2.2 Protein expression and purification
The extracellular domain of human PD-L1 (amino acids 19-228) and the anti-PD-L1 scFvs atezolizumab, D12, and A1 (comprised of the variable heavy [VH] chain followed by the variable light (VL) chain of the respective antibodies) were expressed via transient transfection of HEK 293F cells. The encoding DNA was cloned into the gWiz mammalian expression vector (Genlantis) containing a 6x His-tag for purification. The PD-L1 construct also contained a biotin acceptor peptide (BAP) GLNDIFEAQKIEWHE sequence immediately prior to the His-tag. HEK 293F cells were grown to 1.2×106cells/mL and diluted to 1.0×106 cells/mL on the day of transfection. Midiprepped DNA and polyethyleneimine (PEI, Polysciences) were independently diluted to 0.05 and 0.1 mg/mL in OptiPro medium (Thermo Life Technologies), respectively, and incubated at room temperature for 15 min. Equal volumes of DNA and polyethyleneimine were mixed and incubated at room temperature for an additional 15 min. Subsequently, the diluted HEK 293F cells and the DNA/PEI (40 mL/L) mixture were added to a shaking flask and incubated at 37˚C and 5% CO2 with rotation at 125 rpm for 72 hr. Secreted protein was harvested from HEK 293F cell supernatants after 72 hr by Ni-NTA (Expedeon) affinity chromatography. PD-L1 was biotinylated overnight at 4ºC using BirA ligase enzyme in 0.5 mM Bicine pH 8.3, 100 mM ATP, 100 mM magnesium acetate, and 500 mM biotin (Avidity). scFvs and biotinylated PD-L1 were further purified using a Superdex 200 sizing column on a fast protein liquid chromatography (FPLC) instrument (GE Healthcare), equilibrated in HEPES-buffered saline (HBS). Purity (>98%) was confirmed by SDS-PAGE analysis.