Fusion proteins composed of mouse IgG2a Fc and mouse beta-2-glycoprotein 1 bind to endothelial cells with exposed phosphatidylserine

AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA 4089 A promising target on 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. To target PS on tumor vasculature, a murine monoclonal antibody (3G4) was developed. 3G4 specifically localizes to the vasculature of solid tumors and inhibits growth of murine and human tumor xenografts. A chimeric version of 3G4 (bavituximab) is currently in clinical trials for treatment of solid tumor malignancies. Recently we determined that 3G4 recognizes PS through the formation of a complex with plasma protein beta-2-glycoprotein 1 (β2GP1). β2GP1 is a 50 kDa glycoprotein composed of five domains, including the positively charged domain V that binds anionic phospholipids. However, the interaction of β2GP1 with anionic phospholipids is weak under physiological conditions. Importantly, we demonstrated that 3G4 enhances the avidity of β2GP1 for membrane surfaces with exposed PS through the formation of multivalent 3G4/β2GP1/PS complexes. This increased avidity of β2GP1 for PS is likely responsible for targeting of 3G4 to tumor EC with exposed PS. We report here the construction of recombinant fusion proteins composed of mouse IgG2a Fc and mouse β2GP1. The Fc region is fused to the N-terminus of full-length β2GP1 or domain V only. This orientation was chosen to avoid interference with the lipid binding region in domain V of β2GP1, which is located at the extreme C-terminus of the protein. These compounds are generated as dimers, linked via disulfide bonds in the hinge region of the Fc tags. The dimeric construction provides the increased avidity necessary for these compounds to bind EC with exposed PS; thus, they behave in vitro much like the 3G4/β2GP1 complexes described previously. These Fc-β2GP1 compounds have potential as tumor therapeutic or imaging agents.