CNTO 859, a humanized anti-tissue factor monoclonal antibody, is a potent inhibitor of breast cancer metastasis and tumor growth in xenograft models
Article first published online: 27 DEC 2006
Copyright © 2006 Wiley-Liss, Inc.
International Journal of Cancer
Volume 120, Issue 6, pages 1261–1267, 15 March 2007
How to Cite
Ngo, C. V., Picha, K., McCabe, F., Millar, H., Tawadros, R., Tam, S. H., Nakada, M. T. and Anderson, G. M. (2007), CNTO 859, a humanized anti-tissue factor monoclonal antibody, is a potent inhibitor of breast cancer metastasis and tumor growth in xenograft models. Int. J. Cancer, 120: 1261–1267. doi: 10.1002/ijc.22426
- Issue published online: 30 JAN 2007
- Article first published online: 27 DEC 2006
- Manuscript Accepted: 4 SEP 2006
- Manuscript Received: 11 APR 2006
- CNTO 859;
- tissue factor;
Thromboembolic complications are frequently associated with advanced cancer. Interestingly, one of the major initiators of blood coagulation, tissue factor (TF), is reported to be overexpressed in several tumor types and can be found on both tumor cells and tumor vasculature. Although the exact mechanisms have yet to be elucidated, TF expressed on tumor cells can trigger intracellular signaling events through various pathways that can lead to tumor angiogenesis, proliferation, and metastasis. There exists preclinical evidence that disruption of TF dependent signaling can effectively inhibit tumor cell migration, metastasis, and angiogenesis. Here, we report for the first time that an antibody to tissue factor can also prevent tumor growth in vivo. Prophylactic administration of CNTO 859, a humanized anti-human TF antibody, was shown to inhibit experimental lung metastasis of MDA-MB-231 human breast carcinoma cells by over 99% compared to a control antibody. Furthermore, therapeutic doses of CNTO 859 were shown to reduce tumor incidence and growth of orthotopically implanted MDA-MB-231 cells. © 2006 Wiley-Liss, Inc.
Since the discovery of Trousseau's syndrome, it has been well documented that cancer patients often experience a state of hypercoagulation, leading to the formation of circulating blood clots that further complicate their disease and contribute significantly to morbidity and mortality.1, 2 In many cases, the association between cancer and coagulation can be traced to dysregulation of tissue factor (TF). TF is a transmembrane receptor for the plasma cofactor Factor VII (FVII) and initiates a proteolytic clotting cascade leading to the generation of thrombin and ultimately blood coagulation.
TF is normally expressed at low levels on cells surrounding blood vessels and in some stratified epithelial cells. It has also been shown to be highly overexpressed in a wide variety of tumor types. These include the most common malignant tumor types such as breast, colon, prostate and pancreatic cancers.3, 4, 5, 6, 7 Several lines of evidence suggest that TF expression can promote tumor growth and progression. Enforced overexpression of TF dramatically promoted the growth of xenografted human pancreatic cancer cells8 and increased both growth and blood vessel density of tumors generated with transfected syngeneic mouse tumor cell lines.9 Yu et al. recently demonstrated that TF expression is increased in tumor cells engineered to contain activating K-ras or p53 inactivating mutations. RNAi knockdown experiments in these cells showed that TF overexpression is a critical mediator of the tumor growth promoting and pro-angiogenic effects of K-ras mutation.10
TF may promote tumor growth and survival by a number of distinct mechanisms. The activated proteases that result from TF activity, including FVIIa, Factor Xa (FXa) and thrombin, can activate protease activated receptors (PARs).11, 12, 13, 14, 15, 16, 17 PAR signaling can induce NF-κB activation, prevent anoikis, and promote proliferation and metastasis of PAR expressing cells, including tumor cells. For example, whereas colonic epithelial cells normally do not express PAR-1, colon cancer cells frequently overexpress PAR-1 and consequently gain the ability to proliferate in response to thrombin.18 TF also appears to promote tumor angiogenesis and its activity may be important for both tumor cells themselves as well as angiogenic endothelial cells.1, 19, 20, 21, 22, 23, 24 Additional mechanisms by which TF may promote tumor cell growth and migration have been recently reviewed.8, 25, 26
These observations suggest that TF may be an important target for specific cancer therapy. Targeting of TF with anti-TF antibodies has been shown to inhibit metastasis in experimental models in mice26 but has not been reported to inhibit the growth of primary tumors. TF8-5G9 is a function blocking murine anti-human TF antibody that inhibits coagulation27 by acting as a competitive inhibitor of FX binding to the TF:FVIIa binary complex. Here we report the ability of CNTO 859, a humanized version of TF8-5G9, to potently inhibit both metastasis and tumor growth in xenograft models of human breast cancer.
