A function blocking anti-mouse integrin α5β1 antibody inhibits angiogenesis and impedes tumor growth in vivo
© Bhaskar et al; licensee BioMed Central Ltd. 2007
Received: 04 September 2007
Accepted: 27 November 2007
Published: 27 November 2007
Integrins are important adhesion molecules that regulate tumor and endothelial cell survival, proliferation and migration. The integrin α5β1 has been shown to play a critical role during angiogenesis. An inhibitor of this integrin, volociximab (M200), inhibits endothelial cell growth and movement in vitro, independent of the growth factor milieu, and inhibits tumor growth in vivo in the rabbit VX2 carcinoma model. Although volociximab has already been tested in open label, pilot phase II clinical trials in melanoma, pancreatic and renal cell cancer, evaluation of the mechanism of action of volociximab has been limited because this antibody does not cross-react with murine α5β1, precluding its use in standard mouse xenograft models.
We generated a panel of rat-anti-mouse α5β1 antibodies, with the intent of identifying an antibody that recapitulated the properties of volociximab. Hybridoma clones were screened for analogous function to volociximab, including specificity for α5β1 heterodimer and blocking of integrin binding to fibronectin. A subset of antibodies that met these criteria were further characterized for their capacities to bind to mouse endothelial cells, inhibit cell migration and block angiogenesis in vitro. One antibody that encompassed all of these attributes, 339.1, was selected from this panel and tested in xenograft models.
A panel of antibodies was characterized for specificity and potency. The affinity of antibody 339.1 for mouse integrin α5β1 was determined to be 0.59 nM, as measured by BIAcore. This antibody does not significantly cross-react with human integrin, however 339.1 inhibits murine endothelial cell migration and tube formation and elicits cell death in these cells (EC50 = 5.3 nM). In multiple xenograft models, 339.1 inhibited the growth of established tumors by 40–60% (p < 0.05) and this inhibition correlates with a concomitant decrease in vessel density.
The results herein demonstrate that 339.1, like volociximab, exhibits potent anti-α5β1 activity and confirms that inhibition of integrin α5β1 impedes angiogenesis and slows tumor growth in vivo.
Angiogenesis is the process by which nascent blood vessels form from existing vasculature to supply new tissue with nutrients. This process was proposed by Folkman over three decades ago to drive tumor growth beyond a few millimeters [1, 2]. Since that time, numerous angiogenesis inhibitors have been shown to inhibit vessel growth in models of neovascularization and restrict the growth of tumors in pre-clinical models of cancer. Currently, anti-angiogenic agents that are approved for the treatment of cancer include sunitinib [3, 4], sorafenib [5, 6] and bevacizumab [7, 8], and many others are in late stage clinical testing.
Tumors secrete multiple growth factors that drive activation, migration and proliferation of vascular endothelial cells (EC's), including TGF-α, bFGF and VEGF . These agents bind their respective receptors on EC's to initiate signaling cascades that culminate in pro-angiogenic events. To date, the majority of angiogenesis inhibitors have focused on disrupting growth factor signalling, including the aforementioned clinically approved agents, which primarily target the VEGF axis [4, 5, 8]. However, growth factors are not the only pro-angiogenic molecules that influence the tumor microenvironment. For example, tumor cells and tumor-associated macrophages are known to secrete matrix metalloproteinases, such as MMP-9 and MMP-2 . These enzymes degrade the basement membrane, exposing components of the extracellular matrix, including fibronectin. These exposed ECM proteins drive angiogenesis by ligating integrins, which play a central role in the angiogenic program [11–14].
Integrins are heterodimeric signalling and adhesion molecules consisting of an alpha chain and a beta chain. Ligands for these receptors bind integrins and induce EC shape change, motility and growth [12, 14]. One well-characterized example of an integrin-ECM interaction is that between fibronectin and integrin α5β1, an integrin that is up-regulated in proliferating EC's [15, 16]. Ligation of α5β1 has been shown to promote cell survival through Bcl-2, migration through RhoA and proliferation through ERK, Akt and FAK-dependent mechanisms [13, 15–20]. Activation of these and other cellular programs through integrin α5β1 in endothelial cells results in angiogenesis. Conversely, blockade of α5β1 ligation has been demonstrated to inhibit angiogenesis, at least in part through the inhibition of signalling and the induction of the cell death program through caspases [15–21].
