Document resource
Background Ewing sarcoma (ES) is the second most common pediatric bone cancer, characterized by the EWSR1-FLI1 gene fusion in 85% of cases which drives oncogenesis. Survival beyond first-line therapy is dismal and innovative therapeutics are lacking. 1 Our mRNA lipid particle aggregate (LPA) vaccine targeting total tumor lysate has produced promising preclinical and clinical results.2 We aim to describe the immunogenicity of our vaccine platform when targeting the EWSR1-FLI1 fusion oncoprotein.Methods A sarcoma cell line was transduced with the EWSR1-FLI1 fusion, cultured, and enriched for fusion expression via green fluorescent protein signaling. Tumor cells were cultured at 37C on a shaker plate to form spheroidic cell clusters (spheroids). 3 Three naïve BALB/c mice were vaccinated with total tumor or fusion targeted mRNA-LPA x3 every 2 days. Splenocytes were harvested and isolated from red blood cells and stained for imaging identification. Spheroids were printed into a T cell media/hydrogel matrix with subsequent printing of the harvested splenocytes circumferentially around the spheroid. Spheroid and splenocyte positioning were recorded under confocal microscopy to produce time-lapse imaging over 72 hours with proper controls. Experimentation was performed in triplicates. Supernatant was collected for interferon-gamma (IFN-γ) analysis via ELISA.Results Time-lapse imaging illustrated a radical change in splenocyte positioning with evidence of splenocyte and spheroid co-localization after 72 hours in co-culture while initial positioning did not overlap. Remarkably, time-lapse imaging conveys radical tumor destruction over time. The median IFN-γ release in the group treated with fusion-targeted vaccinated splenocytes was almost 3x that of the group treated with unvaccinated splenocytes (219 pg/ml vs. 622 pg/ml, p=0.11).Conclusions Splenocytes trained with the fusion-targeted mRNA-LPA vaccine in naïve mice effectively produced an adaptive immune response against fusion positive tumor spheroids. Further quantification of immune cell mobilization and identification of targeted antibodies will be performed. This data provides crucial evidence to support FDA application for an investigational new drug for clinical translation toward targeting tumor-driving fusion oncoproteins via mRNA-LPA vaccination.References Womer RB, West DC, Krailo MD, et al. Randomized controlled trial of interval-compressed chemotherapy for the treatment of localized Ewing sarcoma: a report from the Children’s Oncology Group. J. Clin. Oncol. 2011;30:4148–4154.Mendez-Gomez HR, DeVries A, Castillo P, et al. RNA aggregates harness the danger response for potent cancer immunotherapy. Cell. 2024;187: 2521–2535.Masiello T, Dhall A, Hemachandra LPM, et al. A dynamic culture method to produce ovarian cancer spheroids under physiologically-relevant shear stress. Cells. 2018;7(12):277.