Post-transplant relapse of acute myeloid leukemia (AML) remains a primary cause of treatment failure after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Anti-CD33 chimeric antigen receptor (CAR) T cell therapies provide a promising new approach to treat post-transplant relapse; however, these therapies may be associated with on-target off-tumor toxicity because CD33 is also expressed on healthy myeloid cells in the blood and bone marrow. While leukemia-specific antigens are not available, the expression of human leukocyte antigen DR (HLA-DR) is reportedly lost or downregulated in up to 50% of post-transplant relapse, presumably a mechanism to escape donor T cell-mediated graft-versus-leukemia immunity. Based on these reported findings, we propose to enhance the specificity of CAR-T cells against relapsed AML by activating them in response to CD33 while inhibiting them in response to HLA-DR. Our central hypothesis is that dual CAR-T cells, which express both a CD33-targeted CAR and an HLA-DR-targeted inhibitory CAR (iCAR), will eradicate relapsed AML with HLA-DR loss but spare normal myeloid cells. We will test our hypothesis by pursuing two specific aims: 1) Develop, validate, and optimize a pair of anti-CD33 CAR and anti-HLA-DR iCAR in human primary T cells in vitro; and 2) Examine the selectivity and potency of dual CAR-T cells in xenograft mouse models of both cell lines and primary cells of human AML with HLA-DR loss. The successful completion of the proposed study will lead to the development of a novel prototype of dual CAR-T cells that can specifically target relapsed AML with HLA-DR loss. Our proposed approach, if proved successful, can potentially be broadly used to engineer safer CAR-T cells against other hematologic malignancies and solid tumors with loss of HLA class I or II.