Epigenetic age prediction using costal cartilage for the investigation of disaster victims and missing persons.

The DNA intelligence tool, DNA methylation-based age prediction, can help identify disaster victims and suspects in criminal investigations. In this study, we developed a costal cartilage-based age prediction tool that uses massive parallel sequencing (MPS) of age-associated DNA methylation markers. Costal cartilage samples were obtained from 85 deceased Koreans, aged between 26 and 89 years. An MPS library was prepared using two rounds of multiplex polymerase chain reaction of nine genes (TMEM51, MIR29B2CHG, EDARADD, FHL2, TRIM59, ELOVL2, KLF14, ASPA, and PDE4C). The DNA methylation status of 45 CpG sites was determined and used to train an age prediction model via stepwise regression analysis. Nine CpGs in MIR29B2CHG, FHL2, TRIM59, ELOVL2, KLF14, and ASPA were selected for regression model construction. A leave-one-out cross-validation analysis revealed the high performance of the age prediction model, with a mean absolute error (MAE) and root mean square error of 4.97 and 6.43 years, respectively. Additionally, our model showed good performance with a MAE of 6.06 years in the analysis of data of 181 costal cartilage samples collected from Europeans. Our model effectively estimates the age of deceased individuals using costal cartilage samples; therefore, it can be a valuable forensic tool for disaster victim and missing person investigation.

Using Oncolytic Virus to Retask CD19-Chimeric Antigen Receptor T Cells for Treatment of Pancreatic Cancer: Toward a Universal Chimeric Antigen Receptor T-Cell Strategy for Solid Tumor.

BACKGROUND: Chimeric antigen receptor (CAR) T cells targeting the B-cell antigen CD19 are standard therapy for relapsed or refractory B-cell lymphoma and leukemia. CAR T cell therapy in solid tumors is limited due to an immunosuppressive tumor microenvironment and a lack of tumor-restricted antigens. We recently engineered an oncolytic virus (CF33) with high solid tumor affinity and specificity to deliver a nonsignaling truncated CD19 antigen (CD19t), allowing targeting by CD19-CAR T cells. Here, we tested this combination against pancreatic cancer. STUDY DESIGN: We engineered CF33 to express a CD19t (CF33-CD19t) target. Flow cytometry and ELISA were performed to quantify CD19t expression, immune activation, and killing by virus and CD19-CAR T cells against various pancreatic tumor cells. Subcutaneous pancreatic human xenograft tumor models were treated with virus, CAR T cells, or virus+CAR T cells. RESULTS: In vitro, CF33-CD19t infection of tumor cells resulted in >90% CD19t cell-surface expression. Coculturing CD19-CAR T cells with infected cells resulted in interleukin-2 and interferon gamma secretion, upregulation of T-cell activation markers, and synergistic cell killing. Combination therapy of virus+CAR T cells caused significant tumor regression (day 13): control (n = 16, 485 ± 20 mm 3 ), virus alone (n = 20, 254 ± 23 mm 3 , p = 0.0001), CAR T cells alone (n = 18, 466 ± 25 mm 3 , p = NS), and virus+CAR T cells (n = 16, 128 ± 14 mm 3 , p < 0.0001 vs control; p = 0.0003 vs virus). CONCLUSIONS: Engineered CF33-CD19t effectively infects and expresses CD19t in pancreatic tumors, triggering cell killing and increased immunogenic response by CD19-CAR T cells. Notably, CF33-CD19t can turn cold immunologic tumors hot, enabling solid tumors to be targetable by agents designed against liquid tumor antigens.

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.

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 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.

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.

Development of the oncolytic virus, CF33, and its derivatives for peritoneal-directed treatment of gastric cancer peritoneal metastases.

