4.7. In Vitro ADCC and ADCP Assays

For the antibody-dependent cellular cytotoxicity (ADCC) assays, human NK-cells were isolated from healthy donor PB-MNCs by negative selection using a magnetic bead-based separation kit (StemCell Technologies, Vancouver, BC, Canada) and cultured in MyeloCult media (StemCell Technologies) with 500 IU/mL human recombinant IL-2 for 24 h. NK-cells were cocultured in a 96-well V-bottom plate with J558 target cells (mouse MM cell line) labeled with calcein at a 10:1 ratio, in the presence of anti-mCD38 hIgG1 or hIgG1 control mAb. After a 2-h incubation at 37 °C, specific lysis in the supernatant was measured by the fluorescence (485 nm excitation and 515 nm emission) using a Perkin Elmer Envision device. Percentage of specific lysis was calculated using the formula:

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For antibody-dependent cellular phagocytosis (ADCP) assays, U937 effector cells were cocultured with J558 target cells labeled with PKH26 (Millipore Sigma, Burlington, MA, USA) at a 1:4 effector-to-target ratio in the presence of a fixed concentration of control hIgG1 mAb or increasing doses of anti-mCD38 hIgG1 mAb. After a 1-h incubation at 37 °C, cells were washed and stained with APC labeled anti-CD89 mAb (Biolegend, San Diego, CA, USA) and percent phagocytosis (% PKH26 among CD89⁺ cells) was measured by flow cytometry.