Oncolytic Research Articles

An IRF2-Expressing Oncolytic Virus Changes the Susceptibility of Tumor Cells to Antitumor T Cells and Promotes Tumor Clearance.

IFN regulatory factor 1 (IRF1) can promote antitumor immunity. However, we have shown previously that in the tumor cell, IRF1 can promote tumor growth, and IRF1-deficient tumor cells exhibit severely restricted tumor growth in several syngeneic mouse tumor models. Here, we investigate the potential of functionally modulating IRF1 to reduce tumor progression and prolong survival. Using inducible IRF1 expression, we established that it is possible to regulate IRF1 expression to modulate tumor progression in established B16-F10 tumors. Expression of IRF2, which is a functional antagonist of IRF1, downregulated IFNγ-induced expression of inhibitory ligands, upregulated MHC-related molecules, and slowed tumor growth and extended survival. We characterized the functional domain(s) of IRF2 needed for this antitumor activity, showing that a full-length IRF2 was required for its antitumor functions. Finally, using an oncolytic vaccinia virus as a delivery platform, we showed that IRF2-expressing vaccinia virus suppressed tumor progression and prolonged survival in multiple tumor models. These results suggest the potency of targeting IRF1 and using IRF2 to modulate immunotherapy.

Abstract 5396: Vascularized gastric cancer 3D co-culture model using a microphysiological system as an oncolytic virus testing platform

Abstract Introduction: Angiogenesis is a critical aspect of cancer growth and metastasis. Consequently, inhibiting angiogenesis has garnered substantial attention in oncology research. JX-594 (pexastimogene devacirpvec; Pexa-Vec®), engineered from the Wyeth vaccine strain, has emerged as an attractive oncolytic virotherapy and demonstrated oncolytic, immunotherapeutic, and tumor vasculature-disrupting capacities. In our previous study, we found that JX-594 reduced the amount of vasculature in gastric tumors using a xenograft mouse model. In the present study, we investigate the effect of JX-594 on tumor vasculature using an in vitro human cell 3D co-culture model of vascularized gastric cancer, to determine if this tool provides improved accuracy in evaluating the mechanism of action and biological activity of JX-594 as compared to traditional animal models. Methods: The Curio-Spheroid chip is a Microphysiological System (MPS) that can grow tumor organoids or spheroids in 3D using physiologically relevant hydrogels and culturing them with other human cell types such as fibroblasts, endothelial cells, and immune cells. We evaluated nine human gastric cancer cell lines (YCC-18, YCC-19, YCC-22, YCC-30, YCC-32, YCC-38, Hs746T, NUGC-3, SNU-620) for their ability to form spheroids and induce angiogenesis inside Curio-Spheroid chips. We selected YCC-32 for the oncolytic virus testing. Serial increases of M.O.I (multiplicity of infection) of JX-594 disrupted the tumor-induced angiogenesis in a dose-dependent manner. Results: JX-594 treatment of the YCC-32 spheroid resulted in decreased levels of Ki-67 and cytopathic effects in a dose-dependent manner inside the Curio-Spheroid MPS system. Angiogenesis was observed surrounding the spheroid when co-cultured with endothelial cells and fibroblasts. JX-594 decreased YCC-32 spheroid-induced angiogenesis in a dose-dependent manner in the vascularized gastric cancer 3D co-culture model. Conclusion: JX-594 had an inhibitory effect on tumor-induced angiogenesis in our vascularized gastric cancer in vitro 3D human cell co-culture model, demonstrating the utility of the platform as a tool to interrogate the effects of an oncolytic virus on both tumor cell killing and tumor-induced angiogenesis. Citation Format: YeLin Kim, Youngsook Song, Yeongmin Choi, Jinhwan Jang, Suryong Kim, Tsung-Li Liu, Kyusuk Baek, Keunhee Oh, Seunghyun Ma, Tae Soo Kim, Woo Sun Kwon, Sanghee Yoo, Jee Hung Kim, Sun Young Rha. Vascularized gastric cancer 3D co-culture model using a microphysiological system as an oncolytic virus testing platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5396.

Abstract 6658: TcE armed oncolytic virus VSV-GP combines oncolysis and T-cell retargeting for improved efficacy

Abstract Background: Oncolytic viruses, such as the chimeric vesicular stomatitis virus variant VSV-GP have dual modes of action, direct tumor-cell killing, and activation of tumor-specific immunity. While tumor cells deficient in type I interferon (IFN) response are permissive to VSV-GP replication and killing, IFN competent tumor cells are protected from VSV-GP-mediated oncolysis. Our previous data demonstrated that VSV-GP therapy turns immunologically ‘cold’ tumors ‘hot’ by increasing infiltration of T cells into the tumor. The aim of this study was to use the VSV-GP platform to locally express a T cell engager (TcE) molecule to redirect these T cells to destroy non-permissive IFN competent tumor cells expressing the target of the TcE on their cell surface. Methods: We selected EpCAM as the target antigen and used a bispecific EpCAM-TcE molecule consisting of two variable single chain fragments against EpCAM and CD3, respectively. VSV-GP encoding EpCAM-TcE (VSV-GP-EpCAM-TcE) or an invariant control TcE (VSV-GP-TNP-TcE) were generated as previously described. T cells were co-cultured with human target cells expressing EpCAM on their cell surface, either infected with the VSV-GP-EpCAM-TcE virus or with supernatant from VSV-GP-EpCAM-TcE infected cells. T cell activation, proliferation, degranulation and cytokine secretion were analyzed by flow cytometry and multiplex cytokine assays as a measure of TcE mediated cross-linking of T cells to their target on cancer cells. Patient-derived head and neck squamous cell carcinoma (HNSCC) tumors were sectioned into approximately 200 µM slices and infected with the virus. T cell activation was measured by cytokine secretion. The therapeutic potential of VSV-GP-EpCAM-TcE was tested in vivo in cell-line derived xenograft (CDX) models implanted in immunodeficient mice humanized with donor-derived PBMCs or CD34+ cells. Results: Virus fitness was maintained for VSV-GP encoding the TcE molecule. Using co-culture assays, we demonstrated that EpCAM-TcE molecule is successfully expressed by VSV-GP and can activate both CD4+ and CD8+ T cells resulting in proliferation, degranulation, and cytokine secretion. Furthermore, we validated the concept using patient-derived HNSCC tumor slices which are representative of complex human tumor ecosystems. We could confirm that cancer cells infected by VSV-GP-EpCAM-TcE virus express functional TcE molecule and activates T cells to secrete IFNγ and TNFα. Finally, we tested the concept in two different CDX models where VSV-GP-EpCAM-TcE showed improved tumor growth inhibition due to a combined effect of oncolysis and T cell activation by EpCAM-CD3 TcE. Summary: The oncolytic virus VSV-GP can be successfully armed with a TcE to redirect tumor-infiltrating T cells to destroy tumor cells which are not permissive to viral replication. This resulted in an enhanced therapeutic efficacy compared to just the oncolytic effect. Citation Format: Aparna Ponnurangam, Theresa Schwaiger, Sophie Walter, Simone Bartl, Melissa Mayr, Monika Petersson, Tanja Schoenberger, Fabian Heinemann, Peter Gross, Wolfgang Rist, Srinath Kasturirangan, Charlotte Lempp, Jutta Petschenka, Krishna Das, Nolden Tobias. TcE armed oncolytic virus VSV-GP combines oncolysis and T-cell retargeting for improved efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6658.

