C/EBPβ-induced alternative splicing of RCAN1 generates a potent TCR-T target in mesenchymal glioblastoma

Glioblastoma (GBM) is an aggressive brain tumor with limited treatment options and a dismal prognosis. While immunotherapy has shown promise in treating some solid tumors, the treatment of GBM has been mostly unsuccessful because of a lack of targetable tumor antigens and high tumor heterogeneity. Here, we report RCAN1-4 as a novel tumor antigen derived from alternative splicing induced by the transcription factor C/EBPβ. Both C/EBPβ and RCAN1-4 are highly expressed in GBM and glioma stem cells as mesenchymal subtype hallmarks. We report an immunogenic HLA-A24-specific splicing junction epitope within exon 4 and exon 5 that is unique to RCAN1-4. This epitope was validated for its ability to stimulate T cell responses in HLA-A24⁺ donors and GBM patients, leading us to identify RCAN1-4-reactive T cell receptors (TCRs) for the construction of TCR-engineered T cells (TCR-T cells). Functional studies of TCR-Ts demonstrated the in vitro and in vivo killing of RCAN1-4^pos GBM tumor cells, highlighting its potential as an immunotherapeutic target in mesenchymal GBM.