IMMU-02. NEOANTIGENS ARISING FROM ALTERNATIVE SPLICING EVENTS MAY BE TARGETED BY TUMOR INFILTRATING LYMPHOCYTES IN GLIOBLASTOMAS

INTRODUCTION Alternative splicing, the cellular process that converts premature mRNA to mature mRNA and allows for single genes to produce multiple protein products, is frequently dysregulated in many cancers, including glioblastoma. However, along with non-synonymous mutations in the DNA, altered splicing mechanisms in cancers may produce novel antigens (so-called neoantigens) that distinguish cancer cells from healthy cells and can thus be targeted by the immune system. METHODS We developed a new computation pipeline (IRIS – Isoform peptides from RNA splicing for Immunotherapy targets Screening) that took bulk RNA-sequencing data from 23 glioblastoma patient tumor samples and predicted neoantigens that may arise from alternative splicing events. We prioritized predicted neoantigens that arose in HLA*A02:01 and HLA*A03:01 patients and selected 8 potential neoantigens to generate peptide:MHC Class 1 dextramers. We tested PBMCs and/or ex vivo expanded tumor infiltrating lymphocytes (TIL) from 6 of our glioblastoma patients against these dextramers, sorted for any neoantigen-reactive T cells, and performed single-cell RNAsequencing on the sorted population to determine the TCR sequence. RESULTS Among the 8 predicted neoantigens tested, 7 of the neoantigens were recognized by at least 1 patient’s T cells. 1 HLA*A03:01 epitope was recognized in 3 of the 4 HLA*A03:01 patients tested and this epitope was highly positive in an expanded TIL population, representing 1.7% of all CD3+ CD8+ cells. When we sorted for those neoantigen reactive T cells from the expanded TIL population and performed single-cell RNAsequencing, we found 325 unique T cell clonotypes, but the top 10 clonotypes represented 83.6% of all TCR clonotypes. The most frequent TCR clonotype represented 39.1% of the repertoire and suggests that clonal expansion of a select few TCR clones occurred within the tumor. CONCLUSIONS In total, our data indicates that neoantigens arising from alternative splicing events may represent a potential target for immunotherapy in glioblastoma.