BACKGROUND: Gastric cancer (GC) that metastasizes to the peritoneum is fatal. CF33 and its genetically modified derivatives show cancer selectivity and oncolytic potency against various solid tumors. CF33-hNIS and CF33-hNIS-antiPDL1 have entered phase I trials for intratumoral and intravenous treatments of unresectable solid tumors (NCT05346484) and triple-negative breast cancer (NCT05081492). Here, we investigated the antitumor activity of CF33-oncolytic viruses (OVs) against GC and CF33-hNIS-antiPDL1 in the intraperitoneal (IP) treatment of GC peritoneal metastases (GCPM). METHODS: We infected six human GC cell lines AGS, MKN-45, MKN-74, KATO III, SNU-1, and SNU-16 with CF33, CF33-GFP, or CF33-hNIS-antiPDL1 at various multiplicities of infection (0.01, 0.1, 1.0, and 10.0), and performed viral proliferation and cytotoxicity assays. We used immunofluorescence imaging and flow cytometric analysis to verify virus-encoded gene expression. We evaluated the antitumor activity of CF33-hNIS-antiPDL1 following IP treatment (3×105 pfu × 3 doses) in an SNU-16 human tumor xenograft model using non-invasive bioluminescence imaging. RESULTS: CF33-OVs showed dose-dependent infection, replication, and killing of both diffuse and intestinal subtypes of human GC cell lines. Immunofluorescence imaging showed virus-encoded GFP, hNIS, and anti-PD-L1 antibody scFv expression in CF33-OV-infected GC cells. We confirmed GC cell surface PD-L1 blockade by virus-encoded anti-PD-L1 scFv using flow cytometry. In the xenograft model, CF33-hNIS-antiPDL1 (IP; 3×105 pfu × 3 doses) treatment significantly reduced peritoneal tumors (p<0.0001), decreased amount of ascites (62.5% PBS vs 25% CF33-hNIS-antiPDL1) and prolonged animal survival. At day 91, seven out of eight mice were alive in the virus-treated group versus one out of eight in the control group (p<0.01). CONCLUSIONS: Our results show that CF33-OVs can deliver functional proteins and demonstrate effective antitumor activity in GCPM models when delivered intraperitoneally. These preclinical results will inform the design of future peritoneal-directed therapy in GCPM patients.

Simply structured polarization-independent high efficiency multilayer dielectric gratings.

A polarization-independent multilayer dielectric diffraction grating with a low aspect ratio and high diffraction efficiency was designed and fabricated. The diffraction grating designed with a grating density of 1200 lines/mm had an aspect ratio of 0.59, mean polarization-independent diffraction efficiency in the Littrow angle of ±2.5∘, and 1030-1080 nm wavelength range of 97.2%. The designed grating was fabricated using ion assisted deposition and reactive ion etching techniques. The mean polarization-independent diffraction efficiency of the fabricated grating was 96.1%, and its standard deviation was 0.68%. The fabricated diffraction grating was irradiated with a 1064 nm cw laser, with a power density of 30kW/cm2, for 1 min to measure the temperature change before and after the laser application. It was verified that the temperature variation of the diffraction grating without heat treatment was 8.8°C, and the temperature variation after heat treatment at 400°C decreased to 2.3°C.

Priming stroma with a vitamin D analog to optimize viroimmunotherapy for pancreatic cancer.

Pancreatic cancer resistance to immunotherapies is partly due to deficits in tumor-infiltrating immune cells and stromal density. Combination therapies that modify stroma and recruit immune cells are needed. Vitamin D analogs such as calcipotriol (Cal) decrease fibrosis in pancreas stroma, thus allowing increased chemotherapy delivery. OVs infect, replicate in, and kill cancer cells and recruit immune cells to immunodeficient microenvironments. We investigated whether stromal modification with Cal would enhance oncolytic viroimmunotherapy using recombinant orthopoxvirus, CF33. We assessed effect of Cal on CF33 replication using pancreas ductal adenocarcinoma (PDAC) cell lines and in vivo flank orthotopic models. Proliferation assays showed that Cal did not alter viral replication. Less replication was seen in cell lines whose division was slowed by Cal, but this appeared proportional to cell proliferation. Three-dimensional in vitro models demonstrated decreased myofibroblast integrity after Cal treatment. Cal increased vascular lumen size and immune cell infiltration in subcutaneous models of PDAC and increased viral delivery and replication. Cal plus serial OV dosing in the syngeneic Pan02 model caused more significant tumor abrogation than other treatments. Cal-treated tumors had less dense fibrosis, enhanced immune cell infiltration, and decreased T cell exhaustion. Calcipotriol is a possible adjunct for CF33-based oncolytic viroimmunotherapy against PDAC.