Abstract 6664: In situ vaccination with hyper-interferon sensitive influenza induces adaptive immune response and sensitizes murine non-small cell lung cancer to PD-1 blockade

Abstract Despite recent advances in immunotherapy with immune checkpoint inhibitors (ICI), many patients with non-small cell lung cancer (NSCLC) fail to respond or develop resistance after an initial response. The exclusion of T cells from the tumor or the presence of a dysfunctional T cell compartment within the tumor microenvironment (TME) constitute two central hallmarks of resistance to ICI in patients with NSCLC. Seminal studies have identified that loss of LKB1 in KRAS-mutant NSCLC drives resistance to ICI, potentially by suppressing STING, thereby leading to dysregulation of the interferon (IFN) signaling. There is a pressing demand for the development of innovative immune therapies designed to bolster anti-tumor T cell responses to overcome resistance to ICI. In situ vaccination (ISV) with oncolytic viruses has emerged as a promising, antigen-agnostic strategy capable of conditioning the TME and amplifying anti-tumor T cell responses. We engineered a live attenuated viral vaccine, Hyper-Interferon Sensitive virus (HIS), by conducting a genome-wide functional screening and introducing eight interferon (IFN)-sensitive mutations in the influenza A genome. We utilized multiple syngeneic murine models of NSCLC with distinct driver mutations [KrasG12D/P53+/−/Lkb1−/− (KPL); KrasG12D/P53+/− (KP); and KrasG12D (K)] and varying tumor mutational burden to assess the safety and efficacy of intratumoral HIS virotherapy. Compared to wild-type (WT) influenza, HIS replication was attenuated, enhancing its potential as a safe option for cancer therapy. HIS ISV elicited robust yet transient type I IFN responses in murine NSCLCs, leading an influx of polyfunctional effector Th1 CD4 and cytotoxic CD8 T cells into the tumor. HIS ISV demonstrated enhanced anti-tumor efficacy compared to WT in multiple syngeneic murine models of NSCLC. This efficacy was dependent on host type 1 IFN responses and T lymphocytes. HIS ISV synergized with anti-PD-1 to eradicate immune resistant LKB-1 deficient NSCLC in a subset of mice, resulting in improved overall survival and enduring systemic tumor-specific immunity. These studies provide compelling evidence to support further clinical evaluation of HIS as a novel ‘off-the-shelf’ ISV strategy for patients with NSCLC refractory to ICI. Citation Format: Ramin Salehi-Rad, Yushen Du, Tianhao Zhang, Yuan Shi, Bin Liu, Sun Ren. In situ vaccination with hyper-interferon sensitive influenza induces adaptive immune response and sensitizes murine non-small cell lung cancer to PD-1 blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6664.

Abstract 6851: The genomic, transcriptomic, and immunologic landscape of TEM8 (ANTXR1) in neuroendocrine neoplasms (NENs)

Abstract Introduction: TEM8 (coded by ANTXR1) is overexpressed in malignant tissues, with oncolytic viruses such as SVV-01 uniquely binding to this receptor on tumor-associated angiogenic endothelial cells, pericytes, fibroblasts, and immune inflammatory cells. Recent pre-clinical data suggest that TEM8-targeting therapies may convert immunologically “cold” tumor microenvironments (TME) into “hot” milieu more amenable to treatment with immune checkpoint inhibitors (ICIs). Methods: NextGen sequencing of DNA (592 genes or WES) and RNA (WTS) was performed on neuroendocrine neoplasms (NENs; N = 1724), excluding small-cell lung cancer, submitted to Caris Life Sciences (Phoenix, AZ). Mutations were defined as pathogenic SNVs/indels. ANTXR1 expression was divided by ANTXR1-expression quartiles (transcripts per million [TPM];Q4:H;Q1:L). PD-L1 expression (SP142; Positive (+): ≥2+,≥%5) was assessed by IHC. High tumor mutational burden (TMB-H) was defined as ≥10 mutations per MB. Cell infiltration in the TME was estimated by QuantiSEQ. Gene expression profiles were analyzed for transcriptional signatures predictive of response to immunotherapy (T cell-inflamed) and MAPK pathway activation score (MPAS). Overall survival (OS) data was obtained from insurance claims, and Kaplan-Meier estimates were calculated for molecularly defined subpopulations of patients. Mann-Whitney U and X2/Fisher-Exact tests were applied where appropriate, with p-values adjusted for multiple comparisons (p<0.05). Results: NENs from the adrenal gland had the highest median ANTXR1 expression (50 TPM), followed by those from the colon and rectum (43.5), genitourinary organs (43.3), and small bowel (excluding appendix) (39.2). The lowest median ANTXR1 expression was observed in NENs from the pancreas (31.5). No pathogenic mutations were significantly associated with ANTXR1H vs L tumors (p>0.05 for all), along with no differences in the prevalence of TMB-H (8.6% vs 12.6%, p=1) or PD-L1+ (5.8%: vs 3.1%, p=1). A greater proportion of B cells (5.34 % vs 3.78%, p<0.001), M1 (1.46% vs 0.15%, p<0.001) and M2 macrophages (3.92% vs 2.67%, p<0.001), and T-regulatory cells (1.77% vs 1.08%, p<0.001) was observed in ANTXR1H TMEs, which were more frequently classified as T cell-inflamed compared to ANTXR1L (42% vs 8%, p<0.001). ANTXR1H tumors also had higher MPAS compared to ANTXR1L (1.16 vs -1.61 arbitrary units, p<0.001). There was no significant difference in median OS between ANTXR1H vs L tumors (18.8 mo. vs 21.2 months, Hazard Ratio:1.18, p=0.114). Conclusion: The increased immune cell infiltrate and prevalence of T-cell inflamed status among ANTXR1H NENs suggests these tumors might be more responsive to treatment with ICIs. As trials incorporating intratumoral injections of SVV-01 in combination with ICIs are underway, further investigation of clinical and molecular associations with ANTXR1 expression in NENs is warranted. Citation Format: Samuel A. Kareff, Harris Krause, Andrew Elliott, Peter Hosein, Emil Lou, Heloisa Soares, Matthew Oberley, Aman Chauhan. The genomic, transcriptomic, and immunologic landscape of TEM8 (ANTXR1) in neuroendocrine neoplasms (NENs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6851.

