MXPA05012974A - Method of inhibiting tumor growth with anti-tissue factor antibodies - Google Patents

Abstract

A method of using tissue factor antagonists to treat proliferative diseases characterized by neovascularization such as cancer, rheumatoid arthritis, psoriasis, or proliferative retinopathy. or macular degeneration. Tissue factor antagonists capable of rapid prevention of blood clotting via the extrinsic pathway are also capable of inhibiting tumor growth in mammals.

Description

METHOD FOR INHIBITING TUMOR GROWTH WITH TISSUE ANTI-FACTOR ANTIBODIES BACKGROUND OF THE INVENTION FIELD OF THE INVENTION The present invention relates to a method for utilizing tissue factor (TF) antagonists for cancer treatment, specifically by preventing or inhibiting the growth of tumor cells. The invention more specifically relates to methods for the treatment of such diseases by the use of TF antagonists such as antibodies directed to TF, including specific portions or variants thereof, specific for at least one TF protein or fragment thereof, in an amount effective to inhibit the growth of tumors.

Tissue factor (TF) Blood coagulation involves a series of cascade reactions that lead to the formation of fibrin. The coagulation cascade consists of two superimposed routes, both of which are required for hemostasis.

The intrinsic pathway comprises protein factors present in circulating blood, while the extrinsic pathway requires tissue factor (TF), which is expressed on the cell surface of a variety of tissues in response to vascular injury (Davie et al. , 1991, Biochemistry 30: 10363). When exposed to blood, the TF sets in motion a potentially explosive cascade of activation steps that results in the formation of a nonsoluble fibrin clot. TF has been investigated as a target for anticoagulant therapy. TF is a 263-amino acid, single-chain membrane glycoprotein that functions as a receptor for the factor VII and Vlla and thus initiates the extrinsic pathway of the coagulation cascade in response to vascular injury. TF is a transmembrane cell surface receptor that serves as both the receptor and cofactor for the factor VIIa form a complex TF: Vlla proteolytically active on cell surfaces (Ruf et al, (1992) J. Biol Chem 267. : 6375-6381). In addition to its role in the maintenance of hemostasis, excess TF has been implicated in pathogenic conditions. Specifically, the synthesis and cell surface expression of TF has been implicated in vascular disease (Wilcox et al, 1989, Proc Natl Acad Sci, 86:.... 2839) and gram-negative septic shock (Warr et al ., 1990, Blood 75: 1481).

TF Antagonists Various anti-TF antibodies are known. For example, Carson et al, (1987, Blood 70: 490-493) describes a hybridoma producing monoclonal antibody prepared by immunizing mice with purified TF by affinity chromatography on immobilized factor VII. Ruf et al, (1991, Thrombosis and Haemostasis 66: 529) characterized the anticoagulant potential of murine monoclonal antibodies against human TF. The ability of monoclonal antibodies directed binding site FVII on TF is dependent on its ability to compete with FVII for binding to TF and the formation of the TF / VIIa, which is rapidly formed when TF contacts with the plasma. Therefore, said antibodies were relatively slow inhibitors of TF in plasma. A monoclonal antibody, TF8-5G9, was able to inhibit the TF / Vlla complex, thus providing an immediate anticoagulant effect in the plasma. This antibody is described in the Patents of E.U.A. Nos. 6,001,978, 5,223,427, and 5,110,730. Ruf et al, suggested (previously mentioned) that the mechanisms that inactivate the TF / Vlla complex, rather than prevent its formation, can provide strategies for the interruption of coagulation in vivo. In contrast to other antibodies that inhibit the binding of factor VII to TF, TF8-SG9 shows only subtle and indirect effects on the binding of factor VII or factor Vlla to the receptor. TF8-5G9 binds to the extracellular domain of TF with a nanomolar binding constant to block the formation of the tertiary initiation complex TF: F.VIIa: F.X (Huang et al., J. Mol. Biol. 275: 873-894, 1998). It has been shown that anti-TF monoclonal antibodies inhibit TF activity in various species (Morissey et al., 1988, Thromb Res.52: 247-260) and neutralizing anti-TF antibodies have been shown to prevent death in a baboon sepsis model (Taylor et al, Cira Shock, 33: 127 (1991)), and attenuate DIC-induced endotoxin in rabbits (Warr et al, (1990) Blood 75: 1481). WO 96/40921 describes anti-TF antibodies grafted with CDRs derived from TF8-5G9 antibody. Other humanized or humanized anti-TF antibodies are described in Presta et al, Thromb Haemost 85: 379-389 (2001), EP1069185, WO 01/70984 and WO 03/029295.

