e14512 Background: Glypican 3 (GPC3), a cell surface protein associated with early development, is an established biomarker of hepatocellular carcinoma (HCC). In a vast compendium of work, HN3, a human GPC3 nanobody (VH) isolated from a phage library, was shown to abrogate HCC tumors and block the distally located Wnt binding domain of GPC3. To date, it remains challenging how to precisely engineer CAR T cells for improved efficacy in solid tumors. In the context of hepatocellular carcinoma, we determined how hinges and transmembrane portions of varying structures and sizes affect CAR T cell function. Here, we show how rationally designed, engineered Chimeric Antigen Receptor (CAR) T cells containing HN3, demonstrate enhanced CAR T activity in HCC. Methods: We generated and compared multiple permutations of GPC3 targeted CAR T cells containing HN3, CD8, CD28, IgG4 and Fc domains. Incorporation of HN3, into multiple CAR formats yielded nine constructs, which were screened for antigen-specific and antigen-independent signaling using a luciferase reporter system in Hep3B, Huh7 and HepG2 cell lines. For in vivo assessment, immunodeficient NSG mice were intraperitonially injected with Hep3B or Huh7 cells and treated with a single dose (5 or 15 million) of engineered HN3 CAR T cells. Blood was collected at 2, 4 and 5 weeks to assess proliferation, T cell subtypes and exhaustion. Results: In vitro, the HN3-IgG4H-CD28TM CAR T cells induced high specificity and cytotoxic activity. HN3-IgG4H-CD28TM CAR T cells markedly improved HN3 cell killing activity by 30-40% in low (1.6:1) and high (25:1) effector to target ratios. Similarly, in vivo, in a high antigen environment, two determinative changes in the hinge region and transmembrane domains led to complete tumor eradication in immunodeficient mice bearing HCC tumors within 7-10 days. Moreover, HN3-IgG4H-CD28TM CAR T cells maintained specific tumor killing and averted exhaustion by producing a clear T cell response signature of enriched cytotoxic-memory (Temra) CD8+ along with a subset of naïve T cells. Conclusions: Engineered nanobody based CAR T cells containing the appropriate hinge and transmembrane domains can lead to determinative T cell signaling capable of inducing swift and durable eradication of HCC tumors. Altogether, we show that engineered HN3 CAR T cell therapy demonstrates strong potential for treatment of aggressive HCCs enabled by Wnt dysregulation.