A new strategy for immunotherapy of microsatellite-stable (MSS)-type advanced colorectal cancer: Multi-pathway combination therapy with PD-1/PD-L1 inhibitors.

Colorectal cancer (CRC) is a frequent gastrointestinal malignancy with high rates of morbidity and mortality; 85% of these tumours are proficient mismatch repair (pMMR)-microsatellite instability-low (MSI-L)/microsatellite stable (MSS) CRC known as 'cold' tumours that are resistant to immunosuppressive drugs. Monotherapy with programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) inhibitors is ineffective for treating MSS CRC, making immunotherapy for MSS CRC a bottleneck. Recent studies have found that the multi-pathway regimens combined with PD-1/PD-L1 inhibitors can enhance the efficacy of anti-PD-1/PD-L1 in MSS CRC by increasing the number of CD8+ T cells, upregulating PD-L1 expression and improving the tumour microenvironment. This paper reviews the research progress of PD-1/PD-L1 inhibitors in combination with cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) inhibitors, oncolytic virus, intestinal flora, antiangiogenic agents, chemotherapy, radiotherapy and epigenetic drugs for the treatment of pMMR-MSI-L/MSS CRC.

Backflow reduction in local injection therapy with gelatin formulations

The local injection of therapeutic drugs, including cells, oncolytic viruses and nucleic acids, into different organs is an administrative route used to achieve high drug exposure at the site of action. However, after local injection, material backflow and side effect reactions can occur. Hence, this study was carried out to investigate the effect of gelatin on backflow reduction in local injection. Gelatin particles (GPs) and hydrolyzed gelatin (HG) were injected into tissue models, including versatile training tissue (VTT), versatile training tissue tumor-in type (VTT-T), and broiler chicken muscles (BCM), using needle gauges between 23 G and 33 G. The backflow material fluid was collected with filter paper, and the backflow fluid rate was determined. The backflow rate was significantly reduced with 35 μm GPs (p value < .0001) at different concentrations up to 5% and with 75 μm GPs (p value < .01) up to 2% in the tissue models. The reduction in backflow with HG of different molecular weights showed that lower-molecular-weight HG required a higher-concentration dose (5% to 30%) and that higher-molecular-weight HG required a lower-concentration dose (7% to 8%). The backflow rate was significantly reduced with the gelatin-based formulation, in regard to the injection volumes, which varied from 10 μL to 100 μL with VTT or VTT-T and from 10 μL to 200 μL with BCM. The 35 μm GPs were injectable with needles of small gauges, which included 33 G, and the 75 μm GPs and HG were injectable with 27 G needles. The backflow rate was dependent on an optimal viscosity of the gelatin solutions. An optimal concentration of GPs or HG can prevent material backflow in local injection, and further studies with active drugs are necessary to investigate the applicability in tumor and organ injections.

Abstract 3401: Uncovering virus induced dysregulated processes in cancer and their impact on outcomes

Abstract The National Cancer Institute has designated several viruses such as EBV, HBV, HCV, HIV, HPV, and HTLV as agents that either cause cancer or increase the risk of developing cancer. However, extensive analyses of tumor genome and transcriptome datasets have identified numerous additional virus types, the functional implications of which remain largely unknown. Viral infection often dysregulates unique molecular processes within tumor cells and alters the composition of immune cells in the tumor microenvironment. For instance, HPV-positive tumors have been shown to exhibit a significantly elevated expression of the DNA editing enzyme APOBEC3B compared to HPV-negative tumors. Furthermore, HPV positive tumors display and increased number of C-to-T mutations induced by this protein. The genome and transcriptome of tumor tissue biopsies contain a wealth of information about these virus-associated signatures. However, advanced signal processing approaches are needed to extract these viral signatures from complex tumor datasets, and elucidate their specific roles in cancer. Here, we developed a quantitative approach to investigate the link between viral infection and dysregulation of a seven-membered A3 enzyme family (A3A/B/C/D/F/G/H). The primary role of these enzymes is to restrict viral infections by inducing C-to-U mutations in viral genomes. However, at least three members of this enzyme family (A3A/B/H) can also induce mutations in cellular DNA, and drive tumor formation and evolution. We used NMF (Nonnegative Matrix Factorization) to deconvolute the expression profiles of A3 enzymes in &amp;gt;10,000 bulk RNAseq datasets from diverse TCGA tumors. This analysis revealed the expression profiles of A3 enzymes within tumor and tumor microenvironment cells including various immune cells. Next, we conducted an association analysis to identify the link between the presence of viral infection and the proportion of each of these tumor and tumor microenvironment expression profiles. As expected, our analyses revealed strong associations between tumor-specific A3 expression profiles and infections by oncolytic viruses such as HPV. Notably, and somewhat unexpectedly, our analyses also revealed associations with viruses about which little is known in the field of cancer. For example, in certain tumors, we identified a robust association between bacteriophages and a tumor-specific A3 expression profile characterized by A3B upregulation. Taken together, our analyses provide evidence for the link between A3 dysregulation and infection by viruses including bacteriophages. Citation Format: Mohadeseh Soleimanpour moghadam, Azad Khosh, Diako Ebrahimi. Uncovering virus induced dysregulated processes in cancer and their impact on outcomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3401.

