Luciferase reporter assays were performed with the human Jurkat cell line that has been genetically modified to express hCD137 and a luciferase reporter driven by an NF-κB-responsive element that can respond to CD137 ligand/agonist antibody stimulation (4-1BB Bioassay, JA2351, Promega). Jurkat cells were stimulated for 6 hours with mAb and CD137L-Fc coated beads and lysed in dual reporter lysis buffer (Promega). 12.5 × 104 wild-type, Trans1 and Trans2 HCT116 cells were seeded in a 96-well plate. After overnight culture, CD137-Jurkat cells were added in the presence of 1, 0.5 and 0.1 µg/ml CD3-EpCAM and 1.5 μg/ml of ALG.APV-527 BsAbs for 6 hours and lysed in dual reporter lysis buffer (Promega). Firefly luciferase signal was detected in an Orion L Microplate Luminometer (Berthold Detection System)
Otano I., Azpilikueta A., Glez-Vaz J., Alvarez M., Medina-Echeverz J., Cortés-Domínguez I., Ortiz-de-Solorzano C., Ellmark P., Fritzell S., Hernandez-Hoyos G., Nelson M.H., Ochoa M.C., Bolaños E., Cuculescu D., Jaúregui P., Sanchez-Gregorio S., Etxeberria I., Rodriguez-Ruiz M.E., Sanmamed M.F., Teijeira Á., Berraondo P, & Melero I. (2021). CD137 (4-1BB) costimulation of CD8+ T cells is more potent when provided in cis than in trans with respect to CD3-TCR stimulation. Nature Communications, 12, 7296.
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