Metastasis and chemoresistance in colorectal cancer are mediated by certain poorly differentiated cancer cells, known as cancer stem cells, that are maintained by Notch downstream signaling initiated upon Notch cleavage by the metalloprotease ADAM10. It has been shown that ADAM10 overexpression correlates with aberrant signaling from Notch, erbBs, and other receptors, as well as a more aggressive metastatic phenotype, in a range of cancers including colon, gastric, prostate, breast, ovarian, uterine, and leukemia. ADAM10 inhibition, therefore, stands out as an important and new approach to deter the progression of advanced CRC. For targeting the ADAM10 substrate-binding region, which is located outside of the catalytic domain of the protease, we generated a human anti-ADAM10 monoclonal antibody named 1H5. Structural and functional characterization of 1H5 reveals that it binds to the substrate-binding cysteine-rich domain and recognizes an activated ADAM10 conformation present on tumor cells. The mAb inhibits Notch cleavage and proliferation of colon cancer cell lines in vitro and in mouse models. Consistent with its binding to activated ADAM10, the mAb augments the catalytic activity of ADAM10 towards small peptide substrates in vitro. Most importantly, in a mouse model of colon cancer, when administered in combination with the therapeutic agent Irinotecan, 1H5 causes highly effective tumor growth inhibition without any discernible toxicity effects. Our singular approach to target the ADAM10 substrate-binding region with therapeutic antibodies could overcome the shortcomings of previous intervention strategies of targeting the protease active site with small molecule inhibitors that exhibit musculoskeletal toxicity.

ADAM10 is a transmembrane metalloprotease that sheds a variety of cell surface proteins, including receptors and ligands that regulate a range of developmental processes which re-emerge during tumour development. While ADAM10 is ubiquitously expressed, its activity is normally tightly regulated, but becomes deregulated in tumours. We previously reported the generation of a monoclonal antibody, 8C7, which preferentially recognises an active form of ADAM10 in human and mouse tumours. We now report our investigation of the mechanism of this specificity, and the preferential targeting of 8C7 to human tumour cell xenografts in mice. We also report the development of novel 8C7 antibody–drug conjugates that preferentially kill cells displaying the 8C7 epitope, and that can inhibit tumour growth in mice. This study provides the first demonstration that antibody–drug conjugates targeting an active conformer of ADAM10, a widely expressed transmembrane metalloprotease, enable tumour-selective targeting and inhibition.

The Eph receptor tyrosine kinases and their ephrin ligands direct axon pathfinding and neuronal cell migration, as well as mediate many other cell–cell communication events. Their dysfunctional signaling has been shown to lead to various diseases, including cancer. The Ephs and ephrins both localize to the plasma membrane and, upon cell–cell contact, form extensive signaling assemblies at the contact sites. The Ephs and the ephrins are divided into A and B subclasses based on their sequence conservation and affinities for each other. The molecular details of Eph–ephrin recognition have been previously revealed and it has been documented that ephrin binding induces higher-order Eph assemblies, which are essential for full biological activity, via multiple, distinct Eph–Eph interfaces. One Eph–Eph interface type is characterized by a homotypic, head-to-tail interaction between the ligand-binding and the fibronectin domains of two adjacent Eph molecules. While the previous Eph ectodomain structural studies were focused on A class receptors, we now report the crystal structure of the full ectodomain of EphB2, revealing distinct and unique head-to-tail receptor–receptor interactions. The EphB2 structure and structure-based mutagenesis document that EphB2 uses the head-to-tail interactions as a novel autoinhibitory control mechanism for regulating downstream signaling and that these interactions can be modulated by posttranslational modifications.