Abstract 7258: Combinatorial effects of viral oncolysis and miRNA modulation as a novel therapeutic of pancreatic ductal adenocarcinoma

Abstract Pancreatic cancer is a devastating illness that affects numerous individuals each year. Unfortunately, pancreatic ductal adenocarcinoma (PDAC) is an aggressive form of this disease that accounts for a disproportionate number of cancer-related deaths. Despite representing only 3.3% of all cancer cases in the United States, pancreatic cancer is responsible for a staggering 8.3% of cancer-related fatalities. In 2023, 64,050 patients were diagnosed with PDAC alone. Current treatment options, which are limited to surgery, radiation, and chemotherapy, have failed to improve clinical outcomes. Recent research has revealed miRNA (miR) as a contributor to the suppression and progression of various cancers, including PDAC. Specifically, dysregulation of miR-7 has been associated with PDAC. Downregulation of miR-7 is linked to more aggressive cases of the disease. Another potential therapeutic approach to treating cancer is the use of oncolytic viruses (OV). These viruses can replicate in cancerous cells while leaving healthy cells unharmed, making them an attractive treatment option. In this study, we utilized exosomes isolated from human Umbilical Cord stem cells (hUCs) enriched with miR-7 and combined with OHSV to examine their effect on established PDAC cell lines. We utilized cell viability assays, microscopy, and western blot analysis to evaluate the impact of the combinatorial approach on the underlying molecular mechanisms. Our findings suggest that the successful transfer of miR cargo and treatment with oHSV into the target PDAC cells arrested cell proliferation by targeting apoptotic pathways. Citation Format: Monique Kanitz Ruschel, Isaiah Davis, Hannah Taylor Mills, Karthik Gourishetti, Deepak Bhere. Combinatorial effects of viral oncolysis and miRNA modulation as a novel therapeutic of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7258.

Abstract 6657: Oncolytic Vaccinia Virus Carrying OPCML Tumor Suppressor is active in Epithelial Ovarian Cancer

Abstract Objective: Epithelial Ovarian cancer (EOC) has the highest mortality rate among gynecologic malignancies, necessitating novel therapeutic approaches. Viral oncolytic immunotherapy with recombinant oncolytic vaccinia virus (OVV) offers promise for advanced-stage cancer treatment. OPCML is a GPI-anchored 3 Ig-domain tumor suppressor inactivated by somatic methylation in 87% of EOC. OVV with OPCML (OVV-OPCML) gene insertion may have potential as a therapeutic agent. This study explores the oncolytic, signaling-inhibitory, anti-angiogenic and synergistic immunotherapeutic properties of OVV-OPCML in EOC treatment, evaluating its therapeutic potential. Methods: To create OVV-OPCML, the virulent vaccinia virus Copenhagen strain F3L site was modified with OPCML cDNA, confirmed by qPCR and western blot. Oncolytic potential was assessed through in vitro and in vivo EOC cell infection with OVV-OPCML. Real-time cell analysis (RTCA) assessed cell proliferation. Xenograft tumor models in nude mice were used to evaluate in vivo oncolytic activity, while OVV-OPCML combined with anti-PD-1 treatment was tested using a syngeneic orthotopic EOC mouse model (ID8) in C57BL/6 immunocompetent mice. Serum IFN-γ and TNF-α were quantified via ELISA. Receptor Tyrosine Kinases, angiogenesis and phosphorylation together with immune markers were examined by western blotting and by multiplex immunohistochemistry (mIHC) analysis of tumor sections. Results: OVV-OPCML selectively infected OC cells, leading to viral replication and tumor cell death, while sparing normal ovarian cells in-vitro. OPCML protein expression increased post-infection. OVV-OPCML reduced ERBB2, EGFR, and FGFR1 expression in SKVO3 OV cells. Intratumoral or intraperitoneal (IP) OVV-OPCML administration in human ovarian cancer nude mouse models reduced tumor growth and improved survival. IP in-vivo OVV-OPCML in combination with anti-PD-1 treatment in vivo, in the IP ID8 syngeneic model demonstrated synergy with survival improvement. H&amp;E staining showed tumor cell damage without significant harm to normal tissues. Protein expression of ERBB2, EGFR, FGFR1, and their phosphorylated forms decreased. CD31, VEGFR2, p-VEGFR2 (Tyr1175), and p-VEGFR2 (Tyr1214) downregulation suggested tumor angiogenesis disruption. OVV-OPCML treatment elevated serum TNF-α and INF-γ levels and increased CD8+ T cells and PD-L1 expression in OVV-OPCML treated syngeneic tumors. Conclusions: OPCML gene enhances OVV selectivity against OC cells through direct oncolysis and OPCML protein production, inhibiting specific RTKs, suppressing angiogenesis, and activating antitumor immune responses synergistically with anti-PD-1. OVV-OPCML therapy shows promise as a targeted strategy for ovarian cancer treatment. Citation Format: Yong Tang, Yingzhao Liu, Mingjing Deng, Fei Huang, Jiahu Wang, Paul Blake, Hani Gabra, Qi Wang. Oncolytic Vaccinia Virus Carrying OPCML Tumor Suppressor is active in Epithelial Ovarian Cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6657.

Abstract 2808: Levels of isobutyric acid from gut butyrate-producing taxa are associated to Delta-24-RGDOX efficacy against malignant glioma

