Abstract 1383: Characterization of CD8 T cell responses to DNAJB1-PRKACA fusion neoantigens in fibrolamellar carcinoma
Authors: Kirk Allison M. , Chou Ching-Heng , Crawford Jeremy Chase , Awad Walid , Allen E. Kaitlynn , Zhang Xiaoyu , Zamora Anthony E. , Strome Scott E. , Thomas Paul G. 
Published: June 2022

Fibrolamellar carcinoma (FLC) is a rare liver malignancy that has no known cure. Novel therapies for FLC are urgently needed to improve patient outcomes. The key genetic event in FLC tumorigenesis is a highly conserved gene fusion between the first exon of DNAJB1 and the last nine exons of PRKACA. This fusion gene is present in all FLC tumors, and more than 90% of patients express fusion proteins with identical amino acid sequences. Genetic mutations like this gene fusion can serve as important therapeutic vulnerabilities, in part because they may be processed and presented on the surface of tumor cells as neoantigens that can activate an anti-tumor T cell response. The purpose of our study was to test the hypothesis that DNAJB1-PRKACA fusion neoantigens could elicit T cell responses against FLC and serve as targets for novel immunotherapies. Spatial transcriptomic analyses of human FLC tumor samples indicated that CD8+ T cells could infiltrate these tumors, but often expressed markers of dysfunction and exhaustion, such as TOX. Nevertheless, we successfully expanded tumor-infiltrating T cells from FLC ex vivo and used intracellular cytokine staining (ICS) to identify a subset of FLC patient T cells that produced IFNγ and TNFα in response to stimulation with a fusion neoantigen. We used both peptide-MHC tetramer staining and functional response after stimulation to identify T cells in FLC patients and healthy donors that recognize fusion neoantigens. We then determined the paired T cell receptor (TCR) sequences using single-cell sequencing and expressed the TCRs in primary human T cells to test their potential utility in TCR-engineered immunotherapies. We validated the specificity and functionality of these TCRs using peptide-MHC tetramer staining, ICS, and in vitro cytotoxicity assays on the xCelligence and Berkeley Lights Lightning platforms. Primary T cells expressing fusion-specific TCRs bound to their cognate peptide-MHC tetramer, produced multiple cytokines in response to stimulation with their cognate fusion peptide, and specifically killed fusion-presenting target cells in vitro. Ongoing experiments will test whether T cells expressing these fusion-specific TCRs can control growth of fusion-presenting tumors in vivo. Collectively, these studies have defined the first reported fusion-specific T cell response in FLC, as well as fusion-specific TCRs that hold promise for use in adoptive T cell therapies. Our spatial transcriptomic analyses have also begun to illuminate the immune microenvironment in FLC and will inform future efforts to develop immunotherapies for this disease.

Citation Format: Allison M. Kirk, Ching-Heng Chou, Jeremy Chase Crawford, Walid Awad, E. Kaitlynn Allen, Xiaoyu Zhang, Anthony E. Zamora, Scott E. Strome, Paul G. Thomas. Characterization of CD8 T cell responses to DNAJB1-PRKACA fusion neoantigens in fibrolamellar carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1383.