PET imaging and treatment of pancreatic cancer peritoneal carcinomatosis after subcutaneous intratumoral administration of a novel oncolytic virus, CF33-hNIS-antiPDL1.

Peritoneal carcinomatosis of gastrointestinal malignancies remains fatal. CF33-hNIS-antiPDL1, a chimeric orthopoxvirus expressing the human sodium iodide symporter (hNIS) and anti-human programmed death-ligand 1 antibody, has demonstrated robust preclinical activity against pancreatic adenocarcinoma (PDAC). We investigated the ability of CF33-hNIS-antiPDL1 to infect, help detect, and kill peritoneal tumors following intratumoral (i.t.) injection of subcutaneous (s.c.) tumors in vivo. Human PDAC AsPC-1-ffluc cells were inoculated in both the s.c. space and the peritoneal cavity of athymic mice. After successful tumor engraftment, s.c. tumors were injected with CF33-hNIS-antiPDL1 or PBS. We assessed the ability of CF33-hNIS-antiPDL1 to infect, replicate in, and allow the imaging of tumors at both sites (immunohistochemistry [IHC] and 124I-based positron emission tomography/computed tomography [PET/CT] imaging), tumor burden (bioluminescence imaging), and animal survival. IHC staining for hNIS confirmed expression in s.c. and peritoneal tumors following virus treatment. Compared to the controls, CF33-hNIS-antiPDL1-treated mice showed significantly decreased s.c. and peritoneal tumor burden and improved survival (p < 0.05). Notably, 2 of 8 mice showed complete regression of disease. PET/CT avidity for 124I uptake in s.c. and peritoneal tumors was visible starting at day 7 following the first i.t. dose of CF33-hNIS-antiPDL1. We show that CF33-hNIS-antiPDL1 can help detect and kill both s.c. and peritoneal tumors following s.c. i.t. treatment.

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.

CF33-hNIS-antiPDL1 virus primes pancreatic ductal adenocarcinoma for enhanced anti-PD-L1 therapy.

Immunotherapeutic strategies that combine oncolytic virus (OV) and immune checkpoint inhibitors have the potential to overcome treatment resistance in pancreatic ductal adenocarcinoma (PDAC), one of the least immunogenic solid tumors. Oncolytic viral chimera, CF33-hNIS-antiPDL1 genetically modified to express anti-human PD-L1 antibody and CF33-hNIS-Δ without the anti-PD-L1 gene, were used to investigate the immunogenic effects of OVs and virus-delivered anti-PD-L1 in PDAC in vitro. Western blot, flow cytometry, and immunofluorescence microscopy were used to evaluate the effects of CF33-hNIS-Δ and IFNγ on PD-L1 upregulation in AsPC-1 and BxPC-3 cells, and CF33-hNIS-antiPDL1 production of anti-PD-L1 and surface PD-L1 blockade of AsPC-1 and BxPC-3 with or without cocultured activated T cells. The cytosolic and cell surface levels of PD-L1 in PDAC cell lines varied; only BxPC-3 showed high cell surface expression. Treatment of these cells with CF33-hNIS-Δ and IFNγ significantly upregulated PD-L1 expression and translocation of PD-L1 from the cytosol onto the cell surface. Following coculture of activated T cells and BxPC-3 with CF33-hNIS-antiPDL1, the cell surface PD-L1 blockade on BxPC-3 cells by virus-delivered anti-PD-L1 antibody increased granzyme B release and prevented virus-induced decrease of perforin release from activated CD8+ T cells. Our results suggest that CF33-IOVs can prime immune checkpoint inhibition of PDAC and enhance antitumor immune killing.