Material and methods
DMEM, RPMI 1640, FBS, L-glutamine, sodium pyruvate, HBSS, PBS and non-essential amino acids were from Invitrogen (Carlsbad, CA). Simplastin HTF was obtained from BioMerieux (Durham, NC). FVIIa, FX and FXa were obtained through Haematologic Technologies (Essex Junction, VT). S2765 substrate was purchased from DiaPharma (West Chester, OH). Control human IgG and PE-conjugated goat anti-human IgG (H + L) antibodies were purchased from Jackson ImmunoResearch (West Grove, PA). 125I-sodium iodide and PD-10 columns were purchased from Amersham Biosciences (Piscataway, NJ). CNTO 859 is a humanized IgG4 antibody derived from the previously described murine antibody TF8-5G9.27
B16F10 murine melanoma and MDA-MB-231 human breast carcinoma cells were obtained from ATCC (Manassas, VA). Cells were maintained in DMEM supplemented with 10% FBS, 2mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids under 5% CO2 in a 37°C humidified chamber.
Increasing concentrations of CNTO 859 (0–50 μg/ml) were added to citrated normal human plasma and mixed gently for 30 sec on an orbital plate shaker. Coagulation was initiated by the addition of either Simplastin HTF (1:100) or 3 × 104 MDA-MB-231 cells in HBSS with 2.2 mM or 5 mM CaCl2, respectively. Samples were read immediately following the addition of reagents at 405 nm, every 15 sec for 2 hr on a kinetic plate reader. Clotting time was plotted as a function of antibody concentration. All samples were run in triplicate.
Human brain extract (50 μl of 0.6 mg/ml) was plated in a 96 well plate, and FVIIa (50 μl of 3 nM) was added for 10 min to allow FVIIa to bind to tissue factor. At t = 0, increasing concentrations of FX (50 μl of 0–1.5 μM) with or without CNTO 859 (10 nM, final conc.) were added, and the reactions were quenched with EDTA (50 μl of 0.5 M) at various times (0.5–5 min). S2765, a substrate of FXa, was added (50 μl of 3 mM), and the conversion of S2765 substrate to a chromogenic product was monitored at 405 nm every 12 sec for 10 min. The amount of FXa product was extrapolated from a standard curve of known FXa concentrations and plotted as a function of time in order to determine the rate of FXa production. The rate of FXa production was plotted as a function of FX concentration.
To show that CNTO 859 was specific for human TF, B16F10 and MDA-MB-231 cells (3 × 105) were allowed to bind to 5 μg/ml of either control human IgG or CNTO 859 for 1 hr at 4°C. Cells were washed twice with PBS and incubated with a PE-goat anti-human IgG secondary antibody (2.5 μg/ml) for 30 min.
To measure antibody affinity, MDA-MB-231 cells were stained with log-fold dilutions of CNTO 859 ranging from 10 μg/ml to 1 pg/ml in 200 μl serum-free RPMI 1640 on ice for 1 hr. Cells were washed twice with PBS and bound CNTO 859 was detected with a PE-goat anti-human IgG (2 μg/ml) in 200 μl serum-free RPMI 1640 for 30 min at 4°C.
Cells were washed twice with PBS and resuspended in FACSFlow buffer and analyzed for fluorescence on a FACSCalibur (Becton Dickinson, San Jose, CA). Unstained, control human IgG-stained and secondary antibody only-stained cells served as appropriate controls to account for nonspecific fluorescence and as a reference for examining shifts in mean fluorescence intensity (MFI). The total fluorescence was calculated as the product of the percentage of gated cells and the MFI of the gated population.
Iodination and radioligand binding assay
CNTO 859 (400 μg) and control Human IgG were iodinated by the direct method in 200 μl phosphate buffer (20 mM, pH 8) using Iodo-Gen tubes according to manufacturer's instructions (Pierce, Rockford, IL). For determining antibody affinity, 105 MDA-MB-231 cells were seeded on a 96 well filter plate (HBSS with 1% BSA, 0.25% sodium azide) and allowed to bind to titrated amounts of radiolabeled CNTO 859 (0.04–40 μg/ml) in the presence or absence of a 10-fold excess of cold antibody for 2 hrs at room temperature. Cells were rinsed 3x with 300 μl PBS and unbound antibody was aspirated from the filter plate using a vacuum manifold. Individual filters were removed using a Multiscreen multiple punch apparatus and measured in a gamma counter (Millipore, Bedford, MA). Specific binding was calculated by subtracting the residual binding on MDA-MB-231 cells in the presence of cold competitor. The specific activity of the labeled antibody was determined as cpm/μg protein. To calculate the number of CNTO 859 binding sites per cell, the following equation was used:
Female C.B-17/IcrCrl-scid-bgBR (SCID Beige) mice 5–6 weeks of age were purchased from Charles River Laboratories (Wilmington, MA). Mice were acclimated for 2 weeks and housed under specific pathogen-free conditions. All studies were carried out according to the guidelines of the Institutional Animal Care and Use Committee of Centocor, Inc.
