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Research Articles: Therapeutics, Targets, and Development
Direct targeting of
vß3 integrin on tumor cells with a monoclonal antibody, AbegrinTM
MedImmune, Inc., Gaithersburg, Maryland
Requests for reprints: David A. Tice, MedImmune, Inc., One Medimmune Way, Gaithersburg, MD 20878. Phone: 301-398-4592; Fax: 301-398-9592. E-mail: ticed{at}medimmune.com
Abstract
The humanized monoclonal antibody AbegrinTM, currently in phase II trials for treatment of solid tumors, specifically recognizes the integrin 
vß3. Due to its high expression on mature osteoclasts, angiogenic endothelial cells, and tumor cells, integrin vß3 functions in several pathologic processes important to tumor growth and metastasis. Targeting of this integrin with AbegrinTM results in antitumor, antiangiogenic, and antiosteolytic activities. Here, we exploit the species specificity of AbegrinTM to evaluate the effects of direct targeting of tumor cells (independent of targeting of endothelia or osteoclasts). Flow cytometry analysis of human tumor cell lines shows high levels of vß3 on many solid tumors, including cancers of the prostate, skin, ovary, kidney, lung, and breast. We also show that tumor growth of vß3-expressing tumor cells is inhibited by AbegrinTM in a dose-dependent manner. We present a novel finding that high-dose administration can actively impair the antitumor activity of AbegrinTM. We also provide evidence that antibody-dependent cellular cytotoxicity contributes to in vitro and in vivo antitumor activity. Finally, it was observed that peak biological activity of AbegrinTM arises at serum levels that are consistent with those achieved in clinical trials. These results support a concept that AbegrinTM can be used to achieve selective targeting of the many tumor cells that express vß3 integrin. In combination with the well-established concept that vß3 plays a key role in cancer-associated angiogenesis and osteolytic activities, this triad of activity could provide new opportunities for therapeutic targeting of cancer. [Mol Cancer Ther 2006;5(12):3122–9]
Introduction
Biological therapy is providing new weapons for oncologists that allow the selective targeting of cancer. In particular, monoclonal antibodies represent a successful and validated approach for targeting cells within the tumor environment (1). Examples include direct targeting of tumor cells (e.g., cetuximab) or components of the microenvironment (e.g., bevacizumab). Importantly, monoclonal antibodies have proven to be generally safe and effective.
In the search for potential targets for monoclonal antibodies, one could seek to identify either direct or indirect tumor targets. Examples of direct tumor targets include HER-2, epidermal growth factor receptor, CD20, and other molecules that are preferentially overexpressed in tumor cells. Alternatively, indirect tumor targets could include molecules within the tumor environment that nonetheless critically control tumor growth or survival, the most well-known being targets on angiogenic blood vessels.
AbegrinTM (etaracizumab; previously known as Vitaxin® or MEDI-522) is a monoclonal antibody that was chosen for its unique ability to selectively target multiple and different cell types, all of which are relevant to cancer pathophysiology. The target for AbegrinTM is vß3, an integrin that is overexpressed on tumor cells, angiogenic blood vessels, and osteoclasts. Antagonists of vß3 (including antibodies and small molecules) have been studied most extensively for their antiangiogenic properties (2–5). In addition, vß3 is expressed on tumor cells and osteoclasts and is believed to play an important role in bone metastasis and subsequent resorption (6). These findings suggest a potential role for vß3 in the pathology of osteolytic diseases, including breast cancer, prostate cancer, and multiple myeloma. Finally, vß3 is overexpressed on a variety of different tumor types. For example, a preponderance of data suggests that vß3 is found to be overexpressed in metastatic melanoma, glioma, multiple myeloma, ovarian, renal, and breast cancer (7–13).
The murine monoclonal antibody, LM609, binds human vß3 and was humanized and affinity matured based on its abilities to mediate antiangiogenic effects in preclinical models (14). The resulting antibody AbegrinTM has been tested in clinical trials of various solid tumors where it appears to be without significant toxicity. The trials also indicated that AbegrinTM may have effects on tumor perfusion and may exhibit clinical activity in renal cell cancer (15). Based on these findings, AbegrinTM is presently being investigated in clinical trials of androgen-independent prostate cancer and metastatic melanoma. In our present report, we seek to understand further mechanisms by which AbegrinTM functions in cancer. In particular, we have returned to preclinical investigation to address the question of whether AbegrinTM could provide additional opportunities to directly target cancer cells that overexpress vß3. In addition, our studies reveal new opportunities for cancer cell targeting and identify novel mechanisms and dosing properties that could provide a wider understanding for targeting of cancer with monoclonal antibodies.
