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Antibody-Based Targeting of Angiogenesis

Cornelia Halin¹, Luciano Zardi² and Dario Neri¹

¹ Institute of Pharmaceutical Sciences, Department of Applied Biosciences, Swiss Federal Institute of Technology, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland; and
² Laboratorio di Biologia Cellulare, Istituto Nazionale per la Ricerca sul Cancro, Largo Rosanna Benzi 10, 16132 Genova, Italy

	Abstract

 
The selective targeting of neovasculature opens new avenues for the diagnosis and therapy of angiogenesis-related diseases such as cancer, blinding ocular disorders, and rheumatoid arthritis. Here we review recent advances in the identification of markers of angiogenesis as well as in the isolation and use of antibodies (and their derivatives) for the in vivo targeting of both tumoral and nontumoral neovasculature.

	Introduction

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At the end of the 19th century, Paul Ehrlich envisioned the use of antibodies as "magic bullets" that would deliver toxic agents to a tumor site. The idea gained momentum in the late 1970s after the development of hybridoma technology, which allowed the production of antibodies of single specificity. Since then, monoclonal antibodies directed against tumor-associated markers have been used to target solid tumors in animal models and in patients. Initial clinical studies were hindered by the immunogenicity of rodent antibodies, a problem that could be overcome by advances in protein engineering such as the production of chimeric antibodies, antibody humanization, and the construction of fully human antibodies (9). At present, 19 antibody-based biopharmaceuticals have been approved either in the United States or in Europe. Six of them are indicated for the scintigraphic detection of tumors, and three have been approved for the treatment of certain types of B-cell lymphoma (Rituxan), breast cancer (Herceptin), and acute myeloid leukemia (Mylotarg). It is estimated that more than 700 monoclonal antibodies are currently in clinical trials sponsored by over 200 biotech companies (19).

In spite of these promising developments, solid tumors have proven to be relatively resistant to antibody-based therapies. This is due, in part, to the relative inaccessibility of tumor cells and to the poor penetration of antibodies into the tumor tissue. Since tumor cells are separated from the blood by endothelial cells and extracellular matrix (ECM) components surrounding the vasculature, tumor uptake is highly limited by the antibody's ability to cross this layer. An abundance of tumor antigen in the perivascular region, a dense network of ECM components, and an elevated interstitial pressure further limit antibody diffusion to distant tumor cells. In fact, in patients, typically only 0.001–0.01% of the injected antibody dose accumulates localized per gram of solid tumor.

	Vascular targeting

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Considering the limitations of antibody-based targeting of individual tumor cells, recent research has focused on the development of antibodies that selectively target the tumor neovasculature while sparing mature blood vessels and healthy tissues. Vascular targeting approaches are interesting for a number of reasons: 1) markers on the tumor neovasculature are readily accessible to intravenously administered antibody derivatives; 2) markers of neovasculature are typically produced by endothelial cells and/or by fibroblasts, and such cells are genetically more stable than tumor cells; 3) there is growing evidence that the selective damage of tumor neovasculature may lead to massive death of tumor cells, which rely on blood vessels to supply them with nutrients and oxygen to satisfy their metabolic needs (7) (it is estimated that >100 tumor cells rely on one endothelial cell for survival); and 4) therapeutic strategies directed against tumor neovasculature appear to reduce the tumor's ability to develop metastases and may overcome multidrug resistance (10).

	Angiogenesis

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Angiogenesis, i.e., the formation of new blood vessels from preexisting ones, is accompanied by the neosynthesis of antigens on tumor endothelial cells and of novel ECM components. This process is a characteristic feature not only of aggressive solid tumors but also of other diseases, including rheumatoid arthritis, psoriasis, and ocular disorders such as the exudative form of age-related macular degeneration and diabetic retinopathy. Angiogenesis is a rare phenomenon in healthy adults, occurring only locally and transiently under distinctive physiological conditions such as wound healing, inflammation, and the female reproductive cycle.

