Anti-PTK7 Monoclonal Antibodies Inhibit Angiogenesis by Suppressing PTK7 Function

SIMPLE SUMMARY: PTK7 is a catalytically defective receptor protein tyrosine kinase. We previously demonstrated that PTK7 enhances angiogenesis by interacting with KDR, a vascular endothelial growth factor (VEGF) receptor important for angiogenesis, and activating it through oligomerization. To control angiogenesis by inhibiting PTK7 function, we developed anti-PTK7 monoclonal antibodies (mAbs). The selected PTK7 mAbs reduced VEGF-induced angiogenic phenotypes of endothelial cells and angiogenesis ex vivo and in vivo. The PTK7 mAbs also inhibited VEGF-induced KDR activation in endothelial cells and its downstream signaling and PTK7–KDR interaction. Our results show that the PTK7 mAbs inhibit angiogenesis by blocking PTK7 function. Therefore, PTK7 mAbs could be applied as therapeutics to control angiogenesis-associated diseases such as metastatic cancers. ABSTRACT: PTK7, a catalytically defective receptor protein tyrosine kinase, promotes angiogenesis by activating KDR through direct interaction and induction of KDR oligomerization. This study developed anti-PTK7 monoclonal antibodies (mAbs) to regulate angiogenesis by inhibiting PTK7 function. The effect of anti-PTK7 mAbs on vascular endothelial growth factor (VEGF)-induced angiogenic phenotypes in human umbilical vascular endothelial cells (HUVECs) was examined. Analysis of mAb binding with PTK7 deletion mutants revealed that mAb-43 and mAb-52 recognize immunoglobulin (Ig) domain 2 of PTK7, whereas mAb-32 and mAb-50 recognize Ig domains 6–7. Anti-PTK7 mAbs inhibited VEGF-induced adhesion and wound healing in HUVECs. mAb-32, mAb-43, and mAb-52 dose-dependently mitigated VEGF-induced migration and invasion in HUVECs without exerting cytotoxic effects. Additionally, mAb-32, mAb-43, and mAb-52 inhibited capillary-like tube formation in HUVECs, and mAb-32 and mAb-43 suppressed angiogenesis ex vivo (aortic ring assay) and in vivo (Matrigel plug assay). Furthermore, mAb-32 and mAb-43 downregulated VEGF-induced KDR activation and downstream signaling and inhibited PTK7–KDR interaction in PTK7-overexpressing and KDR-overexpressing HEK293 cells. Thus, anti-PTK7 mAbs inhibit angiogenic phenotypes by blocking PTK7–KDR interaction. These findings indicate that anti-PTK7 mAbs that neutralize PTK7 function can alleviate impaired angiogenesis-associated pathological conditions, such as cancer metastasis.