Poster Presentation 14th International Biennial Conference on Metastasis Research 2012

Antibodies binding the ADAM10 substrate recognition pocket inhibit Eph function (#63)

Lakmali Atapattu 1 , Peter W Janes 1 , Nayanendu Saha 2 , Carmen Llerena 1 , Dimitar Nikolov 2 , Andrew M Scott 3 , Martin Lackmann 1
  1. Monash University, Clayton, VIC, Australia
  2. Structural Biology Program, Memorial Sloan-Kettering Cancer Centre, New York, USA
  3. Austin Branch, Ludwig Institute for Cancer Research, Heidelberg, Victoria, Australia

The function of a number of cell surface receptors implicated in oncogenesis relies on shedding of their ligands by members of the ADAM metalloprotease family. Important ADAM substrates include ligands for Eph and EGFR (erbB) receptor tyrosine kinases (RTKs), which are active in a wide range of chronic diseases, including cancer. ADAMs are thus considered promising targets for multi-pathway therapeutic intervention strategies1,2. However, to date considerable effort to develop specific inhibitors of ADAM protease activity has failed to yield specific and effective agonists.

Previously, our discovery that ADAM substrate recognition occurs within a non-catalytic, extracellular domain of ADAM10 allowed elaborating the mechanism regulating the release of Eph-RTK ligands (ephrins), which specifies that only ligands bound to catalytically-active RTKs are cleaved3,4. This regulatory motif thus provides a promising molecular target to inhibit key pathways in tumour progression, and prompted us to develop α-ADAM10 monoclonal antibodies (mAbs) that may block substrate access to this site. From a panel of α-ADAM10 mAbs, the specificity of ‘8C7’ and ‘3A8’ for the substrate recognition domain was confirmed by Biomolecular Interaction Analysis with recombinant ADAM10 fragments and Western blot analysis of ADAM10 KO mouse embryonic fibroblasts expressing human ADAM10 and derived substrate recognition site mutants. Functional analysis demonstrates that mAb 8C7 is rapidly internalised into ADAM10 expressing cells, and reveals 8C7 dose-dependent attenuation of Eph+ cell segregation away from surfaces of high ephrin density, a model considered a paradigm of Eph function5. In addition, 8C7 treatment of tumour cells dampens Eph and EGF receptor phosphorylation, suggesting its capacity to modulate RTK downstream signalling. Together, our results illustrate the potential of our mAbs for further development as novel anti-cancer therapeutics.

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  4. Janes, P. W. et al. Cytoplasmic relaxation of active Eph controls ephrin shedding by ADAM10. PLoS Biol 7, e1000215 (2009)
  5. Knoll, B., Weinl, C., Nordheim, A., and Bonhoeffer, F. (2007) Stripe assay to examine axonal guidance and cell migration, Nat. Protocols 2, 1216-1224