Oncolytic virotherapy represents one of the most advanced strategies to treat otherwise untreatable types of cancer. Despite encouraging developments in recent years, the limited fraction of patients responding to therapy has demonstrated the need to search for new suitable viruses. Coxsackievirus B3 (CVB3) is a promising novel candidate with particularly valuable features. Its entry receptor, the coxsackievirus and adenovirus receptor (CAR), and heparan sulfate, which is used for cellular entry by some CVB3 variants, are highly expressed on various cancer types. Consequently, CVB3 has broad anti-tumor activity, as shown in various xenograft and syngeneic mouse tumor models. In addition to direct tumor cell killing the virus induces a strong immune response against the tumor, which contributes to a substantial increase in the efficiency of the treatment. The toxicity of oncolytic CVB3 in healthy tissues is variable and depends on the virus strain. It can be abrogated by genetic engineering the virus with target sites of microRNAs. In this review, we present an overview of the current status of the development of CVB3 as an oncolytic virus and outline which steps still need to be accomplished to develop CVB3 as a therapeutic agent for clinical use in cancer treatment.

The coxsackievirus B3 strain PD-0 has been proposed as a new oncolytic virus for the treatment of colorectal carcinoma. Here, we generated a cDNA clone of PD-0 and analyzed the virus PD-H, newly generated from this cDNA, in xenografted and syngenic models of colorectal cancer. Replication and cytotoxic assays revealed that PD-H replicated and lysed colorectal carcinoma cell lines in vitro as well as PD-0. Intratumoral injection of PD-H into subcutaneous DLD-1 tumors in nude mice resulted in strong inhibition of tumor growth and significantly prolonged the survival of the animals, but virus-induced systemic infection was observed in one of the six animals. In a syngenic mouse model of subcutaneously growing Colon-26 tumors, intratumoral administration of PD-H led to a significant reduction of tumor growth, the prolongation of animal survival, the prevention of tumor-induced cachexia, and the elevation of CD3⁺ and dendritic cells in the tumor microenvironment. No virus-induced side effects were observed. After intraperitoneal application, PD-H induced weak pancreatitis and myocarditis in immunocompetent mice. By equipping the virus with target sites of miR-375, which is specifically expressed in the pancreas, organ infections were prevented. Moreover, employment of this virus in a syngenic mouse model of CT-26 peritoneal carcinomatosis resulted in a significant reduction in tumor growth and an increase in animal survival. The results demonstrate that the immune status of the host, the route of virus application, and the engineering of the virus with target sites of suitable microRNAs are crucial for the use of PD-H as an oncolytic virus.

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

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.

Oncolytic coxsackievirus B3 (oCVB3) strain PD-H has shown potent oncolytic efficacy and a remarkable safety profile in the treatment of colorectal cancer in vivo after intratumoral (i.t.) injection. In this study, we investigated the safety and efficiency of PD-H following intravenous (i.v.) virus administration. When injected i.v. into Balb/C mice bearing subcutaneous Colon-26 tumors, PD-H led to slightly reduced tumor progression and a significant increase in animal survival, but it also caused multi-organ infection and tissue damage. To improve the safety profile of PD-H, we inserted microRNA target sites (miR-TS) of the heart-specific miR-1, pancreas-specific miR-375, liver-specific miR-122, and brain-specific miR-124 or the tumor-suppressor miR-145 into the genome of PD-H and generated the viruses PD-622TS and PD-145TS. Both viruses replicated similarly and induced cytotoxicity comparable to that of PD-H in the colorectal carcinoma cell lines Colon-26 and CT-26Luc. Their replication was inhibited in HEK293T cells transiently transfected with the cognate microRNAs. In vivo, i.v. administration of PD-145TS and PD-622TS to healthy Balb/C mouse resulted in significantly lower viral titers in the organs of mice and led to significantly less-intense pathological alterations compared to PD-H. PD-622TS injected i.v. into Balb/C mice with CT-26Luc-induced peritoneal carcinomatosis did not induce off-target alterations in normal organs, but it failed to induce a therapeutic effect. These data indicate that PD-H or microRNA-regulated PD derivatives exhibit only limited therapeutic efficacy following i.v. injection in colorectal tumor-bearing mice. However, the newly engineered microRNA-regulated PD-H variants demonstrate improved safety profiles.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer characterized by a dense desmoplastic stroma and a highly immunosuppressive tumor microenvironment (TME). The focal adhesion kinase (FAK), a non-receptor tyrosine kinase, is considered a critical regulator of various cellular processes involved in cancer development. FAK inhibitors (FAKi) have proven to be promising therapeutics for cancer treatment including for pancreatic cancer. As monotherapy, however, FAKi showed only a modest effect in clinical studies. In this study, we investigated the cytotoxicity of six FAKi (Defactinib, CEP-37440, VS-4718, VS-6062, Ifebemtinib and GSK2256098) used in clinical trials on five pancreatic tumor cell lines. We further examined whether their anti-tumor activity can be enhanced by combination with the oncolytic coxsackievirus B3 (CVB3) strain PD-H. IC₅₀ analyses identified Defactinib and CEP-37440 as the most potent inhibitors of tumor cell growth. VS-4718, VS-6062, and Ifebemtinib showed slightly lower activity, while GSK2256098 was largely ineffective. The combination of Defactinib, CEP-37440, VS-4718, and VS-6062 with PD-H resulted in varying effects on cytotoxicity, depending on the cell line and the specific FAKi, ranging from no enhancement to a pronounced increase. Using the Chou–Talalay method, we determined combination indices (CI), revealing synergistic, additive, but also antagonistic interactions between the respective FAKi and PD-H. Considering both oncolytic efficacy and the CI, the greatest enhancement in oncolytic activity was achieved when VS-4718 or CEP-37440 was combined with PD-H. These findings indicate that co-treatment with PD-H can potentiate the therapeutic activity of the selected FAKi and may represent a novel strategy to improve treatment outcomes in PDAC.