Panitumumab and Anti-PD-L1-F(ab')2 Conjugation

Panitumumab (6.8 nmol) was incubated with IR700 NHS-ester (30.8 nmol, LI-COR Biosciences) in 0.1 mol/L Na₂HPO₄ (pH 8.6) at 25°C for 1 hour. Anti-PD-L1-F(ab′)₂ or control-F(ab′)₂ (9.1 nmol) was incubated with IR700 NHS-ester (63.7 nmol, LI-COR Biosciences) in 0.3 mL of 0.1 M Na₂HPO₄ (pH 8.6) at 25°C for 1 hour.22 (link) The mixture was separated and purified with a Sephadex G50 column (PD-10; GE Healthcare, Piscataway, New Jersey, USA).23 (link) The protein concentration was confirmed with a Coomassie Plus protein assay kit (Thermo Fisher Scientific Inc) by measuring absorption at 595 nm with spectroscopy (UV1900, Shimadzu, Japan).24 25 (link) The IR700 concentration was measured via absorption at 689 nm with spectroscopy to confirm the number of fluorophore molecules conjugated to mAb (dye–mAb ratio).26 (link) Bioactivity of the conjugated products was determined by testing its binding on LL/2-luc-GFP-PD-L1 cells. The cells (1×10⁵) were incubated with pan-700 (10 µg/mL) or anti-PD-L1-F(ab′)₂-IR700 (10 µg/mL) in medium for 6 hours at 37°C. For confirming the binding specificity of the new conjugates, a competition assay was performed by adding excess untreated anti-PD-L1 antibody (1 µg). Cells were analyzed with flow cytometry (Gallios, BD Biosciences) using Kalulza2.1 software (BD Biosciences).