The role of TF in cancer Tissue factor is also over-expressed in a variety of malignancies and tumor cell lines isolated from human, suggesting a role in tumor growth and survival. TF is not produced by healthy endothelial cells that line the normal blood vessels but is expressed in these cells in the tumor blood vessels. It seems to play a role both in vasculogenesis, formation of new blood vessels, as in animal development and angiogenesis, generation of new capillaries from existing arteries, in normal and malignant adult tissues. Therefore, inhibition or targeting of TF can be a useful anti-tumor strategy that could affect the survival of tumor cells that over-express TF directly by inhibiting cell signaling mediated by TF or other activities. In addition, this method can indirectly prevent tumor growth via an anti-angiogenic mechanism by inhibiting the growth or function of intra-tumor endothelial cells expressing TF.

TF and angiogenesis Angiogenesis is the process of generating new capillary blood vessels, and results from the activated proliferation of endothelial cells. Neovascularization is tightly regulated, and only occurs during embryonic development, tissue remodeling, scarring and periodic cycle of corpus luteum development (Folkman and Cotran, Relation of vascular proliferation to tumor growth, Int. Rev. Exp. Pathol. , 207-248 (1976)). There is currently considerable evidence that tumor growth and cancer progression require angiogenesis and neovascularization, growth and extension of blood vessels, in order to provide the tumor tissue with nutrients and oxygen, to take away waste products and act as conduits for cancer. metastasis of tumor cells to distant sites (Folkman, et al., N Engl J Med 285: 1181-1186, 1971 and Folkman, et al., N Engl J Med 333: 1757-1763, 1995). However, angiogenesis and neovascularization of tissue and tumors represent complex processes mediated by the interaction of cellularly produced factors: including TNFalpha, VEGF, and tissue factor. Studies show that the routes leading to upregulation of VEGF and TF overlap (Chen J. et al. (2001) Thromb. Haemost, 86-334-5), two major participants in the onset of blood vessel formation new. Endothelial cells normally proliferate much more slowly than other types of cells in the body. However, if the rate of proliferation of these cells becomes dysregulated, pathological angiogenesis can occur. Pathological angiogenesis is implicated in many diseases. For example, cardiovascular diseases such as angioma, angiofibroma, vascular deformity, atherosclerosis, synechia and endemic sclerosis; and ophthalmological diseases such as neovascularization after corneal implant, neovascular glaucoma, diabetic retinopathy, corneal angiogenic disease, macular degeneration, pterygium, retinal degeneration, retrolateral fibroplasias, and granular conjunctivitis are related to angiogenesis. Chronic inflammatory diseases such as arthritis; Dermatological diseases such as psoriasis, telangiectasis, pyogenic granuloma, seborrhoeic dermatitis, venous ulcers, acne, rosacea (acne rosacea or erythematosa), small tumors (warts), eczema, hemangiomas, lymphangiogenesis are also dependent on angiogenesis. Vision can be altered or lost due to various eye diseases in which the vitreous humor is infiltrated by capillary blood. Diabetic retinopathy can take one of two forms, nonproliferative or proliferative. Proliferative retinopathy is characterized by abnormal formation of new blood vessels (neovascularization), which grow on the vitreous surface or extend into the vitreous cavity. In advanced disease, neovascular membranes may occur, resulting in retinal traction separation. Vitreous hemorrhages can result from neovascularization. The visual symptoms vary. A sudden severe vision loss can occur when there is an intravitreal hemorrhage. Visual prognosis with proliferative retinopathy is further monitored if it is associated with severe retinal ischemia, extensive neovascularization, or extensive formation of fibrous tissue. Similarly, macular degeneration takes two forms, dry and wet. In exudative macular degeneration (wet form), which is much less common, there is formation of a subretinal network of choroidal neovascularization frequently associated with intraretinal hemorrhage, subretinal fluid, separation of the pigment epithelium, and hyperpigmentation. Eventually, this complex contracts and leaves a raised scar distinguished at the posterior pole. Both forms of macular degeneration related to age are often bilateral and are preceded by inflammation in the macular region. Another cause of vision loss related to angiogenic etiologies is damage to the iris. The two most important situations that result in the iris being pushed towards the angle are the contraction of a membrane such as in neovascular glaucoma in patients with diabetes or central retinal vein