Abstract Glioblastoma (GBM) is the most aggressive malignant primary brain tumor worldwide. Current standard-of-care treatments allow for a median survival of 12 to 18 months. GBM has a poor survival prognosis, observing less than 6% of patients surviving 5 years post-diagnosis. Among recent treatment advancements, the oncolytic adenovirus Delta-24-RGDOX armed with the T-cell activator OX40L is promising. Studies have unveiled a relationship between the gut microbiome and high levels of short-chain fatty acids (SCFA), such as isobutyric and propionic acid, to immunotherapy response. Changes in the gut microbiome and metabolome associated to viroimmunotherapy efficacy remain unclear. Here, we assessed the gut microbial changes in addition to isobutyric and propionic acid levels regarding viroimmunotherapy efficacy. Immunocompetent C57BL/6 mice were intracranially implanted GL261-5 GBM cells and received intratumoral injections of PBS (control) or Delta-24-RGDOX. A naïve group with no tumor nor treatment was also included. Fecal pellets were collected at three different time points: (1) before tumor implantation, (2) before treatment, and (3) 14 days after the first dose of treatment. Genomic DNA was isolated with the QIAGEN DNeasy Powersoil Pro Kit from fecal pellets (n=75), followed by sequencing with the Illumina platform of the 16S ribosomal RNA V4 region. Quality assessment of the sequences was executed in QIITA. Bacterial community analysis with a sequencing depth of 1,470 reads, while using the taxonomic database SILVA, was followed downstream with QIIME2 and R. Metabolic profiling of SCFA of the fecal pellets was performed through Gas Chromatography. Results showed significant differences in bacterial community structure of viroimmunotherapy-treated mice with survival of &amp;gt;100 days compared to controls (ANOSIM p-value=0.006). Alpha diversity metrics showed significant differences in richness between the naive and tumor-implanted mice of the PBS (KW p-value=0.049) and Delta-24-RGDOX (KW p-value=0.019) groups, highlighting that tumor presence alters the gut microbiota. Additionally, viroimmunotherapy-treated mice with survival of &amp;gt;100 days had a significant increase of Lactobacillus and the butyrate-producing bacterias Erysipelatoclostridium and Ruminococcaceae. Also, we found significantly higher levels of isobutyric acid and lower levels of propionic acid in viroimmunotherapy-treated mice with survival of &amp;gt;100 days (p-value&amp;lt;0.05). These findings suggest that butyrate-producing bacteria coupled with higher levels of isobutyric acid may be important in the efficacy of Delta-24-RGDOX. This highlights the microbial and metabolic changes associated with oncolytic viral therapy efficacy. Sponsored by an award within The University of Puerto Rico/UT MD Anderson Cancer Center Partnership for Excellence in Cancer Research Grant (2U54CA096297-17). Citation Format: Natalie M. Meléndez-Vázquez, Xuejun Fan, Juan Fueyo, Candelaria Gomez-Manzano, Filipa Godoy-Vitorino. Levels of isobutyric acid from gut butyrate-producing taxa are associated to Delta-24-RGDOX efficacy against malignant glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2808.

Abstract 588: Preclinical evaluation of novel chimeric vesiculovirus mediated expression of proteolytic enzymes targeting two major stromal components of pancreatic ductal adenocarcinoma

Abstract The vesiculovirus genus has recently garnered interest as an oncolytic virotherapy for pancreatic ductal adenocarcinoma (PDAC). A new synthetic chimeric vesiculovirus, VMG, demonstrated efficacy and safety against PDAC both in vitro and in vivo. The therapeutic effectiveness of virotherapies against PDAC is often hindered by immunosuppressive factors in the tumor environment, as well as the presence of hyaluronic acid (HA) and collagen (CN), which increase tumor stiffness and interstitial fluid pressure, leading to reduced drug/oncolytic virus and immune cell penetration into tumor. To address this, we engineered VMG viruses incorporating enzymes PH20, collagenase (CNase), and novel proteolytic enzyme SMC, the latter exhibits both PH20 and CNase-like activity. We characterized these modified viruses for their cytotoxicity and performance in vitro. Various PDAC cell lines displayed varied sensitivity to VMG vectors, yet they showed consistent oncolytic effects relative to the parent VMG-GFP virus. Our results revealed no compromise in viral replication or apoptosis-inducing ability after transgene insertion, which is evident as infectious viral progenies were produced to approximately similar level of magnitude of around 108 TCID50/ml in all the cell line tested. Additionally, the VMG-SMC and VMG-hPH20-CNase variants demonstrated notable enzyme activity. To enhance clinical relevance, we utilized an ex vivo live tumor platform, which indicated around 20% tumor cell death using VMG vectors compared to that of the control, signifying tumor viability and virus replicability 72 hours post-infection. Fluorescent imagery highlighted the viruses' ability to spread and express functional enzymes within tumors. In summary, VMG vectors expressing stroma-degrading proteolytic enzymes can effectively express PH20, CNase and SMC enzymes that degrade stromal barrier of HA and CN without interrupting the replicability, performance, and oncolytic characteristics of the VMG vectors. Future research should prioritize the safety of these vectors and their potential to augment neoantigen expression and immune cell infiltration in vivo, which are the hallmarks of antitumor efficacy. Citation Format: Khandoker Usran Ferdous, Mulu Z. Tesfay, Camila C. Simoes, Aleksandra Cios, Bolni Marius Nagalo. Preclinical evaluation of novel chimeric vesiculovirus mediated expression of proteolytic enzymes targeting two major stromal components of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 588.

Abstract 2025: Zeb1 downregulation sensitizes pancreatic cancer-associated fibroblasts to killing by oncolytic reovirus through upregulation of the reovirus receptor junction adhesion molecule A

Abstract Pancreatic tumors display an abundance of cancer-associated fibroblasts (CAFs), which negatively affect prognosis and therapy response. Oncolytic virotherapy exploits viruses that preferentially lyse epithelial cancer cells as opposed to normal cells. Interestingly, we have observed that oncolytic reoviruses are able to infect and lyse CAFs, in addition to epithelial cancer cells. Targeting CAFs, in addition to cancer cells, could be advantageous to increase therapy effectiveness. It could serve as a conduit for viral spread and simultaneously disrupt the desmoplastic barrier around tumors, thereby also accelerating the influx of other therapeutics and immune cells. We previously found that the proneness of CAFs to lysis by oncolytic reovirus correlates with the cell surface expression levels of the reovirus entry receptor junction adhesion molecule A (JAM-A). However, most pancreatic CAFs do not express JAM-A. Therefore, a genome-wide CRISPR/Cas9 screen was employed to identify the genes regulating JAM-A expression on fibroblasts, which can subsequently be targeted to sensitize CAFs to reovirus. Pancreatic stellate cells with a moderate JAM-A expression level were transduced with a gRNA library making a knockout of one gene per cell. The highest and lowest JAM-A expressing cells were sorted and sequenced to identify the gRNAs that regulate JAM-A expression. Clonal CRISPR/Cas9-generated knockouts of a top negative regulator were generated and infected with reovirus, followed by cell viability assays to quantify their susceptibilities to reovirus-induced cell death. F11R, the gene encoding JAM-A, was identified as the top positive regulator of JAM-A expression in the CRISPR/Cas9 screen, verifying the validity of the screen. The top negative regulators identified were Fibroblast Growth Factor Receptor 1 (FGFR1) and Zinc finger E-box Binding homeobox 1 (Zeb1), thereby serving as potential therapeutic targets to sensitize CAFs to reovirus treatment. Using clonal Zeb1 knock-outs, Zeb1 was confirmed as a strong regulator of JAM-A expression. Zeb1 knockout in JAM-A negative pancreatic fibroblasts caused a robust upregulation of JAM-A and sensitized these inherently resistant fibroblasts to reovirus-directed cytolysis. Additionally, the clinically approved drug Mocetinostat, previously described to inhibit Zeb1, upregulated JAM-A expression on CAFs and increased cell lysis by reovirus. Altogether, our data show that Zeb1 is a strong negative regulator of JAM-A expression on fibroblasts and that Zeb1 inhibition can sensitize CAFs to reovirus-induced cell death. This research provides a rationale for combining Zeb1 inhibitory drugs with oncolytic reovirus treatment to improve killing of CAFs, which in turn could boost overall tumor eradication. Citation Format: Nicole Dam, Tom J. Harryvan, Bernhard Schmierer, Lukas J.A.C. Hawinkels, Vera Kemp. Zeb1 downregulation sensitizes pancreatic cancer-associated fibroblasts to killing by oncolytic reovirus through upregulation of the reovirus receptor junction adhesion molecule A [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2025.

