Anti-Tumor Immunogenicity of the Oncolytic Virus CF33-hNIS-antiPDL1 against Ex Vivo Peritoneal Cells from Gastric Cancer Patients.

We studied the immunotherapeutic potential of CF33-hNIS-antiPDL1 oncolytic virus (OV) against gastric cancer with peritoneal metastasis (GCPM). We collected fresh malignant ascites (MA) or peritoneal washings (PW) during routine paracenteses and diagnostic laparoscopies from GC patients (n = 27). Cells were analyzed for cancer cell markers and T cells, or treated with PBS, CF33-GFP, or CF33-hNIS-antiPDL1 (MOI = 3). We analyzed infectivity, replication, cytotoxicity, CD107α upregulation of CD8+ and CD4+ T cells, CD274 (PD-L1) blockade of cancer cells by virus-encoded anti-PD-L1 scFv, and the release of growth factors and cytokines. We observed higher CD45-/large-size cells and lower CD8+ T cell percentages in MA than PW. CD45-/large-size cells were morphologically malignant and expressed CD274 (PD-L1), CD252 (OX40L), and EGFR. CD4+ and CD8+ T cells did not express cell surface exhaustion markers. Virus infection and replication increased cancer cell death at 15 h and 48 h. CF33-hNIS-antiPDL1 treatment produced functional anti-PD-L1 scFv, which blocked surface PD-L1 binding of live cancer cells and increased CD8+CD107α+ and CD4+CD107α+ T cell percentages while decreasing EGF, PDGF, soluble anti-PD-L1, and IL-10. CF33-OVs infect, replicate in, express functional proteins, and kill ex vivo GCPM cells with immune-activating effects. CF33-hNIS-antiPDL1 displays real potential for intraperitoneal GCPM therapy.

An oncolytic poxvirus encoding hNIS, shows anti-tumor efficacy and allows tumor imaging in a liver cancer model.

Oncolytic viruses (OVs) are live viruses that can selectively replicate in cancer cells. We have engineered an OV (CF33) to make it cancer-selective through the deletion of its J2R (thymidine kinase) gene. Additionally, this virus has been armed with a reporter gene, human sodium iodide symporter (hNIS), to facilitate non-invasive imaging of tumors using positron emission tomography (PET). In this study we evaluated the oncolytic properties of the virus (CF33-hNIS) in liver cancer model, and its usefulness in tumor imaging. The virus was found to efficiently kill liver cancer cells and the virus-mediated cell death exhibited characteristics of immunogenic death based on the analysis of 3 damage associate molecular patterns (DAMPs): calreticulin, ATP and HMGB1. Furthermore, local or systemic administration of a single dose of the virus showed anti-tumor efficacy against a liver cancer xenograft model in mice and significantly increased survival of treated mice. Lastly, PET scanning was performed following injection of the radioisotope I-124, for imaging of tumors, and a single dose of virus as low as 1E03 pfu, administered intratumorally (I.T.) or intravenously (I.V.), allowed for PET imaging of tumors. In conclusion, CF33-hNIS is safe and effective in controlling human tumor xenografts in nude mice, and it also facilitates non-invasive imaging of tumors.

An Oncolytic Poxvirus Encoding hNIS, Shows Antitumor Efficacy and Allows Tumor Imaging in a Liver Cancer Model.

Oncolytic viruses (OV) are live viruses that can selectively replicate in cancer cells. We have engineered an OV (CF33) to make it cancer-selective through the deletion of its J2R (thymidine kinase) gene. In addition, this virus has been armed with a reporter gene, human sodium iodide symporter (hNIS), to facilitate noninvasive imaging of tumors using PET. In this study, we evaluated the oncolytic properties of the virus (CF33-hNIS) in liver cancer model, and its usefulness in tumor imaging. The virus was found to efficiently kill liver cancer cells and the virus-mediated cell death exhibited characteristics of immunogenic death based on the analysis of 3 damage-associated molecular patterns: calreticulin, ATP, and high mobility group box-1. Furthermore, local or systemic administration of a single dose of the virus showed antitumor efficacy against a liver cancer xenograft model in mice and significantly increased survival of treated mice. Finally, PET scanning was performed following injection of the radioisotope I-124, for imaging of tumors, and a single dose of virus as low as 1E03 pfu, administered intra-tumorally or intravenously, allowed for PET imaging of tumors. In conclusion, CF33-hNIS is safe and effective in controlling human tumor xenografts in nude mice, and it also facilitates noninvasive imaging of tumors.

Peritoneal-directed chimeric oncolytic virus CF17 prevents malignant ascites and improves survival in gastric cancer peritoneal metastases.

Gastric cancer (GC) peritoneal metastasis (PM) is fatal without effective therapy. We investigated CF17, a new replication-competent chimeric poxvirus, against GC cell lines in vitro and PM in an aggressive GCPM mouse model. We performed viral proliferation and cytotoxicity assays on intestinal-type and diffuse-type human GC cell lines following CF17 treatment. At lower MOIs of 0.01, 0.1, there was >80% killing in most cell lines, while in the more aggressive cell lines, killing was seen at higher MOIs of 1.0 and 10.0. We observed reduced peritoneal tumor burden and prolonged survival with intraperitoneal (i.p.) CF17 treatment in nude mice implanted with the resistant GC cell line. At day 91 after treatment, seven of eight mice were alive in the CF17-treated group vs. one of eight mice in the control group. CF17 treatment inhibited ascites formation (0% vs. 62.5% with PBS). Thus, CF17 efficiently infected, replicated in, and killed GC cells in a dose- and time-dependent manner in vitro. In vivo, i.p. CF17 treatment exhibited robust antitumor activity against an aggressive GCPM model to decrease tumor burden, improve survival, and prevent ascites formation. These preclinical results inform the design of future clinical trials of CF17 for peritoneal-directed therapy in GCPM patients.

A comprehensive preclinical study supporting clinical trial of oncolytic chimeric poxvirus CF33-hNIS-anti-PD-L1 to treat breast cancer.

CF33-hNIS-anti-PD-L1 is an oncolytic chimeric poxvirus encoding two transgenes: human sodium iodide symporter and a single-chain variable fragment against PD-L1. Comprehensive preclinical pharmacology studies encompassing primary and secondary pharmacodynamics and biodistribution and safety studies were performed to support the clinical development of CF33-hNIS-anti-PD-L1. Most of the studies were performed in triple-negative breast cancer (TNBC) models, as the phase I trial is planned for patients with TNBC. Biological functions of virus-encoded transgenes were confirmed, and the virus demonstrated anti-tumor efficacy against TNBC models in mice. In a good laboratory practice (GLP) toxicology study, the virus did not produce any observable adverse effects in mice, suggesting that the doses proposed for the clinical trial should be well tolerated in patients. Furthermore, no neurotoxic effects in mice were seen following intracranial injection of the virus. Also, the risk for horizontal transmission of CF33-hNIS-anti-PD-L1 was assessed in mice, and our results suggest that the virus is unlikely to transmit from infected patients to healthy individuals. Finally, the in-use stability and compatibility of CF33-hNIS-anti-PD-L1 tested under different conditions mimicking the clinical scenarios confirmed the suitability of the virus in clinical settings. The results of these preclinical studies support the use of CF33-hNIS-anti-PD-L1 in a first-in-human trial in patients with TNBC.