Concurrent binding of anti-EphA3 antibody and ephrin-A5 amplifies EphA3 signaling and downstream responses: potential as EphA3-specific tumor-targeting reagents.
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.
Vearing
Christopher
C
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia. Martin.Lackmann@med.monash.edu.au
Lee
Fook-Thean
FT
Wimmer-Kleikamp
Sabine
S
Spirkoska
Violeta
V
To
Catherine
C
Stylianou
Con
C
Spanevello
Mark
M
Brechbiel
Martin
M
Boyd
Andrew W
AW
Scott
Andrew M
AM
Lackmann
Martin
M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism* / Q000493:pharmacokinetics / Q000494:pharmacology; D045744:Cell Line, Tumor; D036386:Ephrin-A5 / Q000378:metabolism*; D006801:Humans; D018796:Immunoconjugates / Q000493:pharmacokinetics* / Q000494:pharmacology; D007205:Indium Radioisotopes / Q000493:pharmacokinetics; D008545:Melanoma / Q000000981:diagnostic imaging / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D015452:Precursor B-Cell Lymphoblastic Leukemia-Lymphoma / Q000000981:diagnostic imaging / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011877:Radionuclide Imaging; D020794:Receptor Protein-Tyrosine Kinases / Q000276:immunology / Q000378:metabolism*; D036121:Receptor, EphA3; D011961:Receptors, Fc / Q000378:metabolism; D015398:Signal Transduction; D013379:Substrate Specificity; D014018:Tissue Distribution; D014183:Transplantation, Heterologous
D000911:Antibodies, Monoclonal; D036386:Ephrin-A5; D018796:Immunoconjugates; D007205:Indium Radioisotopes; D011961:Receptors, Fc; C491718:EPHA3 protein, human; D020794:Receptor Protein-Tyrosine Kinases; D036121:Receptor, EphA3