Experimental metastasis model
Animals were given i.v. injections of PBS, CNTO 859 or control Ig antibody (20 mg/kg). Three hours later, 2 × 105 MDA-MB-231 cells were injected into the lateral tail vein. An additional group received vehicle (without cells) and served as a normal lung weight control. The mice were separated into 2 cohorts; 1 received a single prophylactic dose and the other received a maintenance therapy of 10 mg/kg, q7d. This dose was determined from a previous single dose PK study where a dose of 10 mg/kg resulted in a serum antibody concentration of 50 μg/ml after 7 days. At termination, lungs were removed and scored for metastatic lesions.
Orthotopic breast cancer model
Mice were anesthetized with ketamine/xylazine (90/10 mg/kg) in PBS. 2.5 × 106 MDA-MB-231 cells were injected into the right no. 2 axillary mammary fat pad in 50 μl of serum-free DMEM. Tumor growth was measured once a week and calculated using the formula V = LW2/2 (where V = volume, L = length and W = width). Unless indicated, antibody therapy (i.v., q7dx8) commenced 3 days post tumor implantation.
Data analysis was performed using GraphPad Prism software (GraphPad Software, San Diego, CA). Significance was defined as p< 0.05 in an unpaired t-test analysis, and repeated measures ANOVA.
CNTO 859 binds to human TF but not rodent TF
In order to define the effects of CNTO 859 in xenograft models expressing both human (hTF) and mouse TF (mTF) on the implanted tumor cells and on host stromal cells, respectively, it was important to first determine the species reactivity of the molecule. The reactivity of CNTO 859 was determined by flow cytometry (Fig. 1) using murine and human carcinoma cell lines known to overexpress TF. B16F10 mouse melanoma cells have previously been shown to express mTF and we confirmed this by flow cytometry with an anti-mouse TF antibody (Ref.28; unpublished data). Briefly, PHD126, a surrogate murine IgG2a antibody specific for murine TF was generated in-house using phage display. It was engineered to have properties similar to TF8-5G9, such that it could inhibit murine FX binding to murine TF. (An abstract on the antibody engineering efforts and characterization of the antibodies was presented at the Cambridge Healthtech Institute's Sixth Annual Phage Display Technologies Engineering Protein Therapeutics meeting on April 26–27, in Cambridge, Massachusetts). B16F10 cells were incubated with titrated amounts of PHD126, or an isotype control antibody. A FITC-goat anti-mouse IgG secondary antibody was used for detection. By flow cytometry, we did not see specific binding of PHD126 to B16F10 cells compared to the control antibody. CNTO 859 did not bind to mTF expressed on B16F10 melanoma cells but did bind hTF expressed on MDA-MB-231 cells. Therefore, the direct effects of CNTO 859 in murine models utilizing human tumor cells xenografts can be attributed to binding of CNTO 859 to the tumor cells and not to the host stromal cells.
