Cy5.5-Anti-ephrin receptor B4 (EphB4) humanized monoclonal antibody hAb47

Excerpt

The ephrin (Eph) receptors constitute the largest group in the receptor tyrosine kinase family (1, 2). The Eph receptors and their ligands (ephrins) mediate numerous biological processes in normal development, particularly in the nervous and cardiovascular systems (3-5). On the basis of their structures and sequence relationships, ephrins are divided into two classes: the ephrin-A class, Ephs that are anchored to the cell membrane by a glycosylphosphatidylinositol linkage, and the ephrin-B class, Ephs that are transmembrane proteins. The Eph family of receptors is divided into two groups, EphA and EphB, on the basis of the similarity of their extracellular domain sequences and their affinities for binding ephrin-A and ephrin-B ligands. The Eph receptors transmit forward signals via their kinase domains and reverse signals via their transmembrane ephrin ligands (6). EphB–ephrin-B interactions are capable of mediating bidirectional signaling events upon cell–cell contact, either into the receptor-expressing cell as "forward signaling" or into the ligand-expressing cell as "reverse signaling" (7).

Eph-2 is expressed on arterial and activated endothelial cells, whereas EphB4 is normally expressed on venous endothelial cells and various blood cells (8). EphB4 selectively binds to ephrin-2 to promote cell signaling and angiogenesis. EphB4 has been implicated in cancer progression and in pathological forms of angiogenesis. Overexpression of EphB4 has been observed in cancer cells and is associated with tumorigenesis via forward signaling and with angiogenesis via reverse signaling through Eph-2 interaction (9). EphB4 forward signaling stimulates cellular proliferation. Koolpe et al. (10) identified a 15-mer peptide, Tyr-Asn-Tyr-Leu-Phe-Ser-Pro-Asn-Gly-Pro-Ile-Ala-Arg-Ala-Trp (TNYL-RAW), to be a selective antagonist of EphB4 with the use of phage display screening. Xiong et al. (11) reported the development of ⁶⁴Cu-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid-TNYL-RAW (⁶⁴Cu-DOTA-TNYL-RAW) for use with positron emission tomography (PET) imaging of EphB4 in nude mice bearing tumor xenografts. A monoclonal antibody mAb47 was found to have high affinity (0.8 nM) for the fibronectin-like extracellular domain of both human and mouse EphB4. Li et al. (12) conjugated Cy5.5 to the humanized Ab47 (hAb47) antibody to produce Cy5.5-hAb47 for use with near-infrared (NIR) fluorescence imaging of EphB4 expression in tumors.