Toward comprehensive imaging of oncolytic viroimmunotherapy.

Oncolytic viruses infect, replicate in, and kill cancer cells, leaving normal cells unharmed; they also recruit and activate immune cells against tumor cells. While clinical indications for viroimmunotherapy are growing, barriers to widespread treatment remain. Ensuring real-time tracking of viral replication and resulting anti-tumor immune responses will overcome some of these barriers and is thus a top priority. Clinically optimizing trackability of viral replication will promote safe dose increases, guide serial dosing, and enhance treatment effects. However, viral delivery is only half the story. Oncolytic viruses are known to upregulate immune checkpoint expression, thereby priming otherwise immunodeficient tumor immune microenvironments for treatment with checkpoint inhibitors. Novel modalities to track virus-induced changes in tumor microenvironments include non-invasive measurements of immune cell populations and responses to viroimmunotherapy such as (1) in situ use of radiotracers to track checkpoint protein expression or immune cell traffic, and (2) ex vivo labeling of immune cells followed by nuclear medicine imaging. Herein, we review clinical progress toward accurate imaging of oncolytic virus replication, and we further review the current status of functional imaging of immune responses to viroimmunotherapy.

The epigenetic DNA modification 5-carboxylcytosine promotes high levels of cyclobutane pyrimidine dimer formation upon UVB irradiation.

In mammals, DNA methyltransferases create 5-methylcytosines (5mC) predominantly at CpG dinucleotides. 5mC oxidases convert 5mC in three consecutive oxidation steps to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and then 5-carboxylcytosine (5caC). Upon irradiation with UV light, dipyrimidines containing C, 5mC and 5hmC are known to form cyclobutane pyrimidine dimers (CPDs) as major DNA photolesions. However, the photobiology of 5fC and 5caC has remained largely unexplored. Here, we tested a series of oligonucleotides with single or multiple positions carrying cytosine (C), 5mC, 5hmC, 5fC or 5caC and irradiated them with different sources of UV irradiation. While UVC radiation produced CPDs near dipyrimidines containing all types of modified cytosine bases, UVB radiation produced by far the highest levels of CPDs near 5caC-containing sequences. Dipyrimidines one or two nucleotide positions adjacent to 5caC but not always those involving this modified base directly were the major sites for these prominent UVB photoproducts. This selectivity did not depend on whether 5caC was present on one or both DNA strands at CpG sequences. We also observed a tendency of the 5caC-containing DNA strands to undergo apparent covalent crosslinking. This reaction occurred with UVB or UVC but not with UVA irradiation. Our data show that 5-carboxylcytosine, although generally a rare base in the genome, can nonetheless make a strong contribution to sequence-specific DNA damage perhaps by acting as a DNA-intrinsic photosensitizer.

Recombinant Orthopoxvirus Primes Colon Cancer for Checkpoint Inhibitor and Cross-Primes T Cells for Antitumor and Antiviral Immunity.

Although it is known that oncolytic viruses can inflame and recruit immune cells to otherwise immunosuppressed tumor microenvironments, the influence of the antiviral immune response on antitumor immunity is less clear across viral platforms and tumor types. CF33 is a recombinant orthopoxvirus backbone effective against colon cancer. We tested derivatives of CF33 with and without immune-checkpoint inhibition (anti-PD-L1) in mouse models of colon cancer. Results showed that the efficacy of CF33 backbone with J2R deletion (single-deleted) against colon cancer is not altered by additional deletion of F14.5L in vitro or in vivo CF33 infection upregulated PD-L1 expression on tumor cells and led to an increased influx of lymphocytes and macrophages in tumors. Also, the levels of active CD8+ (IFNγ+) T cells in the virus-treated tumors were higher than those in control-treated tumors. Furthermore, a combination of CF33 derivatives with anti-PD-L1 resulted in durable tumor regression and long-term survival, resistant to tumor rechallenge. Analysis of immune cells from the treated mice showed that tumor-specific T cell activation occurred more robustly in tumors treated with the virus and that T cells were more strongly activated against the virus than against tumor, in an MHC-I-dependent manner. Our findings warrant further studies on the role of cross-priming of T cells against viral and tumor antigens, in the overall success of viroimmunotherapy.

