Fig. 3.

Anti-hinge antibodies rescued antibody dependent cellular cytotoxicity (ADCC) activity for single hinge cleaved pertuzumab (scIgG-P). a-b Purified human anti-protease-induced, anti-hinge autoantibodies (AHA) using peptide analogues representing hinge-immunoglobulin G-degrading enzyme S (IdeS) cleavage sites, 1981 or F(ab’)₂ generated by digesting immunoglobulin G (IgG-P) with IdeS as the absorbent, restored ADCC activity for scIgG-P. SKOV-3 cell (5000 cells/well) was seeded on the E-plate 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 in complete cell culture medium containing scIgG-P (30 nM). The percentage of cell lysis was defined as: (cell index of control group – cell index of treatment group)/cell index of control group) × 100. c Flow cytometry showing binding results for AH-mAb with IgG-P or scIgG-P on surfaces of high human epidermal growth factor receptor 2-expressing cancer cells. Biotinylated 2095–2 and streptavidin-PE conjugate were used for cell staining. d-e 2095–2 ADCC rescuing effect for scIgG-P at varying concentrations. A fixed concentration of 30 nM for IgG-P with threefold dilutions from 30 nM for 2095–2 were used in the ADCC assay. SKOV-3 cells (5000 cells/well) and SKBR3 cell (7000 cells/well) were used as the target cells and PBMCs isolated from a single donor were used as the immune effector cells at an effector (E)-target (T) ratio of 25:1