Abstract 7271: SENTI-202 CD33 OR FLT3 NOT EMCN logic-gated gene circuit components selectively target AML while protecting human HSC/HPCs from off-tumor toxicity in a humanized mouse model

Abstract There are no approved chimeric antigen receptor (CAR) natural killer (NK) or T cell therapies for acute myeloid leukemia (AML), due in-part to the scarcity of tumor-specific cell surface targets, which creates potential safety challenges with on-target off-tumor toxicity. SENTI-202 is an innovative CAR NK cell therapy that expresses a potent CD33 OR FLT3 NOT EMCN logic-gated gene circuit, in conjunction with IL-15. The bivalent CD33 OR FLT3 activating CAR targets 2 AML antigens, CD33 & FLT3, enabling targeting of AML stem cells & blasts (Gonzalez, 2023; Kaveri, 2024) and venetoclax-resistant AML cells (Muftuoglu, 2023). The NOT EMCN inhibitory CAR (iCAR) protects hematopoietic stem and progenitors (HSC/HPCs) from off-tumor toxicity (even if the healthy cells are FLT3+/CD33+), which is a major technological advancement that could enhance safety and hematopoietic recovery in patients. The iCAR recognizes the healthy cell marker endomucin (EMCN), which is expressed on up to 76% of HSCs (Reckzeh, 2018) but not AML cells. Here, we used CD33 OR FLT3 NOT EMCN CAR NK cells to validate iCAR-driven HSC/HPC protection in a humanized mouse model by comparing CD33 OR FLT3 NOT EMCN NK (88% CAR+) to (1.) Non-engineered NK and (2.) CD33 OR FLT3 NOT HER2 (iCAR control) NK (89% CAR+). First, we confirmed expected in vitro anti-AML activity (observed 50-70% killing; E:T=1:1) and iCAR-mediated model healthy cell preservation activity (observed ∼50% preservation; E:T=1:1), using EMCN- and EMCN+ cell lines, consistent with our prior results showing the EMCN iCAR preserves primary human HSCs from off-tumor toxicity in vitro (Garrison, 2022). Here, using a humanized mouse model we demonstrated iCAR-mediated in vivo protection of human HSC/HPCs from off-tumor toxicity for the first time in the field. Following successful human HSC/HPC engraftment, mice were given 2 CAR NK cell injections on d0 & d7, then HSC/HPC frequency was assessed on d14. While treatment with the iCAR control NK cells led to a significant decrease in HSC/HPC frequency compared to non-engineered NK (18.15% versus 28.6% of hCD45+; p value=0.00015), treatment with CD33 OR FLT3 NOT EMCN NK cells resulted in a significantly increased HSC/HPC frequency versus iCAR control NK cells (34.2% versus 18.15% of hCD45+; p value=0.00000355). This demonstrates that the EMCN iCAR protects human HSC/HPCs from off-tumor toxicity in vivo, and suggests that the EMCN iCAR may increase the SENTI-202 therapeutic window. In conclusion, the SENTI-202 CD33 OR FLT3 NOT EMCN logic-gated gene circuit enables exceptional efficacy against AML while protecting HSC/HPCs from off-tumor toxicity, demonstrating the potential of SENTI-202 as a targeted and precise therapy for AML. SENTI-202 is currently in Phase 1 clinical development in patients with relapsed/refractory hematologic malignancies including AML (NCT06325748). Citation Format: Muharrem Muftuoglu, Li Li, Mahesh Basyal, Jiangxing Lv, Lauren B. Ostermann, Han Deng, Priscilla Wong, Tim Lu, Brian Garrison, Kanya Rajangam, Michael Andreeff. SENTI-202 CD33 OR FLT3 NOT EMCN logic-gated gene circuit components selectively target AML while protecting human HSC/HPCs from off-tumor toxicity in a humanized mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7271.