CNTO 859 inhibits hTF-induced coagulation and FX conversion in vitro
TF8-5G9 was shown to competitively inhibit FX binding to the TF:FVIIa complex, thus inhibiting the coagulation cascade. To ensure that CNTO 859 retained TF function blocking activity after humanization, inhibition of coagulation by CNTO 859 was examined. Coagulation was measured by adding recombinant hTF (Simplastin hTF) to human plasma in the presence of CaCl2. Inhibition of coagulation by CNTO 859 was measured by adding the antibody to the plasma immediately prior to addition of hTF. CNTO 859 inhibited coagulation in a dose-dependent manner (Fig. 2a). The data were fit to a sigmoidal-dose response equation, providing an EC50 of 4.8 nM. Similar results were observed when TF-expressing MDA-MB-231 human breast cancer cells were used to initiate coagulation (Fig. 2b; EC50 = 13.3 nM). A normal clotting time of 750 sec was observed with a PBS control group. At concentrations above 3 μg/ml, CNTO 859 delayed coagulation time by over 2-fold. The mechanism of CNTO 859 inhibition of coagulation was demonstrated to be due to competitive inhibition of FX binding to the pre-formed TF:FVIIa complex, and hence FX activation. A chromogenic FX conversion assay was used to measure the rate at which TF:FVIIa converted FX to FXa. The rate was plotted against the concentration of FX, and the data were fit to a hyperbola, providing a KM for FX of 30 nM (Fig. 2c). When the experiment was repeated in the presence of 10 nM CNTO 859, the VMax was not changed, but the KM shifted to 80 nM. These results are consistent with the properties of competitive inhibition, and mirror those previously reported for the parental mouse monoclonal antibody TF8-5G9.27
CNTO 859 binds to hTF on cancer cells with high affinity
In order to test the in vivo antitumor efficacy of CNTO 859, it was desirable to identify a tumor cell line with high levels of TF expression. Flow cytometry was used to show binding of CNTO 859 to TF on MDA-MB-231 cells. To determine the affinity of CNTO 859 for TF, the MFI was plotted against CNTO 859 concentration, providing an EC50 of 1.7 nM (Fig. 3a). To confirm the data obtained by flow analysis, CNTO 859 was radiolabeled with 125I and binding studies to MDA-MB-231 cells were performed. Increasing concentrations of CNTO 859 were mixed with cells, and the amount of radioactivity bound to the cells was measured. Assuming a 1:1 ratio of one bivalent antibody molecule binding to one TF molecule, the amount of radioactivity could be converted to the number of receptors per cell. This was plotted against the concentration of CNTO 859 to provide an EC50 of 1.7 nM (Fig. 3b), remarkably similar to that obtained by FACS. In addition, the maximum binding was measured to be 259,000 antibody accessible TF molecules per cell.
CNTO 859 inhibits lung colonization in an experimental metastasis model
To determine whether functional inhibition of TF in vivo could inhibit coagulation-dependent late-stage metastatic events such as tumor cell adhesion and lung colonization,29 a tail vein lung metastasis model using MDA-MB-231 cells was utilized. A single bolus of CNTO 859 (20 mg/kg) injected 3 hr prior to tumor cell inoculation was able to completely prevent lung metastasis compared to either PBS or human IgG controls, reducing the median number of foci from 135 lesions to zero (p = 0.004). Representative images of lungs harvested from a human IgG treated animal and a CNTO 859 treated animal are shown in Figure 4a. A second cohort of animals remained on antibody therapy, receiving a maintenance regimen of 10 mg/kg weekly until the end of the study. Control (saline-treated) animals had a combined median tumor lesion count of 173. Those treated with control antibody had 156 lung metastases. Prolonged CNTO 859 treatment dramatically and significantly reduced the median number of lung metastases to zero (Fig. 4b). Because the initial injection was so efficacious it was impossible to detect any additional benefit of further weekly maintenance dose injections of CNTO 859.
CNTO 859 monotherapy inhibits tumor progression of orthotopic MDA-MB-231 breast carcinoma xenografts
To investigate the efficacy of CNTO 859 in inhibiting the growth of primary tumors, the MDA-MB-231 orthotopic model was utilized. In an initial study, CNTO 859 profoundly inhibited tumor growth. CNTO 859 (20 mg/kg) dosed 3 days after tumor cell implantation and once weekly thereafter, significantly reduced final tumor volumes by roughly 95% (Fig. 5a). Control antibody and saline treated animals had average tumor burdens of 350 mm3 whereas animals treated with CNTO 859 had final tumor volumes of only 20 mm3. In a subsequent study, animals were dosed starting on day 3 with increasing concentrations of CNTO 859 (0.1–20 mg/kg). Weekly therapy of 1, 5, 10 and 20 mg/kg significantly inhibited tumor growth by over 95%. At doses as low as 0.1 mg/kg, an 88% tumor inhibition was observed compared to the saline control (p = 0.0012) (Fig. 5b). CNTO 859 also inhibited the growth of established tumors. Xenografts were allowed to seed and animals were randomized once palpable (20 or 50 mm3) tumors were present. In cohorts bearing established xenografts, CNTO 859 therapy (10 mg/kg, weekly) reduced the median final tumor volumes of early stage and established tumors by 64 and 33%, respectively (Fig 5c).
TF is frequently overexpressed in many tumor types. This observation, coupled with experimental evidence that TF plays a role in tumor proliferation, metastasis and angiogenesis, suggests that TF may be an important molecular target for anti-cancer therapies.16, 18, 20, 25, 30, 31, 32 We have characterized the functional activity of the humanized anti-tissue factor antibody CNTO 859 and demonstrated profound antitumor and antimetastatic efficacy of this agent in animal models of human breast cancer.
