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By Fang Liao, PhD, Elisabetta Dejana, PhD, Peter Bohlen, PhD, and Daniel J. Hicklin, PhD
Over the past decade, the concept of angiogenesis has come of age since it was first described by Dr. Judah Folkman in the early 1970s.1,2 The complex nature of the cellular and molecular events associated with angiogenesis provides an abundant source of molecular targets for therapeutic design.
It is now widely accepted that unnecessary angiogenesis contributes to the pathogenesis of a variety of diseases, including solid tumors. Blocking angiogenesis has been shown to be a promising way of treating cancer. A significant number of angiogenic inhibitors have been reported that show promise in animals or clinical trials.3 Some of these inhibitors act via known mechanisms, e.g., endothelial cell growth/proliferation (inhibitors of vascular endothelial growth factor [VEGF] and its receptors), adhesion and migration/invasion (integrins and matrix metalloproteinase inhibitors), as well as the intracellular signaling pathways mediating cell growth and survival (kinase inhibitors).3 However, many others act via undefined mechanisms, e.g., angiostatin and endostatin, and may or may not be specific to endothelium. Herein, we will describe a novel target/mechanism for blocking angiogenesis, i.e., blocking assembly of capillary tube structures by preventing vascular endothelial (VE)-cadherin-mediated adherens junction formation among endothelial cells.
Endothelial cell-specific adhesion molecules play a crucial role in cell-cell and cell-extracellular matrix interaction and thus, have important vascular functions related to angiogenesis, such as mediation of cell migration and invasion. Certain members of the integrin family (e.g., avb3 and avb5) gained the attention of tumor biologists due to their restricted tissue distribution and differential regulation in tumor endothelium.4,5 Another type of adhesive molecule, the cadherins, also are involved in facilitating tumor growth, metastasis, and angiogenesis. Cadherins, a rapidly growing family of molecules that mediate calcium-dependent homophilic adhesion between cells of the same type, are transmembrane glycoproteins that typically contain five ectodomains and a short, highly conserved cytoplasmic portion anchoring the cadherin to the cytoskeleton. The ectodomains mediate homotypic cell-cell interaction and clustering.6 In addition to the classical roles that the cadherins play in "sorting" and maintaining the integrity of tissues,7 a number of cadherins are implicated in tumor biology. For example, loss or mutation of E-cadherin has been associated with increased invasiveness and metastasis in certain human tumors;8 switching of gene expression from E- to N-cadherin in melonocytes is a turning point in the development of malignant melanomas;9 and differential levels of H/T cadherin are observed in tumors exhibiting various amounts of vascularization.10 More recently, our studies showed that VE-cadherin (VE-cad) is involved in tumor angiogenesis.11
VE-cad is an endothelial-specific molecule and mediates adherens junction formation.12 Accumulating evidence implicates VE-cad in various aspects of vascular biology related to angiogenesis, most notably, endothelial cell assembly into tubular structures.13 (See Figure 1.) VE-cad null mouse embryos exhibit severely impaired assembly of vascular structures, which results in embryonic lethality at day E9.5,14 implicating VE-cad as an important mediator in developmental angiogenesis. Its restricted distribution and unique biological function distinguish VE-cad as a potential target for inhibition of endothelial cell-specific events, such as angiogenesis.
Monoclonal Antibody to VE-Cad Blocks Angiogenesis, Tumor Growth, and Metastasis
We tested one monoclonal antibody (BV13) directed against the extracellular region of murine VE-cad as a novel anti-angiogenic strategy designed to inhibit the assembly of capillary structures and, thus, tumor angiogenesis.15 This antibody has been studied extensively in mouse models of angiogenesis, mouse tumors, and human tumor xenografts, and has demonstrating potent anti-angiogenic and antitumor activity.11 Moreover, BV13 treatment inhibited the growth of metastases following removal of the primary tumor. Typically, treatment of human xenograft tumors with BV13 resulted in nearly complete inhibition of tumor growth and prevented tumor metastases in lungs. Histological examination of BV13-treated tumors showed evidence of decreased microvessel density, tumor cell apoptosis, decreased tumor cell proliferation, and extensive tumor necrosis. All these effects were observed as early as two weeks after treatment and gradually increased as antibody treatment continued. The decrease in tumor cell proliferation and necrosis in BV13-treated tumors likely reflects the lack of neovasculature needed to supply the rapidly growing tumor mass. Interestingly, antibody BV13-induced apoptosis was observed only in the tumors (mostly tumor cells and certain tumor endothelium), and not in normal endothelium.16 This selective effect of the anti-VE-cad antibody on tumor cell apoptosis may be due to its ability to inhibit tumor angiogenesis. Taken together, these findings indicate that VE-cad plays a crucial role in post-natal angiogenesis, and thus, validates VE-cad as a target for anti-angiogenic therapy.
