Proc Amer Assoc Cancer Res, Volume 47, 2006 2179 A promising target for tumor vasculature is phosphatidylserine (PS), an anionic phospholipid that resides exclusively on the inner leaflet of the plasma membrane under normal conditions. We have previously shown that PS becomes exposed on the surface of viable endothelial cells (EC) in solid tumors, likely in response to oxidative stresses that exist in the tumor microenvironment. To target PS on tumor vasculature, a murine monoclonal antibody (3G4) was developed. 3G4 specifically localizes to the vasculature of solid tumors. Treatment of mice with 3G4 inhibits growth of murine and human tumors and enhances the anti-tumor effects of standard radio- and chemo-therapeutic strategies. Therefore, 3G4 is a promising new therapeutic agent for treatment of solid tumor malignancies. We demonstrate here that the interaction between 3G4 and PS is dependent on antibody binding to the plasma glycoprotein beta2-glycoprotein I (β2GPI). When purified from a hybridoma converted to grow under serum-free conditions, 3G4 does not bind PS-coated ELISA plates unless serum is added to the binding reaction A similar phenomenon was described in the early 1990s, when “anti-cardiolipin antibodies” from patients with Anti-Phospholipid Syndrome (APS) were shown to require various plasma proteins (such as β2GPI or prothrombin) for binding to cardiolipin. Screening a panel of serum proteins for reactivity with 3G4 by ELISA and western blot identified β2GPI as the primary antigen for 3G4. β2GPI is a 50 kDa glycoprotein composed of five domains, including the positively charged domain V that binds anionic phospholipids. However, the interaction with anionic phospholipids is rather weak under physiological conditions. We demonstrate that 3G4 binds domain II of β2GPI and enhances the binding of β2GPI to EC induced to expose PS (PS-positive) 40- to 50-fold. We also show that binding of 3G4 to PS-positive EC is dependent on the lipid binding domain of β2GPI (domain V), since enzymatic cleavage of the lipid binding site abrogates both binding to PS and to EC. Finally, we demonstrate that the 3G4/β2GPI/PS binding interaction requires multivalent binding of 3G4 to β2GPI, since 3G4 Fab’ fragments do not bind PS-positive ECs. Together, the data suggest that 3G4 targets tumor vessels by increasing the binding affinity of β2GPI to PS exposed on tumor EC.