Volociximab is a chimeric, function blocking antibody that targets integrin α5β1. We have previously shown that this antibody elicits cell death in dividing endothelial cells, inhibits angiogenesis in a cynomolgus monkey model of choroidal neovascularization  and slows tumor growth in a rabbit VX2 carcinoma model . Volociximab, however, does not cross-react with rodent α5β1, precluding its use in standard mouse xenograft models of cancer. Although commercially available antibodies against mouse α5 exist, we and others have found that these monoclonals do not elicit cell death or inhibit tumor progression in vivo [23, 24].
To determine whether blockade of α5β1 in murine disease models results in inhibition of angiogenesis and tumor growth, we sought to generate a function blocking antibody that closely mirrored the known properties of volociximab. To this end, we generated a panel of rat-anti-mouse integrin α5β1 antibodies and subjected them to a rigorous screening strategy designed to identify antibodies that reproduce the known properties of volociximab that are associated with its efficacy in vitro and in vivo.
Purification of mouse placental α5β1
Mouse placental integrin α5β1 was purified by affinity chromotagraphy using rat anti-mouse α5β1 antibody BMC5 (Chemicon) essentially as described . Briefly, antibody was coupled to CNBr-coupled Sepharose (Pharmacia) and mouse placenta was homogenized using a Polytron tissue homogenizer in lysis buffer [200 mM octyl-β-gluocopyranoside, 1 mM phenylmethylsulfonyl fluoride, 1 mM MgCl2, 0.5 mM CaCl2, 150 mM NaCl, 25 mM HEPES (pH 7.0)], followed by incubation on ice. After centrifugation, the cleared lysate was loaded onto a BMC5-Sepharose column and washed with 20 column volumes of lysis buffer with the octyl-β-gluocopyranoside substituted with 0.1% Nonidet P-40. Integrin was eluted at low pH in a buffer containing 10 mM EDTA and dialyzed against phosphate buffered saline.
Cloning of mouse integrin and purification of α5β1-Fc fusion protein
Total RNA (1 μg) isolated from mouse tissue was solubilized in Trizol™ (Invitrogen) and cDNA was produced using SuperScript II (Invitrogen). Full-length α5 and β1 were amplified by PCR using gene-specific primers (Qiagen N.V.). PCR products were subcloned into the pCR4 TOPO vector (Invitrogen) and verified by sequencing. To generate the α5 and β1-Fc fusion genes, regions encoding the extracellular domains of the α5 and β1 integrin genes were isolated by restriction digest and individually subcloned into the expression vector DEF38 (ICOS) to create an in-frame fusion between each of the extracellular domains and the constant region of the human γ1 immunoglobulin heavy chain gene. A stable cell line was generated by co-transfecting 293 cells with a 50:50 mix of α5-Fc and β1-Fc plasmid DNAs using FuGene (Roche). Conditioned medium was harvested and Fc fusion protein was purified by standard methods using Protein G Sepharose.
Generation of anti-mouse integrin α5β1 antibodies
Female Sprague-Dawley rats (Simonsen) were immunized intraperitoneally with α5β1 purified from mouse placenta or with a mouse α5β1-Fc fusion protein. Monoclonal antibodies were generated by standard techniques fusing spleen cells from immunized mice with an NSO-derived fusion partner (American Type Culture Collection). A panel of α5β1-specific antibodies was identified by ELISA using the Fc-fusion protein and by flow cytometry analysis of binding to mouse endothelial cells and cell lines.
Integrin α5β1 binding ELISA
Purified α5β1-Fc (100 ng) was plated into wells in 50 mM Na2CO3 (pH 8.6), 0.5 mM each of CaCl2, MgCl2, and MnCl2 at 4°C overnight. Plates were blocked with 5% BSA in PBS for 1 hr with shaking. Following washes in PBS with 0.05% Tween-20, wells were incubated for 1 hr with serial dilutions of candidate antibodies or control rat IgG (Jackson ImmunoResearch) in 1% BSA in PBS with 0.05% Tween-20. Wells were then washed and incubated with 100 μL/well of the goat anti-rat IgG-HRP (1:5,000) for 1 hr. Washed plates were incubated with TMB substrate (Sigma), developed with 650 Stop Reagent (BioFX), and read at A650.