Vitamin D as a Primer for Oncolytic Viral Therapy in Colon Cancer Models.

Oncolytic viroimmunotherapy is an exciting modality that can offer lasting anti-tumor immunity for aggressive malignancies like colon cancer. The impact of oncolytic viruses may be extended by combining them with agents to prime a tumor for viral susceptibility. This study investigates vitamin D analogue as an adjunct to oncolytic viral therapy for colon cancer. While vitamin D (VD) has historically been viewed as anti-viral, our in vitro investigations using human colon cancer cell lines showed that VD does not directly inhibit replication of recombinant chimeric poxvirus CF33. VD did restrict growth in HT29 but not HCT116 human colon cancer cells. In vivo investigations using HCT116 and HT29 xenograft models of colon cancer demonstrated that a VD analogue, calcipotriol, was additive with CF33-based viral therapy in VD-responsive HT29 but not in HCT116 tumors. Analyses of RNA-sequencing and gene expression data demonstrated a downregulation in the Jak-STAT signaling pathway with the addition of VD to viral therapy in HT29 models suggesting that the anti-inflammatory properties of VD may enhance the effects of viral therapy in some models. In conclusion, VD may prime oncolytic viral therapy in certain colon cancers.

Effective combination immunotherapy using oncolytic viruses to deliver CAR targets to solid tumors.

Chimeric antigen receptor (CAR)-engineered T cell therapy for solid tumors is limited by the lack of both tumor-restricted and homogeneously expressed tumor antigens. Therefore, we engineered an oncolytic virus to express a nonsignaling, truncated CD19 (CD19t) protein for tumor-selective delivery, enabling targeting by CD19-CAR T cells. Infecting tumor cells with an oncolytic vaccinia virus coding for CD19t (OV19t) produced de novo CD19 at the cell surface before virus-mediated tumor lysis. Cocultured CD19-CAR T cells secreted cytokines and exhibited potent cytolytic activity against infected tumors. Using several mouse tumor models, delivery of OV19t promoted tumor control after CD19-CAR T cell administration. OV19t induced local immunity characterized by tumor infiltration of endogenous and adoptively transferred T cells. CAR T cell-mediated tumor killing also induced release of virus from dying tumor cells, which propagated tumor expression of CD19t. Our study features a combination immunotherapy approach using oncolytic viruses to promote de novo CAR T cell targeting of solid tumors.

Oncolytic poxvirus CF33-hNIS-ΔF14.5 favorably modulates tumor immune microenvironment and works synergistically with anti-PD-L1 antibody in a triple-negative breast cancer model.

Triple-negative breast cancer is the most aggressive subtype of breast cancer and is difficult to treat. Breast cancer is considered to be poorly immunogenic and hence is less responsive to immunotherapies. We tested whether the oncolytic poxvirus CF33-hNIS-ΔF14.5 could modulate tumor immune microenvironment and make the tumors responsive to the immune checkpoint inhibitor anti-PD-L1. We found that virus infection causes the upregulation of PD-L1 levels on triple-negative breast cancer cells in vitro as well as in vivo in mice. In a mouse model of orthotopic triple-negative breast cancer, the virus was found to increase tumor infiltration by CD8+ T cells. Likewise, in mice treated with CF33-hNIS-ΔF14.5 high levels of proinflammatory cytokines IFNγ and IL-6 were found in the tumors but not in the serum. The levels of immune modulation were even higher in mice that were treated with a combination of the virus and anti-PD-L1 antibody. While CF33-hNIS-ΔF14.5 and anti-PD-L1 antibody failed to exert significant anti-tumor effect as a single agent, a combination of the two agents resulted in significant anti-tumor effect with 50% mice experiencing complete tumor regression when both agents were injected intra-tumorally. Furthermore, the 'cured' mice did not develop tumor after re-challenge with the same cancer cells suggesting that they developed immunity against those cancer cells. Taken together, our study shows that CF33-hNIS-ΔF14.5 favorably modulates tumor immune microenvironment in triple-negative breast cancer model making them responsive to the immune checkpoint inhibitor anti-PD-L1, and hence warrants further studies to determine the clinical applicability of this combination therapy.

