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. 2016 Jul 11;8(2):2781–2799. doi: 10.18632/oncotarget.13157

Figure 1. The 3A2 Fab is a selective, low nanomolar inhibitor of MT1-MMP.

Figure 1

A. The clone, the sequence and the length of the CDR-H3 region in the selected Fab binders of MT1-MMP. B. Fab ELISA with the selected Fab binders of MT1-MMP. Left, the biotin-labeled catalytic domain of MT1-MMP (MT1-CAT) was captured onto streptavidin-coated wells of a 96-well plate. The Fab antibodies (0-8,000 nM) were allowed to bind to MT1-CAT. The bound antibodies were detected using HRP-conjugated anti-human Fab and a TMB/E substrate. Data are means ± SE from three individual experiments performed in triplicate. Right, the Kd values were calculated from the reactions in which a half of MT1-CAT was complexed with the added Fab. C. Inhibition of the MT1-MMP cleavage activity by the selected Fab antibodies. Left, the dose-response inhibition by the Fab fragments. The cleavage activity of MT1-CAT (5 nM) was measured in the presence of the increasing concentrations of the Fab antibodies (0-5,000 nM) using a Mca-PLGL-Dpa-AR-NH2 substrate (1 μM). The residual cleavage activity was expressed in percent relative to a “no Fab” control. Data are means ± SE from 3 individual experiments conducted in triplicate. Right, the IC50 values for the selected Fab antibodies. *a and *b, the weak inhibitory and non-inhibitory Fabs, respectively. D. The 3A2 Fab antibody is a selective inhibitor of MT1-MMP. The individual CAT of MT-MMPs (5 nM, each) were co-incubated with the increasing concentrations of the 3A2 Fab antibody (0-5,000 nM). The residual cleavage activity was measured using a Mca-PLGL-Dpa-AR-NH2 substrate (1 μM). Left, the representative dose-response curves of the 3A2 Fab antibody against MT1-CAT. Right, the IC50 values of the 3A2 Fab antibody in the individual MT-MMPs. RFU, relative fluorescence unit; *c, no inhibition at the highest Fab concentration used. E. The 3A2 Fab antibody inhibits MT1-MMP proteolysis of AAT. AAT (2 μM) was co-incubated with MT1-CAT alone (40 nM, no inhibitor) or jointly with the 3A2 or DX2400 Fab fragments (20 and 200 nM, each), TIMP-1 (1,000 nM), TIMP-2 (20 nM) or GM6001 (1,000 nM). The reactions were analyzed by SDS-PAGE followed by Coomassie staining. DX, DX2400.