Fibronectin binding inhibition ELISA
Cellular mouse fibronectin-coated plates (250 ng in coating buffer overnight at 4°C) were washed with PBS containing 0.05% Tween-20, and blocked as described above. Plates were incubated with α5β1-Fc fusion protein in the presence of various concentrations of the indicated antibodies for 1 hr at room temperature. Following 2 washes, plates were further incubated with goat anti-human IgG Fc-HRP (1:5000; Jackson ImmunoResearch) at room temperature for 1 hr. Plates were then washed 5 times and incubated with TMB substrate, developed, and read at A650.
Cells were removed with 20 mM EDTA in Tris-HCl (pH 8.0) and blocked by centrifugation in HBSS containing 3% heat-inactivated FBS, 1% normal goat serum (Sigma) and 1% BSA at 4°C for 5 min. Cells were incubated for 1 hr at 4°C with the indicated supernatant or antibody (10 μg/mL) in FACS buffer (PBS containing 0.1% BSA). Excess mAb was removed by centrifugation and cells were resuspended in FACS buffer containing anti-rat IgG-PE secondary antibody (Southern Biotech). After an additional wash, fluorescence intensity was measured on a FACSCalibur flow cytometer (Becton Dickinson).
BD HTS FluoroBlok 96-well plates (top plate) were coated with mouse plasma fibronectin (10 μg/ml; Upstate) in PBS and air-dried. 200 μl migration medium (MM; RPMI + 0.1% BSA) with or without antibody was dispensed into a BD Falcon 96-Square Well Flat Bottom Assay plate (bottom plates) and 10,000 cells/well in 50 μl was added to the top plate in MM. After lowering the top plate into the lower plate, cells were incubated at 37°C at 5% CO2 for 4 hr. Cells were stained with 2 μM Calcien-AM (Invitrogen) in MM and visualized using the Discovery-1 High Content Screening System (Molecular Devices).
Surface plasmon resonance
Affinities between α5β1-Fc and anti-integrin antibodies were analyzed using a BIAcore 3000 essentially as described [26, 27]. Goat anti-rat Fc antibodies were immobilized on a Research Grade CM5 chip using an amine coupling kit (BIAcore). Rat-anti-mouse α5β1 antibodies were captured onto goat anti-rat Fc surfaces, followed by injection of mouse α5β1 in running buffer [(10 mM HBS, 2 mM CaCl2, 1 mM MnCl2, 700 mM NaCl (pH 7.4)] at a flow rate of 30 μL/min at 25°C. Association phase occurred over 3 min and dissociation over 1.5 hr. Kinetics of binding was calculated from data at 6 different concentrations of analyte (512 nM, 128 nM, 32 nM, 8 nM, 2 nM, 0.5 nM), using the BIAevaluation program. Each goat anti-rat Fc surface was regenerated at the end of each cycle by a quick injection of 30 mM HCl. Double-referencing was applied to eliminate responses from the reference surface and buffer-only control. Affinity constant (KD) was obtained by simultaneously fitting the association and dissociation phases of the sensorgram from the analyte concentration series using the 1:1 Langmuir model from the BIAevaluate software.
Annexin V cell death assay
Primary mouse endothelial cells were incubated with anti-mouse α5β1 antibodies at 10 μg/ml for 16 hr, after which cell membrane phosphatidylserine was detected using Oregon Green 488 conjugated Annexin V. Harvested cells were washed with Annexin binding buffer [(ABB; 10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2 (pH 7.4]) and incubated in ABB (100 μl) containing Annexin V (10 μl). Following washing, cells were suspended in ABB containing 0.5 μg/mL propidium iodide (200 μl) and assessed by flow cytometry.