Novel Chimeric Immuno-Oncolytic Virus CF33-hNIS-antiPDL1 for the Treatment of Pancreatic Cancer.

BACKGROUND: Peritoneal carcinomatosis (PC) from pancreatic ductal adenocarcinoma (PDAC) is fatal. Our preclinical study presents an effective treatment against PDAC PC using a novel oncolytic viral agent, CF33-hNIS-antiPDL1. STUDY DESIGN: CF33-hNIS-antiPDL1 is a genetically engineered chimeric orthopoxvirus, CF33, armed with the human Sodium Iodide Symporter (hNIS) and anti-PD-L1 antibody (anti-PD-L1). The in vitro cytotoxic ability of this virus against 5 PDAC cell lines was tested at various doses (multiplicity of infection [MOI] = 0.01, 0.1, 1, 10). Production and blockade function of virus-encoded anti-PD-L1 antibody were verified using immunoblot, immunoprecipitation, and PD-1/PD-L1 bioassay. In vivo mouse models of PC, with or without subcutaneous (SC) tumors, created by injecting AsPC-1-ffluc cells into nude mice, were treated with PBS or a single dose (1×105 plaque-forming units) of either intraperitoneal (IP) or IV injection of CF33-hNIS-antiPDL1. Mice with PC tumors were treated on days 0, 2, or 14 after tumor implantation. RESULTS: CF33-hNIS-antiPDL1 killed PDAC cells in a dose-dependent manner, achieving >90% cell killing by day 8. Cells infected with CF33-hNIS-antiPDL1 produced bioactive anti-PD-L1 antibody, which blocked PD-1/PD-L1 interaction. In vivo, a single dose of virus reduced tumor burden and prolonged survival of treated mice. It was observed that IP administration of CF33-hNIS-antiPDL1 was more effective than IV administration. CONCLUSIONS: CF33-hNIS-antiPDL1 virus is effective in infecting and killing human PDACs and producing functional anti-PD-L1 antibody. Intraperitoneal delivery of CF33-hNIS-antiPDL1 effectively reduces peritoneal tumor burden and improves survival after only 1 dose and is superior to IV delivery.

Complete mitochondrial genome of two shorebirds (Charadriiformes: Scolopacidae), great knot (Calidris tenuirostris) and bar-tailed godwit (Limosa lapponica).

The mitochondrial genome of Calidris tenuirostris and Limosa lapponica were described using the whole mitochondrial genome obtained from Illumina Next-Generation Sequencing (NGS) technology. Total length of the mitogenome of C. tenuirostris was 16,732bp with slight A+T bias (55.3%). Genome size of L. lapponica was 16,773bp long and A+T biased (56.3%). Both gemones consisting of 2 rRNAs, 13 protein-coding genes, 22 tRNA genes and 1 non-coding regions. This is the first report of complete mitogenomes of these two shorebird species, (C. tenuirostris and of L. lapponica). We observed paraphyletic relationship among the species in the Family Scolopacidae. Also our result showed analogous patterns with the previous studies on the parallel relationships of shorebird species. This study provides basic genetic information for help in understanding phylogenetic relationships . within the Charadriiformes.