OS05.5.A RADIAL GLIAL CELLS AS SOURCE ANTIGEN FOR ADOPTIVE CELLULAR THERAPY IN GLIOBLASTOMA Article Swipe

BACKGROUND Glioblastoma (GBM) is a highly lethal brain cancer with limited treatment options and poor long-term survival. Adoptive cellular therapy (ACT) has shown promise in targeting GBM, but many ACT strategies rely on patient tumor tissue to derive tumor-specific antigens, limiting their use in inoperable tumors. Further, tumor heterogeneity—primarily driven by glioma stem cells (GSCs) that survive standard treatments—complicates recurrence. Radial glial cells (RGCs), neurodevelopmental progenitors transcriptionally similar to a subpopulation of GSCs, express developmental antigens (DAs) also found in GBM. We hypothesized that RGCs harbor immunogenic antigens shared with GBM and can serve as a renewable, tumor-independent antigen source for ACT to target GSC cells. MATERIAL AND METHODS Using single-cell RNA sequencing, we profiled RGCs cultured from post-natal day 4 (P4) murine cortices and compared their transcriptomes with tumors from murine GBM model KR158B. Shared antigens were screened for immunogenicity using our O.R.A.N. antigen prediction and validation platform. We developed a cDNA antigen library from identified RGC immunogens and used this to generate in vitro transcribed RNA for ACT. T cells were expanded ex vivo using dendritic cells (DCs) pulsed with RGC RNA or total tumor RNA (ttRNA) and tested for cytokine production and tumor-specific killing. These RGC T cells were incorporated into our ACT platform alongside hematopoietic stem cell rescue and serial DC vaccinations. Survival was assessed in GBM-bearing mice. We also tested human RGC antigens against patient-derived xenograft (PDX) GBM models using human T cells and 3D bioprinted immune-tumor interaction assays. RESULTS scRNA-seq analysis revealed a subset of immunogenic antigens shared between RGCs and GBM tumors. RGC T cells demonstrated robust IFNγ and TNFα release, and selectively killed tumor cells in vitro. In vivo, ACT incorporating RGC T cells significantly prolonged survival (p=0.0058) in KR158B GBM murine model, matching the efficacy of ttRNA ACT. Preliminary toxicity studies revealed no overt signs of neurotoxicity or systemic inflammation. In human models, RGC T cells recognized and killed a smaller subset of GBM PDX cells when compared to ttRNA T cells, but showed enriched targeting of GSC-like subpopulations in 3D bioprinted assays. CONCLUSION RGC-derived antigens represent a potent source of targets for ACT in GBM that can be used to generate a renewable antigen library. This library bypasses the need for tumor resection while maintaining therapeutic efficacy. It offers a novel approach to overcoming initiation of tumor heterogeneity through targeting of neurodevelopmental cell types.