Materials and Methods
Cell Lines
All cell lines used in this study were obtained from the American Type Culture Collection (Manassas, VA) and cultured according to specifications.
Radioligand Binding Assay
For the concentration dependence of binding, [125I]AbegrinTM was added at various concentrations to M21 cells at 107 cells/mL and incubated with cells at 4°C for 30 min with shaking every 3 to 5 min. Duplicate 75 µL aliquots were layered over 200 µL phthalate oil mixture [Dibutylphthalate/bis(2-ethylhexyl)phthalate = 60:40; both from Sigma, St. Louis, MO] and immediately centrifuged for 1 min at 13,000 rpm in a microfuge. Two to 50 µL of the supernatant were removed and measured for 125I radioactivity to determine the concentration of free ligand. The tubes were then snap frozen in dry ice, and the tips containing the pellet were cut and counted for radioactivity. Duplicate samples were within 10% of the mean for the data reported. Nonspecific binding was measured in samples, in which >20-fold excess unlabeled AbegrinTM was added 20 min before adding [125I]AbegrinTM; it was typically 
10% of the total bound. Average KD and binding sites per cell are calculated from nine independent experiments.
Flow Cytometry
Adherent cells were harvested using enzyme-free dissociation buffer (Invitrogen, Carlsbad, CA), washed once, and incubated with AbegrinTM (1 µg/106 cells) at 4°C for 1 h in fluorescence-activated cell sorting buffer (PBS + 2% fetal bovine serum). Cells were then washed in fluorescence-activated cell sorting buffer and stained with a goat F(ab')2 anti-human IgG R-phycoerythrin secondary antibody (used at 1:250 dilution; Southern Biotech, Birmingham, AL) at 4°C for 45 min. Subsequently, cells were washed twice and resuspended in 500 µL fluorescence-activated cell sorting buffer for analysis. Live whole cells were gated using propidium iodide (Roche, Indianapolis, IN) staining, and 10,000 events were acquired using a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). Data were analyzed using BD CellQuest software (BD Biosciences).
Immunohistochemistry
Placental tissue, a rich source of vß3, was collected either immediately after parturition or from freshly sacrificed, late-term pregnant animals. Tissue fragments, 
1 cm3, were frozen in optimal cutting temperature, and thin sections were stained with AbegrinTM (10 µg/mL) at Pathology Associates, Inc. (Frederick, MD).
Epitope Mapping
Human, rat, mouse, and rabbit ß3 integrin sequences corresponding to residues 164 to 202 were aligned. Specific residues within human ß3 integrin were mutated to the corresponding residue from the rat sequence using QuikChange Site-Directed Mutagenesis kit from Stratagene (La Jolla, CA) as per the manufacturer's instructions. The resulting mutant human ß3 integrin-containing plasmids were transfected into HEK-293 cells, using the endogenous human v chain for dimerization of the integrin receptor. The mutant ß3 clones were analyzed for binding to AbegrinTM by flow cytometry as described above. A human ß3 integrin-specific antibody, clone PM6/13, from Chemicon (Temecula, CA) was used as a control to compare transient expression levels of the mutants.
In vivo Tumor Xenograft Studies
Athymic nu/nu (Harlan, Somerville, NJ), scid (Harlan), or scid/nod (Taconic, Germantown, MD) female mice 4 to 6 weeks of age were injected s.c. with 5 x 106 tumor cells. Treatments of PBS, IgG control, bevacizumab (McKesson Bioservices Corp., Rockville, MD), or AbegrinTM were injected two or three times weekly in the i.p. cavity as indicated in the figure legends and continued throughout the study. Only scid and scid/nod mice with <100 ng/mL of mouse IgG as assessed by ELISA (Pierce Biotechnology, Rockford, IL) were used for the study.