Antibody BV13 Increases Vascular Permeability
It should be noted that BV13 has potent in vivo anti-tumor activity at 10-fold lower doses (50 mcg/dose) than other anti-angiogenic antibodies, such as those that block the functions of VEGF, VEGF receptor (VEGFR), or avb3.3 However, higher doses of BV13 (> 75 mcg, i.p.) resulted in increased vascular permeability and edema in the lung and was followed by the death of some animals within 24-48 hours. Moreover, the BV13-induced permeability effect is long-lasting and irreversible as compared to other permeability agents such as thrombin and histamine. Histological examination of BV13-treated lungs showed a noticeable pathology: bleb formations of endothelial and alveolar epithelial cells, aggregates of degranulated platelets, and the formation of platelet microthrombi, as well as leukocyte activation and infiltration resulting from endothelial cell retraction.15 These events were likely the consequence of vascular leakage caused by BV13 and further compound the vascular damage. Interestingly, BV13-induced vascular leakage could be alleviated by treating mice with an antibody (DC101)3,16 against VEGFR-2/Flk-1 due to the anti-permeability effect of this antibody (Liao and associates, unpublished data). Furthermore, we demonstrated that antibody DC101 abolishes the VEGF-induced tyrosine phosphorylation on both VEGFR-2 and VE-cad. These findings link the two pathways mediated by VE-cad and VEGF/VEGFR-2, and provide direct evidence for the notion that increased vascular permeability is the primary cause of the pathologic effect of antibody BV13. It remains to be determined whether a combination of these two antibodies will be beneficial therapeutically.
The permeability effect of BV13 on normal tissues is not entirely unexpected, since VE-cad is expressed equally in tumor and normal vasculature and BV13 does not
preferentially distribute to tumor blood vessels, but also binds to vessels in several tissues (Liao et al, unpublished data), including lung, kidney, and heart).17 Therefore, anti-VE-cad antibodies may not only prevent the formation of adherens junctions in nascent vasculature (junction formation), but also may interfere with established adherens junctions and, thus, cause increased permeability of the affected vasculature (junction disruption). We hypothesize that antibody blockade of VE-cad molecules on tumor vasculature is more likely to result in a therapeutic effect due to the tumor vasculature’s poor structural integrity (fenestration) and active angiogenesis. Indeed, we did not observe significant increased permeability in the lung or other tissues at the therapeutically efficacious dose of 50 mcg, nor did we observe other overt signs of toxicity during the course of treatment in a number of animals studies.
Is It Possible to Identify Antibodies That Selectively Inhibit Tumor Angiogenesis Without Affecting Existing Vasculature?
Our results indicate that antibody BV13 would not be an appropriate agent for therapeutic use due to its narrow therapeutic window. Thus, we aimed to identify a more desirable VE-cad inhibitor that preferentially affects ongoing angiogenesis. As for other cadherins, VE-cad clustering involves multiple adhesive contacts between different ectodomains. 18,19 Therefore, it may be possible to target regions of the VE-cad accessible only in growing vessels where junctions are not fully organized ("angiogenic epitopes"), but not exposed in existing vessels with mature junctions (see Figure 2). Indeed, in preliminary work, we have identified unique VE-cad antibodies that appear to inhibit adherens junction
formation but do not disrupt existing junctions.19 Detailed analyses of these antibodies in animal models, and of their corresponding epitopes, may lead to identification of reagents with a more selective anti-angiogenic potential.
Antibodies represent a unique class of therapeutics due to their high specificity toward defined antigens. The recent success of commercial, antibody-based cancer therapeutics has greatly revitalized enthusiasm in this field. With the advance and maturation of antibody engineering technologies, fully humanized antibodies with desired specificity can be readily obtained for cancer therapeutics. The use of a therapeutic antibody against VE-cad will be particularly appealing because of the unique localization of these target molecules on endothelium. It seems likely that an antibody to VE-cad with desired activity may be generally applied to all angiogenic settings, regardless of the angiogenic stimuli or tumor type. (Dr. Liao is Senior Scientist, Dr. Hicklin is Associate Vice President, and Dr. Bohlen is Senior Vice President, ImClone Systems, Inc., New York, NY; Dr. Dejana is a Consultant and Professor of Vascular Biology, Institute of Pharmacological Research, Milan, Italy.)
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