NeuroMab™ Anti-Clusterin BBB Shuttle Antibody(NRZP-1022-ZP3574)

Figure 1. Kinetic analysis of 16B5 murine and humanized anti-clusterin antibodies.

Figure 2. h16B5 blocks the migration of cancer cell lines. In a scratch test against mouse breast cancer cells, h16B5 was at least as active as mouse 16B6. This figure shows the ability of various configurations of antibodies to block cell migration in vitro.

Figure 3. Inhibit h16B5 and reduce the invasiveness of human prostate cancer cells.

The bottom of 12-well plates was covered with 200 μL Growth factor reduced matrigel (Becton Dickinson). Cells (2.5×104) were resuspended in 200 μL matrigel, which was layered on top of this, and finally 500 μL of cell specific growth medium was added on top of the matrigel. h16B5 was added to every layer of matrigel as well as the medium in a concentration of 8 μg/mL. Plates were incubated at 37° C. for up to 3 weeks during which the growth medium (+/−h16B5) was replenished weekly. The arrows indicate the areas of epithelial-like cells.

Figure 4. Treatment of prostate cancer tumors with h16B5 reduces tumor growth and increases their response to chemotherapy.

DU145 prostate cancer cells (2×106) were implanted s.c. into SCID mice and allowed to grow until the tumor sizes were approximately 100 mm3. Tumors were measured bi-weekly with a digital caliper and tumor volumes were calculated as L×W×H. Each group contained 8 animals that were randomized prior to the beginning off the treatments. The results were expressed as mm3±SEM. The P values were calculated using the Student's T test.

Figure 5. h16B5 inhibits the growth of PC-3 prostate tumors.

PC-3 prostate cancer cells (2×106) were implanted s.c. into SCID mice and allowed to grow until the tumor sizes were approximately 100 mm3. Tumors were measured bi-weekly with a digital caliper and tumor volumes were calculated as L×W×H. Each group contained 8 animals that were randomized prior to the beginning off the treatments. The results were expressed as mm3±SEM.

Figure 6. Clusterin is expressed in human pancreatic tumors.

Tumors derived from pancreatic cancer cell lines (as indicated) were grown in SCID mice, harvested, fixed in formalin, sections and examined using immunohistochemistry with h16B5. Positive staining was visualized by standard methods using a HRP-conjugated secondary antibody. The negative control was performed under identical conditions with an isotype control antibody.

Figure 7. H16B5 inhibits the migration of pancreatic cancer cell lines.

PANC-1 cells were seeded in serum-free medium in the upper chamber of a 24-well Transwell plate containing a Matrigel barrier. The lower wells were filled with medium containing 10% FBS as a chemo-attractant. After a 24 h incubation in the presence or absence of TGFβ and/or h16B5 (as indicated), the number of cells in the Matrigel layer were stained and counted.

Figure 8. H16B5 inhibits the internalization of clusterin secreted in cancer cells.

The experiment was performed H16B5 was added at 10 μg/ml.

Figure 9. Shown is the binding of h16B5 (labeled AB-16B5 in the figure) to mouse clusterin, which is expressed in fixed frozen sections generated from 4T1 mouse mammary tumors.

The experiment was performed using immunohistochemistry and detection was accomplished using a HRP-conjugated secondary antibody.