Project Abstract Fibrolamellar hepatocellular carcinoma (FLC) is a rare and often lethal form of liver cancer that primarily affects children and young adults without cirrhosis. There are no approved systemic therapies for FLC, and it is usually refractory to treatment approaches developed for other forms of liver cancer. A chimeric transcript between DNAJB1 and PRKACA was identified as a signature genomic event in FLC and leads to constitutive activation of PKAc, but pharmacological inhibition of PKAc for FLC with traditional small molecule inhibitors has been infeasible due to on-target toxicity. Prior work from our group and others has demonstrated that neoantigens derived from gene fusions, including the DNAJB1-PRKACA fusion in FLC, can stimulate strong T cell responses. Furthermore, all patients with FLC share an identical amino acid sequence at the fusion junction, allowing a single “off the shelf” neoantigen-specific vaccine to be utilized nearly universally for this cancer. Neoantigen- specific vaccines are most effective in combination with other immunomodulatory agents including ICIs to prevent T cell exhaustion. Our overall hypothesis is that a neoantigen-specific vaccine targeting the DNAJB1- PRKACA chimera will synergize with ICIs to elicit a specific antitumor immune response in FLC. We will conduct a clinical trial of a vaccine targeting the DNAJB1-PRKACA chimera (FLC-Vac), in combination with nivolumab and ipilimumab, in patients with unresectable FLC. We will further determine if FLC-Vac combined with ICIs increases the number of neoepitope-specific T cells with specificity for the DNAJB1-PRKACA chimera in the peripheral blood that traffic to the tumor. Multiplex immunohistochemistry (IHC) on paired pretreatment and on- treatment biopsies will further define the mechanisms of immune response and resistance to immunotherapy in FLC. We will use these samples to identify T cell receptors (TCRs) specific for the FLC fusion protein in the context of the patient's HLA. Using TCRs from our trial and from endogenous responses identified in untreated specimens, we will use humanized mouse orthotopic models to determine the relative efficiency of processing and presenting specific epitopes from the DNAJB1-PRKACA fusion in diverse MHC contexts. These models will involve the endogenous presentation of the FLC fusion within tumor lines that we will treat with primary cells transduced with our identified TCRs, allowing us to compare the targeting efficiency of TCRs specific for the corresponding fusion epitopes. This work may advance a novel treatment paradigm for FLC, a tumor for which there is no standard or effective systemic therapy, and will have important implications for targeting recurrent ”undruggable” driver genes in other immune-resistant tumor types. Identifying optimal peptide-HLA-TCR combinations for targeting the DNAJB1-PRKACA fusion will also lay the groundwork for the next generation clinical trials for FLC, including adoptive...