PROJECT SUMMARY Fibrolamellar carcinoma (FLC) is a rare liver tumor with poor outcomes: 5-year overall survival is reported at only 35-45%, and relapse rates are reported at 57-100%. Surgical resection is currently the mainstay of treatment, so novel therapies are desperately needed for FLC. A potential therapeutic vulnerability in this cancer is a characteristic gene fusion between DNAJB1 and PRKACA that is highly specific for FLC, expressed in all FLC tumors, and necessary to drive FLC tumorigenesis in a mouse model. Genetic mutations that alter amino acid sequence, including such gene fusions, can be presented as neoantigens on the surface of cancer cells by human leukocyte antigen (HLA) class I molecules and thereby elicit anti-tumor immune responses mediated by cytotoxic T cells. T cell receptor (TCR) recognition of neoantigens is the basis of several types of immunotherapy, which could serve as novel treatments for FLC. The goal of this study is to test the hypothesis that FLC neoantigens, including the conserved DNAJB1-PRKACA fusion, can elicit T cell responses in FLC patients and serve as targets for novel T cell-based immunotherapies for FLC. Understanding which FLC neoantigens elicit immune responses and which TCRs best recognize those neoantigens is crucial for both identifying and prioritizing potential targets and therapeutic agents for immunotherapy. This study will therefore first seek to define the neoantigen landscape in human FLC patients and identify TCRs specific for putative neoantigens in each patient. The presence of both a conserved mutation (the DNAJB1-PRKACA fusion) and patient-specific mutations in FLC offers the opportunity to compare anti-tumor immune responses against conserved and patient-specific neoantigens. This study will additionally determine which neoantigens are preferentially targeted by the immune response and which TCRs dominate the anti-neoantigen response to facilitate prioritization of neoantigen targets and neoantigen-specific TCRs for future study and therapeutic development. Further, these experiments may uncover previously unknown principles of neoantigen recognition that may be applied to other cancer types. Preliminary experiments have already identified both a functional T cell response directed against the DNAJB1-PRKACA fusion and DNAJB1-PRKACA-specific TCRs in one FLC patient. This study will therefore also evaluate this conserved fusion protein as a potential target for TCR-engineered immunotherapy approaches in FLC. TCRs specific for DNAJB1-PRKACA neoantigens will be identified and tested for their ability to mediate responses against fusion-expressing cells in both in vitro functional assays and proof-of- principle in vivo experiments using xenograft mouse models. Together, these studies will provide key foundational data for clinical pursuit of neoantigen-targeted immunotherapies in FLC.