Abstract 244: Patient-derived organoids: A pre-clinical model to assess infection and killing capacity of viral vectors for cancer therapy

Abstract HUB’s patient-derived organoids (PDOs) are in vitro models that preserve tissue morphology and original cellular complexity. Additionally, PDOs are compatible with (high) throughput screening methods, enabling informed decision-making early in the drug development pipeline and significantly improving the odds of positive outcomes in clinical trials. Akamis Bio has developed experimental viral vector-based tumour gene therapies that have the potential to treat a variety of solid tumours by driving the expression of immunologically active biomolecules and therapeutic proteins hampering tumour immune evasion. These vectors are based on a replication competent, chimeric group B adenovirus backbone that selectively replicates and expresses transgene payloads in tumor cells, and additionally exerts oncolytic activity. To evaluate the efficacy of such a virus-based gene delivery strategy, HUB set up a proof-of-concept study to assess the propagation capability of Akamis Bio’s viral vectors in tumour PDOs. Intact PDOs from primary pancreatic tumours were infected with several viral loads, ranging from 0.1 viral particles per cell (PPC) to 100 PPC. The viral vector encoded a GFP reporter gene to identify infected cells using fluorescence imaging. The cultures were imaged daily for 14 days, allowing the assessment of cytotoxicity and infection efficiency and dynamics. Independently of PPC, infections always started in the exterior of PDOs in a few GFP-positive cells. The gradual increase over time in GFP-signal toward the interior of PDOs suggests the high likelihood of secondary infection events. Crucially, GFP intensity was correlated with cell death in PDOs, as evidenced by morphological features detected in brightfield images. Moreover, the infection dynamics were dose-dependent, as the GFP intensity peak arose later with decreasing PPC. These results show the capacity of HUB PDOs to study the infection dynamics of Akamis viral vectors. This pre-clinical platform permits rapid and efficient screening and dose evaluation for oncolytic virotherapies. Unique access to extensive tumour PDO biobanks with characterized genomic landscapes allows best-in-class stratification of treatment response based on tumour type and genetic heterogeneity. Citation Format: Francisco Morales-Rodriguez, Maria Stella Sasso, Taya Bezhaeva, Samantha Bailey-Bucktrout, Ka Wai Lai, Foteini Gkogkou, Sylvia Boj. Patient-derived organoids: A pre-clinical model to assess infection and killing capacity of viral vectors for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 244.

Abstract 6653: Oncolytic virotherapy induces a long-term immune antitumoral response leading to glioma eradication

Abstract Oncolytic herpes simplex viruses (oHSVs) have shown promise as effective treatments against various cancers. Our study evaluated the efficacy of R-115, an oHSV retargeted to human Her2 (hHer2), fully virulent in its target cells and expressing murine interleukin 12 (mIL12), in a high-grade, immunocompetent murine glioma model. This model is based on orthotopically transplant of cells derived from PDGFB-induced primary gliomas in syngeneic mice, engineered to express hHer2. Our experimental design compared the impact of R-115 on gliomas where all cells expressed hHer2 against a setup where only about 50% of cells expressed hHer2. The latter aimed to replicate the heterogeneity of patient’s high-grade gliomas, where not all cells express the target molecule. Strikingly, results in terms of overall survival and tumor eradication were similar in both setups, with about a quarter of animals showing complete glioma eradication. This suggests that mIL12-armed oHSV's impact is less about direct viral-induced cell death and more about the immune response elicited by the mIL12-expressing virus. This response effectively targets cells lacking the viral-targeted protein but likely sharing other neoantigens with hHer2-positive cells. Mice cured in both experimental setups were rechallenged with purely hHer2-negative cells. Almost all did not develop gliomas, indicating a strong immune response capable of rejecting the tumor. Plasma analysis revealed that R-115-treated mice from both setups were more immunoreactive against both hHer2-positive and negative cells compared to untreated mice. Further, co-culturing long-surviving mice's splenocytes with glioma cells showed increased cell division in CD8, CD4 and CD19 populations, and elevated IFN-γ, granzyme B, and TNF-α production. These findings underscore R-115's profound anti-cancer potential, especially its capacity to elicit an immune response effective against glioma cells, irrespective of their expression of the target molecule. This outcome, coupled with the results of the rechallenge experiments, is pivotal. It suggests that in a clinical context, an interleukin-12-expressing oHSV could effectively target not only residual cells post-surgical resection but also infiltrating cells that are distant from the site of virus administration. Encouraged by these promising findings, we are currently advancing our research with a novel oHSV that expresses mIL12 and targets EGFRvIII, a mutation found exclusively in tumor cells. This is aimed at enhancing the safety and translatability of this therapeutic approach. Citation Format: Francesca Piaggio, Francesco Alessandrini, Chiara Riviera, Irene Appolloni, Davide Ceresa, Daniela Marubbi, Tatiana Gianni, Gabriella Campadelli-Fiume, Paolo Malatesta. Oncolytic virotherapy induces a long-term immune antitumoral response leading to glioma eradication [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6653.