occlusion or inflammatory precipitates associated with uveitis that pushes the iris towards the angle (Chapter 99. The Merck Manual 17th Ed. 1999). Rheumatoid arthritis, an inflammatory disease, also results in inappropriate angiogenesis. The growth of vascular endothelial cells in the synovial cavity was activated by inflammatory cytokines, and resulted in cartilage destruction and replacement with pannus in the joint (Koch AK, Polverini PJ and Leibovich SJ, Arthritis Rheum, 29, 471-479 ( 1986); Stupack DG, Storgard CM and Cheresh DA, Braz. J. Med. Biol. Res., 32, 578-581 (1999); Koch AK, Arthritis Rheum, 41, 951-962 (1998)). Psoriasis is caused by the uncontrolled proliferation of skin cells. Rapidly growing cells require adequate blood supply, and abnormal angiogenesis is induced in psoriasis (Folkman J., J. Invest, Dermatol., 59, 40-48 (1972)). WO 94/05328 describes the use of anti-TF antibodies to inhibit the onset and progression of metastases by suppressing prolonged adhesion of metastasis cells in the microvasculature thus inhibiting metastasis, but does not disclose any effect on the growth of the established tumor cells. Given the complexity in the factors that regulate tumor vascularization as well as in the incomplete understanding of the role of tissue factor as a receptor that mediates cell growth in both tumor growth and healing, it is possible that TF blockage may already play is a critical role or a redundant role in the pathogenesis of cancer or other diseases characterized by inappropriate angiogenic activity. Therefore, it could be beneficial to understand whether an antibody to TF could be used as a primary therapy or adjunct in the treatment of human cancers as well as in other proliferative diseases accompanied by mechanisms of neovascularization and angiogenic mechanisms.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to a method for using TF antagonists, including antibodies directed to TF, and specified portions or variants thereof specific for at least one TF protein or fragment thereof, to inhibit the growth of tumors in mammals. Such TF antagonists such as antibodies can act through their ability to bind to TF in a manner that prevents events associated with the growth of cancerous tissue, particularly solid tumors. In another aspect, the invention provides a method for the treatment of a disease characterized by an increase in vascularized tissue, which comprises administering a tissue factor antagonist in an amount effective to inhibit the increase of said tissue.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph showing tumor growth rates (volume change) of human breast cancer cells implanted subcutaneously in the flanks of nude and dosed mice with CNTO 859, irrelevant hlg, or HBSS once a week starting to day 0. Figure 2 is a graph showing the data from the same experiment as in Figure 1 for groups of mice dosed with CNTO 859 or hlg once a week starting at day 14. Figure 3 is a graph that shows the change in tumor volumes either from control animals, animals treated with either PBS or Ig control of human and animals treated with CNTO 859. Figure 4 is a bar graph representing the mean and standard deviation of the volumes Tumor endings either from control animals, animals treated either with PBS or with control IgG and animals treated with CNTO 859. Figure 5 shows the tumor incidence rate in anima they were treated either with PBS, Ig control or CNTO 859 starting on the same day that the tumor cells were implanted. Figure 6 shows the tumor progression of MDA MB 231 xenografts as measured by volume in animals treated with either PBS, human control Ig or various doses of CNTO 859. CNTO 859 was able to inhibit tumor growth at all concentrations . The tumor inhibition had an interval of 90% at 0.1 mg / kg (p = 0.0012 and p = 0.0106, respectively, test of two Wilcoxon samples using t distribution) up to 95% at any concentration above this. Figure 7 is a scatter plot showing the distribution of final tumor volumes from animals treated with either PBS, human control Ig or various doses of CNTO 859 (0.1, 1, 5, 10 and 20 mg / kg) . Figure 8 is a graph of tumor volumes over time for an experiment using xenografts of MDA MB 231 human breast cancer cells implanted in mice orthotopically (in breast tissue) and wherein the mice were treated with either PBS, Ig human control, CNTO 859Ala / Ala or various doses (0.01, 0.1 and 1 mg / kg) of CNTO 859 and CNTO 860. Figure 9 shows the means and standard deviations of four of the groups from the same experiment as shown in Figure 8, showing only the controls and CNTO 859 and CNTO 860 at 0.1 mg / kg. Figure 10 is a graphic representation of each of the individual final tumor volumes and the means from each group in the same experiment shown in Figure 8. Figure 11 shows the tumor incidence data from the same experiment shown in figure 8.

