Work

1047 Alternative splicing-derived tumor-specific antigens as putative immunotherapeutic targets for glioblastoma

Year: 2026

Type: conference-abstract

Abstract: Background Glioblastoma (GBM) remains refractory to targeted immunotherapy because its tumor antigens are heterogeneous, variably expressed, and susceptible to immune escape. Alternative splicing is a non-mutational source of MHC-I ligands and is broadly dysregulated in cancer. In GBM, phosphoproteomic and transcriptional profiling revealed that splicing programs may be coupled to tumor development, making resulting splicing derived antigens potentially less vulnerable to immune editing. Here, we sought to identify naturally presented, recurrent, tumor-selective splicing-derived antigens (SDAs) as translational candidates for MHC-I antigen-directed immunotherapy.Methods We integrated spatially resolved phosphoproteomics, bulk RNA-seq alternative-splicing analysis, and LC-MS/MS immunopeptidomics across murine GBM models, human GBM patient-derived xenografts (PDX), and primary patient tumors. GBM-enriched skipped exons, alternative splice sites, and retained introns were translated into custom databases containing tumor-enriched sequence fragments and flanking residues. Candidate SDAs were identified from MHC-I immunopeptidomes, prioritized by population coverage, manual spectral validation, MHC binding predictions, and tumor enrichment, then confirmed using SureQuant targeted mass spectrometry against normal tissues or paired PBMC controls. Murine immunogenicity was tested using circular mRNA vaccination.Results Phosphoproteomics revealed spatial and temporal enrichment of RNA-metabolism pathways, spliceosomal factors activation, and SRPK-associated signaling during GBM progression, providing a mechanistic rationale for SDA discovery. In murine GBM, 159 SDAs were identified, including predicted H2-Kb/H2-Db binders, where 8 prioritized peptides were validated by targeted MS, and 4 of which were GBM-restricted across normal tissue panels. Circular mRNA vaccination induced antigen-specific T-cell responses in GL261 and CT2A tumor-bearing mice and significantly delay in CT2A tumor growth. In human analyses, 251 SDAs were detected across 10 GBM PDX lines and 405 across 16 primary GBM tumors. Targeted validation confirmed recurrent and tumor specific presentation of several SDAs in HLA-restricted populations.Conclusions This integrated discovery and validation workflow demonstrates that GBM-associated alternative splicing generates naturally presented and broadly applicable MHC-I antigens, many arising from tumor enriched splice isoforms. Validated SDAs show tumor selectivity, HLA-restricted sharing, and in vivo immunogenicity, supporting translation into off-the-shelf or semi-personalized immunotherapy targets. Splicing-derived antigen offer a new pool of targets linked to GBM progression programs, expanding therapeutic options for tumors with limited actionable neoantigens.

Source: Journal for ImmunoTherapy of Cancer

DOI: 10.1136/jitc-2026-sitc2026.1047