Fig. 4

Pertuzumab variants with Fc engineered to withstand protease attack, protease-resistant variant of pertuzumab (PRIgG-P) and PR2095–2, restored lost antibody dependent cellular cytotoxicity (ADCC) activity for immunoglobulin G (IgG-P) in an immunoglobulin G-degrading enzyme S (IdeS)-rich environment. a The hinge cleavage profiles are shown for IgG-P, Protease-resistant variant of trastuzumab (PRIgG-T), and PRIgG-P for the SKOV3 ovarian cancer cell line overexpressing the IdeS protease (SKOV3-IdeS), and for the BT474 breast cancer cell line overexpressing IdeS protease-stable cell lines (BT474-IdeS). The SKOV3-IdeS and BT474-IdeS cancer cell lines were treated with 10 μg/ml of IgG-P/PRIgG-P/PRIgG-T for 24 h at 37 °C, 5% CO₂ in serum-free medium. IgG-P and scIgG-P generated by digesting IgG-P with IdeS were used as standards. Protein A magnetic beads were used to pull down the IgG hinge proteolytic products, which were visualized by western blotting. b IgG-P and PRIgG-P binding affinity to human epidermal growth factor receptor 2 (HER2) receptor by ELISA. Microtiter plate wells were coated with recombinant human HER2 extracellular domain (ECD) at a concentration of 2 μg/ml as the antigen. IgG-P/PRIgG-P was used as the primary antibody then detected by goat anti-human Fc-HRP conjugate. c Flow cytometry showing the association between PRIgG-P or IgG-P and HER2 ECD on the cell surface. R-PE conjugated F(ab’)₂ goat anti-human IgG Fcγ was used for detection. d Comparison of ADCC activity between IgG-P and PRIgG-P. SKOV-3-IdeS ovarian cancer cell line (5000 cells /well) was used as the target cell and peripheral blood mononuclear cells (PBMCs) (25,000 cells/well) isolated from a single donor were used as the immune effector cell. The percentage of cell lysis was defined as: (cell index of control group – cell index of treatment group)/cell index of control group) × 100. e Comparison of ADCC activity between 2095 and 2 and PR2095–2 in an IdeS-rich environment. The SKOV-3-IdeS cell (5000 cells/well) was used as the target cell and PBMCs (25,000 cells/well) isolated from a single donor were used as the immune effector cells. Fixed concentrations of 30 nM of IgG-P and 60 nM of 2095–2/PR2095–2, respectively, were used in the ADCC assay. Experiments were conducted in triplicate and the error bars in the graphs correspond to SDs obtained in three independent experiments