Summary/Abstract Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer with dismal survival for patients with unresectable disease. Recent approval for PD-L1 inhibitor atezolizumab (atezo) and VEGF inhibitor bevacizumab (bev) in frontline treatment is a landmark advance; however further improvement is needed with a median progression free survival of less than 7 months. We have found through a multi-center cooperative study that tumor transcriptome signature high in interferon-gamma and MHC-II signaling (INFAP signature) is correlated with increased response and survival to PD-1 inhibitor. We propose to add high dose external beam radiotherapy (EBRT) followed by intra-tumor injection of autologous dendritic cells (DC) to atezo/bev to further enhance the immune stimulatory effect. Radiation can induce inflammatory tumor cell death that can be favorable for tumor neoantigen presentation. Injection of autologous DC after EBRT would be a novel method of boosting in vivo tumor antigen uptake and presentation to expand tumor-reactive cytotoxic T cells. We have treated subjects with unresectable liver tumors (HCC and cholangiocarcinoma) in a pilot study with this EBRT and DC approach with promising response and acceptable toxicity (no grade ≥3 toxicity). Three of the five subjects had partial response, including a patient with ongoing response beyond 1 year. Both emergence of new TCR clones and expansion of existing TCR clones, including clones with tumor reactive and cytotoxic profile, have been observed, suggesting this combination could enhance tumor reactive cytotoxic T cell response. However, many of the TCR clones also have early exhaustion signal with upregulation of multiple checkpoint receptors. Thus, combining DC injection with atezo/bev may help further enhance the cytotoxic functions of these TCR clones. We hypothesize that combining EBRT followed by intratumor DC injections with atezolizumab and bevacizumab can improve the PFS for patients with unresectable HCC and that the effect is mediated by systemic expansion of a tumor reactive T cell repertoire. We will test the hypothesis through 2 aims. 1) Assess the clinical efficacy of this combination therapy in a phase II study with a safety run-in phase. Increased PFS rate at 1 year will be the primary endpoint. 2) identify the effect of this novel combination immunotherapy on tumor reactive T cell repertoire. We will use scRNAseq and TCRseq to identify TCR clonal expansion and transcriptome profile of the TCR clones in the blood and tumor, with a focus on tumor reactive TCR clones. We will also use scRNAseq and flow to profile the changes of other immune cells in the tumor and blood, with a focus on the changes in expression of the INFAP signature. Finally, we will use imaging cytometry to examine the tumor and immune spatial relationship in the tumor. Our study will not only identify the clinical activities of this novel combination therapy but also use state-of-the-...