Abstract 1605: Oncolytic hsv-1(vc2) gm-csf regulates the proinflammatory profile suggestive of an immunomodulatory effect in PDAC cells

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease that is projected to be the second leading cause of cancer-associated deaths by 2030. Owing to its intrinsic immune evasive tumor microenvironment (TME), PDAC is regarded as an immunologically “cold” tumor with a significant therapeutic challenge. Although 5-fluorouracil or gemcitabine-based chemotherapy is considered as standard of care, harnessing immunological pathways may potentially convert PDAC to a “hot” tumor, thereby providing survival advantage to patients. Lately, the use of oncolytic virus has proven to be an alternative strategy to activate the innate immune response within the TME coupled with its cytotoxic effect against the cancer cells. The FDA approval of T-VEC for the treatment of stage IIIB/IV melanoma validates the preceding argument. In this study, we have investigated the effect of herpes simplex virus type-1 (HSV-1) (VC2) overexpressing granulocyte-macrophage colony-stimulating factor (GMCSF), HSV-1VC2GM-CSF in the murine KRAS, p53, and Cre (KPC), and human MIA PaCa-2 pancreatic carcinoma cells. Following a 24h infection of the KPC and MIA PaCa-2 cells with the HSV-1VC2GM-CSF, the observed cytopathic effects (CPE) included cell rounding, detachment, and aggregation as compared to the wild-type HSV-1VC2 treated or the uninfected control cells. The CPE effect of HSV-1VC2GM-CSF was proven to be a more direct and dynamic response against these cells. In the next steps, the viral titers were determined to be 9.6E7 pfu/ml, 1.26E7 pfu/ml for HSV-1VC2 and VC2GM-CSF respectively. The infection of VC2GM-CSF 0.1, 1.0 and 10 MOI in KPC cells resulted in a significant increase in the expression of proinflammatory markers including interleukin 10 (IL-10), cyclooxygenase -2 (COX-2) and iNOS at the gene level in contrast to the uninfected controls. This was coupled with an increase in the expression of Fas ligand (FasL) in the treated cells. Intriguingly, the treated cells showed a significant upregulation of programmed death ligand (PD-L1). Interestingly, in a 3D-coculture of KPC and RAW264.7 cells the expression of PD-L1 was significantly upregulated. Based on the evidence presented here, it is speculated that VC2GM-CSF-induced proinflammatory profile in KPC and Mia PaCa-2 cells converts to an immunomodulatory one characterized by the regulation of FasL and PD-L1. The effect of VC2GM-CSF in a PDAC organoid model to understand the interplay between the tumor and the stromal cells in response to the virus is currently underway. Citation Format: Naveen Chintala Ramulu, Shilpa Thota, Brent Stanfield, Konstantin Kousoulas, Joseph Francis. Oncolytic hsv-1(vc2) gm-csf regulates the proinflammatory profile suggestive of an immunomodulatory effect in PDAC cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1605.

Abstract 3720: Utility of oncolytic virus as an immunostimulants and potential for combination therapy with ICIs

Abstract Background: Anti-PD-1 antibodies have been approved for a variety of carcinomas, but their efficacy in gastrointestinal cancers is limited. CD8-positive T cells (CD8) of tumor-infiltrating lymphocytes (TILs) and resident memory T cells (TRMs) with long-term anti-tumor immune function have been reported to be associated with the therapeutic efficacy of anti-PD-1 antibodies. Among our oncolytic adenoviruses (OVs), OBP-301, in which the human telomerase reverse transcriptase (hTERT) promoter element drives the expression of the viral E1A and E1B genes, is currently undergoing clinical trials. In the present study, we investigated the usefulness of OVs as immunostimulants and their potential efficacy in combination with anti-PD-1 antibodies. Methods: The cell lines used were the mouse colon cancer cell line CT26 and the mouse pancreatic cancer cell line PAN02. In vitro, the cytotoxic activity of the OVs and extracellular ATP in the culture supernatant were determined. OBP-502, an OBP-301 variant with the gene cassette of the RGD peptide, and anti-PD-1 antibody were used to examine the antitumor effect on subcutaneous tumors. OBP-702, another OBP-301 variant with the gene cassette of the wild-type p53 tumor suppressor gene, was used to examine the effect of long-term anti-tumor immunity. The effects on memory T-precursor cells (MPEC), CD8 and TRM were immunologically assessed in a rechallenge study. Results: OBP-502 and OBP-702 showed cytotoxic activity against CT26 and PAN02 cells in vitro, and significantly increased ATP in the culture supernatant. In vivo, the anti-PD-1 antibody/OBP-502 combination therapy significantly increased intratumor CD8 in CT26 and PAN02 subcutaneous tumors compared to each monotherapy. Complete response was achieved in 4 out of 12 mice by the combination therapy against CT26 tumors. When CT26 cells was re-seeded into tumor-free mice cured by the combination therapy, 2 out of 4 mice remained tumor-free. In the CT26 and PAN02 subcutaneous tumor model, OBP-702 increased CD44-positive CD62L-negative MPECs that differentiated into TRMs in the spleen, and this effect was blocked by ATP receptor inhibition. In CT26 and PAN02 bilateral subcutaneous tumor models, OBP-702 had a significant antitumor effect on both OBP-702-treated and -non-treated sides, and TIL analysis showed a significant increase in CD8 and TRM. In a rechallenge study, OBP-702-pretreatment showed significant growth inhibition of re-inoculated tumors. Conclusion: The OVs showed potent and long-term immune activation by inducing CD8 and TRM in tumors as well as direct anti-tumor effects, and synergistic effects with ICI. Citation Format: Daisuke Kadowaki, Shinji Kuroda, Masashi Hashimoto, Nobuhiko Kanaya, Yoshihiko Kakiuchi, Satoru Kikuchi, Kazuhiro Noma, Hiroshi Tazawa, Shunsuke Kagawa, Yasuo Urata, Toshiyoshi Fujiwara. Utility of oncolytic virus as an immunostimulants and potential for combination therapy with ICIs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3720.