HUVEC and murine EC in vitro angiogenesis models
HUVEC or murine lung EC's (5 × 105 cells/ml) were seeded in Hank's balanced salt solution (HBSS) containing 3 mg/ml fibrinogen and 200 μg/ml aprotinin (Roche) at 37°C, as described previously . For each condition, EC/fibrinogen/aprotinin mixture (500 μL) was quickly transferred to a 24-well tissue culture plate containing α-thrombin (1 U) and gently mixed. The resulting fibrin matrices were polymerized at 37°C for 20 min. Antibody was added in Medium 200 [1 ml, without LSGS and containing 20% human or mouse serum (Rockland Immunochemicals), and rhTGF-α (0.01 μg/ml), rhHGF (0.1 μg/ml) and rhVEGF and/or rmVEGF (0.1 μg/ml)]. Tubes formed over 6 days, with one medium change (Medium 200 plus serum only) at day 3. For visualization of tubes, matrices were fixed with 4% formaldehyde for 4 hr and stained overnight with 1 ml phalloidin-Alexa 488 (1.75 U/ml) in 50% fetal calf serum (FCS) containing 0.25% saponin. Vessel formation was visualized using the Discovery-1 High Content Screening System (Molecular Devices) and quantified using the accompanying software.
Xenograft tumors derived from cells inoculated subcutaneously in ICR-SCID or SCID-Beige mice (Taconic Farms) were frozen in OCT compound and stored at -70°C. Cryostat tissue sections (4-5 μm) were fixed in acetone for 10 min, air dried and incubated in 0.03% H2O2 for 10 min. After successive incubations with Avidin block, Biotin block and Protein block solutions for 15 min each, samples were treated with the indicated antibodies or biotin-conjugated rat anti-mouse CD31 mAb MEC 13.3 or biotin-conjugated IgG2a isotype control (2.5 μg/ml; BD Pharmingen) for 60 min. Sections were developed using the Vectastain Elite ABC kit (Vector Laboratories) and stable diaminobenzidine (Dako). All staining procedures were performed using a Dako Autostainer at room temperature.
In vivo xenograft studies
Six- to eight-week-old ICR SCID or SCID-Beige female mice, obtained from Taconic Farms and maintained in micro-isolator cages, received subcutaneous injections on the right flank of 5 × 106 A673 human rhabdomyosarcoma or 1 × 107 SVR murine angiosarcoma cells. Tumors were allowed to establish for 7–18 days, reaching an average of 50–100 mm3, as determined by caliper measurement. Animals were distributed into groups of ten and received vehicle (PBS) or rat anti-mouse integrin α5β1 antibody (200 μl at 1.0 mg/ml). Reagents were delivered by intraperatoneal injection twice or thrice weekly for the duration of the studies. Tumor volume was measured twice weekly, and clinical and mortality observations were performed daily according to Institutional Animal Care and Use Committee regulations.
Generation and characterization of rat anti-mouse integrin α5β1 antibodies
To generate antibodies directed against murine integrin α5β1, Sprague-Dawley rats were immunized with mouse α5β1-Fc fusion protein or with affinity-purified integrin from mouse placenta. Supernatants from the resulting hybridomas were subjected to a number of assays designed to identify clones that produced antibodies encompassing characteristics that define volociximab, including heterodimer specificity, blocking activity, high affinity binding to the Fc fusion protein and binding to endothelial cells . Over sixty clones were screened for these functional properties.
Volociximab has a very high affinity for human α5β1 . We were therefore interested in identifying antibodies with similarly high affinity for mouse α5β1. Selected antibodies were purified from supernatants using Protein A affinity chromatography and assessed for binding to mouse and human α5β1 by BIAcore. Of the human cross-reactive set, 517-2 had the highest affinity (KD = 0.21 nM) and of the non-cross-reactive set, antibody 339.1 bound tightest (KD = 0.59 nM). The affinities of both antibodies were sub-nanomolar, comparing favorably with volociximab (KD = 0.32 nM).
Cell death assays
Tube formation assays
The strategy of targeting angiogenesis to inhibit cancer progression has received increasing attention in recent years. Despite the recent approval of targeted therapies in this area, optimizing the use of anti-angiogenic drugs in the clinic has been difficult. Challenges that face anti-angiogenic agents that are currently under development include choosing disease areas that might benefit most, optimizing combination strategies with existing standards of care and defining patient populations that might respond best to therapy. Preclinical models of disease provide the best opportunity for addressing these issues, therefore appropriate reagents for use in these systems are essential for driving drugs through development.