Figure 12 is a graph showing tumor growth rates (volume change) of human BxPC-3 pancreatic tumor cells implanted in treated mice starting the next day with CNTO 859. Tumor growth was inhibited by 46.9% (p <); 0.0012). Figure 13 is a graph showing tumor growth rates (volume change) of human BxPC-3 pancreatic tumor cells implanted in mice when established tumors were treated with CNTO 859. Tumor growth was inhibited by 35% (p. < 0.0001). Figure 14 is a bar graph showing that angiogenesis induced by human pancreatic tumor cells PANC-1, as measured by the length of blood vessels in MATRIGEL in mice, is reduced by 88% (p <0.05) by a human murine anti-TF antibody (PHD 127).

DETAILED DESCRIPTION OF THE INVENTION The TF antagonists of the invention are useful in the inhibition and prevention of tumor growth. Numerous pathologies involving various forms of solid primary tumors are improved by treatment with TF antagonists in the method of the present invention.

Tumors Both benign and malignant tumors, including various cancers such as cervical, anal and oral cancers, stomach, colon, bladder, rectal, liver, pancreatic, lung, breast, cervix uteri, body cancers of the uterus, ovary, prostate, testicle, kidney, brain / snc (eg, gliomas), head and neck, eye or eye, throat, skin melanoma, acute lymphocytic leukemia, acute myeloid leukemia , Ewing's sarcoma, Kaposi's sarcoma, basal cell carcinoma and squamous cell carcinoma, small cell lung cancer, choriocarcinoma, rhabdomyosarcoma, angiosarcoma, hemangioendothelioma, Wilms tumor, neuroblastoma, mouth / pharynx, esophageal, laryngeal cancer, of kidney and lymphoma, among others can be treated using anti-TF antibodies of the present invention. Therefore, the present invention provides a method for the modulation or treatment of at least one malignant disease in a cell, tissue, organ, animal or patient, including, but not limited to, at least one of: carcinoma of the kidney, carcinoma colorectal, renal cell carcinoma, pancreatic carcinoma, prostatic carcinoma, nasopharyngeal carcinoma, malignant histiocytosis, paraneoplastic syndrome / hypercalcemia of malignancy, solid tumors, adenocarcinomas, sarcomas, malignant melanoma, hemangioma, metastatic disease, and the like. Said method can optionally be used in combination with, by prior administration, concurrently or after the administration of said TF antagonist, radiation therapy, an anti-angiogenic agent, a chemotherapeutic agent, a farnesyl transferase inhibitor, an inhibitor. of prothesis or the like.

TF Antagonists As used in the present invention, the term "TF antagonists" refers to a substance which inhibits or neutralizes the activity of TF. Such antagonists achieve this effect in a variety of ways. One class of TF antagonists will bind to the TF protein with adequate affinity and specificity to neutralize the effect of TF. This class of molecules includes antibodies and antibody fragments (such as, for example, the F (ab) or F (ab ') 2 molecules). Therefore, as used in the present invention "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules. Another class of TF antagonists are fragments of the TF protein, muteins or small organic molecules for example peptidomimetics, which will bind TF ligands, thus inhibiting the activity of TF or its ability to cause intracellular signaling. The TF antagonist can be any of these classes as long as it is a substance that inhibits the anti-tumor activity of TF or anti-angiogenic activity. TF antagonists include TF antibodies, modified TF, antisense TF and partial TF peptides.

In a particularly preferred embodiment, the TF antagonist is a murine, chimeric, humanized or human monoclonal antibody or a fragment thereof which has one of the following properties: it prevents the binding of factor Vlla to TF thus preventing it from proceeding coagulation, prevents the formation of the TF: FVIIa: FX complex, or prevents TF signaling via its intracellular domain. Such antibodies are known in the art and can be used in the method of the present invention. Monoclonal antibodies of murine to TF are known in, for example, Patents of E.U.A. Nos. 6,001, 978, 5,223,427, and 5,110,730. WO 96/40921 discloses CDR-grafted anti-TF antibodies derived from TF8-5G9 antibody in which the complementarity determining regions (CDRs) from the variable region of TF8-5G9 mouse antibody are transplanted into the variable region of a human antibody and bind to the constant region of a human antibody. Other humanized anti-TF antibodies capable of preventing anti-coagulant TF and receptor-mediated activities are described in Presta et al., Thromb Háemost 85: 379-389 (2001) and EP1069185. Each of the foregoing references is incorporated by reference into the present application.

