success threshold" above which infection prevalence reaches 100% and the tumor shrinks to its smallest size. Moreover, we explain how changes in the virulence of the virus alter the success threshold and the minimum tumor size. Our study suggests that the optimal virulence of an oncolytic virus depends on the timescale of virus dynamics. We identify a threshold for the virulence of the virus and show how this threshold depends on the timescale of virus dynamics. Our results suggest that when the timescale of virus dynamics is fast, administering a more virulent virus leads to a greater reduction in the tumor size. Conversely, when the viral timescale is slow, higher virulence can induce oscillations with high amplitude in the tumor size. Furthermore, we introduce the concept of a "Hopf bifurcation Island" in the parameter space, an idea that has applications far beyond the results of this paper and is applicable to many mathematical models. We elucidate what a Hopf bifurcation Island is, and we prove that small Islands can imply very slowly growing oscillatory solutions.

Cancer immunotherapies have reshaped the paradigm for cancer treatment over the past decade. Among them, therapeutic cancer vaccines that aim to modulate antigen-presenting cells and subsequent T cell priming processes are among the first FDA-approved cancer immunotherapies. However, despite showing benign safety profiles and the capability to generate antigen-specific humoral and cellular responses, cancer vaccines have been limited by the modest therapeutic efficacy, especially for immunologically cold solid tumors. One key challenge lies in the identification of tumor-specific antigens, which involves a costly and lengthy process of tumor cell isolation, DNA/RNA extraction, sequencing, mutation analysis, epitope prediction, peptide synthesis, and antigen screening. To address these issues, in situ cancer vaccines have been actively pursued to generate endogenous antigens directly from tumors and utilize the generated tumor antigens to elicit potent cytotoxic T lymphocyte (CTL) response. Biomaterials-based in situ cancer vaccines, in particular, have achieved significant progress by taking advantage of biomaterials that can synergize antigens and adjuvants, troubleshoot delivery issues, home, and manipulate immune cells in situ. This review will provide an overview of biomaterials-based in situ cancer vaccines, either living or artificial materials, under development or in the clinic, and discuss the design criteria for in situ cancer vaccines.