P08.13.a Identification of Vaccine-Induced TCRS Against Glioblastoma-Associated Antigens

BACKGROUND TCR-engineered T cell (TCR-T) therapy holds significant promise in cancer treatment, allowing precise targeting of intracellular antigens. However, its application in neuro-oncology, particularly in glioblastoma (GB), faces unique challenges. In addition to the restrictive nature of the blood-brain barrier and a profoundly immunosuppressive tumor microenvironment, the comparatively low mutational burden of GB limits the availability of immunogenic targets, collectively hampering the success of immunotherapy in primary brain tumors. Despite these challenges, recent vaccine studies have demonstrated that both tumor-specific neoantigens and non-mutated, overexpressed glioma-associated antigens (GAAs) can induce robust and durable T cell responses in patients with GB. In tumors exhibiting high clonal heterogeneity and a limited number of shared mutations, the ability to exploit a broader antigenic repertoire may be critical for therapeutic success. To broaden the clinical applicability of TCR-T cell therapy for GB, there is an urgent need to develop TCRs targeting diverse GAAs across multiple HLA backgrounds. MATERIAL AND METHODS To this end, we aimed to identify and functionally validate GAA-specific TCRs from vaccinated GB patients. PBMCs from a single patient enrolled in the GAPVAC-101 vaccine trial were expanded in vitro using peptide pools spanning immunogenic GAA epitopes. Following peptide-specific expansion, GAA-reactive T cells were enriched by FACS and subjected to single-cell V(D)J sequencing to retrieve paired TCR α/β chain information. Dominant clonotypes were selected based on frequency, and corresponding TCRs were cloned into a lentiviral backbone via Golden Gate Assembly. The cloned TCRs were expressed in CD4⁺/CD8⁺ Jurkat reporter cells for functional evaluation. Following TCR expression, Jurkat cells were co-cultured overnight with patient-derived B-lymphoblastoid cell lines (B-LCLs) to assess TCR reactivity. Reactivity was evaluated the next day by flow cytometry through staining for CD69 surface-expression and analysis of NFAT-GFP reporter activity. RESULTS Analysis of the TCR repertoire from peptide-expanded, FACS-sorted CD4⁺ and CD8⁺ T cells revealed an oligoclonal pattern, consistent with antigen-driven expansion. Utilizing material from the GAPVAC-101 trial, we identified both CD4⁺ and CD8⁺ TCRs reactive to GAAs. Selected TCRs were successfully cloned and lentivirally expressed in Jurkat reporter cells, where they exhibited antigen-specific activation in vitro, confirming functionality. CONCLUSION These findings demonstrate the feasibility of isolating functional, GAA-specific TCRs from vaccinated GB patients. While further validation in vitro and in vivo is necessary, this approach provides a foundation for expanding the repertoire of TCRs available for personalized TCR-T cell therapy in GB.