IMMU-55. TUMOR-WIDE RNA SPLICING ABERRATIONS GENERATE IMMUNOGENIC PUBLIC NEOANTIGENS IN GLIOBLASTOMAS AND OTHER CANCER TYPES
Authors: Darwin Kwok, Nicholas Stevers, Inaki Etxeberria, Takahide Nejo, Maggie Colton Cove, Lee Chen, Jangham Jung, Kaori Okada, Senthilnath Lakshmanachetty, Marco Gallus, Abhilash Barpanda, Chibo Hong, Gary Chan, Jerry Liu, Samuel Wu, Emilio Ramos, Akane Yamamichi, Payal Watchmaker, Hirokazu Ogino, Atsuro Saijo, Aidan Du, Nadia Grishanina, James Woo, Aaron Diaz, Susan Chang, Joanna Phillips, Arun Wiita, Christopher Klebanoff, Joseph Costello, Hideho Okada
Journal: Neuro-Oncology (2024)

Abstract BACKGROUND Immunotherapy in gliomas is limited by tumor heterogeneity and low mutational burden. Aberrant RNA-splicing (neojunctions) offers a new source for targets, and our neoantigen discovery platform (SNIPP) characterizes a novel class of clonally-expressed splicing-derived neoantigens that elicit a CD8+ T-cell-mediated tumor killing response. METHODS SNIPP identified public neojunctions expressed in TCGA RNA-seq (positive sample rate (PSR) &amp;gt; 10%) and not in GTEx normal tissue RNA-seq data (PSR &amp;lt; 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 obtained RNA-seq data from each intratumoral site. Two independent algorithms then predicted peptide processing likelihood and HLA-binding affinity of ASN candidates. Neoantigen-specific TCR sequences characterized from subsequent PBMC in vitro sensitization and 10x V(D)J scRNA-seq were transduced into CD8+ T-cells for immunogenicity and cytotoxicity assays against glioma cell lines. RESULTS Our pipeline identified 789 public neojunctions, with 32 neojunctions 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 identified TCR clonotypes reactive against neojunctions in RPL22 (n=7) and GNAS (n=1), the latter being highly intratumorally-conserved (detected in &amp;gt; 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 multiple GBM PDX cell lines, and co-culturing these T-cells against both glioblastoma and melanoma cell lines demonstrated tumor-specific cytotoxicity. 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. CONCLUSION Our unique SNIPP neoantigen discovery platform identified novel public tumor-wide splice-derived neoantigens and reactive TCRs, and its pan-cancer application characterized candidates that are targetable across multiple diseases.