Victor x4 luminometer

Dynabeads (Invitrogen) were coated with 0.8 µg/mL mouse anti-hCD3 (clone UCHT1, BD Biosciences), 10 µg/mL mouse anti-hCD28 (clone 28.2, BD Biosciences), and 10 µg/mL hPD-L1 mIgG1 Fc chimera (generated at Boehringer Ingelheim) or mIgG1 (Sigma), and stored at 4°C. Jurkat cells expressing hPD-1 and containing an NFAT-activation reporter gene (luciferase; generated by Boehringer Ingelheim) were incubated at 1.5 × 10⁵ cells/well with serially diluted ezabenlimab or control at a final volume of 90 µL in 96-well plates. After 10 min incubation at 37°C, 10 µL Dynabeads/well were added, cells were mixed and cultured for 6 hours at 37°C. 100 µL of Steady-Glo® Luciferase Reagent was added to the wells per manufacturer’s instructions and measured on a Victor X4 luminometer (Perkin Elmer). For EC₉₀ calculations, 0% activation was defined as the luminescence value of cells incubated with the CD3/CD28/PD-L1 beads and no antibody. One hundred percent activation was defined as the signal obtained with cells incubated with CD3/CD28/mIgG1 control beads and no antibody. Antibody activity was calculated for the antibody concentration of 166 nM in the presence of CD3/CD28/PD-L1 beads, with 100% defined as the signal obtained with CD3/CD28/mIgG1 beads.