Anti-tumour effects of antibodies targeting the extracellular cysteine-rich region of the receptor tyrosine kinase EphB4

Abstract

EphB4 is a membrane-bound receptor tyrosine kinase (RTK) commonly over-produced by many epithelial cancers but with low to no expression in most normal adult tissues. EphB4 over-production promotes ligand-independent signaling pathways that increase cancer cell viability and stimulate migration and invasion. Several studies have shown that normal ligand-dependent signaling is tumour suppressive and therefore novel therapeutics which block the tumour promoting ligand-independent signaling and/or stimulate tumour suppressive ligand-dependent signaling will find application in the treatment of cancer. An EphB4-specific polyclonal antibody, targeting a region of 200 amino acids in the extracellular portion of EphB4, showed potent in vitro anti-cancer effects measured by an increase in apoptosis and a decrease in anchorage independent growth. Peptide exclusion was used to identify the epitope targeted by this antibody within the cysteine-rich region of the EphB4 protein, a sequence defined as a potential ligand interacting interface. Addition of antibody to cancer cells resulted in phosphorylation and subsequent degradation of the EphB4 protein, suggesting a mechanism that is ligand mimetic and tumour suppressive. A monoclonal antibody which specifically targets this identified extracellular epitope of EphB4 significantly reduced breast cancer xenograft growth in vivo confirming that EphB4 is a useful target for ligand-mimicking antibody-based anti-cancer therapies.

The Role of EphB4 and Ephrin-B2 Interactions in Prostate Cancer Models of Intravasation and Extravasation

“…The first model, simulated a primitive endothelium using a HUVEC monolayer to investigate the invasiveness of 22Rv1 EphB4 over-expressing cells. Two rare, events were observed where HUVEC cells formed a circular enclosure around a 22Rv1 EphB4 over-expressing cell, and a similar event was also found for 22 Rv1 (2013) shows is that knockdown of EphB4 inhibits the invasiveness and migratory capability of tumour cells, which has been shown in multiple studies to date [16][17][118][119]135]. However, we show that CIL is restored not because the invasiveness of EphB4 overexpressing tumour cells has been suppressed, but that the interaction of with EphB4-Fc [242].…”

“…Studies have shown that the balance between EphB4 and ephrin-B2 is disrupted in many cancers [117,118]. In cancer, the normal balance is altered when EphB4 is over-expressed, disrupting the normal ligand-dependent signalling and instead activating pro-tumorigenic ligand-independent signalling [119]. A multitude of studies have reported that EphB4 is over-expressed in many epithelial cancers including, prostate (66% of cases) [118,120] [124][125][126], breast (58-64%) [127][128][129], endometrial (100%) [130], pancreatic (100%) [131], lung (100%) [18] and cervical cancer (95%) [132,133].…”

“…A multitude of studies have reported that EphB4 is over-expressed in many epithelial cancers including, prostate (66% of cases) [118,120] [124][125][126], breast (58-64%) [127][128][129], endometrial (100%) [130], pancreatic (100%) [131], lung (100%) [18] and cervical cancer (95%) [132,133]. Data collated to 2015 reported that EphB4 protein levels had been examined in 1,318 tumour samples and were increased above the normal level in 1,046 (82%) of these [119]. Also studies have reported amplification of the EphB4 gene in clinical samples [127] [136].…”

“…Another interesting study developed an EphB4-specific monoclonal antibody that targets a region of 200 amino acids in the extracellular portion of EphB4 [119]. The antibody showed potent in vitro anti-cancer effects including an increase in apoptosis and a decrease in anchorage independent growth through phosphorylation and subsequent degradation of the EphB4 protein, suggesting a tumour suppressive mechanism of signalling [119]. In vivo application of the antibody significantly reduced breast cancer xenograft growth, thereby confirming that EphB4 may be a useful target for ligand-mimetic based antibody-cancer therapies [119].…”

EphB4 localises to the nucleus of prostate cancer cells

“…Generation of 22Rv1 prostate cancer cells over-expressing EphB4 (22Rv1-B4) has been described previously [26]. Murine monoclonal antibodies specifically targeting amino acids 218 to 239 in the cysteine-rich region of the human EphB4 extracellular domain (PVAGSCVVDAVPAPGPSPSLYC) (mAb 11H4, mAb C1) were provided by BenEphex Biotechnologies Pty Ltd (Adelaide, Australia) [27]. The EphB4 specificity of these monoclonal antibodies is reflected in the data shown in Supplementary Fig.…”