Background
High tumor heterogeneity and low mutational burden in cancers pose significant challenges for immunotherapy. To address this, we developed a novel in silico pipeline to characterize cancer-specific splicing events (neojunctions) expressed ubiquitously across ten cancer types. This approach successfully identified tumor-wide, public, alternatively splicing neoantigens (ASNs) that elicit CD8+ T-cell-mediated cytotoxicity across multiple cancers.
Methods
Our SSNIP pipeline identified recurring public neojunctions expressed in TCGA RNA-seq data (positive sample rate (PSR) > 10%) but not in GTEx normal tissue RNA-seq data (PSR < 1%) across 10 cancer types. We utilized multi-site RNA sequencing data to characterize intratumorally conserved neojunctions. Our in-house glioma dataset included 56 patients with approximately 10 spatially distinct intratumoral biopsy sites per patient (n=535). Two independent algorithms predicted peptide processing likelihood and HLA-binding affinity of ASN candidates. In vitro sensitization (IVS) of CD8+ T-cells from healthy donors against high-confidence ASN candidates, followed by 10x VDJ scRNA-seq, identified ASN-specific TCR sequences. These TCRs were transduced into triple-reporter Jurkat76 cells and co-cultured with ASN and HLA-expressing COS7 cells and glioma cell lines to evaluate TCR functionality. TCR-transduced donor-derived CD8+ T-cells were cultured against neojunction-expressing glioma and melanoma cell lines to evaluate tumor-specific killing.
Results
Our pipeline identified 789 public neojunctions, with 32 neojunctions concurrently identified in transcriptomic and proteomic data and predicted to be presented by HLA-A*02:01 with high confidence. We captured 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%)). Mutant GNAS-derived ASN-specific CD8+ T-cell clones were detectable from glioma patient PBMC by IVS. TCR-transduced T-cells demonstrated recognition and tumor-specific killing against endogenously processed and presented ASNs in multiple glioblastoma and melanoma cell lines. Furthermore, IDH1-mutant oligodendroglioma samples demonstrated significantly elevated expression of neojunctions over IDH1-mutant astrocytoma and IDH1wt subtypes. Differential gene expression (DESeq2) identified decreased expression of splicing factors due to oligodendroglioma-specific co-deletion of Chromosomes 1p/19q. siRNA knockdown of these splicing factors (e.g. SF3A3, SNRPD2) in IDH1wt glioma cells resulted in significantly increased expression of corresponding neojunctions. Performing DESeq2 on the remaining cancer types revealed similar splicing gene set-associated differences in neojunction expression across hepatocellular carcinoma and lung adenocarcinoma iCluster subtypes.
Conclusions
SSNIP identified novel public tumor-wide splice-derived neoantigen candidates and ASN-specific TCRs, offering a promising off-the-shelf immunotherapy approach for diverse cancer types. Characterization of intratumorally conserved neoantigens addresses the critical challenge of intratumoral heterogeneity in immunotherapy resistance.