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Background KMT2A rearrangements (KMT2Ar) are commonly observed in pediatric acute myeloid leukemia (AML) and treatment-related AML, resulting in a highly aggressive and treatment-resistant subtype. While CAR T cell (CART) therapy has shown remarkable success in B-cell malignancies, its efficacy in AML has been limited, largely due to the overlap of antigen expression between AML cells and normal hematopoietic cells. To identify novel, leukemia-specific targets, we analyzed the AML transcriptome and identified CLEC2A as a promising candidate. CLEC2A is highly expressed in KMT2Ar AML but remains silent in normal hematopoiesis.Methods Following validation of CLEC2A surface expression on AML cells, we developed a novel fully human anti-CLEC2A antibody, engineered CLEC2A-specific CART cells, and assessed their preclinical efficacy using both cell line-derived (CDX) and patient-derived xenograft (PDX) models.Results In an AML cell-derived xenograft model, NSG mice were engrafted with either CLEC2A + OCI-AML2 cells or CLEC2A– MOLM13 cells, followed by treatment with either CLEC2A CART cells or unmodified T cells. All mice treated with unmodified T cells developed rapidly progressive leukemia and required euthanasia by Day +31. In contrast, CLEC2A CART treatment significantly extended survival (mean survival: 76 days; range: 46–127 days vs. 31 days in controls), resulting in improved overall (p=0.0009) and leukemia-free survival (p=0.0012) (figure 1). CLEC2A CART cells showed robust expansion and persistence, with detectable cells in peripheral blood at Day 27 and in bone marrow at endpoint. In the CLEC2A– MOLM13 model, all mice developed leukemia, confirming the antigen specificity of CLEC2A CART cells.In an aggressive KMT2Ar PDX model, CLEC2A CART treatment completely eradicated leukemia in all treated mice by Day 42, whereas all control mice developed progressive leukemia and required euthanasia by Day +36. One treated mouse developed CLEC2A– leukemia, likely due to antigen escape, while the remaining four mice remained alive through Day +140 (p=0.0002). Two CART treated mice developed extramedullary disease around Day +140 with one mouse having low level (<5%) disease in the bone marrow, while all other mice remained disease-free in the bone marrow (figure 2).Conclusions CLEC2A is a novel, leukemia-specific immunotherapeutic target that is enriched in KMT2Ar AML and not expressed in normal hematopoietic cells. CLEC2A CART cells demonstrated potent anti-leukemic activity and significantly prolonged survival in both cell line-derived and patient-derived AML models. Based on these promising preclinical results, a Phase 1 clinical trial of CLEC2A CART cells for relapsed/refractory AML is currently in development.Ethics Approval This study was conducted with IACUC approval (#51068) from Fred Hutch Cancer Center.Abstract 238 Figure 1Abstract 238 Figure 2