Human Anti-HPN Recombinant Antibody (clone hH35) (CAT#: PABL-125)

Figure 2 Comparison of inhibition and affinity of hepsin antibodies

(A) Inhibition of hepsin activity by increasing amounts of antibody mH35 () displayed in log scale, chH35 () and hH35 (). After pre-incubation of the antibodies with hepsin for 30 min, the hydrolysis reaction was started by adding the acetyl-KQLR-AMC peptide. Fluorescence increase was measured after 40 min of incubation. The IC50 values were calculated after fitting the percentage inhibition data to a four-parameter equation. Error bars represent the S.D. for three experiments. (B) Adjusted SPR (Biacore) sensogram analysing the binding of hepsin to the immobilized hH35 antibody. Hepsin was injected at concentrations ranging from 0 to 200 nM. Curve fittings using a 1:1 Langmuir binding model are shown by black lines. (C) Comparison of binding and dissociation constants for the mH35, chH35 and hH35 antibodies, analysed by SPR measurement and displayed in log scale.

Koschubs, T., Dengl, S., Dürr, H., Kaluza, K., Georges, G., Hartl, C.,... & Huang, K. S. (2012). Allosteric antibody inhibition of human hepsin protease. Biochemical Journal,442(3), 483-494.

Figure 3 (A) HEK-293 cell lines that stably overexpress full-length hepsin with a C-terminal GFP fusion tag were analysed by flow cytometry. Specific and saturable surface staining could be detected by incubating with increasing amounts of hH35

Koschubs, T., Dengl, S., Dürr, H., Kaluza, K., Georges, G., Hartl, C.,... & Huang, K. S. (2012). Allosteric antibody inhibition of human hepsin protease. Biochemical Journal,442(3), 483-494.

Figure 4 (B) Hepsin surface staining by hH35 was confirmed by confocal laser-scanning microscopy analysis (red). The intrinsic fluorescence of the hepsin–GFP fusion protein is shown in green. Untransfected cells (HEK wt) did not display any detectable binding.

Koschubs, T., Dengl, S., Dürr, H., Kaluza, K., Georges, G., Hartl, C.,... & Huang, K. S. (2012). Allosteric antibody inhibition of human hepsin protease. Biochemical Journal,442(3), 483-494.

Figure 5 mAb hH35 is protease- and species-specific and exhibits non-linear inhibition

(A) Activity of hepsin antibodies against other serine proteases. The same test conditions were used for assessing the inhibitory potential of the antibodies on other serine proteases as were used for hepsin. Hepsin (1 nM), matriptase (2 nM), HAT (2 nM), enteropeptidase (0.7 nM) and trypsin (1.8 nM) were incubated with antibody (500 nM) for 30 min and the reaction was started with 5 μM acetyl-KQLR-AMC peptide. Data are shown for three replicate experiments. (B) Activity of antibody hH35 analysed in a FRET activity assay against human, cynomolgus and mouse hepsin. Cross reactivity was found with cynomolgus, but not with mouse hepsin. IC50 values are stated accordingly. (C) Progress curve of hepsin inhibition by hH35 mAb. Hepsin (0.5 nM) was added to a mixture of acetyl-KQLR-AMC peptide (10 μM) and 0, 18, 55, 166 and 500 nM hH35 mAb. The increase in fluorescence was monitored at 3-s intervals over 2 min. Rates for initial (v 0) and steady-state (vs) reactions were calculated using linear regression analysis. (D) Eadie–Hofstee plot of hepsin inhibition by hH35 mAb. Hepsin (1 nM) was pre-incubated without or with hH35 mAb (20–0.31 nM in 2-fold dilution steps) for 15 min. After the addition of acetyl-KQLR-AMC peptide (40, 20, 10, 5 and 2.5 μM) the linear rates of the increase in fluorescence were measured on a kinetic microplate reader.

Koschubs, T., Dengl, S., Dürr, H., Kaluza, K., Georges, G., Hartl, C.,... & Huang, K. S. (2012). Allosteric antibody inhibition of human hepsin protease. Biochemical Journal,442(3), 483-494.