Abstract 3907: Novel public and tumor-wide neoantigens arising from clonal aberrant splicing events drive tumor-specific T-cell responses across diverse cancer types

Abstract BACKGROUND: Immunotherapy in gliomas is limited by intratumor heterogeneity (ITH) and low mutational burden. We developed a novel comprehensive in silico pipeline for detecting tumor-specific splicing events (neojunctions), and across multiple cancer types, we successfully identified a new class of 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 ten cancer types. To characterize tumor-wide neojunctions, we utilized available multi-site RNA sequencing across diverse cancer types. With gliomas, our in-house dataset comprised of 56 patients with approximately 10 maximally-distanced intratumoral biopsy sites per patient (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=447). Two independent algorithms then predicted peptide processing likelihood and HLA-binding affinity of ASN candidates. TCRs identified by in vitro sensitization (IVS) of donor-derived CD8+ T-cells followed by 10x VDJ scRNA-seq, were transduced into CD8+ T-cells and co-cultured with tumor cells to evaluate TCR functionality and cytotoxicity. RESULTS: Pan-cancer analysis identified large subsets of neojunctions that were interpatiently and intratumorally conserved. Our glioma-specific analysis identified 789 public neojunctions, and 8 ASNs were validated 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 PBMC-derived CD8+ populations cultured against ASN-pulsed dendritic cells 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 in 17/56 patients (26.78%)). TCR-transduced T-cells demonstrated recognition and immunogenic activation against endogenously processed and presented neoantigens in both pan-cancer cell lines and transfected COS7 cells and cytotoxicity against glioma cell lines. Pan-cancer analysis revealed the detection of both neojunctions in various tumor types beyond gliomas, with the GNAS neojunction notably found tumor-wide in mesothelioma, prostate cancer, and liver hepatocellular carcinoma spatial data. CONCLUSION: Our unique integrative pipeline identified a novel class public tumor-wide splice-derived neoantigen candidates and ASN-specific TCRs, offering a promising off-the-shelf immunotherapy approach for diverse cancer types and tackling the critical challenge of ITH in immunotherapy resistance. Citation Format: Darwin Kwok, Nicholas O. Stevers, Takahide Nejo, Lee H. Chen, Inaki Etxeberria, Jangham Jung, Kaori Okada, Maggie Colton Cove, Senthilnath Lakshmanachetty, Marco Gallus, Abhilash Barpanda, Chibo Hong, Gary KL Chan, Samuel H. Wu, Emilio Ramos, Akane Yamamichi, Jerry YZ Liu, Payal Watchmaker, Hirokazu Ogino, Atsuro Saijo, Aidan Du, Nadia Grishanina, James Woo, Aaron Diaz, Susan M. Chang, Joanna J. Phillips, Arun P. Wiita, Christopher A. Klebanoff, Joseph F. Costello, Hideho Okada. Novel public and tumor-wide neoantigens arising from clonal aberrant splicing events drive tumor-specific T-cell responses across diverse cancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3907.