Fig. 4: Induction of NK cell reactivity and cytotoxicity by Fc-optimized CD276 antibody against primary AML cells.

PBMC from healthy donors were co-cultured with primary AML cells in the presence or absence of 8H8_SDIE or MOPC_SDIE control (both 1 µg/mL) (E:T 2.5:1). A–D The left panels show representative flow cytometry results obtained from an AML patient sample and a PBMC donor, the right panel shows combined data from AML patients (n = 6) and PBMC from healthy donors (n = 4). A NK cell activation was analyzed based on the CD69 expression after 24 h. B NK cell activation was determined by the CD25 expression after 72 h. C NK cell degranulation was determined by the CD107a expression after 4 h. D Intracellular IFNγ expression in NK cells within PBMC, characterized by CD3^-CD56⁺CD16⁺ counterstaining, was determined by FACS after 4 h. E Culture supernatants were analyzed after 4 h for release of immunoregulatory molecules TNF, IL-2, IFNγ and for the effector molecules granzyme A (GrzA), granzyme B (GrzB), perforin (PFN), granulysin (Grly) by Legendplex assays. The heatmap plots show results for AML patients (n = 2) with PBMC from healthy donors (n = 4). F Targeted lysis of primary AML cells was determined by Europium cytotoxicity assays after 2 h of incubation. In the left panel exemplary data obtained from a healthy PBMC donor and a primary AML sample at different E:T ratios is shown. In the right panel, pooled data for primary AML cells (n = 3) were obtained with PBMC from healthy donors (n = 3) (E:T 80:1). G Survival of primary AML cells was determined using a live cell imaging system. The primary AML sample was cultured with PBMC from 4 healthy donors (E:T 80:1) for 140 h. The live target cell areas were normalized to the respective initial target cell area’s initial measurement at T = 4 h. H Lysis of primary AML cells with PBMC from healthy donors (n = 4) was analyzed by flow cytometry after 72 h (E:T 20:1). In the left panel, separate data for each AML patient with PBMC from healthy donors (n = 4), in the right panel pooled data for all AML patients (n = 4) (E:T 20:1) are shown.