In tumors, the switch to an angiogenic phenotype is known to be critical for disease progression. Unless a tumor can stimulate the formation of new blood vessels, it remains restricted to a microscopic size. Inflammation and hypoxia are widely accepted as key elements in the induction of angiogenesis.

In tissues undergoing angiogenesis, the ECM is remodeled by proteolysis and neosynthesis of its components, providing a more permissive and instructive environment for endothelial cells to migrate. There endothelial cells proliferate, differentiate, and align to form new vessels. During these processes, which may be common in different types of cancer and in other angiogenesis-related disorders, new antigens are formed, which are undetectable in mature vascular structures. In this light, antibodies directed against common markers of neovasculature, expressed in different diseases, may open up a very general and widely applicable approach for diagnostic and therapeutic interventions.

	Antibodies directed against markers of angiogenesis

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To date, only few good-quality markers of angiogenesis, either on endothelial cells or in the modified ECM, are known. The biggest problem with many of the markers is that they lack sufficient specific expression or significant upregulation in tissues undergoing angiogenesis. However, recent advances in new technologies, such as proteomics and genomics, are now facilitating the identification of such differentially expressed molecules (14). Furthermore, phage display and protein engineering are powerful tools that complement hybridoma technology in the generation of high-affinity antibodies. In the following pages, some of the most prominent markers currently known (and the corresponding antibodies) shall briefly be outlined.

Integrins vß3 and vß5.
Some integrins, in particular vß3 and vß5, have been proposed both as markers and as functional mediators of angiogenesis in tumors and in ocular neovascular disorders. Expression of integrin vß3 was also shown to be increased in synovial blood vessels from patients with rheumatoid arthritis. However, in recent immunohistochemical studies, the vasculature in apparently normal tissue as well as several extravascular cell types were shown to stain positive for vß3, even though at lower intensity than in tissues undergoing angiogenesis. A phase I clinical trial in tumor patients has recently been completed with the anti-vß3 antibody Vitaxin, which interferes with blood vessel formation by inducing apoptosis in newly generated endothelial cells. The treatment was considered safe and potentially active, suggesting that vascular integrin vß3 may represent a clinically relevant antiangiogenic target for prolonged cancer therapy (8).

Endoglin.
Many recent studies have described endoglin (CD105), a component of the transforming growth factor-ß receptor complex, as an attractive marker of neovascularization. Endoglin shows considerably increased expression on proliferating endothelium, but it also weakly stains endothelial cells in the majority of normal, healthy adult tissues of both human and mouse origin. Several monoclonal antibodies to endoglin have been characterized and have recently been tested as targeting agents for therapy and imaging of tumors. Unexpectedly, the targeting results obtained in mice were relatively modest (2), in spite of the accessible localization of the antigen on endothelial cells.

VEGF and VEGF-receptor complex.
Vascular endothelial growth factor (VEGF) is an angiogenic growth factor that is a primary stimulant of the vascularization of solid tumors. In the tumor microenvironment, an upregulation of both VEGF and its receptors occurs, leading to a high concentration of occupied receptor on tumor vascular endothelium. VEGF-receptor complexes were shown to be a specific target on tumoral endothelium for antibodies in vivo. In a recent study, a monoclonal antibody (2C3) was shown to have potent antitumor activity in tumor xenografts in mice (3). Other studies have also demonstrated that antibodies to VEGF selectively stain tumoral blood vessels after injection into tumor-bearing mice in vivo.

