Batda

NK cell cytotoxicity was evaluated by lanthanide (Europium, Eu) fluorescence assay. Briefly, target cells were labeled with BATDA (PerkinElmer) for 30 minutes at 37°C in the complete medium. Labeled cells were transferred to 96-well polystyrene plates (U-bottom, Nunc), mixed with NK cells at different E/T ratios, and incubated for 2 hours at 37°C. For ADCC, NK cells were incubated with SKOV-3 target cells in the presence of 50 ng/mL of either anti-HER2 antibody or control IgG. After cytolysis, the cell supernatant, containing the released TDA ligand, was added to the Eu solution to generate the fluorescent Eu-TDA chelate. The fluorescence of Eu-TDA was measured using EnSpire multimode plate reader (PerkinElmer). The amount of released TDA ligand in cell supernatants was regarded as the experimental TDA release. Total TDA release was measured after complete lysis of target cells by 1% NP-40. Lysis percentage was calculated using the following equation: (experimental TDA release – spontaneously released TDA) / (total TDA release – spontaneously released TDA).

Cytolytic activity of NK cells was measured in a 2 h BATDA [bis(acetoxymethyl)2,2:6,2-terpyridine-6,6-dicarboxylate] europium release assay. Cryopreserved primary childhood AML blasts and the erythroblastoid cell line K562 (M6 leukemia) were used as target cells. No enrichment of primary childhood AML blasts was done: purity > 80% blasts. K562 was used as positive control to exclude functional NK cell inactivity. Target cells (leukemic blasts) were labeled with 3 μL of the fluorescence enhancing ligand BATDA (Perkin Elmer, Waltham, MA, USA) for 60 min at 37°C. After five washing steps, the target cell suspension was adjusted to 2 × 10⁵ cells/well and seeded into microplates (5000 cells/well). Four different effector to target (E : T) ratios were tested with and without IL2 preincubation overnight (Proleukin, Basel, Switzerland). The assays were done as published [16 (link)]. Specific lysis was calculated as follows: %-specific lysis = (experimental release − spontaneous release)/(maximum release − spontaneous release)  ∗  100.

For experiments with KHYG-1 cells, 5 × 10³ target cells/well were incubated with KHYG-1 cells with/without CC-96191 or other TriNKET molecules. After 2 days, cell numbers and NK cell- as well as drug-induced cytotoxicity, using DAPI to detect non-viable cells, were determined flow cytometrically. For experiments with other cytotoxic agents, AML cells were incubated with/without GO (Pfizer, New York, NY, USA) or mitoxantrone (Sigma-Aldrich, St. Louis, MO, USA) for 3 days, after which, cell numbers and drug-induced cytotoxicity were quantified. For experiments with primary human NK cells, target cells were labeled with BATDA (PerkinElmer, Shelton, CT, USA) and then incubated with NK cells with/without TriNKET molecules for 2–3 h before time-resolved fluorescence (TRF) analysis. For cytotoxicity with primary PBMCs, EOL-1 cells were labeled with CellTrace Violet (Thermo Fisher) before co-culturing with PBMCs and either CC-96191, lintuzumab, or the CD33/CD3 BsAb, and, in some experiments, recombinant soluble CD33-His (amino acids 18–259; Creative Biomart, Shirley, NY, USA) or recombinant MICA Fc (R&D Systems, Minneapolis, MN, USA). After 24 h, live EOL-1 cells were enumerated by flow cytometry using CountBright™ Absolute Counting Beads.