Novel EphB4 Monoclonal Antibodies Modulate Angiogenesis and Inhibit Tumor Growth

EphB4 receptor tyrosine kinase and its cognate ligand EphrinB2 regulate induction and maturation of newly forming vessels. Inhibition of their interaction arrests angiogenesis, vessel maturation, and pericyte recruitment. In addition, EphB4 is expressed in the vast majority of epithelial cancers and provides a survival advantage to most. Here, we describe two anti-EphB4 monoclonal antibodies that inhibit tumor angiogenesis and tumor growth by two distinct pathways. MAb131 binds to fibronectin-like domain 1 and induces degradation of human EphB4, but not murine EphB4. MAb131 inhibits human endothelial tube formation in vitro and growth of human tumors expressing EphB4 in vivo. In contrast, MAb47 targets fibronectin-like domain 2 of both human and murine EphB4 and does not alter EphB4 receptor levels, but inhibits angiogenesis and growth of both EphB4-positive and EphB4-negative tumors in a mouse s.c. xenograft model. Combination of MAb47 and bevacizumab enhances the antitumor activity and induces tumor regression. Indeed, humanized antibodies hAb47 and hAb131 showed similar affinity for EphB4 and retained efficacy in the inhibition of primary tumor development and experimental metastasis.Angiogenesis is a process of new blood vessel sprouting from pre-existing vessels. This process includes primary capillary sprouting, branching, and remodeling into a mature blood vessel network. In the embryo, angiogenesis is preceded by vasculogenesis and represents one of the earliest processes in organ development.^1,2 In adulthood, angiogenesis is induced at sites of tissue repair, tissue remodeling such as during menstruation, and in diseases constituting enhanced angiogenesis.¹ Angiogenesis is also induced to varying degrees in cancers.¹ Angiogenesis is triggered by protease-mediated degradation of matrix proteins releasing angiogenic factors such as vascular endothelial growth factor (VEGF), epidermal growth factor, and fibroblast growth factor, followed by the migration of endothelial cells to sprout new vessels.^1,3 One critical step in vessel maturation is the interaction between arterial and venous capillaries leading to the fusion and lumen formation across the two cell types. This step is dependent on the interaction between EphB4 receptor tyrosine kinase expressed on venous endothelial cells and the trans-membrane ligand EphrinB2 expressed on arterial endothelial cells.^4,5EphB4 belongs to the Eph family, the largest family of receptor tyrosine kinases. To date, sixteen members of this receptor family have been characterized.^6,7 Eph receptors are divided into two subgroups: EphA members (10 in number) that bind to ligands (EphrinA1-6) that lack trans-membrane domains and localize to the cell membrane via glycosylphosphatidyl inositol linkage. EphB receptors (6 members) bind to ligands (EphrinB1-3) that contain a trans-membrane domain.^6,7 Receptor-ligand interaction leads to dimerization and phosphorylation of both receptor and ligand, resulting in bidirectional signaling. On stimulation with EphrinB2, Eph receptor “forward” signaling is triggered by autophosphorylation of its intracellular tyrosine kinase domain and downstream signaling.^8,9,10 Ephrin-B “reverse” signaling also depends on tyrosine phosphorylation of the Ephrin cytoplasmic region.¹⁰Targeted disruption of the EphB4 gene in mice is embryonically lethal secondary to the failure of blood vessel maturation and capillary arrest.¹¹ EphrinB2 is the only known ligand for EphB4. Targeted disruption of the EphrinB2 gene has a phenotype similar to EphB4 knockout.^5,11 The role of EphB4 in adult vertebrates is not fully understood, in part because of a lack of appropriate conditional knock-out lines. EphB4 and EphrinB2 are induced in newly forming venous and arterial vessels, respectively, and are thus expected to play an important role in adult angiogenesis.^9,12,13 This hypothesis is supported by the observation that the soluble monomeric form of the extracellular domain of EphB4 receptor (sEphB4) that blocks EphrinB2 interaction with its cognate receptors, blocks bidirectional signaling and inhibits angiogenesis at sites of neovascularization in the adult.^14,15,16Human tumor tissue analysis has shown that EphB4 is overexpressed in many tumor types: breast,¹⁷ colon,^18,19,20 bladder,²¹ endometrium,²² head and neck,²³ prostate,^24,25 and ovary.^26,27 EphB4 directly supports tumor cell survival by inhibiting apoptosis in many of these cancers.^{17,18,21,22,23,24,27} Tumor cell–expressed EphB4 also induces angiogenesis by direct interaction with EphrinB2 on tumor vessels.²⁸ In this study, we describe novel EphB4-specific monoclonal antibodies that inhibit formation and maturation of newly forming vessels and inhibit tumor growth in vivo.