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Background Advanced solid tumors remain a significant clinical challenge due to their heterogeneity and immunosuppressive tumor microenvironment, which undermines the efficacy of approved therapies. Building on promising clinical trial outcomes with the oncolytic adenovirus (OA) Delta-24-RGD in patients with recurrent glioblastoma, 1 we previously reported that Delta-24-RGDOX, using Delta-24-RGD as a vector to express the immune co-stimulatory molecule OX40 ligand (OX40L), amplifies both local and systemic anti-tumor immunity through an in situ autovaccination effect in immunocompetent mice with orthotopic intracranial (i.c.) gliomas and disseminated subcutaneous (s.c.)/i.c. melanomas.2–4 Since IL-15 enhances NK and T cell activation and supports the persistence of CD8+ memory T cells, to further improve OA-mediated anti-tumor immunity, we developed Delta-24-RGDOX-IL15, a novel OA co-expressing IL-15 and OX40L.Methods Murine GD2 CAR T cells were generated by transduction of mouse T cells with retrovirus encoding the mouse CAR. Oncolytic activity of T cells was assessed using a luciferase-based assay in co-cultures of T cells and cancer cells expressing luciferase and corresponding T cell targets. Therapeutic effect was evaluated in C57BL/6 mice bearing syngeneic GD2-expressing solid tumors. Flow cytometry was employed to analyze the expression of cell surface markers.Results Delta-24-RGDOX-IL15 retained strong viral replication and oncolytic activity, while more effectively stimulating T cell responses, including that of CAR T cells, against tumor cells expressing corresponding tumor-associated antigens. In mice with syngeneic GD2-positive tumors (i.c. gliomas derived from DMG 26C-2 cells or disseminated s.c./i.c. melanomas derived from B16-GD2-Luc cells), intratumoral administration of Delta-24-RGDOX-IL15 into the i.c glioma or s.c. melanoma significantly reduced the frequency of microglia and myeloid cell frequency while increasing T cell frequency and activation of NK, dendritic, B and T cells within the tumor-bearing brain hemisphere from the mice of the two mouse models ( figures 1 and 2). Importantly, combination treatment of the virus with GD2 CAR T cells led to increased CAR T cell infiltration into both treated and distant, untreated brain tumors, resulting in long-term survival in 20% of mice with s.c./i.c. melanomas. Optimization of dosing regimens is ongoing in both tumor models to further improve outcomes.Conclusions These findings highlight the potent immunostimulatory and therapeutic properties of Delta-24-RGDOX-IL15 and support its potential to synergize with CAR T cell therapy for the treatment of advanced primary or metastatic brain tumors.References Lang FF, et al. Phase I study of DNX-2401 (delta-24-RGD) oncolytic adenovirus: replication and immunotherapeutic effects in recurrent malignant glioma. J Clin Oncol. 2018;36(14):1419–1427.Jiang H, et al. Oncolytic adenovirus and tumor-targeting immune modulatory therapy improve autologous cancer vaccination. Cancer Res. 2017;77(14):3894–3907.Jiang H, et al. Localized treatment with oncolytic adenovirus delta-24-RGDOX induces systemic immunity against disseminated subcutaneous and intracranial melanomas. Clin Cancer Res. 2019;25(22):6801–6814.Jiang H, et al. Adjuvant therapy with oncolytic adenovirus delta-24-RGDOX after intratumoral adoptive T-cell therapy promotes antigen spread to sustain systemic antitumor immunity. Cancer Res Commun. 2023;3(6):1118–1131.Abstract 308 Figure 1Immune landscape within the TME of i.c. glioma derived from 26C-2 DMG cells. A, Schedule and route of tumor implantation and treatments. B, The immune cells from the brain hemispheres with tumor were profiled with flow cytometry. Shown are tSNE plots (20,000 events/group) (PBS, CAR T: n = 5; RGDOX-IL15, combination: n = 4). C and D, the frequency and density of the indicated cell populations at the tumor site. Note that Delta-24-RGOX-IL15 caused the microglia and myeloid cells frequency dramatically decreased while the density of T cells, including CAR T cells, greatly increased within the tumor. RGDOX-IL15: Delta-24-RGDOX-IL15Abstract 308 Figure 2Immune landscape within the TME of brain tumor derived from B16-GD2-Luc melanoma cells. A, Schedule and route of tumor implantations and treatments. B, The immune cells from the brain hemispheres with tumor were profiled with flow cytometry. Shown are tSNE plots (20,000 events/group) of the immune cells from indicated tumors of treatment groups (PBS, RGDOX-IL15 : n = 5; CAR T, combinaation: n = 7). C and D, the frequency and density of the indicated cell populations at the tumor site. Note that Delta-24-RGOX-IL15 caused the microglia and myeloid cell frequency decreased while the density of T cells, including CAR T cells, greatly increased within the tumor. RGDOX-IL15: Delta-24-RGDOX-IL15