Fig. 6
Anti-hinge cleavage site antibodies rescued antibody dependent cellular cytotoxicity (ADCC) activity for a mixture of single hinge cleaved trastuzumab (scIgG-T) and single hinge cleaved pertuzumab (scIgG-P). SKOV-3 cells (5000 cells/well) were seeded on the E-plate as the target cell and peripheral blood mononuclear cells (25,000 cells/well) isolated from a single donor were used as the immune effector cells in complete cell culture medium containing a mixture of intact pertuzumab (IgG-P) (30 nM) and intact trastuzumab (IgG-T) (30 nM), or scIgG-P (30 nM) and scIgG-T (30 nM) with and without anti-hinge antibody (AHA) (120 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. a ADCC activity for a combination of IgG-T and IgG-P (black bar), a combination of scIgG-P and scIgG-T (white bar), and a combination of scIgG-T and scIgG-P using human anti-protease-induced AHA using peptide analogues representing hinge-immunoglobulin G-degrading enzyme S (IdeS) cleavage sites, 1981B (dark gray bar) or F(ab’)2 generated by digesting IgG-P with IdeS as the absorbent (light gray bar). b ADCC activity for a combination of IgG-T and IgG-P (black bar), a combination of scIgG-P and scIgG-T (white bar), and a combination of scIgG-T and scIgG-P using the anti-hinge mAb 2095–2 (dark gray bar). c ADCC cell lysis of the IdeS-expressing SKOV3-IdeS cell line by a combination of IgG-T and IgG-P (black bar), a combination of IgG-T and IgG-P + anti-hinge mAb 2095–2 (white bar), and a combination of IgG-T, IgG-P, and protease-resistant PR2095–2 (dark gray bar)