G7940

The ADE assays were performed using Raji, THP-1, K562, SC and Daudi cell lines. 25 µL of serially diluted mAbs or mAbs combinations were mixed with 25 µL supernatant containing 750 TCID₅₀ pseudovirus. The mixture was incubated for 60 min at 37 °C, supplied with 5% CO₂. 50 µL cells at the density of 2 × 10⁶ cells/mL were added to the mixtures of pseudoviruses and mAbs for an additional 24 h incubation. Then, the same volume of luciferase detecting regents (Promega G7940) was added to each well. After 2 min of incubation, the luciferase activity was measured using a microplate luminometer (SpectraMax i3x MolecularDevices).

The ADE inhibition assay were performed using Raji cells. Diluted mAbs (MW05: 750 ng/mL and MW05/Mu: 200 ng/mL) were mixed with pseudovirus. The mixture was incubated for 60 min at 37 °C, supplied with 5% CO₂. 25 µL of serially diluted inhibitors of micropinocytosis and endocytosis. (Dynole, Abcam ab120346; methyl-β-cyclodextrin, Sigma C4555-1G, Thioridazine, Sigma T9025-5G; Apilimod, MCE HY-14644; Cytochalasin D, MCE HY6682; Fillipin, CAYMAM CHEMICAM COMPANY 70400; Baf.A1,Cell Signaling 56545 S) were mixed with 25 µL cells at the density of 4 × 10⁶ cells/mL. Then add the mixtures of pseudoviruses and mAbs to the cell plate for an additional 24 h incubation. Then, the same volume of luciferase detecting regents (Promega G7940) was added to each well. After 2 min of incubation, the luciferase activity was measured using a microplate luminometer (SpectraMax i3x MolecularDevices). For evaluation whether FcγRI expression on B cells could impact the ADE mediated by MW05 or not, the construct FcγRI-P2A-gamma chain driven by SFFV promoter was generated. Raji cells were used for ADE measurement after transfected by FcγRI-P2A-gamma chain construct with electroporation strategy.