Abstract 3152: Targetable public neoantigens are generated by tumor-wide cancer-specific splicing events in gliomas

Abstract

BACKGROUND: The efficacy of immunotherapy in gliomas is limited by tumor heterogeneity and low mutational burden. Recent studies revealed increased alternative splicing (AS) in various cancer types that potentially translate into targetable neoantigens. We developed a novel comprehensive in silico pipeline for detecting tumor-specific AS events (neojunctions), and successfully identified tumor-wide, public, alternatively spliced neoantigens (ASNs) that elicited CD8+ T-cell-mediated immune responses. METHODS: Our computational pipeline first identified recurring splice junctions in high purity TCGA LGG/GBM bulk RNA-seq samples (n=429, PSR > 10%) and not in GTEx normal tissue bulk RNA-seq data (n=9166, PSR < 1%). We subsequently employed two independent algorithms to predict the peptide processing likelihood and the HLA-binding affinity of ASN candidates before validating neojunction and ASN expression in RNA-seq data from patient-derived glioma cell lines (n=68) and spatially-mapped glioma samples (n=535), and in the Clinical Proteomic Tumor Analysis Consortium (CPTAC) GBM mass spectrometry data (n=99). In vitro sensitization of healthy donor-derived CD8+ T-cells against our top ASN candidates followed by 10x VDJ scRNA-seq was performed to identify ASN-specific TCR sequences. We transduced these TCRs into Jurkat76s and co-cultured them with ASN-pulsed T2 cells and ASN and HLA-expressing COS7 cells to determine whether the TCR recognized the presented HLA:ASN. RESULTS: Our analysis identified 249 putative neojunctions that translated into 222 cancer-specific peptides sequences. Iterating across these peptides yielded 17,562 glioma-specific n-mer sequences between 8 to 11 amino acids in length. Both prediction algorithms concurrently identified 636 n-mers likely to be presented by demographically common HLA haplotypes. Through transcriptomic and proteomic validation with patient-derived samples and cell lines, we identified 8 ASNs that were tumor-wide, 4 of which were predicted to be HLA*A0201-presented. In vitro sensitization of healthy-donor derived CD8+ T-cells against these 4 ASNs resulted in an expansion of reactive CD8+ T-cells against ASNs derived from neojunctions within S100A6, RPL22, and GNAS. Subsequent 10x VDJ scRNA-seq on the expanded CD8+ population identified mutRPL22-reactive TCR clonotypes with neoantigen-mediated increases in IFNG and GZMB signatures. When transduced and expressed in Jurkat76 cells, one particular TCR clonotype demonstrated recognition and immunogenic activation against mutRPL22-pulsed T2 cells and COS7 cells endogenously expressing both HLA*A0201 and mutRPL22. CONCLUSION: Our unique integrative pipeline detected novel tumor-wide splice-derived neoantigen candidates, and ASN-reactive TCRs identified through our pipeline offer a new avenue for TCR-based therapies across cancer types. Citation Format: Darwin W. Kwok, Takahide Nejo, Nicholas Stevers, Lee H. Chen, Kaori Okada, Chibo Hong, Gary K. Chan, Akane Yamamichi, Aidan Du, Maggie Colton, James Woo, Joseph Costello, Hideho Okada. Targetable public neoantigens are generated by tumor-wide cancer-specific splicing events in gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3152.