Concurrent binding of anti-EphA3 antibody and ephrin-A5 amplifies EphA3 signaling and downstream responses: potential as EphA3-specific tumor-targeting reagents.

Abstract

The Eph receptor tyrosine kinases and their membrane-bound ephrin ligands form a unique cell-cell contact-mediated system for controlling cell localization and organization. Their high expression in a wide variety of human tumors indicates a role in tumor progression, and relatively low Eph and ephrin levels in normal tissues make these proteins potential targets for anticancer therapies. The monoclonal antibody IIIA4, previously used to isolate EphA3, binds with subnanomolar affinity to a conformation-specific epitope within the ephrin-binding domain that is closely adjacent to the "low-affinity" ephrin-A5 heterotetramerization site. We show that similar to ephrin-A5, preclustered IIIA4 effectively triggers EphA3 activation, contraction of the cytoskeleton, and cell rounding. BIAcore analysis, immunoblot, and confocal microscopy of wild-type and mutant EphA3 with compromised ephrin-A5 or IIIA4-binding capacities indicate that IIIA4 binding triggers an EphA3 conformation which is permissive for the assembly of EphA3/ephrin-A5-type signaling clusters. Furthermore, unclustered IIIA4 and ephrin-A5 Fc applied in combination initiate greatly enhanced EphA3 signaling. Radiometal conjugates of ephrin-A5 and IIIA4 retain their affinity, and in mouse xenografts localize to, and are internalized rapidly into EphA3-positive, human tumors. These findings show the biological importance of EphA3/ephrin-A5 interactions and that ephrin-A5 and IIIA4 have great potential as tumor targeting reagents.

• Authors: Christopher Vearing, Fook-Thean Lee, Sabine Wimmer-Kleikamp, Violeta Spirkoska, Catherine To, Con Stylianou, Mark Spanevello, Martin Brechbiel, Andrew W Boyd, Andrew M Scott, Martin Lackmann
• Journal: Cancer research (Cancer Res) Vol. 65 Issue 15 Pg. 6745-54 (Aug 01 2005) ISSN: 0008-5472 [Print] United States
• PMID: 16061656 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Antibodies, Monoclonal
• Ephrin-A5
• Immunoconjugates
• Indium Radioisotopes
• Receptors, Fc
• EPHA3 protein, human
• Receptor Protein-Tyrosine Kinases
• Receptor, EphA3

Topics

• Animals
• Antibodies, Monoclonal (metabolism, pharmacokinetics, pharmacology)
• Cell Line, Tumor
• Ephrin-A5 (metabolism)
• Humans
• Immunoconjugates (pharmacokinetics, pharmacology)
• Indium Radioisotopes (pharmacokinetics)
• Melanoma (diagnostic imaging, metabolism)
• Mice
• Mice, Inbred BALB C
• Mice, Nude
• Precursor B-Cell Lymphoblastic Leukemia-Lymphoma (diagnostic imaging, metabolism)
• Protein Structure, Tertiary
• Radionuclide Imaging
• Receptor Protein-Tyrosine Kinases (immunology, metabolism)
• Receptor, EphA3
• Receptors, Fc (metabolism)
• Signal Transduction
• Substrate Specificity
• Tissue Distribution
• Transplantation, Heterologous