Antibody-Dependent Cellular Cytotoxicity Assay
Antibody-dependent cellular cytotoxicity (ADCC) was assayed in a 4-h nonradioactive lactate dehydrogenase release assay (Promega Corp., Madison, WI). Briefly, target cells were resuspended in RPMI 1640 containing 5% fetal bovine serum at 2 x 105 per mL and distributed into 96-well U-bottomed plates (1 x 104 per well). The target cells were preincubated with serial dilution of antibodies (50 µL/well) for 20 min on ice before the human effector cells (100 µL/well) were added. Human effector cells were peripheral blood mononuclear cells purified from healthy donors using lymphocyte separation medium (MP Biomedicals, Solon, OH). After a 4-h incubation at 37°C in 5% CO2, plates were centrifuged. Fifty microliter supernatant was transferred to a 96-well flat-bottomed plate and incubated with 50 µL lactate dehydrogenase substrate for 30 min at room temperature. The reaction was stopped by adding a 50-µL stop solution. The samples were measured at 490 nm, and the percentage cytotoxicity was calculated according to the formula: % specific lysis = 100 x (EX – Espon – Tspon) / (Tmax – Tspon), where EX represents the release from experimental wells, Espon is the spontaneous release of effector cells alone, Tspon is spontaneous release of target cells alone, and Tmax is the maximum release from lysed target cells.
Human IgG1 Quantitation ELISA
Three mouse serum samples per treatment group were collected 4 days following the final treatment of the A498 tumor xenograft studies. They were diluted in PBS/T-BSA [1x PBS (pH 7.4) + 0.1% Tween 20 + 0.5% bovine serum albumin] and added in duplicate to wells of BD BioCoat anti-human IgG ELISA plates (BD Biosciences). AbegrinTM serially diluted in PBS/T-BSA served as the standard. Plates were incubated at room temperature for 60 min and then washed thrice with PBS/T [1x PBS (pH 7.4) + 0.1% Tween 20]. Goat anti-human IgG horseradish peroxidase conjugate (Pierce Biotechnology) diluted 1:60,000 in PBS/T-BSA was added to each well and incubated at room temperature for 60 min. Subsequently, plates were washed thrice with PBS/T and the binding of human IgG was detected using TMB substrate (BioFX Laboratories, Owing Mills, MD). Color development was analyzed by adding 0.18 mol/L (1%) H2SO4 to each well and reading the plate at 450 nm.
Results
Antibody Targeting of vß3 on Tumor Cells
Based on evidence that vß3 is overexpressed on many tumor types, we asked if vß3 might provide a role in direct tumor targeting independent of its established role in angiogenesis. The successful accomplishment of these studies required further characterization of vß3 expression and AbegrinTM binding to vß3. RIAs revealed that AbegrinTM bound to cell surface vß3 with high affinity (KD of 2.29 nmol/L; Fig. 1A
). Scatchard analysis on M21 melanoma cells revealed an average of 223,000 available binding sites per cell. Flow cytometric analyses revealed strong AbegrinTM immunoreactivity on multiple cell models that were representative of many different tumor types, including carcinomas of the breast, prostate, kidney, and skin (Fig. 1B). Melanoma models were emphasized based on a wealth of published evidence that has linked vß3 overexpression in clinical specimens of metastatic melanoma. Flow cytometric analyses revealed that the estimated density of vß3 receptors on melanoma cells ranged from 83,000 (SK-MEL-5) to >250,000 (A375) molecules per cell.
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vß3 is present on the surface of many tumor-derived cell lines. To focus on the role of vß3 on tumor cells, it would be necessary to identify antibodies that would not interact with cells of the host microenvironment (e.g., endothelial cells). For this, the species specificity of AbegrinTM was determined via immunohistochemical staining of placental trophoblasts. This cell type proved to be particularly useful because vß3 is ubiquitously expressed on mammalian placental trophoblasts. Immunohistochemical analyses revealed strong AbegrinTM immunoreactivity with human and moderate to weak binding to guinea pig, hamster, or rabbit vß3 (Fig. 1C). In contrast, AbegrinTM was completely unable to react with either mouse or rat vß3.
The species-dependent binding of AbegrinTM provided an opportunity to further characterize the epitope. Based on knowledge that binding to LM609 is imparted by residues 164 to 202 of the ß3 chain (16), the homologues of ß3 integrin were aligned from multiple species. Using the species specificity information, we were able to focus on several residues in the ß chain that distinguish human from mouse or rat vß3. Each difference was introduced into human ß3 integrin by site-directed mutagenesis, and the resulting constructs were expressed in HEK-293 cells, which endogenously express the human v chain. The human vß3 integrin that contained a mutation in residue 171 of the ß3 chain was not recognized by AbegrinTM, suggesting that this residue is critically important in the binding and species specificity of AbegrinTM (Fig. 1D). Equal cell surface expression of ß3 integrin was determined by staining transfected cells with a ß3 integrin-specific antibody (data not shown).