Volociximab has been shown to inhibit the growth of new blood vessels in preclinical models of ocular angiogenesis . This effect was found to translate into decreased tumor growth in the rabbit VX2 carcinoma model . These experiments provided a strong proof of concept demonstration of volociximab activity in vivo and defined a novel mechanism of action for angiogenesis inhibition. However, the VX2 model is limited in that it represents a very aggressive tumor, must be passaged in vivo, is carried out in immunocompetent animals (resulting in antibody clearance) and requires large amounts of antibody. To further define volociximab mechanism of action and identify appropriate settings for its use in tractable animal models of cancer, it was therefore imperative a similar reagent with activity in mouse be generated.
A number of antibodies against mouse α5β1 are available commercially. We have found that although some of these antibodies inhibit binding of α5β1 to fibronectin, none inhibited other biological functions, such as migration, in vitro angiogenesis or tumor growth in vivo (unpublished observations; [23, 24]). However, the α5 knockout mouse is embryonically lethal due to gross defects in vascular architecture , suggesting that in mice, as in humans, α5β1 is important for blood vessel formation and/or integrity. The new panel of reagents described herein represents a number of α5- and β1-specific antibodies. Of note, Fc-fusion protein-based immunizations resulted in a higher proportion of α5-specific antibodies, whereas placenta-based immunization resulted in a higher proportion of heterodimer-specific antibodies, including 339.1 (data not shown). As the overall number of antibodies produced by each method was similar, this suggests that the purified material may have resulted in similar immunogenicity while maintaining a more native quaternary structure in vivo. In either case, many of the antibodies that bound α5 or were specific for α5β1 heterodimer blocked binding to fibronectin and competed, at least in part, with one another in ELISA or FACS assays (data not shown). Of these antibodies, one group cross-reacted with human integrin, while another did not, suggesting that at least two distinct epitopes were represented. This implies that inhibition of binding to fibronectin can be achieved through blocking at multiple sites, possibly through steric hindrance. Importantly, not all antibodies that block binding to fibronectin have equivalent biological function in vitro or in vivo. 517-2 and 339.1, for example, each bind with high affinity (0.21 nM and 0.59 nM, respectively) block binding to fibronectin and inhibit migration. Moreover, both antibodies have rat IgG1 constant regions, which like volociximab, a human IgG4, would be predicted to lack significant effector activity. However, only 339.1, which does not cross-react with human α5β1, elicits significant cell death in vitro and inhibits angiogenesis and tumor progression in vivo. This finding suggests that although these antibodies have similar biological functionality and similar affinities, initiation of the cell death program requires binding to a highly specific epitope. This result also suggests that 339.1 binds to the murine cognate of the epitope recognized by volociximab, which would be predicted to be non-homologous between mouse and human α5β1, since volociximab does not cross-react with mouse integrin. A corollary of this hypothesis is that an antibody that recognizes both human and mouse integrin would not bind this important epitope, and therefore might not elicit cell death, as is the case with 517-2.
339.1 inhibits tumor growth in an A673 rhabdosarcoma model. This model was chosen to evaluate anti-α5β1 activity because it was reported to be sensitive to the mouse parent antibody of bevacizumab, A.4.6.1, suggesting that its growth is highly dependent on angiogenesis . However, A.4.6.1 and bevacizumab do not inhibit tumor growth in other xenograft models to the same extent as in the A673 model [29, 30]. The reasons for this are not fully understood; 339.1 is currently being evaluated in additional xenograft models to determine if similar differences in sensitivities are observed with this antibody. Comparing xenograft models that respond to 339.1 to varying degrees may reveal molecular mechanisms that will help stratify patients and illuminate combination strategies that might best slow disease progression. In addition, the combination of 339.1 and volociximab is currently being assessed in xenograft models to determine the effect of targeting both tumor and host α5β1 in vivo.
The results described in this report represent the first description of a mouse-specific α5β1 inhibitor that blocks EC function, angiogenesis and tumor growth in vivo. These results demonstrate that blocking α5β1 function in mouse models results in an inhibition of tumor growth through a decrease in vessel density. The identification and characterization of anti-α5β1 antibody 339.1 will facilitate further exploration of α5β1 function and the utility of integrin blocking strategies in disease.
The authors wish to thank Dr. Patty Culp, Dr. Ernst Lengyel and Dr. Sanford Shattil for critical reading of the manuscript.
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