Engineering Hybridoma Cells for PD-L1 Targeting

Previously, MIH5 hybridoma cells were engineered to express mIgG1, mIgG2a_silent or Fab fragments against PD-L1 with a Sortag and a Histag at the C-terminus of the heavy chain(s) (plasmids available at www.addgene.org/, ID: 124802, 124807 and 124810) [27 (link)]. Other cell lines that were used throughout this study were 4T1 (ATCC CRL-2539) and Renca (ATCC CRL-2947). All cells were cultured in RPMI-1640 (Gibco, 11875-093, Thermo Fisher Scientific) supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mM UltraGlutamine (BE16-605E/U1, Lonza) and 1 × antibiotic–antimycotic (15240-062, Thermo Fisher Scientific). In addition, Hybridoma medium contained 50 μM Gibco 2-mercaptoethanol (2-ME) (21985-023, Thermo Fisher Scientific). Renca cell medium additionally contained 0.1 mM non-essential amino acids (NEAA) (11140-035, Thermo Fisher Scientific) and 1 mM Sodium Pyruvate (Gibco, 11360-070), Thermo Fisher Scientific). (Semi-) adherent cells were washed with phosphate-buffered saline (PBS) (Fresenius Kabi) and detached by incubation with 0.025% trypsin and 0.01% ethylenediaminetetraacetic acid (EDTA) in PBS (TE) (Thermo Fisher, R001100) for 10 min. at 37 °C, or by using a cell scraper.