AbegrinTM Antitumor Activity and Mechanism of Action
Based on the studies above, we considered that human tumors implanted within a murine background could provide the opportunity to evaluate direct targeting of tumor cells by AbegrinTM. The use of human tumor cells that expressed vß3, in combination with the species specificity of AbegrinTM, provided an opportunity to ask if AbegrinTM could confer antitumor effects independent of its role in angiogenesis. Biodistribution studies with labeled AbegrinTM showed specific targeting of the antibody to vß3-expressing human tumor cells in mouse xenografts (17). Analysis of xenografts revealed that AbegrinTM targeting of tumor cells is sufficient to decrease tumor growth in vivo. For example, treatment of mice with AbegrinTM prevented the subsequent growth of tumors by M21 melanoma cells (Fig. 2A
). These effects were not unique to any particular model as AbegrinTM decreased the growth of A375 melanoma cells (Fig. 2B) as well as experimental models of renal and prostate cancer.
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vß3 antagonists (14, 18, 19). This finding is consistent with the well-established concept that vß3 integrin confers an important cell adhesion function. We also considered that host effector mechanisms (e.g., ADCC and complement-dependent cytotoxicity) could contribute to the antitumor activity observed in xenograft models. To examine the relative importance of ADCC, xenograft analyses were done using scid/nod mice, which have a deficiency in monocytes and natural killer cells, thereby compromising their ADCC activity. Bevacizumab (Avastin) provided a control as it binds a soluble ligand and thus does not efficiently mediate ADCC. As expected, AbegrinTM treatment of scid mice decreased A375 melanoma tumor growth by at least 70% relative to isotype-treated controls (P < 0.0001; Fig. 3A ). In contrast, tumor inhibition was marginal (<25%) when similar studies were conducted within a scid/nod background (Fig. 3B). As expected, the antitumor effects of bevacizumab were not altered in the nod background. Altogether, these results suggest that host effector mechanisms, and ADCC in particular, critically contribute to the antitumor activity of AbegrinTM in vivo.
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vß3 integrin.
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vß3-deficient A549 lung carcinoma cells. 3F2 was again used for ADCC assays in the presence or absence of various doses of AbegrinTM. The inclusion of AbegrinTM, even at concentrations as high as 100 µg/mL, did not decrease 3F2-mediated ADCC (Fig. 5B). Controls confirmed that AbegrinTM itself also did not mediate ADCC, which is consistent with the absence of vß3 on the target cells. These results indicate that high-dose AbegrinTM does not function as a dominant inhibitor of ADCC by acting on effector cells and that free unbound effector cells are in excess even at high antibody concentrations. For our final set of studies, we considered that high-dose antibody administration might alter the serum levels of AbegrinTM. For example, bolus administration might cause antibody aggregation in vivo, thereby accelerating its clearance and limiting its availability. We determined that the concentrated AbegrinTM stocks used for these experiments were free of aggregates (data not shown) but could not exclude the possibility that such an event might occur in vivo. A greater understanding of the serum levels was also important as AbegrinTM is currently being investigated in clinical trials of melanoma and prostate cancer. To ask how "dose-limited activity" in xenograft models (at 30 mg/kg, given twice weekly) relates to the situation encountered in the clinic (at 8 mg/kg given weekly), the serum level of AbegrinTM in mice was compared with the known pharmacokinetic profile in humans. The trough serum concentration of human IgG (AbegrinTM) is defined by the serum concentration just before the administration of the next dose and was measured by ELISA in mice during week 8 of a xenograft study using A498 tumor cells (Fig. 6 ). The circulating levels of AbegrinTM were proportional to the dose administered, with mean trough concentrations of 11 and 192 µg/mL in animals that had been treated with 1 or 10 mg/kg. Trough levels of 574 µg/mL were attained in animals treated with 30 mg/kg AbegrinTM, indicating that lack of antitumor activity at this dose was not due to increased clearance of the antibody. Tumor homogenates also showed a comparable increase in IgG levels with increasing dose, suggesting that diffusion into the tumor was not altered at the high dose (data not shown). Notably, the serum levels of AbegrinTM from human trials at a dose of 8 mg/kg (peak and trough of approximately 300 and 100 µg/mL, respectively) coincided with doses associated with optimal antitumor activity in our in vivo tumor studies (15).
