Generation of CAR-NK cells with iMC and IL-15 targeting CD123. (A) Schematic retroviral vector encoding signal peptide, CD123-targeting single-chain variable fragment variable light (V_L) and heavy (V_H) domains, the minimal CD34 epitope, CD8α stalk and transmembrane region, and the CD3ζ signaling domain. (B) Activated NK cells were doubly transduced with γ-retroviral vector encoding CD123.ζ (CAR) and/or dual-switch iRC9-IL15-ΔCD19-iMC. Flow cytometric analysis to determine transduction efficiency using anti-CD34 (CD123.ζ) and anti-CD19 (iRC9-IL15-ΔCD19-iMC) antibodies compared with nontransduced (NT) or single transduced NK cells. NK cells were first gated out as CD56⁺ populations. (C) Gene-modified NK cells (n = 4 donors) were cocultured with THP-1–eGFPFfluc cells at dilutive effector/target (E:T) ratios in the presence or absence of 1 nM of rimiducid (Rim) for 24 hours. Tumor-cell killing percentages were calculated by luciferase activity relative to tumor cells alone. Multiple Student t tests were used to compare iRC9-IL15-ΔCD19-iMC Rim and iRC9-IL15-ΔCD19-iMC plus CD123.ζ Rim. (D-F) NSG mice (n = 5 per group) were engrafted with 10⁶ THP-1–eGFPFFluc tumor cells and, 3 days later, treated with 10⁷ NK cells, NT or transduced with CD123.ζ, DS.IL15, or CD123.ζ plus DS.IL15. Mice were subsequently administered weekly IP vehicle (Veh) or 1 mg/kg of Rim. Tumor BLI was assessed by IVIS. Multiple Student t tests were used to compare CD123.ζ plus iRC9-IL15-ΔCD19-iMC Rim group with NT group. (G) At day 53 after NK therapy, CD123.ζ plus DS.IL15 Rim group was euthanized. Human NK cells were identified in spleen, bone marrow, and peripheral blood by flow cytometric analysis as hCD56⁺mCD45⁻ populations. All groups were euthanized at time point day 35, except for the DS.IL15 plus CD123.ζ Rim group that was obtained at day 53; 2-way analysis of variance was performed for comparisons (P = .059). **P < .01, ***P < .001.