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

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.