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Fig. 5 | Breast Cancer Research

Fig. 5

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

Fig. 5

Intact trastuzumab (IgG-T) and intact pertuzumab (IgG-P) combination treatment increased IgG-P cleavage. aThe binding affinity to human epidermal growth factor receptor 2 (HER2) extracellular domain (ECD) for IgG-P, IgG-T and F(ab’)2 fragments of IgG-T and IgG-P. Microtiter plate wells were coated with HER2 ECD at a concentration of 2 μg/ml as the antigen. Threefold dilutions of IgG-P, IgG-T and the F(ab’)2 fragments of IgG-T and IgG-P were each applied to microtiter wells coated with recombinant human HER2 ECD. Goat anti-human kappa light chain-HRP conjugate was used as the detection antibody. b IgG-T and IgG-P proteolytic cleavage profile with/without addition of IgG-P-F(ab’)2 fragment and IgG-T-F(ab’)2 fragment, respectively. BT474 breast cancer cell line or SKOV3 ovarian cancer cell line were treated with IgG-T (10 μg/ml) with/without F(ab’)2 fragment of IgG-P (10 μg/ml) or vice versa 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

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