Identification of T cells specific for DNAJB1-PRKACA fusion neoantigens in fibrolamellar carcinoma

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Abstract Fibrolamellar carcinoma (FLC) is a rare liver tumor with poor outcomes and for which novel therapies are urgently needed. A potential therapeutic vulnerability in FLC is a gene fusion between DNAJB1 and PRKACA that is expressed in all FLC tumors and has been shown to drive FLC tumorigenesis. Genetic mutations that alter amino acid sequence, including gene fusions, can be presented as neoantigens by MHC class I molecules on tumor cells and thereby elicit anti-tumor CD8 T cell responses. We hypothesize that DNAJB1-PRKACA fusion neoantigens can elicit T cell responses in FLC patients and serve as targets for T cell-based immunotherapies for FLC. To test this hypothesis, T cells isolated and expanded from an FLC patient tumor were stimulated with DNAJB1-PRKACA fusion peptides, which reproducibly induced cytokine production measured by intracellular cytokine staining. Single cell gene expression and T cell receptor (TCR) repertoire analysis of patient tumors using the 10X Genomics platform identified clonally expanded TCR clonotypes that were associated with expression of cytokines and activation markers in response to fusion peptide stimulation. These TCRs were cloned and expressed in Jurkat 76 cells to test their fusion specificity and their ability to mediate in vitro functional responses to fusion neoantigens. When stimulated with their cognate fusion neoepitope, TCR-expressing cells produced cytokines, expressed the early activation marker CD69, and downregulated surface expression of TCR and CD3. These studies have therefore identified T cell responses against the DNAJB1-PRKACA fusion for the first time, as well as identified DNAJB1-PRKACA-specific TCR clonotypes that could be used to develop novel immunotherapies for FLC.