Prostate-specific membrane antigen. Prostate-specific membrane antigen (PSMA) is a type II transmembrane protein expressed by virtually all prostate cancers. In recent studies, monoclonal antibodies against PSMA were described that also strongly react with vascular endothelium within a wide variety of carcinomas (including lung, colon, breast, and others) but not with normal vascular endothelium. However, anti-PSMA antibodies have also been reported to recognize epithelia in benign tissue of breast, renal, duodenal, or prostate origin (6). The 7E11.C5 murine monoclonal antibody has been characterized in immunohistochemistry as well as in imaging studies. In fact, the 7E11.C5 antibody has been approved by the American Food and Drug Administration as an immunoscintigraphic agent (ProstaScint, Cyt-356) for the imaging of metastatic prostate cancer. However, this antibody is known to bind an intracellular epitope. A number of other anti-PSMA monoclonal antibodies, recognizing extracellular epitopes, have recently been described and have been characterized in vitro. Evaluation of their targeting performance in vivo is still missing.

CD44.
Different studies have shown that endothelial cells from solid tumors display an enhanced expression of CD44 compared with endothelial cells from normal tissue. CD44 is a cell adhesion receptor of great molecular heterogeneity due to alternative splicing and posttranslational modifications. It has been implicated in a variety of other responses, including leukocyte homing, activation, and invasion of malignant cells. Recent biodistribution and therapy studies, performed in mice and rats with an antibody against a CD44 splice variant, have characterized this antigen as an interesting vascular target expressed in different tumor types of various animal species (17).

ED-B of fibronectin.
The extradomain B (ED-B) of fibronectin is a sequence of 91 amino acids identical in mouse, rat, rabbit, dog, and humans that can be inserted into the fibronectin molecule by a mechanism of alternative splicing (20). Fibronectin containing ED-B (B-FN) accumulates around neovascular structures in aggressive tumors and other tissues undergoing angiogenesis, such as the endometrium in the proliferative phase and some ocular structures in pathological conditions. Otherwise it is undetectable in normal adult tissues (5). To date, the production of monoclonal antibodies directly recognizing the ED-B in B-FN has not been possible using hybridoma technology because of tolerance. This problem, however, has been overcome by using antibody phage technology with large synthetic antibody repertoires (13). Several antibody fragments specific for the ED-B of fibronectin have recently been generated. These antibody fragments stain vascular structures in tumor sections (Fig. 1, A and B) and selectively target tumor neovasculature, as shown in tumor-bearing mice by using infrared fluorescence and radioactive techniques (11, 15). Increased binding affinity leads to improved targeting of tumoral angiogenesis, as demonstrated by biodistribution studies performed using the L19 antibody fragment with affinity for the ED-B in the picomolar range and L19 mutants with reduced affinity (18). Figure 1C shows an ex vivo microautoradiographic analysis of the localization of radiolabeled scFv(L19) in a murine F9 teratocarcinoma. The antibody localizes strongly on the tumor vascular structures but does not stain vascular structures of normal tissues (15). The targeting results (Fig. 2) obtained with the scFv(L19) antibody are particularly impressive, considering that the target is a component of the modified ECM, located in the abluminal side of new blood vessels, and that neovasculature represents only a small percentage of the total tumor mass.

View larger version (76K): [in this window] [in a new window]   FIGURE 1. Immunohistochemical analysis of tumor sections by using a scFv fragment against the extradomain B (ED-B) of fibronectin. A: section of glioblastoma multiform showing staining of the typical glomerulus-like vascular structures. Bar = 50 µm. B: immunohistochemical staining performed on a section of murine F9 teratocarcinoma from a subcutaneously grafted tumor-bearing mouse. Bar = 25 µm. C: microautoradiography of an F9 teratocarcinoma removed from a tumor-bearing mouse after injection with a radiolabeled scFv against the ED-B of fibronectin. The radiolabeled antibody accumulates around the neovascular structures in the tumor. Bar = 50 µm.

View larger version (10K): [in this window] [in a new window]   FIGURE 2. Typical biodistribution data as obtained 24 h after injection of radiolabeled scFv(L19) into F9 teratocarcinoma-bearing nude mice. Targeting results are expressed as percent injected dose per gram of tissue (%ID/g). The tumor-to-blood and tumor-to-organ ratios, indicators of targeting performance, are approximately 10:1.

