The
urokinase plasminogen activator receptor (uPAR) plays a role in
tumor progression and has been proposed as a target for the treatment of
cancer. We recently described the development of a novel humanized
monoclonal antibody that targets uPAR and has anti-
tumor activity in multiple xenograft animal
tumor models. This antibody,
ATN-658, does not inhibit
ligand binding (i.e. uPA and
vitronectin) to uPAR and its mechanism of action remains unclear. As a first step in understanding the anti-
tumor activity of
ATN-658, we set out to identify the
epitope on uPAR to which
ATN-658 binds. Guided by comparisons between primate and human uPAR,
epitope mapping studies were performed using several orthogonal techniques. Systematic site directed and
alanine scanning mutagenesis identified the region of aa 268-275 of uPAR as the
epitope for
ATN-658. No known function has previously been attributed to this
epitope Structural insights into
epitope recognition were obtained from structural studies of the
Fab fragment of
ATN-658 bound to uPAR. The structure shows that the
ATN-658 binds to the DIII domain of uPAR, close to the C-terminus of the receptor, corroborating the
epitope mapping results. Intriguingly, when bound to uPAR, the
complementarity determining region (CDR) regions of
ATN-658 closely mimic the binding regions of the
integrin CD11b (αM), a previously identified uPAR
ligand thought to be involved in leukocyte rolling, migration and
complement fixation with no known role in
tumor progression of solid
tumors. These studies reveal a new functional
epitope on uPAR involved in
tumor progression and demonstrate a previously unrecognized strategy for the therapeutic targeting of uPAR.