Abstract 135: Memory-like NK cell targeting to CD123 and CD33 improves killing of acute myeloid leukemia.

Abstract Background: Memory-like natural killer (ML NK) cells are NK cells which upon stimulation with IL12/15/18 undergo a differentiation process to develop improved metabolic fitness and enhanced killing capabilities of different types of malignant cells. They are a promising cellular therapeutic against acute myeloid leukemia (AML), for which the prognosis remains dismal for both pediatric and adult patients who experience relapse of their AML. Phase I studies of adoptive ML NK cell therapy in adult and pediatric patients showed promising results with several patients achieving complete responses, but many go on to relapse after a few months. We investigate two strategies to improve recognition of AML by memory like NK cells by targeting them to AML surface antigens CD33 and CD123. Objective: We target ML NK cells to CD123 by engineering ML NK cells to express a chimeric antigen receptor (CD123 CAR ML NK). We target ML NK cells to CD33 via a trispecific killer engager (CD33 TriKE) that enhances the immune synapse by engaging CD16 on the ML NK cell and CD33 on AML. The CD33 TriKE also contains IL-15 to enhance ML NK activation and survival. We also explore whether both technologies function synergistically to prevent immune escape. Methods: Treatment of AML with ML NK cells vs CD123 CAR ML NK cells, ML NK cells plus CD33 TriKE, or the combination, was compared in series of short and long-term assays. AML targets include MOLM13, THP-1, and Primary AML blasts. Interferon gamma production was assessed in a 6-hour co-incubation. In-vitro killing was assessed at 4-hours using a flow-based killing assay, and at 24 hours using a luciferase-based assay. Tumor control was assessed over 5 days using Incucyte (live cell imaging). Tumor control and survival in vivo was assessed using an THP-1 Xenograft model in NSG mice. Primary AML xenografts using NSG-S mice is under development. Results: Both targeting strategies demonstrate an antigen-specific killing response. Both strategies significantly increase interferon gamma production compared to ML NK cells (CAR vs MOLM13: +16.57%, p=0.0002, Primary AML #34: IFNg +9.1% p=0.0092; CD33 TriKE vs MOLM13: +24.42%, p=.0015, AML34: +9.1% p=0.0092). Both strategies also demonstrated increased AML killing at various E:T ratios (CAR vs MOLM13: p=.0011; AML34: p=.0006; CD33 TriKE vs MOLM13: p.0001; AML34 p=.0026). Both CD123 CAR and CD33 TriKE improved THP-1 control in vivo (bioluminescence at 23 days-CD123 CAR: p=0.004, CD33 TriKE: p.0001). Both therapies improved median survival by about 3 weeks (CD123 CAR: p.0001, CD33 TriKE: p=.0001). Conclusion: CD123 CAR ML NK cells and ML NK cells treated with CD33 TriKE both enhance the abilities of ML NK cells to kill AML in vitro and in vivo, and prolong time of survival of THP-1 bearing mice. Treatment of CD123 CAR ML NK cells with CD33 TriKE may prevent antigen escape. Either of these targeting strategies are highly translatable to the clinical setting. Citation Format: Emily Phillips, Lyra Morina, Lynne Marsala, Wilbur Song, Michelle Becker-Hapak, Yoojin Ahn, Sushanth Pureti, Elizabeth Juarez Diaz, Sadia Afrin, Pamela Wong, Jennifer Tran, Joseph Rueve, Allison Burdi, Nancy Marin, Melissa Marie Berrien-Elliott, Martin Felices, Jeffrey S. Miller, Todd A. Fehniger. Memory-like NK cell targeting to CD123 and CD33 improves killing of acute myeloid leukemia abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 135.