Fig 2: A specific transcriptomic effector program associated with enhanced NK cell responses is induced by ANKET-IL-2v stimulation(A–D and F) Purified human NK cells were activated for 4 h in the presence of the murine huCD20-B16F10 target cells, with 0.1 µg/mL IL-2v/aNKp46/Fc/aCD20, IL-2v/IC/Fc-null/IC, Obinutuzumab, or aNKp46/Fc/aCD20 or a combination of 0.1 µg/mL IL-2v/IC/Fc-null/IC + 0.1 µg/mL aNKp46/Fc/aCD20 molecules. RNA sequencing was performed, and reads were aligned with the human genome, N = 4 to 6 donors.(A) Volcano plot representing the results of the analysis of differential expression between NK cells activated with IL-2v/aNKp46/Fc/aCD20 and those treated with a mixture of IL-2v/IC/Fc-null/IC + aNKp46/Fc/aCD20 molecules. In green: differentially expressed genes (FDR = 0.05); in gray: genes not differentially expressed.(B) Gene sets displaying significant enrichment in NK cells stimulated with IL-2v/aNKp46/Fc/aCD20 relative to cells stimulated with a combination of the trispecific aNKp46/Fc/aCD20 and the IL-2v polypeptide, based on GSEA.(C) Venn diagram representing the number of genes upregulated in NK cells following activation with the indicated molecules, relative to unstimulated conditions (FDR = 0.05).(D) Heatmap representing the expression of a selection of differentially regulated genes.(E) CD69 monitored by flow cytometry on purified human NK cells activated for 24 h in the presence of the murine huCD20-B16F10 target cells with 0.1 µg/mL IL-2v/aNKp46/Fc/aCD20, IL-2v/IC/Fc-null/IC, Obinutuzumab, or aNKp46/Fc/aCD20 or a combination of equal doses of IL-2v/IC/Fc-null/IC + aNKp46/Fc/aCD20. Picture is a boxplot obtained with NK cells from four donors. Whiskers show minimal and maximal value. Symbols are conserved for each donor across the conditions of stimulation.(F) Gene set score for CD56dimCD16a+CD57+ NK1 cells and CIML NK cells regulated by each of the indicated condition of stimulation.

Fig 3: CD123-NKCE displays strong cytotoxic activity against AML cells, strong activation of NK cells and no off-target effects.a, Diagrams showing the molecular organization of the NKCE molecules. The top shows the CD123-NKCE trifunctional molecule built with an unmodified human IgG1-Fc (red), targeting CD123 (orange) and coengaging NKp46 (green) and CD16a on NK cells. The bottom shows the bifunctional NKCE containing a human IgG1-Fc silenced for binding to all FcγRs (Fc null; purple). b, Comparison of the cytotoxicities of NKCEs targeting CD123 and engaging CD16a only (IC-Fc-CD123), NKp46 only (NKp46-Fc null-CD123) or coengaging NKp46 and CD16a (NKp46-Fc-CD123; CD123-NKCE). MOLM-13 cells were used as the targets and purified resting NK cells as effectors. Results for two healthy donors are shown (n = 3). Data are presented as mean values ± s.d. c, Cytotoxicity of CD123-NKCE against the AML cell line MOLM-13. Results for five healthy donors are shown. Data are presented as mean values ± s.d. d, Evaluation, by flow cytometry, of CD107, CD69, TNF-α, IFN-γ and MIP-1β expression by NK cells treated with CD123-NKCE. NK cells alone are compared with NK cells cocultured with MOLM-13 cells. Results for one representative donor are shown (n = 3).