Plasma Protein β-2-Glycoprotein 1 Mediates Interaction between the Anti-tumor Monoclonal Antibody 3G4 and Anionic Phospholipids on Endothelial Cells

Simmons Comprehensive Cancer Center, Hamon Center for Therapeutic Oncology Research, the Department of Pharmacology and the Department of Radiation Oncology, the University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 11/2006; 281(40):29863-71. DOI: 10.1074/jbc.M605252200
Source: PubMed


A promising target on tumor vasculature is phosphatidylserine (PS), an anionic phospholipid that resides exclusively on the inner leaflet of the plasma membrane of resting mammalian cells. We have shown previously that PS becomes exposed on the surface of endothelial cells (EC) in solid tumors. To target PS on tumor vasculature, the murine monoclonal antibody 3G4 was developed. 3G4 localizes to tumor vasculature, inhibits tumor growth, and enhances anti-tumor chemotherapies without toxicity in mice. A chimeric version of 3G4 is in clinical trials. In this study, we investigated the basis for the interaction between 3G4 and EC with surface-exposed PS. We demonstrate that antibody binding to PS is dependent on plasma protein beta-2-glycoprotein 1 (beta2GP1). beta2GP1 is a 50-kDa glycoprotein that binds weakly to anionic phospholipids under physiological conditions. We show that 3G4 enhances binding of beta2GP1 to EC induced to expose PS. We also show that divalent 3G4-beta2GP1 complexes are required for enhanced binding, since 3G4 Fab' fragments do not bind EC with exposed PS. Finally, we demonstrate that an artificial dimeric beta2GP1 construct binds to EC with exposed PS in the absence of 3G4, confirming that antibody binding is mediated by dimerization of beta2GP1. Together, these data indicate that 3G4 targets tumor EC by increasing the avidity of beta2GP1 for anionic phospholipids through formation of multivalent 3G4-beta2GP1 complexes.

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    • "Bavituximab is a human-mouse chimeric IgG1 monoclonal antibody that targets PS by recognizing the PS-binding protein β2 glycoprotein- 1 (β2GP1). Specifically, by cross-linking two β2GP1 molecules at surface of cells with exposed PS, Bavituximab localizes to tumors [10]. Once bound, Bavituximab activates host effector (immune) functions, such as antibody dependent cellular cytotoxicity (ADCC), resulting in tumor vessel destruction. "
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    ABSTRACT: Bavituximab is a chimeric monoclonal antibody with immune modulating and tumor-associated vascular disrupting properties demonstrated in models of non-small cell lung cancer (NSCLC). The molecular target of Bavituximab, phosphatidylserine (PS), is exposed on the outer leaflet of the membrane bi-layer of malignant vascular endothelial cells and tumor cells to a greater extent than on normal tissues. We evaluated the tumor-targeting properties of Bavituximab for imaging of NSCLC xenografts when radiolabeled with (111)In through conjugation with a bifunctional chelating agent, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). In vitro binding of (111)In-DOTA-Bavituximab to PS was determined by enzyme-linked immunosorbent assay (ELISA). Biodistribution of (111)In-DOTA-Bavituximab was conducted in normal rats, which provided data for dosimetry calculation. Single-photon emission computed tomography/computed tomography (SPECT/CT) imaging was performed in athymic nude rats bearing A549 NSCLC xenografts. At the molar conjugation ratio of 0.54 DOTA per Bavituximab, the PS binding affinity of (111)In-DOTA-Bavituximab was comparable to that of unmodified Bavituximab. Based on the quantitative SPECT/CT imaging data analysis, (111)In-DOTA-Bavituximab demonstrated tumor-specific uptake as measured by the tumor-tomuscle ratio, which peaked at 5.2 at 72 hr post-injection. In contrast, the control antibody only presented a contrast of 1.2 at the same time point.These findings may underlie the diagnostic efficacy and relative low rates of systemic vascular and immune-related toxicities of this immunoconjugate. Future applications of (111)In-DOTA-bavituximab may include prediction of efficacy, indication of tumor immunologic status, or characterization of radiographic findings.
    Full-text · Article · Nov 2015 · American Journal of Nuclear Medicine and Molecular Imaging
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    • "We have developed a family of PS-targeting monoclonal antibodies that reactivate tumor immunity and induce immune cell-mediated destruction of tumor vasculature. In vivo screening methods were designed to identify antibodies that bound PS directly, but further characterization of the antibodies revealed that they interact with PS by forming high affinity complexes with the serum protein β2-glycoprotein I (β2GP1) [21]. The family of antibodies is named after a human-mouse chimeric antibody known as bavituximab that is currently being evaluated in clinical trials in cancer patients as an adjuvant to chemotherapy. "
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    ABSTRACT: Phosphatidylserine (PS) is an attractive target for imaging agents that identify tumors and assess their response to therapy. PS is absent from the surface of most cell types, but becomes exposed on tumor cells and tumor vasculature in response to oxidative stresses in the tumor microenvironment and increases in response to therapy. To image exposed PS, we used a fully human PS-targeting antibody fragment, PGN635 F(ab')2, that binds to complexes of PS and β2-glycoprotein I. PGN635 F(ab')2 was labeled with the positron-emitting isotope iodine-124 ((124)I) and the resulting probe was injected into nude mice bearing subcutaneous or orthotopic human PC3 prostate tumors. Biodistribution studies showed that (124)I-PGN635 F(ab')2 localized with remarkable specificity to the tumors with little uptake in other organs, including the liver and kidneys. Clear delineation of the tumors was achieved by PET 48 hours after injection. Radiation of the tumors with 15 Gy or systemic treatment of the mice with 10 mg/kg docetaxel increased localization in the tumors. Tumor-to-normal (T/N) ratios were inversely correlated with tumor growth measured over 28 days. These data indicate that (124)I-PGN635 F(ab')2 is a promising new imaging agent for predicting tumor response to therapy.
    Full-text · Article · Dec 2013 · PLoS ONE
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    • "Confocal microscopy revealed that the FGF1 export domains were PS positive (Figure 1E,F). These results were confirmed with bavituximab (Marconescu and Thorpe, 2008) that binds and stabilizes complexes of PS and β2-glycoprotein I (Luster et al., 2006). Bleb-like structures on the surface of heat shocked cells were coincidently stained with both anti-HA antibodies and bavituximab (Figure 1G). "
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    ABSTRACT: The mechanisms of nonclassical export of signal peptide-less proteins remain insufficiently understood. Here, we demonstrate that stress-induced unconventional export of FGF1, a potent and ubiquitously expressed mitogenic and proangiogenic protein, is associated with and dependent on the formation of membrane blebs and localized cell surface exposure of phosphatidylserine (PS). In addition, we found that the differentiation of promonocytic cells results in massive FGF1 release, which also correlates with membrane blebbing and exposure of PS. These findings indicate that the externalization of acidic phospholipids could be used as a pharmacological target to regulate the availability of FGF1 in the organism.
    Full-text · Article · Mar 2012 · Journal of Cellular Biochemistry
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