2.4. CDC and ADCC assays

The test samples used were pabinafusp alfa and humanized anti-hTfR monoclonal antibody (mAb), the parent antibody for pabinafusp alfa. For the CDC assay, target cells (CCRF-CEM) were cultured, and 5 × 10⁴ cells (50 μL/well) were added to a 96-well clear-bottom plate. The test sample solutions (25 μL) were added to the appropriate wells. Next, 25 μL of diluted normal human serum complement (Quidel, San Diego, CA) was added to each well at a final human serum concentration of 10%, and the cells were incubated at 37 °C in a humidified atmosphere containing 5% CO₂ for 4 h. Cytotoxicity was detected using a CytoTox-Fluor™ Cytotoxicity Assay Kit (Promega, Madison, WI) according to the manufacturer's instructions. Fluorescence (excitation at 485 nm and emission at 520 nm) was measured using a fluorescence microplate reader (Molecular Devices, San Jose, CA).

For ADCC measurement, the ADCC Reporter Bioassay Core kit (Promega) was used according to the manufacturer's instructions. Briefly, the target cells (CCRF-CEM, K562, or HEL92.1.7) were cultured, and 1 × 10⁴ cells (25 μL/well) were added to a 96-well white plate. The test sample solutions (25 μL) were added to the appropriate wells. Next, 25 μL of a separately prepared effector cell suspension (Jurkat cells stably expressing the V158 variant FcγRIIIa receptor and an NFAT response element driving expression of firefly luciferase, provided in the kit) was added to each well and incubated at 37 °C in a humidified atmosphere containing 5% CO₂ for 6 h. Cytotoxicity was determined based on the chemiluminescence intensity using the GloMax® Discover System (Promega) according to the manufacturer's instructions.