Identification of a New Epitope in uPAR as a Target for the Cancer Therapeutic Monoclonal Antibody ATN-658, a Structural Homolog of the uPAR Binding Integrin CD11b (αM)

Abstract

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.

Stabilizing a Flexible Interdomain Hinge Region Harboring the SMB Binding Site Drives uPAR into Its Closed Conformation

“…No crystal structure has so far been determined for unoccupied uPAR wt , which may pertain to its inherent interdomain flexibility [29]. All crystal structures solved so far thus represent a closed, stabilized uPAR conformation, which is attained by ligand occupancy with ATF [4][5][6]8,57], a synthetic antagonist peptide [7,50], or an artificial disulfide constraint introduced between DI and DIII [3,24]. The assembly of an empty and solvent-exposed uPA binding cavity by the constrained uPAR H47C-N259C mutant nevertheless clearly mimics the one we observe in the present uPAR·8B12 Fab complex.…”

“…The crystal structure solved for the uPAR·8B12 complex is shown in (a) where uPAR is depicted in a cartoon representation (DI, yellow; DII, light blue; DIII, red) and the bound Fab fragment of mAb 8B12 in a surface representation colored by elements (C, white; N, blue; O, red; S, yellow). Fab fragments of two mAbs ATN-615[5] and ATN-658[57], used to assist previous structure determinations of uPAR·ATF complexes, are superimposed for comparison. The dominating hotspot residues on uPAR (Arg 89 and Arg 91 ) for the interaction with 8B12 are shown as sticks.…”

uPAR antibody (huATN-658) and Zometa reduce breast cancer growth and skeletal lesions

“…No physical or behavioral abnormalities were noted in the huATN-658-treated animals during this study, consistent with previously published studies using huATN-658. [22][23][24] huATN-658 and Zometa combination decreases cell proliferation and invasion in vitro Since huATN-658 inhibits tumor growth while Zometa is approved for the treatment of bone metastases, we hypothesized that a combination of huATN-658 and Zometa would be more effective in blocking or reducing skeletal tumor burden as compared with huATN-658 or Zometa alone. We first evaluated the effect of the huATN-658 and Zometa combination on a panel of human breast cancer cell lines (MDA-MB-231, Hs578T, MDA-BoM-1833) in vitro.…”

“…The huATN-658 antibody binds to the DIII domain of uPAR near its C-terminal and does not interfere with uPA or vitronectin binding to uPAR. 24 Instead, huATN-658 blocks uPARintegrin interactions. 24 The fact that huATN-658 can bind to and neutralize uPAR functions regardless of its uPA binding status makes it pharmacologically attractive since it does not require overcoming the barrier associated with the displacement of endogenous uPA that is already present in a complex with uPAR.…”

“…24 Instead, huATN-658 blocks uPARintegrin interactions. 24 The fact that huATN-658 can bind to and neutralize uPAR functions regardless of its uPA binding status makes it pharmacologically attractive since it does not require overcoming the barrier associated with the displacement of endogenous uPA that is already present in a complex with uPAR. 22 In the current study, we assessed whether targeting uPAR is a viable anticancer strategy against breast tumor growth using a xenograft model.…”