496 Study of Affinity Tuning in AML-Targeted CAR-NK Cells

Background: CD123 is overexpressed on AML blasts compared to healthy myeloid progenitor cells. There is opportunity to design anti-CD123 therapy to discriminate on antigen density. Our goal is to study affinity modification of the single chain variable fragment (scFv) in the context of chimeric antigen receptor (CAR)-modified NK cell activation and target killing. We hypothesize that lower affinity CAR-NK cells will demonstrate improved discrimination between tumor and healthy tissue. Methods: We designed NK-cell specific CARs to contain anti-CD123 scFvs binding distinct target epitopes (26292 or 7G3) fused to intracellular portions of NK-activating receptors 4-1BB.CD3Z and 2B4.CD3Z. We synthesized novel 26292 and known 7G3 variants to capture a spectrum of CD123 binding affinities (26292: Kd=15nM; variant Kd range 0.29-160nM; 7G3: Kd=8.75nM; variant Kd range 23.9-64.9nM). These scFv were generated using site-directed mutagenesis of the wild type sequences and subcloned into CAR backbones. Retroviral transduction of primary human NK cells was performed, and CAR expression was measured using FACS (range 23.9%-89.6% CAR+ NK cells). Raji target cells (CD123neg) were engineered and sorted to obtain model cell lines with stable low (Raji.Low) and high (Raji. High) CD123 expression, comparable to healthy cells (CD123.Low) and AML blasts (CD123.High). We tested CAR-NK cell activation and antigen-specific cytotoxicity using in vitro co-culture assays. We established a mouse model of disease using implantation of Raji cells engineered to express firefly Luciferase (Raji.ffLuc). Results: Antitumor cytotoxicity of 26292 and 7G3-based CAR-NK cells with a range of binding affinities showed CAR-specific activation but did not differentiate on target antigen density. Similarly, no differences were seen in CAR-NK activation as measured by TNFa and IFNy secretion following coculture with CD123-positive cells. NSG mice implanted with Raji.ffLuc had median survival of 42 days without treatment. Conclusions: There are no significant differences across a broad range of variable affinity 26292 and 7G3-based anti-CD123 CAR-NK cells in effector cell activation or target killing when assayed short-term. These data support the value of additional long-term investigation of the immune synapse and CAR-NK antitumor activity in vivo. This work is supported by the St. Baldrick's Foundation Fellowship Award and the Johns-Hopkins-Allegheny Health Network Cancer Research Fund.