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Fig. 1 | Breast Cancer Research

Fig. 1

From: Proteolytic single hinge cleavage of pertuzumab impairs its Fc effector function and antitumor activity in vitro and in vivo

Fig. 1

Pertuzumab (IgG-P) hinge cleavage was detected when IgG-P was incubated with higher human epidermal growth factor receptor 2 (HER2)-expressing cancer cell lines but not in low HER2 expressing cancer cell line. a Fragments of IgG with a single proteolytic cleavage in the lower hinge region (scIgG) generated under denaturing and reducing conditions, as assessed by western blotting detection. Fc(m) is the Fc monomer from the hinge cleavage and sc-Heavy chain indicates the N-terminal fragment from the hinge cleavage. Western blots showing hinge cleavage of IgG-P for the cell lines: BT474 (b, top panel); SKOV3 (c, top panel); SKBR3 (d, top panel); and MCF7-HER2, a MCF7 breast cancer cell line overexpressing HER2 (e, top panel). Low levels of Fc(m) were detected in MCF7 cells without HER2 expression (f, top panel). Cells were treated with 10 μg/ml of IgG-P for 4 h and 24 h at 37 °C, 5% CO2 in serum-free medium. Protein A magnetic beads were used to pull down the IgG-P proteolytic product. The hinge cleavage product, Fc monomer, was visualized by blotting the membrane using a secondary detection antibody, goat anti-human Fc-HRP antibody. A band shown on the western blotting with a molecular weight of 25 kDa was the Fc(m), which was seen in the scIgG-P enzymatically cleaved at the hinge region by immunoglobulin G-degrading enzyme S (IdeS). The intact IgG-P did not show a detectable band on the western blotting under reduced and denatured gel running conditions. High HER2 expression in BT474 (b, bottom panel), SKOV3 (c, bottom panel), SKBR3 (d, bottom panel), and MCF7-HER2 (e, bottom panel), and no detectable level of HER2 expression in MCF7 cells (f, bottom panel) were measured by FACS. g Detection of IgG-P hinge cleavage in cancer cell culture medium. Cancer cell-conditioned medium from BT474, SKOV3, SKBR3, MCF7, and MCF7-HER2 after treatment with IgG-P were collected after 24-h incubation and subjected to western blotting using a secondary detection antibody, anti-human Fc-HRP antibody: 10 μl of cancer cell-conditioned medium was loaded in each lane

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