CD33–CD123 IF-THEN Gating Reduces Toxicity while Enhancing the Specificity and Memory Phenotype of AML-Targeting CAR-T Cells

Abstract Chimeric antigen receptor (CAR) T-cell therapy has remarkably succeeded in treating lymphoblastic leukemia. However, its success in acute myeloid leukemia (AML) remains elusive because of the risk of on-target off-tumor toxicity to hematopoietic stem/progenitor cells (HSPC) and insufficient T-cell persistence and longevity. Using a SynNotch circuit, we generated a high-precision “IF-THEN” gated logical circuit against the combination of CD33 and CD123 AML antigens and demonstrated antitumor efficacy against AML cell lines and patient-derived xenografts. Unlike constitutively expressed CD123 CAR-T cells, those expressed through the CD33 SynNotch circuit could preserve HSPCs and lower the risk of on-target off-tumor hematopoietic toxicity. These gated CAR-T cells exhibited lower expression of exhaustion markers (PD-1, TIM-3, LAG-3, and CD39), higher frequency of memory T cells (CD62L+CD45RA+), and enhanced expansion. Although targeting AML, the moderated circuit CAR signal also helped mitigate cytokine release syndrome, potentially addressing one of the ongoing challenges in CAR-T immunotherapy. Significance: Our study demonstrates the use of “IF-THEN” SynNotch-gated CAR-T cells targeting CD33 and CD123 in AML reduces off-tumor toxicity. This strategy enhances T-cell phenotype, improves expansion, preserves HSPCs, and mitigates cytokine release syndrome—addressing critical limitations of existing AML CAR-T therapies.