The PDL1 specific VHH A12-, B3-, and irrelevant specificity VHHCTR(1B7)-IFNγ coding sequences were subcloned into the mammalian expression vector pVRC and transiently transfected using polyethyleneimine into HEK293F cells cultured in Freestyle media (ThermoFisher) (3 (link)). Media containing secreted protein was harvested six days following transfection by centrifugation at 8,000 × g for 20 min at 4°C, then loaded onto a HiTrap NiNTA column (GE Healthcare) and washed with 50 mM Tris, pH 8, 150 mM NaCl and 10 mM imidazole. Protein was eluted in 50- mM Tris, pH 8, 150 mM NaCl, 500 mM imidazole and 10% glycerol, then loaded onto a Superdex 200 16/600 column (GE Healthcare) in 50 mM Tris, pH 8, 150 mM NaCl, 10% glycerol. Recombinant VHH purity was assessed by SDS-PAGE, and peak fractions were recovered and concentrated with an Amicon 10,000 KDa MWCO filtration unit (Millipore), and stored at −80°C.