[Proc Amer Assoc Cancer Res, Volume 46, 2005]
Immunology 15: Novel Vaccine and Antibody Approaches
In vitro and in vivo characterization of a monoclonal antibody, ATN-658, targeting the uPA system
Jennifer A. Callahan,
Todd W. Bauer,
Lee M. Ellis,
Andrew P. Mazar and
Graham C. Parry
Attenuon, L.L.C., San Diego, CA and UTMD Anderson Cancer Center, Houston, TX
A substantial body of in vitro and in vivo data has implicated the urokinase plasminogen activator system, comprised of the serine protease urokinase (uPA), its cell surface receptor uPAR and plasminogen activator inhibitor-1 (PAI-1), in the neo-vascularization, invasion and metastasis of many solid tumors. More recent work has suggested that the interaction of uPAR with other cell surface receptors, particularly integrins, may play a significant role in these processes - independent of uPA catalytic activity. The important role of uPA-uPAR in tumor growth combined with its abundant expression within tumor, but not normal tissue makes it an attractive diagnostic and therapeutic target. We previously described the production and characterization of monoclonal antibodies specific for the D2D3 domain of uPAR. We have extensively characterized one of these antibodies, ATN-658, both in vitro and in vivo. ATN-658 bound immobilized recombinant uPAR and uPAR expressed on the surface of HeLa cells with a KD of 1 nM. Importantly, ATN-658 bound recombinant suPAR in which all non-essential glycosylation sites were mutated with a similar affinity - indicating that the antibody is specific for the protein portion of uPAR. Interestingly, although [I-125]-labeled ATN-658 bound to HeLa cells with high affinity, its binding was not competed by uPA. Similarly, binding of [I-125]-labeled scuPA to HeLa cells was unaffected by pre-incubation with unlabeled ATN-658. Unlike other antibodies we have tested, ATN-658 was not internalized upon binding to HeLa cells or MDA MB231 cells. To characterize the functional activity of the antibody, migration assays were performed. Although ATN-658 does not disrupt uPA binding to its receptor, ATN-658 inhibited uPA-induced migration of CHO cells expressing uPAR. Similarly, using a modified Boyden chamber model, ATN-658 inhibited the growth factor (HGF and IGF) induced migration of tumor cells. This data suggests that ATN-658 disrupts the interaction of uPAR with other cell surface receptors. The ability of ATN-658 to inhibit primary tumor growth was investigated in a number of in vivo models. To identify an appropriate dose and schedule for antibody treatment the circulating levels of ATN-658 were measured using a modified ELISA assay. Twice weekly treatment IP at 10 mg/kg resulted in a steady state plasma concentration of antibody of 0.4 µM, or 100-fold higher than the KD measured by in vitro binding assays. Using this dose and treatment schedule, ATN-658 significantly inhibited tumor growth in an A549 NSCLC early treatment model. Similarly, ATN-658 inhibited tumor growth in a staged A2780 ovarian carcinoma model. Taken together these data suggest that ATN-658, a monoclonal antibody specific for the D2D3 domain of uPAR, significantly inhibits tumor growth by a mechanism independent of uPA binding to its receptor.
Copyright © 2005 by the American Association for Cancer Research.