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Background Neuroblastoma is the most common and deadly extracranial pediatric tumor, accounting for up to 15% of childhood cancer deaths annually. The identification of GD2 on the surface of nearly all neuroblastoma cells has provided a target for immunotherapy, including CAR-T cell therapy. Recently published work using anti-GD2 CAR-T cells in relapsed or refractory high-risk neuroblastoma demonstrated signs of clinical efficacy with a 63% overall response rate but only a 36% event free survival after 3 years, implying responses are not durable.Methods We developed a novel 2nd generation anti-GD2-41BB CAR-T cell construct utilizing DAP12 (GD2BBDAP12) in place of the conventional CD3ζ intracellular cytotoxicity domain (GD2BBζ). We assessed the anti-tumor function of DAP12-based CAR-T cells in vitro using IncuCyte live-cell imaging to monitor cytotoxicity against CHLA20, a GD2+ human neuroblastoma cell line. To evaluate sustained killing, we performed serial tumor rechallenges. CAR-T cells were analyzed by flow cytometry 72 hours after tumor exposure to evaluate phenotype, activation status, and expression of exhaustion markers. For in vivo analysis, NSG mice were injected intravenously with CHLA20 cells to establish a metastatic model, then treated with CAR-T cells four days later. Tumor progression was monitored weekly using bioluminescent IVIS imaging. Overall survival and clinical scores were measured.Results GD2BBDAP12 CAR-T cells showed increased cytotoxicity against neuroblastoma compared to GD2BBζ CAR-T cells In vitro ( figure 1). Flow cytometric analysis of GD2BBDAP12 CAR-T cells demonstrated a significant increase in conversion to an effector memory phenotype compared to GD2BBζ CAR-T cells (figure 1). GD2BBDAP12 CAR-T cells also had significantly reduced downregulation of CD62L (figure 1). Further, GD2BBDAP12 CAR-T cells demonstrated downregulation of exhaustive markers including PD-1, LAG-3, and TIM-3 (figure 1). GD2BBDAP12 CAR-T cells demonstrated superior anti-tumor efficacy against metastatic neuroblastoma in vivo, with 100% survival at 60 days post CAR infusion compared to GD2BBζ CAR-T cells with 0% survival at 36 days post CAR infusion (figure 2). No evidence of toxicity was noted clinically.Conclusions In conclusion, GD2BBDAP12 CAR-T cells demonstrate superior anti-tumor efficacy against neuroblastoma compared to traditional GD2BBζ-based constructs both in vitro and in vivo. Further, GD2BBDAP12 CAR-T cells show enhanced conversion to an effector memory phenotype, reduced downregulation of CD62L, and reduced expression of exhaustive markers compared to GD2BBζ CAR-T cells, which may lead to more durable responses.Ethics Approval This study was approved by the University of Wisconsin-Madison Institutional Review Board (protocol #2017-1070) for the use of human peripheral blood mononuclear cells (PBMCs). Informed consent was obtained from all donors prior to sample collection, in accordance with institutional and federal guidelines. All animal procedures were approved by the University of Wisconsin-Madison Institutional Animal Care and Use Committee (protocol #M005915) and conducted in compliance with institutional and national standards for the care and use of laboratory animals.Abstract 218 Figure 1Abstract 218 Figure 2