HtrA3 modulating monoclonal antibodies and their potential therapeutic applications (#289)
HtrA3 is a secreted multidomain serine protease of the HtrA family proteins. It was initially identified in the developing placenta both in the mouse and human. It negatively regulates trophoblast invasion during placental development and abnormal levels of HtrA3 during early pregnancy in women are associated with risks of developing preeclampsia. HtrA3 is proposed to be a tumor suppressor and HtrA3 downregulation is associated with development of lung, endometrial and ovarian cancer. To date, there are no reagents available for treatment of diseases involving HtrA3 dysregulation. We have produced a panel (6G6, 9C9, 2C4, 10H10 and 3E6) of highly specific monoclonal antibodies (mAbs) to HtrA3. We aimed to establish whether some of these HtrA3 mAbs can modulate HtrA3 activity in vitro and in cells.
An in vitro protease activity assay of HtrA3 in the presence or absence of HtrA3 mAbs was performed to hydrolyse p-nitroanaline (-pNA) labelled peptide substrate. When HtrA3 was incubated with the substrate, a progressive release of -pNA resulting from hydrolysis was detected overtime. While mAbs 9C9, 2C4 or 3E6 did not significantly affect HtrA3 activity, 10H10 inhibited whereas 6G6 stimulated HtrA3 protease activity in a dose dependent manner. In addition, 10H10 inhibited the HtrA3 activity following 6G6 stimulation, and the inhibition was also dose-dependent. Both 10H10 and 6G6 modulated the HtrA3activity, but not HtrA1 and HtrA2, confirming high specificity. Using real-time monitoring of cell migration and invasion, we further confirmed that 10H10 mAb, compared to isotype-matched control mAb, significantly increased the migration and invasion of trophoblast HTR8 cells by inhibiting HtrA3.
This data confirmed that 10H10 neutralizes while 6G6 stimulates HtrA3 enzyme activity. Both antibodies are highly specific to HtrA3 and do not affect HtrA1 or HtrA2 activity. These HtrA3 mAbs provide useful tools for studying HtrA3 and treating human diseases associated with HtrA3 dysregulation.