Mouse Anti-PLAU Recombinant Antibody (clone mAb-112) (CAT#: PABL-306)

Figure 1 Functional effects of antibodies on in vitro plasminogen activation initiated by pro-uPA or active uPA.

0.25 nM pro-uPA or 0.25 nM uPA was incubated without (light blue dots) antibody or with 5 nM (pink dots), 10 nM (dark blue dots), 20 nM (yellow dots), 40 nM (green dots), 80 nM (red dots) and 160 nM (black dots) of mAb-112, mAb-PUK, mAb-12E6B10, mAb-101 or anti-PAI-1 mAb-2, as indicated. After incubation for 30 min at room temperature, plasminogen was added to 0.5 µM and S-2251 to 0.5 mM. Substrate hydrolysis was followed at 37°C by measuring the absorbance at 405 nm. Pro-uPA or uPA was omitted in controls (grey curves).

Botkjaer, K. A., Fogh, S., Bekes, E. C., Chen, Z., Blouse, G. E., Jensen, J. M.,... & Declerck, P. J. (2011). Targeting the autolysis loop of urokinase-type plasminogen activator with conformation-specific monoclonal antibodies. Biochemical Journal, 438(1), 39-51.

Figure 2 Effect of the antibodies on cell surface-associated plasminogen activation.

U937 cells were washed with glycine-HCl, pH 3, 0.1 M NaCl to remove pro-uPA and uPA bound to the cell surface. One nM pro-uPA or uPA alone or pre-incubated with monoclonal antibodies were added to the cells together with 0.4 µM α2-antiplasmin and 0.2 µM plasminogen. A, Example of a time course experiment with pro-uPA preincubated alone (black) or with mAb-PUK at 0.2 nM (blue), 2 nM (yellow), 20 nM (green) or 200 nM (red). B, IC50 plots of inhibition of cell surface-associated plasminogen activation by mAb-101 (black), mAb-112 (green) and mAb-PUK (red) in an assay with pro-uPA. C, IC50 plots of inhibition by mAb-101 (black), mAb-112 (green) and mAb-12E106B (yellow) in an assay with active uPA. All curves in B and C were fit to a hyperbolic decay equation, which provided approximate IC50-values.

Botkjaer, K. A., Fogh, S., Bekes, E. C., Chen, Z., Blouse, G. E., Jensen, J. M.,... & Declerck, P. J. (2011). Targeting the autolysis loop of urokinase-type plasminogen activator with conformation-specific monoclonal antibodies. Biochemical Journal, 438(1), 39-51.

Figure 3 Inhibitory effects of antibodies on tumour cell invasion in vitro and dissemination in vivo.

A, Matrigel invasion assay, in which PC-Hi/diss cells were allowed to invade the matrix barrier towards chemoattractants present in chicken embryonic fibroblast conditioned medium in the presence of 333 nM control IgG, mAb-112, mAb-PUK or mAb-12E6B10 or 0.1 TIU/ml aprotinin. The data are presented as percentage of control and are means ± SEM. ** and ***, p<0.005 and 0.0001, respectively. B, Tumour cell escape assay, in which PC-hi/diss cells were allowed to escape the initial collagen droplet and invade a fibrin-enriched collagen matrix. Antibodies and aprotinin were incorporated in final concentrations of 167 nM and 0.1 TIU/ml, respectively. The invasion index was calculated as the number of escaped tumour cells multiplied by the distance invaded. The data are presented as percentage of control and are means ± SEM. * and **, p<0.05 and 0.01, respectively. C and D, Spontaneous dissemination assay in chick embryos. PC-hi/diss cells were inoculated on the chorioallantoic membrane (CAM) of chicken embryos and allowed to form primary tumours, which were excised and weighed after 7 days of growth (C). The numbers of disseminated PC-hi/diss cells were determined by Alu qPCR in the portions of the CAM distal to the site of primary tumour formation (D). Data are presented as numbers of human cells per 10⁶ chicken cells and are means ± SEM.

Figure 4 Activation status of uPA produced by PC-hi/diss cells and its inhibition by serine protease inhibitors and activation-blocking mAb-112.

PC-hi/diss cells were incubated with control IgG, anti-uPA mAb-112, or indicated inhibitors, aprotinin and α2-antiplasmin (a2-AP). Chicken serum was added at 0.1% to the cultured cells as a source of plasminogen to initiate a plasmin-generating cascade. After incubating for 48 hours, samples of CM were analyzed by Western blot for uPA. Lane 1, pro-uPA is almost completely converted to a two-chain enzyme in the IgG control; lanes 2 and 4, aprotinin and a2-AP completely abrogated activation of pro-uPA; lane 3, mAb-112 significantly inhibited conversion of single-chain pro-uPA zymogen into the two-chain uPA enzyme. Positions of the molecular weight markers (kDa) are indicated on the right.