Abstract 6906: Engineered bacteria launch and control an oncolytic virus

Abstract The ability of bacteria and viruses to selectively replicate in tumors has led to synthetic engineering of new microbial therapies. In our system, Salmonella has been programmed to produce Senecavirus A (SVA)-RNA and launch an SVA infection in bacterially-infected cancer cells. Utilizing time-lapse microscopy, we visualize S. typhimurium invasions into cancer cells and their subsequent delivery of multiple picornaviral species. S.-typhimurium-viral-RNA launch in vitro identified a small-cell-lung cancer cell line, H446, as a promising candidate for S.-typhimurium-SVA launch. This viral RNA-carrying Salmonella strain confers complete tumor regression in vivo. Finally, we integrate into this strain a protease-dependent mechanism to confer SVA-infection spatial localization. This work, to the best of our knowledge, is the first instance of bacteria delivering full-length viral RNAs for therapeutic oncolytic purposes. Citation Format: Jonathan Pabon, Zakary Singer, Hsinyen Huang, Charles Rice, Tal Danino. Engineered bacteria launch and control an oncolytic virus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6906.

Abstract 6660: The novel codon-modified Zika virus CDX-602 shows anti-tumor efficacy and remodels the tumor microenvironment in preclinical models

Abstract Multiple oncolytic viruses have been shown to induce multifaceted changes in the tumor microenvironment, that ultimately support induction of anti-tumor immune responses. We have previously demonstrated that CodaLytic, a codon-modified influenza virus, can kickstart the cancer immunity cycle at various steps in several mouse models with differing baseline immune contextures. Here, we are describing the activity of a new codon-modified virotherapeutic derived from the same synthetic viral engineering platform [Coleman et al., Science 2008]. Zika virus strain MR766 was synthetically codon-modified at 664 positions in the envelop gene to yield CDX-602. Virus was originally passaged in Vero cells and working stocks were expanded in BSR-T7 cells for in vivo use after titration on MA104.1 cells. Efficacy after intratumoral injection of 107 PFU/dose CDX-602 alone or in combination with 200 μg/dose αPD-1 blockade (RMP1-14, i.p.) was determined in subcutaneous B16-F10 melanoma and orthotopic EMT6 breast cancer models (n = 10). Changes in the tumor immune infiltrate were characterized using flow cytometry 6 or 8 days after treatment initiation, respectively (n = 5). In the immunotherapy-resistant B16-F10 model, CDX-602 monotherapy led to highly reproducible tumor growth inhibition (TGI, 52%, p &amp;lt; 0.0001 vs vehicle control), resulting in modest, but significant prolongation of median survival (25%, p &amp;lt; 0.01). Anti-tumor efficacy correlated with increased tumor immune infiltration with CD4+ and CD8+ T cells (R2 = 0.68 and 0.67, respectively, p &amp;lt; 0.01). Importantly, frequencies of cross-presenting dendritic cells (cDC) were increased 3.8-fold (p &amp;lt; 0.0001) after virus treatment, while no compensatory influx of CD206+ macrophages was observed, suggesting immune contexture changes conducive to anti-tumor immune induction. Efficacy was confirmed in the EMT6 model, in which monotherapy TGI was 44% and thus on par with αPD-1 blockade (50%, both p &amp;lt; 0.0001 vs control). Combination viroimmunotherapy in this model further improved efficacy, leading to 71% TGI and a doubling in complete regressions to 40%. Inclusion of CDX-602 in the treatment regimen led to similar changes in immune infiltration, in particular recruitment of CD8+ T cells and cDCs (9.7-fold and 6.9-fold increase after combination, respectively). Additional ex vivo analyses to confirm induction of anti-tumor immune responses are ongoing. Taken together, this preclinical data establishes CDX-602 as a novel virotherapeutic candidate that engages multiple mechanisms of action that contribute to anti-tumor activity. CDX-602 is the second virotherapeutic candidate derived from Codagenix’s codon modification platform, further supporting the utility of this synthetic viral engineering technology for viroimmunotherapy of cancer and highlighting its potential across multiple viral species. Citation Format: Nusrat Jahan, Yiwen Zhao, Katarina Blagovic, Ying Wang, J Robert Coleman, Steffen Mueller, Johanna K. Kaufmann. The novel codon-modified Zika virus CDX-602 shows anti-tumor efficacy and remodels the tumor microenvironment in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6660.

Abstract 7229: In situ trans-differentiation therapy effectively terminates the growth and recurrence of glioblastoma

Abstract Background: Glioblastoma (GBM) is the most common and malignant brain tumor in adults, and remains almost invariably lethal due to its aggressive and invasive nature. The standard first-line treatment for GBM is surgical resection, postoperative radiotherapy and chemotherapy with temozolomide (TMZ), but it exhibits short-term effects due to a high recurrence rate of GBM is its (up to almost one hundred percent). This long-existing clinical challenge has demanded development of novel and effective therapeutic strategies. In situ cell trans-differentiation therapy which direct reprograms tumor cells into terminally differentiated benign cells represents a novel therapeutic strategy for tumors. We further explored the application of in situ trans-differentiation therapy for glioblastoma. Results: In the present study, we have conducted a large-scale screen for transcription factors which can reprogram glioblastoma cells into terminally differentiated postmitotic cells. Over-expression of a combo of selected trans-differentiation factors that possess the highest trans-differentiation rate effectively reprogramed human glioma cells into neuronal-like cells with characteristic neuronal morphology and expression of neuronal genes in culture, as a result substantially reducing glioblastoma cell proliferation and tumor development after orthotopic transplantation. With replicable recombinant oncolytic virus vectors, product candidate can specifically replicate in glioma cells, therefore transcription factors can be specifically delivered to all tumor cells rather than normal cells. In either cell line-derived or patient-derived tumor tissue xenograft models, intra-tumoral injection product candidate inhibited glioblastoma cells growth at an exceptionally high efficiency. Remarkably product candidate combination with TMZ resulted in a complete regression of tumor and no recurrence was observed in animal model cases. Conclusion: In summary, our findings have established a novel therapeutic approach for treating glioblastoma by terminating sustained proliferation of glioma cells via transcription factors-induced in situ cell trans-differentiation and supported further clinic investigations in patients. Citation Format: Huihui Zhao, Zhaozhong Li, Xiaohui Zhang, Congjian Zhao, Zhao Zhu, Yueguang Liu. In situ trans-differentiation therapy effectively terminates the growth and recurrence of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7229.