Year: 2026
Type: conference-abstract
Abstract: Background For osteosarcoma patients with metastatic disease, the 5-year survival rate remains below 40%, underscoring the need for novel and more effective therapies. 1 While immunotherapy holds promise, efficacy has been limited by the tumor immunosuppressive microenvironment and lack of well-defined tumor targets. Endogenous virus specific T cells (VSTs) have been found as tumor-infiltrating lymphocytes (TILs) in multiple solid tumors.2–4 We hypothesize that VSTs are similarly present in osteosarcoma and can be repurposed for antitumor activity. To redirect VSTs against osteosarcoma, we propose delivering viral antigens and immune stimulation at the tumor site using CAdVEC, a clinically used binary platform that combines an oncolytic adenovirus (OAd) with a helper-dependent virus (HD) which has a cargo capacity of up to 34 kb, allowing for co-expression of multiple transgenes.5–7Methods To confirm the presence of VSTs in osteosarcoma tumors, we evaluated osteosarcoma patient-derived TILs for Epstein-Barr Virus (EBV) specific responses using interferon-gamma (IFN-γ) Enzyme-Linked Immunospot (ELISpot). To test whether TILs could be redirected for anti-tumor effects, green fluorescent protein (GFP) expressing osteosarcoma 3D spheroids were infected with either the current clinical HD expressing immunostimulatory cytokine IL-12 and immune checkpoint inhibitor α-PD-L1 (HD_Trio), or a modified version additionally expressing EBV antigens (HD_EBV_Trio). TILs were added to partially HLA-matched tumor spheroids for co-culture. Live fluorescence imaging was obtained via IncuCyte and anti-tumor activity was quantified by loss of GFP. Spheroids were dissociated for flow cytometry and T cell counts were quantified as CD3 +/7AAD- population.Results Our results confirm that functional EBV specific T cells (EBVSTs) are present in 6 of 7 TIL samples from primary and metastatic osteosarcoma lesions. When compared to uninfected spheroids, infection with HD_Trio or HD_EBV_Trio resulted in TIL mediated anti-tumor effect as evidenced by loss of green fluorescence over time (fold difference: 0.35 and 0.19 respectively, p < 0.0001 for both). Loss of green fluorescence was not observed in spheroids infected with virus in the absence of effector cells. We also observed that co-culturing TILs with HD_EBV_Trio infected osteosarcoma spheroids increased T cell numbers by 1.5 fold compared to uninfected spheroids.Conclusions Overall, we confirmed functional EBVSTs are present amongst osteosarcoma TILs. These patient-derived TILs have anti-tumor activity against osteosarcoma cells infected with HD_EBV_Trio for both EBV antigen presentation and immune stimulation. We are now incorporating the OAd component of the binary CAdVEC to amplify the anti-tumor activity of HD_EBV_Trio and to evaluate this approach in vivo.8References Kar E, Ammanamanchi A, Yousif M, Geetha SD, Schwartz K, Mishra AS, et al. From bimodal to unimodal: The transformed incidence of osteosarcoma in the United States. J Bone Oncol. 2024 June 6;47:100613.Simoni Y, Becht E, Fehlings M, Loh CY, Koo SL, Teng KWW, et al. Bystander CD8+ T cells are abundant and phenotypically distinct in human tumour infiltrates. Nature 2018 May 1;557(7706):575–9.Rosato PC, Wijeyesinghe S, Stolley JM, Nelson CE, Davis RL, Manlove LS, et al. Virus-specific memory T cells populate tumors and can be repurposed for tumor immunotherapy. Nat Commun. 2019 Feb 4;10(1):567.Leen AM, Bollard CM, Mendizabal AM, Shpall EJ, Szabolcs P, Antin JH, et al. Multicenter study of banked third-party virus-specific T cells to treat severe viral infections after hematopoietic stem cell transplantation. Blood 2013 June 27;121(26):5113–23.Rosewell Shaw A, Porter CE, Watanabe N, Tanoue K, Sikora A, Gottschalk S, et al. Adenovirotherapy Delivering Cytokine and Checkpoint Inhibitor Augments CAR T Cells against Metastatic Head and Neck Cancer. Mol Ther. 2017 Nov 1;25(11):2440–51.Farzad L, Cerullo V, Yagyu S, Bertin T, Hemminki A, Rooney C, et al. Combinatorial treatment with oncolytic adenovirus and helper-dependent adenovirus augments adenoviral cancer gene therapy. Mol Ther Oncolytics. 2014 Dec 17;1:14008.Wang D, Porter CE, Lim B, Rosewell Shaw A, Robertson CS, Woods ML, et al. Ultralow-dose binary oncolytic/helper-dependent adenovirus promotes antitumor activity in preclinical and clinical studies. Sci Adv. 2023;9(13):eade6790.Talbot LJ, Chabot A, Funk A, Nguyen P, Wagner J, Ross A, et al. A novel orthotopic implantation technique for osteosarcoma produces spontaneous metastases and illustrates dose-dependent efficacy of B7-H3-CAR T cells. Front Immunol. 2021 June 15;12.
Language: English
Subfield: Genetics
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $2,490
OpenAlex ID: W7225307221