PROJECT SUMMARY Children with high-risk neuroblastoma (NB) have a significant risk of treatment failure and death emphasizing the need to develop novel targeted therapies, which constitutes the long-term goal of this proposal. The proposed research focuses on the oncoprotein glypican-2 (GPC2), which is glycophosphatidylinositol (GPI)-linked to the cell surface of NBs and other cancers but is not found on vital normal tissues. To capitalize on GPC2’s differential expression, we developed GPC2 chimeric antigen receptor (CAR) T cells and performed Investigational New Drug (IND) application-enabling studies in NB showing that GPC2 CARs induce safe and robust tumor regression. These data led directly to the development of a first-in-human GPC2 CAR T cell Phase 1 clinical trial that opened in May 2023. However, NB cells can escape GPC2 CAR pressure leading to tumor relapse and limiting long-term efficacy. Thus, there is an urgent need to identify the mechanisms of GPC2 CAR T cell resistance in NB and develop next-generation CAR approaches to combat these processes. Towards this goal, our recent profiling of tumors that escaped GPC2 CAR pressure showed that NB relapse is associated with GPC2 downregulation and conversely upregulation of the GPI-cleaving angiotensin-converting enzyme (ACE). We have also found that GPC2 is a major component of NB-associated extracellular vesicles (EVs), modulation of which can significantly change GPC2 cell surface density. Together, our published and unpublished preliminary data suggest the following central hypothesis: A major mechanism of GPC2 CAR T cell resistance in NB is downregulation of cell surface GPC2 driven both by ACE-induced GPC2 cleavage and increased shedding of GPC2+ EVs and can be circumvented by a new bicistronic CAR dual antigen targeting approach. We propose to test this original concept in two integrated but independent Specific Aims. In Aim 1, we focus on validating GPC2 cell surface downregulation as a major mechanism of GPC2 CAR resistance in NB, including defining the role of ACE-induced GPC2 cleavage and the dynamic modulation of GPC2+ EVs under GPC2 CAR pressure. In Aim 2, we will define the efficacy and safety of a new CAR T cell dual antigen targeting approach we designed to overcome GPC2 CAR escape in NB; GPC2 CAR T cells simultaneously targeting the disialoganglioside GD2 via a secreted bispecific innate immune cell engager (BiCE) that also binds CD16a on NK cells and macrophages to activate antitumor innate immunity in a GPC2-independent manner. This innovative CAR.BiCE therapeutic approach synergizes dual antigen targeting and activating adaptive and innate immunity. Our results will yield critical insights into CAR T cell resistance and these new immunotherapeutic principles and technology can be applied to other potent CARs where antigen escape limits long-term efficacy. These studies will also produce IND application-enabling data for the clinical translational of a CAR.BiCE therapeuti...