ACTIVATION MECHANISM AND NOVEL THERAPEUTIC AGENT OF A MEMBRANE RECEPTOR INVOLVED IN PATHOGENIC ANGIOGENESIS
- Author(s): Au, Adrian Chichuen
- Advisor(s): Sun, Hui
- et al.
Pigment epithelial derived factor (PEDF) is a natural ligand with a wide range of therapeutic functions. It was first characterized as a neurotrophic factor secreted by the retinal pigment epithelium (RPE) but later found to be a potent anti-angiogenic factor that acts independent of vascular endothelial growth factor (VEGF). As a result, PEDF garnered significant attention from industry and academia but progress in leveraging PEDF's ability was hampered by the lack of insight in its signaling mechanism. After 20 years from its initial identification, the cell surface receptor responsible for PEDF's anti-angiogenic effect was found to be tumor endothelial marker 7 (TEM7) or plexin-domain containing 1 (PLXDC1). This receptor was found to be highly expressed in the endothelium of a wide range of solid tumors (e.g. pancreatic and colon cancer) and vision disease (e.g. diabetic retinopathy and age-related macular degeneration).
To transform the power of PEDF into an anti-angiogenic therapy that can treat human disease, we focused on two main goals. The first was to elucidate how PLXDC1 receptor activation causes endothelial cell death. The second is to develop antibodies that can diagnose endogenous PLXDC1 expression and that can kill pathogenic blood vessels through PLXDC1 receptor activation. Receptors are ideal therapeutic targets due to their accessibility and ability to regulate cell outcomes. Although no receptor activating antibody is currently used clinically, antibodies are an ideal drug as they are highly specific with minimal off-target toxicity.
With the use of a small molecule PLXDC1 agonist, we found that the mechanism of PLXDC1-mediated endothelial cell death is consistent with anoikis, a detachment induced cell death pathway. We observed that a small molecule PLXDC1 agonist inhibited cell adhesion, which caused cell detachment, re-localization of beta-catenin, de-phosphorylation of focal adhesion kinase, and ultimately cell death. Independently, integrin beta-1 (ITGB1), a transmembrane protein involved in attaching endothelial cells to the extracellular matrix, was identified as a binding partner to PLXDC1 by unbiased affinity purification/mass spectrometry. We also discovered that ITGB1 not only bound to PLXDC1, but at higher levels in the activated form of PLXDC1. This was functionally confirmed by enhanced PEDF mediated receptor activation with co-expression of ITGB1 and inhibition of PEDF induced endothelial cell death with knockdown of ITGB1.
Separately, we produced monoclonal and fragment antigen binding (Fab) antibodies that detect and kill pathogenic blood vessels through hybridoma and phage display strategies. Amongst the 96 mouse monoclonal antibodies, we identified three antibodies that reliably detected PLXDC1 in a wide range of human cancers. We then screened over 200 billion human Fabs for antibodies that could preferentially bind the activated receptor. Amongst those, we developed 30 antibodies that can inhibited cell adhesion, induced cell detachment, and caused endothelial cell death in vitro and ex vivo. Lastly, we demonstrate that a few of those antibodies showed ability to inhibit laser induced choroidal neovascularization progression in vivo with intravitreal injections.
In summary, we have identified the mechanism of PLXDC1 mediated endothelial cell death and developed diagnostic and therapeutic antibodies targeting PLXDC1. Combined this provides further insight into how we can translate PEDF's natural anti-angiogenic potential into a therapy to treat human cancer and vision disease.