For expression of recombinant antibodies, synthetic genes encoding the mAb (B5, 1D8, urelumab analog, and 1A10) or bsAb (HER2×1D8, B5×1D8, and B5×1A10) were subcloned into pcDNA 3.4-TOPO plasmid using the pcDNA 3.4-TOPO TA Cloning Kit (Thermo Fisher Scientific, A14308). To produce recombinant antibodies, plasmids encoding each antibody were transfected to ExpiCHO-S cells (Thermo Fisher Scientific, A29127) using the ExpiCHO Expression System Kit (Thermo Fisher Scientific, A29133, containing ExpiFectamine CHO Reagent, ExpiFectamine CHO Enhancer, and ExpiCHO Feed). ExpiCHO-S cells were maintained in a shaking incubator at 37°C with 8% CO2 and prepared by diluting the ExpiCHO Expression Medium (Thermo Fisher Scientific, A29100) at a cell concentration of 6 × 106 cells/ml on the day of transfection. The ExpiFectamine CHO Reagent/plasmid complex was prepared in OptiPRO SFM (Thermo Fisher Scientific, 12309), incubated for 5 min at room temperature, and then transferred to the prepared cells. After 20 hours of incubation, ExpiFectamine CHO Enhancer (used to enhance protein production) and ExpiCHO Feed (used to support long-term, high-density transient transfection) were added and then further incubated for 7 to 10 days. Recombinant antibodies were purified from the cell culture fluid by protein A affinity chromatography using a HiTrap MabSelect SuRe column (GE Healthcare, 28-4082-55). The column was equilibrated by equilibration buffer [50 mM tris-HCl (pH 7.4), 100 mM NaCl] and then loaded with cell culture fluid. The antibody was eluted using elution buffer (50 mM citrate, pH 3.4) and then neutralized to pH 6.5 using 1 M tris-HCl (pH 9.0). Purified antibodies were concentrated in formulation buffer (20 mM histidine, 7% trehalose) by ultrafiltration using an Amicon Ultra 15 30K device (Merck, UFC903096), and protein concentrations were measured using NanoDrop One (Thermo Fisher Scientific). Endotoxin levels were ≤1.0 endotoxin unit (EU)/mg as measured by Endosafe nexgen-PTS (Charles River Laboratories).

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