We demonstrate here that CNTO 859 is highly effective at preventing experimental metastatic spread of human MDA-MB-231 breast cancer cells to the lungs of SCID Beige mice. Inhibition of metastasis was essentially complete, with a 99.9% reduction in the number of lung lesions in treated mice. Although pharmacologic inhibitors of coagulation such as heparin, LMWHs, or hirudin, have been shown to inhibit metastasis in tail vein models,33, 34, 35, 36, 37, 38, 39 and murine anti-hTF antibodies26 have been used in experimental melanoma metastasis models, this is the first demonstration that a function blocking human antibody can inhibit experimental breast cancer metastasis to the lung.
The mechanism by which the coagulation system promotes metastasis in this model remains unclear. However, components such as circulating functional platelets, and fibrinogen are known to be critical for the establishment of metastatic colonies.40 Possible hypotheses include improved adhesion due to fibrin coating and platelet activation, altered endothelial cell adhesion molecule expression due to coagulation protease activity, and evasion of tumor cell killing as a result of microthrombi physically shielding tumor cells from cells of the innate immune system. The latter idea is supported by the studies of Palumbo et al., who showed that specific depletion of natural killer cells obviated the pro-metastatic effects of platelet or fibrinogen deficiency.40 In our studies SCID Beige mice, known to be deficient for NK cells, were still protected against lung metastasis by the anti-coagulant CNTO 859 antibody. It will be interesting to determine if perhaps other innate immune cell populations, such as macrophages, might be mediating the anti-metastatic effects observed in the SCID Beige host.
Multiple lines of evidence have suggested that TF plays an important role in tumor growth and progression. Enforced overexpression of TF has been demonstrated to promote tumor growth in several studies.8, 24, 26 TF signaling results in increased production of pro-angiogenic factors by tumor cells, promoting tumor growth in vivo.10, 41, 42, 43 TF is also expressed on tumor-associated endothelial cells and is thought to promote angiogenesis in this cell type. In other systems, TF or its downstream products have been shown to promote cancer cell proliferation and inhibit anoikis or apoptosis. This study provides the first evidence that antibody inhibition of TF activity can inhibit the growth of primary tumors.
Hembrough et al. reported inhibition of mouse Lewis lung carcinomas with the TF:FVIIa complex inhibitors TFPI and rNAPc2.24 In contrast, a specific inhibitor of FXa did not show significant anti-tumor effects against mouse B16 melanoma or Lewis lung tumors. These authors concluded that the observed inhibition of tumor growth was due to inhibition of TF:FVIIa signaling, possibly being mediated by FVIIa cleavage of PARs, and was independent of blood coagulation. We demonstrate here that the mechanism of action of CNTO 859 is distinct from TFPI and rNAPc2, in that it is primarily a competitive inhibitor of FX binding to the intact TF:FVIIa complex and hence, activation to FXa. Since FXa and thrombin are also potent activators of PARs,13, 16, 25, 44, 45, 46 and CNTO 859 prevents FXa and thrombin production, inhibition of PAR activation may also play a role in the anti-tumor effects of CNTO 859 treatment. The antitumor efficacy observed with CNTO 859 in these studies could be due to interference with any of the above-mentioned pro-tumor activities of TF or a combination of these effects. Further studies designed to elucidate the mechanisms of the anti-tumor activity we observed with CNTO 859 are in progress.
This study extends previous results demonstrating antimetastatic effects of anti-TF antibodies against melanoma cells to human breast carcinoma cells. The results also show for the first time that anti-TF antibodies such as CNTO 859, by blocking only tumor and not host-derived TF, can have profound antitumor effects against primary tumors. It was not possible to assess the potential adverse effects of CNTO 859 in the mouse model used here due to the lack of rodent cross-reactivity. Potential adverse events might logically include bleeding and this risk will be assessed in reactive species such as human TF knock-in mice or non-human primates or both. The results reported here suggest that CNTO 859 or its derivatives may ultimately be useful as targeted cancer therapies for the clinical treatment of human tumors.
- 23Vascular endothelial growth factor induces tissue factor and matrix metalloproteinase production in endothelial cells: conversion of prothrombin to thrombin results in progelatinase A activation and cell proliferation. Int J Cancer 1998; 75: 780–6., , , , , , .
- 43Factor VIIa and thrombin induce the expression of Cyr61 and connective tissue growth factor, extracellular matrix signaling proteins that could act as possible downstream mediators in factor VIIa x tissue factor-induced signal transduction. J Biol Chem 2000; 275: 14632–41., , , .