	Diagnostic and therapeutic applications

Top Introduction Vascular targeting Angiogenesis Antibodies directed against... Diagnostic and therapeutic... References

 
Antibody-based targeting of angiogenesis is likely to open new important diagnostic and therapeutic opportunities. To date, however, only a few antiangiogenesis antibodies have entered clinical investigations.

Antibodies specific for markers of angiogenesis may be useful for the detection of the corresponding antigen in plasma or for the in vivo imaging of angiogenesis-related diseases by immunoscintigraphic techniques. One possible drawback of neovascular targeting for imaging is the fact that blood vessels only represent a small percentage of the total tumor mass. Thus in vivo detection of new blood vessels crucially depends on the efficiency by which they can be targeted. Ultimately, targeting will depend on the quality of the antibody used, on the abundance and accessibility of the antigen, and on the fenestration and circulation properties of the neovasculature.

Therapeutic strategies employing antibodies targeting angiogenesis can be subdivided into two main categories. In the first, the antibodies are used to deliver therapeutic molecules to the vasculature. The second approach features antibodies that may have an intrinsic antiangiogenic activity, e.g., the blocking of essential mediators of vascular proliferation. Prominent examples (currently in clinical trials) are neutralizing anti-VEGF antibodies and antibodies directed against a VEGF receptor or the vß3 integrin.

Targeting therapeutic molecules to tumor blood vessels often relies on the assumption that destruction and/or occlusion of tumor blood vessels may indirectly cause tumor cell death. A strong proof of this concept has been provided by the work of Thorpe and colleagues (16). The authors used antibodies, directed against an artificially-induced marker on endothelial cells, to deliver a toxin (ricin A) or a procoagulant agent (truncated tissue factor) to the tumor neovasculature. Complete tumor remissions were observed in a significant proportion of the mice treated.

We have recently shown that a fusion protein, consisting of the scFv(L19) antibody fragment specific for the oncofetal ED-B of fibronectin fused to the extracellular domain of tissue factor, selectively targets tumor blood vessels in vivo. Furthermore, this immunoconjugate mediates the complete and selective infarction of three different types of solid tumors in mice. At the highest doses administered, complete tumor eradication was observed in 30% of the mice treated, without apparent side effects (12). These results could be of therapeutic relevance, since the ED-B of fibronectin, a naturally occurring marker of angiogenesis identical in mouse and humans, is expressed in the majority of aggressive solid tumors but is undetectable in normal vessels and tissues. Furthermore, our laboratories have recently demonstrated that fusion proteins consisting of scFv(L19) fused to interleukin-2 or interleukin-12 can significantly improve the therapeutic index of these cytokines.

A further therapeutic approach is the targeted delivery of photosensitizers, i.e., molecules that, on irradiation and in the presence of oxygen, release toxic diffusible agents such as singlet oxygen or reactive radicals. Birchler et al. (1) have recently reported that the anti-ED-B antibody fragment scFv(L19) selectively localizes to newly formed blood vessels in a rabbit model of ocular angiogenesis. When chemically coupled to a photosensitizer and irradiated with red light, this immunoconjugate mediates the complete and selective occlusion of ocular neovasculature and promotes apoptosis of the corresponding endothelial cells. Photosensitizers are already used in the clinic for the photodynamic therapy of certain forms of age-related macular degeneration. It is likely that the targeted delivery of such agents to neovascular sites will increase their therapeutic potential.

In conclusion, recent studies have shown that it is possible to selectively target angiogenesis in vivo by means of specific monoclonal antibodies. The outcome of present and future clinical trials will show how relevant vascular targeting approaches are for the diagnosis and therapy of angiogenesis-related diseases.

	Acknowledgments

 
D. Neri gratefully acknowledges financial support from the Krebsforschung Schweiz and the Eidgenössische Technische Hochshule, Zürich.

	References

Top Introduction Vascular targeting Angiogenesis Antibodies directed against... Diagnostic and therapeutic... References

 

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