Tumor-wide RNA splicing aberrations and their potential as therapeutic neoantigen targets.

2519 Background: Tumor heterogeneity and low mutational burden limits the availability of effective immunotherapy targets. Aberrant RNA-splicing (neojunctions) represents an underexplored yet promising source of neoantigens. To address this, we developed a neoantigen discovery platform (SNIPP) that characterizes a novel class of clonally-expressed, splicing-derived neoantigens. Furthermore, we validated the immunogenicity of these neoantigens by identifying specific TCRs that drive CD8+ T-cell-mediated tumor killing. Methods: SNIPP identified public neojunctions by analyzing TCGA RNA-seq data, selecting neojunctions with a positive sample rate (PSR) > 10% and filtering out those found in GTEx normal tissue RNA-seq data (PSR < 1%) across 12 cancer types. To characterize intratumorally conserved neojunctions, we performed maximally-distanced multi-site biopsies ( n = 535) within glioma patients ( n = 56) and generated RNA-seq data for each intratumoral site. Two independent algorithms were utilized to predict peptide processing likelihood and HLA-binding affinity of splicing-derived neoantigen candidates. Neoantigen-specific TCR sequences were identified via in vitro sensitization of PBMCs and subsequent 10x V(D)J scRNA-seq. These TCRs were transduced into CD8+ T-cells, which were tested downstream for immunogenicity and cytotoxicity against glioma cell lines. Results: Our pipeline identified 789 public neojunctions, including 32 neojunctions concurrently detected in transcriptomic and proteomic glioma datasets and confidently predicted to be presented by HLA-A*02:01. IVS and subsequent 10x V(D)J scRNA-seq identified TCR clonotypes reactive against neojunctions in RPL22 ( n = 7) and GNAS ( n = 1), with the latter exhibiting high intratumoral conservation (detected in > 90% of spatially-mapped biopsies across 17/56 patients (26.78%)). TCR-transduced CD8+ T-cells recognized and were immunogenically activated and demonstrated cytotoxicity against endogenously processed and presented neoantigens in GBM and melanoma lines. Additionally, IDH1-mutant oligodendrogliomas exhibited significantly higher neojunction expression compared to IDH1-mutant astrocytomas and IDH1wt subtypes. Differential gene expression analysis (DESeq2) revealed reduced expression of splicing factors in oligodendrogliomas, attributed to their specific co-deletion of chromosomes 1p and 19q. CRISPRi-mediated knockdown of these splicing factors (e.g. SF3A3, SNRPD2) in IDH1wt glioma cells resulted in significantly increased expression of corresponding neojunctions. Conclusions: Our study highlights a novel class of neoantigens derived from tumor-wide aberrant RNA splicing. The SNIPP platform effectively identifies public intratumorally-conserved neojunctions with strong therapeutic potential. Furthermore, elevated neojunction expression in oligodendroglioma underscores the mechanistic link between dysregulated splicing factor expression and RNA splicing abnormalities.