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The major finding of our present study is that antibody targeting of vß3 integrin on tumor cells can provide usefulness for targeting cancer independent of, and in addition to, antibody targeting of vß3 on angiogenic blood vessels. We have also increased our understanding of the distribution of vß3 receptors and the mechanisms of action for AbegrinTM. In particular, we provide evidence that AbegrinTM invokes host defense mechanisms and that ADCC critically contributes to AbegrinTM antitumor activity. Finally, we present a novel finding that high-dose administration can actively impair the antitumor activity of AbegrinTM. This new information has important implications not only for the future development of vß3 integrin inhibitors but also for understanding the mechanism of action for monoclonal antibody-based therapies.
Our present findings are novel, in part, because they shed new light about potential therapeutic opportunities for antibody targeting of vß3 in cancer. Previously, much of the interest surrounding this integrin centered on the overexpression and functional relevance of vß3 on tumor-associated endothelial cells. This information triggered considerable interest in vß3 as a target for antiangiogenic cancer therapy and, indeed, this was the original rationale supporting AbegrinTM as a cancer therapeutic. vß3 is also known to be overexpressed on osteoclasts and functionally relevant to bone resorption. This has stimulated considerable interest in the application of antagonists of vß3, including AbegrinTM, to osteolytic tumor types, such as melanoma, multiple myeloma, and breast cancer. Our present study widens this knowledge by presenting evidence for AbegrinTM activity against tumor cells themselves. In some ways, the "triad" of AbegrinTM activity may be likened to combination targeted therapy through simultaneous blocking of tumor growth and survival, angiogenesis, and bone metastasis. All of these strategies can be achieved with a single agent that shows minimal toxicity. Consequently, future studies will be focused on the potential for combining AbegrinTM with known cytotoxic agents to evaluate potential opportunities for synergistic therapies and to ask if AbegrinTM might have applications for drug sensitization in treatment-refractory indications.
Another novel aspect of our present study is the demonstration that ADCC is a major mechanism that contributes to the activity of AbegrinTM directed against the tumor cells. Before this report, vß3 was generally understood to play an important role in cell adhesion and signaling. Indeed, vß3 has been shown to critically control many different aspects of malignant character, including cell migration, invasion, and survival (e.g., via anoikis) and antagonists of vß3 (both antibodies, such as AbegrinTM and small molecules) are presently being investigated in clinical trials (5). Although our present studies do not preclude that these mechanisms contribute to antitumor activity, they do identify an important role for ADCC for targeting vß3. This information is consistent with findings with other antibodies (rituximab and trastuzumab) and also has potential importance for identifying improved means of vß3 targeting (20). Specifically, small-molecule antagonists of vß3 function are incapable of mediating ADCC and this may limit their therapeutic potential.
Perhaps, the most intriguing finding of our present report is the demonstration of "dose-limited" antitumor activity. The decreased activity of AbegrinTM at the highest dose levels has important implications for our understanding of basic mechanistic activities of monoclonal antibodies and conveys potentially important information that is highly relevant to clinical applications. Importantly, this dose-limited activity was not unique to AbegrinTM or vß3 as we have observed comparable effects with other antibodies and with different antigens. The dose-limited activity described herein is reminiscent of the prozone-like effect observed in passive immunization approaches against human pathogens (21). Several studies have shown that sera or purified IgG from patients or infected animals elicited a prozone-like response in ADCC (22–25), complement-dependent cytotoxicity (26), and neutralization (27) assays in vitro. Furthermore, prozone-like effects have been observed in vivo following infection with various pathogens, such as Streptococcus pneumoniae and Cryptococcus neoformans (28, 29). Much less is known about how this phenomenon affects passive immunization approaches in other disease areas, such as oncology.
Regardless of why dose-limited activity occurs, the outcome could have particular importance for designing and applying antibody-based therapeutics. Historically, biologics generally have relatively few side effects and many clinical trials fail to reach a maximum tolerated dose. Consequently, the dose selected for further clinical application may be defined by other considerations (cost and availability). If the findings herein have a more general application to other antibodies, then important changes will be necessary to identify optimal doses, particularly with biological agents that function via ADCC. Within this context, we have conducted extensive pharmacokinetic analyses with AbegrinTM and are fortunate in that the clinical doses presently under investigation in clinical trials seem to fall within the optimal zone of preclinical activity. Nonetheless, our present findings indicate a need to determine and apply similar criteria to other biologics-based therapeutic agents.
Acknowledgments
We thank the animal barrier facility staff at MedImmune, Inc. (Gaithersburg, MD) for their careful attention and assistance in animal handling and the clinical department for their helpful discussions and review of the manuscript.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 6/21/06; revised 10/ 6/06; accepted 10/27/06.
References
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