SUMMARY/ABSTRACT Neuroblastoma (NB) is the only pediatric solid tumor for which immunotherapy has become part of the standard of care, with FDA approval of two anti-GD2 antibodies. Immunotherapy with anti-GD2 is utilized both during upfront therapy and for patients who have relapsed. Despite the availability of these important drugs, approximately 50% of NB patients relapse and eventually die from their disease. Those patients who survive are left with debilitating long-term effects, including growth retardation, hearing loss, infertility, and even secondary malignancies. Many of these secondary effects derive from high dose chemotherapy, while immunotherapy carries a much lower risk of long-term effects. Thus, enhancing the efficacy of immunotherapy for NB could potentially cure more patients while causing fewer debilitating late effects in survivors. This proposal pairs expertise in both antibody and chimeric antigen receptor (CAR) T cell immunotherapy with biological insights into immunotherapy target biology and expression. Our broad hypothesis is that heterogeneous and low surface antigen density of GD2 and other targets can limit the efficacy of immunotherapies in NB, but that this can be overcome through therapeutic maneuvers that increase their expression and/or modulate immune cell function. CARs are synthetic receptors that redirect the specificity of a T cell against a surface molecule expressed on tumor cells. Our laboratories have defined the antigen density threshold for CAR T cell activity, and have demonstrated that low level expression of target antigens can drive resistance to these immunotherapies. We have successfully reengineered CARs to lower their activation threshold and enhance CAR T cell activity at low antigen density. We have also focused on enhancing antibody based immunotherapies in NB focusing on clinically validated anti-GD2 antibodies. We recently discovered that NB cells can lower the density of GD2 on their surface, avoiding immune cell killing. We uncovered the mechanism of GD2 regulation in NB, linked GD2 expression epigenetic cell state, and reversed GD2 downregulation with FDA approved drugs. The work proposed in this renewal application will address the challenges of low target antigen expression to enhance the efficacy of immunotherapies in NB. We will test our central hypothesis in three Specific Aims: 1) Define and modulate the GD2 antigen density threshold necessary for therapeutic success of anti-GD2 based immunotherapies; 2) Optimize approaches to deliver CAR T cells in combination with EZH2 inhibitors to overcome antigen negative/low resistance; 3) Engineer GD2 and B7-H3 CAR T cells to overcome low target antigen density in NB by utilizing two novel platforms alone or in combination. This project is significant because it will result in new immunotherapeutic approaches for the treatment of children with NB, while also uncovering fundamental mechanisms driving successful immunotherapies that can be br...