Abstract 845: Development of enhanced extracellular vesicle-based canine osteosarcoma cancer vaccines via metabolic labeling and click chemistry approach

Abstract Introduction: Osteosarcoma, characterized by its high metastatic potential, remains a prevalent bone malignancy in canines. Current therapeutic approaches, including amputation and chemotherapy, demonstrate limited efficacy and may compromise the patient's quality of life. This study explores a novel immunotherapeutic strategy utilizing metabolically labeled extracellular vesicles (EVs) conjugated with toll-like receptor (TLR) agonists as a potential cancer vaccine for canine osteosarcoma. The approach involves a facile and universal metabolic labeling technique to introduce azido groups onto the surface of mammalian cells, including cancer cells. Subsequently, nanosized EVs released from these cells inherit the azido groups, enabling conjugation with TLR agonists via click chemistry. Upon internalization by dendritic cells (DCs), the conjugated EVs are hypothesized to undergo lysis, facilitating antigen processing and presentation to effector T cells, while simultaneously activating DCs through TLR stimulation. This dual-action mechanism aims to promote a robust cytotoxic T lymphocyte (CTL) response against osteosarcoma cells. Methods: In this study, peripheral blood mononuclear cells (PBMCs) were isolated from canine donor blood samples. CD14+ monocytes were subsequently purified using magnetic-activated cell sorting (MACS) with microbeads. The isolated monocytes were cultured for 6 days in the presence of human granulocyte-macrophage colony-stimulating factor (GM-CSF) and canine recombinant interleukin-4 (IL-4) to induce differentiation into monocyte-derived dendritic cells (moDCs). For the conjugation with EVs, a panel of toll-like receptor (TLR) agonists was selected, comprising imiquimod (TLR7 agonist), motolimod (TLR8 agonist), CpG oligodeoxynucleotides (TLR9 agonist), and polyinosinic:polycytidylic acid (poly(I:C), TLR3 agonist). Results and Conclusion: The selected TLR agonists were successfully conjugated with EVs. Notably, EVs conjugated with imiquimod demonstrated significantly enhanced activation of moDCs compared to EVs alone, imiquimod alone, or a simple mixture of EVs and imiquimod. These findings suggest that the conjugation of TLR agonists, particularly imiquimod, with EVs may potentiate the immunostimulatory effects on moDCs. This approach shows promise for the development of a novel immunotherapeutic strategy against canine osteosarcoma. In vivo evaluation of the vaccine's efficacy is currently in preparation, which will provide crucial insights into its potential clinical application. Citation Format: Yuan Liu, Yusheng Liu, Matthew R. Berry, Hua Wang. Development of enhanced extracellular vesicle-based canine osteosarcoma cancer vaccines via metabolic labeling and click chemistry approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 845.