Public tumor-wide neoantigens derived from clonal aberrant splicing events drive tumor-specific t-cell responses

Abstract BACKGROUND Immunotherapy in gliomas is limited by tumor heterogeneity and low mutational burden. We developed a novel comprehensive in silico pipeline for detecting tumor-specific splicing events (neojunctions) across multiple cancer types, and in gliomas, we successfully identified tumor-wide, public, alternatively spliced neoantigens (ASNs) that elicit CD8+ T-cell-mediated immune responses. METHODS Our pipeline identified recurring public neojunctions expressed in TCGA RNA-seq (positive sample rate (PSR) > 10%) and not in GTEx normal tissue RNA-seq data (PSR < 1%) across six cancer types. To characterize intratumorally-conserved neojunctions, we utilized multi-site RNA sequencing, and with gliomas, we analyzed approximately 10 maximally-distanced intratumoral sites within 56 patients (n=535). Tumor-wide public neojunction expression was subsequently validated in RNA-sequencing and mass spectrometry (MS) data from patient-derived cell lines (n=68) and samples (n=99). Two independent algorithms then predicted peptide processing likelihood and HLA-binding affinity of ASN candidates. In vitro sensitization (IVS) of healthy donor-derived CD8+ T-cells against high-confidence ASN candidates, followed by 10x VDJ scRNA-seq, was performed to identify ASN-specific TCR sequences. TCRs were transduced into triple-reporter Jurkat76s and co-cultured with ASN and HLA-expressing COS7 cells and glioma cell lines to evaluate TCR functionality. RESULTS Our glioma-specific analysis identified 249 public neojunctions with varying intratumoral heterogeneity. 4 ASNs were concurrently identified in transcriptomic and proteomic glioma data and predicted to be presented by HLA-A*02:01 with high confidence. IVS and subsequent 10x VDJ scRNA-seq on expanded CD8+ populations identified TCR clonotypes reactive against neojunctions in RPL22 (n=7) and GNAS (n=1), the latter being highly intratumorally-conserved (detected in > 90% of spatially-mapped biopsies across 17/56 patients (26.78%)). TCR-transduced T-cells demonstrated recognition and immunogenic activation against endogenously processed and presented neoantigens in glioma and transfected COS7 cells. CONCLUSION Our unique integrative pipeline identified novel public tumor-wide splice-derived neoantigen candidates and TCRs, offering a promising off-the-shelf immunotherapy approach for gliomas.