Bekes, E. M., Deryugina, E. I., Kupriyanova, T. A., Zajac, E., Botkjaer, K. A., Andreasen, P. A., & Quigley, J. P. (2011). Activation of pro-uPA is critical for initial escape from the primary tumor and hematogenous dissemination of human carcinoma cells. Neoplasia, 13(9), 806-IN7.

Figure 5 Inhibition of uPA-generated plasmin activity by mAb-112.

CM, containing a mixture of pro-uPA and active uPA produced by PC-hi/diss cells cultured in SF-DMEM for 24 hours, was incubated with control IgG, mAb-112, the chemical inhibitor of uPA activity amiloride or the plasmin inhibitor aprotinin. Plasminogen and the chromogenic S-2251 substrate were added to the CM to final concentrations of 100 and 0.5 mM, respectively, and the activity of the generated plasmin was determined by rate of S-2251 cleavage. Aprotinin completely prevented S-2251 cleavage, and there was no S-2251 cleavage in the presence of plasminogen alone (No CM).

Bekes, E. M., Deryugina, E. I., Kupriyanova, T. A., Zajac, E., Botkjaer, K. A., Andreasen, P. A., & Quigley, J. P. (2011). Activation of pro-uPA is critical for initial escape from the primary tumor and hematogenous dissemination of human carcinoma cells. Neoplasia, 13(9), 806-IN7.

Figure 6 Inhibition of spontaneous metastasis of PC-hi/diss carcinoma cells in a murine orthotopic model of prostate cancer by treatment with mAb-112.

(A) PC-hi/diss cells, tagged with firefly luciferase, were implanted into anterior prostates of NOD-SCID mice. After 7 days, mice were injected intraperitoneally with luciferin, imaged by IVIS and allocated evenly into two treatment groups. Levels of bioluminescence in the prostate region were similar at this time, indicating comparable initial tumor sizes (top panels). After 28 days, tumor-bearing mice treated with control IgG or mAb-112 were imaged again (bottom panels). One of the control mice presented extensive tumor cell ascites (far left bottom panel). However, levels of bioluminescence in the prostate area were similar in the mice from both treatment groups. (B) Gross morphology of primary prostate tumors (T, outlined with dotted line), enlarged inguinal LNs (circled with dotted line), and macroscopic tumor colonies in the mesenterium (yellow arrows). (C) Immunohistochemical staining of consecutive sections of prostate xenografts from mice treated with control IgG or mAb-112 (boxed panels on the left and right, respectively). To discriminate human tumor cells from the mouse stroma, sections were stained (brown) with human-specific anti-CD44 (top panels). Vasculature was highlighted (brown) by staining with anti-CD31 (bottom panels). In the control IgG group, gray arrowheads point to a nest of CD44-positive tumor cells (top), whereas gray arrows in a consecutive section point to the blood vessels associated with tumor nests (bottom). CD44-positive tumor cells, which seem to localize intravascularly, are indicated by black arrowheads (right top panel); the intravascular localization of these cells is confirmed by CD31 staining of capillaries indicated by solid black arrows (right bottom panel). In the mAb-112-treated group, black arrows (right bottom panel) point to blood vessels localized close to the tumor border, but not interacting with or encompassing the nearby tumor cells (brown staining, right top panel). Numbers above scale bars indicate length in micrometers. (D) Primary tumors were excised and weighed, indicating no difference in primary tumor size. Levels of metastasis to the liver, lungs, brain, and LNs (inguinal and axillary) were quantified by Alu-qPCR. Percent inhibition was calculated from pooled data for mAb-112-treated mice (n = 11) in comparison to IgG control mice (n = 14) in three individual experiments. Levels of metastasis in LNs were determined for the nodes positive for human cells; 9 of 14 inguinal and 11 of 14 axillary nodes were positive in the control group, whereas 7 of 11 nodes of both types were positive in the mAb-112 group.

Bekes, E. M., Deryugina, E. I., Kupriyanova, T. A., Zajac, E., Botkjaer, K. A., Andreasen, P. A., & Quigley, J. P. (2011). Activation of pro-uPA is critical for initial escape from the primary tumor and hematogenous dissemination of human carcinoma cells. Neoplasia, 13(9), 806-IN7.