PROJECT 3 - PROJECT SUMMARY The combination of Immune Checkpoint Blockade (ICB) with the α-PD-L1 antibody atezolizumab, and the vascular endothelial growth factor (VEGF)-targeting antibody bevacizumab, has become the new FDA approved Standard of Care (SOC) for advanced Hepatocellular Carcinoma (HCC). However, the 3-year survival for this SOC is still <30%, leaving a clear unmet need for novel therapies for advanced HCC patients who have progressed on first-line ICB therapies. Whilst the immune suppressive liver/HCC tumor microenvironment (TME) restricts the efficacy of anti-tumor T cell responses, the pro-inflammatory nature of oncolytic virus (OV) infection/replication offers a complementary mechanism of action to ICB by promoting immune infiltration (immunological heat) into poorly infiltrated (cold) HCC. Our group has conducted a Phase I first-in-human study of Vesicular Stomatitis Virus (VSV) expressing human interferon beta (hIFN-β) in patients with advanced sorafenib refractory/intolerant HCC. To investigate how OV could best synergize with ICB to treat HCC, murine Sleeping Beauty (SB) models of HCC were established in which α-PD-L1 ICB induced 30- 50% long term cures through re-invigoration of specific α-tumor CD8+T populations. However, a combination of α-PD-L1 ICB with VSV-IFNß to reverse immune suppression in the TME, led to the loss of the survival benefit of α-PD-L1 alone, associated with replacement of a diverse profile of CD8+ T cell populations in α-PD- L1-treated tumors with a dominant population of highly activated, α-viral effector CD8+T cells. However, by expressing an HCC tumor associated antigen (HCCTAA) from VSV, the potent α-viral effector CD8+ T cell response also became an α-tumor T cell response –with highly activated HCCTAA effector cells becoming focused on the tumor. In parallel studies, we showed that a novel VSV platform in which the CSDE1 protein is co-expressed within the virus induced significantly better therapy than the parental VSV-IFNß. Finally, to enhance infiltration of immune-excluded (cold) HCC with effector α-tumor T cells, we developed novel nanobody-based CAR T cells specific for the TAAHCC GPC3. In others studies, we have shown that CAR T cells loaded with oncolytic VSV 1). mediate tumor-specific virus delivery and 2). are significantly more therapeutically potent than conventional, non-loaded CAR T cells. Therefore, to test our overall hypothesis that the highly immune suppressive HCC TME, which inhibits endogenous α-HCC T cell responses, can be reversed in the setting of SOC therapies by administration of appropriately timed and optimally delivered viro/cellular/immunotherapy, we have formulated 3 Specific Aims: 1: To test how appropriately timed VSV- CSDE1-TAAHCC virotherapy can optimally enhance SOC immunotherapy in SB models of HCC; 2: To combine CAR T cell/VSV virotherapy to enhance SOC immunotherapy in murine and human xenograft models of HCC; and 3: To test the safety of adjuvant VSV-CSDE1-GPC...