Composition and its uses The anti-TF neutralizing monoclonal antibody can be used to inhibit tumor growth according to the invention. The individual to be treated can be any mammal and preferably is a human patient in need of such treatment. The amount of monoclonal antibody administered will vary in accordance with the purpose for which it is used and the method of administration. The TF antibodies of the present invention can be administered by any of a number of methods that result in an effect on the tumor tissue in which it is desired that the growth be prevented or stopped. Additionally, the anti TF antibodies of the invention do not need to be present locally to impart an antitumor effect, therefore, these can be administered whenever there is access to the body compartments or that fluids containing TF are obtained. In the case of malignant tissues, these methods may include the direct application of a formulation containing the antibodies. Such methods include intravenous administration of the liquid composition, for subcutaneous or transdermal administration of a liquid or solid, oral formulation, topical administration, or interstitial or inter-operative administration. The administration can also be oral or by local injection into a tumor or tissue but generally, the monoclonal antibody is administered intravenously. Generally, the dose range is from about 0.01 mg / kg to about 12.0 mg / kg. This can be as a bolus or as a slow or continuous infusion which can be controlled by a controlled microprocessor and a programmable pump device.

Alternatively, the DNA that preferably encodes a fragment of said monoclonal antibody can be isolated from hybridoma cells and administered to a mammal. The DNA can be administered in a nude form or can be inserted into a recombinant vector, e.g., vaccinia virus so as to result in expression of the DNA in the patient's cells and administration of the antibody. The monoclonal antibody used in the method of the present invention can be formulated by any of the established methods of formulating pharmaceutical compositions, for example as described in Remington's Pharmaceutical Sciences, 1985. For ease of administration, the monoclonal antibody will typically be combined with a pharmaceutically acceptable vehicle. Such vehicles include water, physiological saline, or oils. Formulations suitable for parenteral administration include sterile aqueous and non-aqueous solutions which may contain anti-oxidants, pH regulators, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. Except when its use is contemplated in any compositions, since any conventional means is incompatible with the active ingredient and its intended use. The formulations can be presented in unit dose or multi-dose containers, for example, sealed s and s, and can be stored in a freeze drying condition (lyophilized) that only requires the addition of the sterile liquid carrier, for example, water for injections, immediately before use.

Combinations with TF antagonists The method can be carried out by combining the TF antagonists of the invention with one or more other agents having anti-tumor effect or a different mechanism for the inhibition of live angiogenesis, including, but not limited to, chemotherapeutic agents. In addition, the TF antibody can be combined with one or more anti-angiogenic agents. The angiogenesis that is characterized by the invasion, migration and proliferation of smooth muscle cells and endothelial cells. It is known that the integrin avß3 (also known as the vitronectin receptor) plays a role in various conditions or disease states including tumor metastasis, solid tumor growth (neoplasia), osteoporosis, Paget's disease, humoral hypercalcemia of malignancy, angiogenesis, including tumor angiogenesis, retinopathy, including macular degeneration, arthritis, including rheumatoid arthritis, periodontal disease, psoriasis, and smooth muscle cell migration (e.g. restenosis). The adhesion receptor integrin avß3 binds to vitronectin, fibrinogen, von Willebrand factor, laminin, thrombospondin, and other similar ligands. It was identified as a marker of angiogenic blood vessels in chicken and man and plays a critical role in angiogenesis or neovascularization. The avß3 antagonists inhibit this procedure by selectively promoting apoptosis of the cells in the neovasculature. Therefore, avß3 antagonists could be useful therapeutic targets for the treatment of such conditions associated with neovascularization (Brooks et al., Science, Vol. 264, (1994), 569-571). Additionally, the invasion of tumor cells occurs through a three-step procedure: 1) binding of the tumor cell to the extracellular matrix; 2) proteolytic solution of the matrix; and 3) movement of the cells through the dissolved barrier. This procedure can occur repeatedly and may result in distant site metastases from the original tumor. The avß3 integrin has been shown to play a role in the tumor cell invasion as well as in angiogenesis. As avß3 antagonists and anti-TF neutralizing antibodies both target tumors act through different mechanisms, the combination of anti-integrin antibodies with anti-TF antibodies could result in a particularly potent and effective combination therapy with little normal tissue toxicity . Therefore, in one embodiment of the present invention, there is provided a method of inhibiting tumor growth which comprises administering a combination of an antagonistic integrin and an anti-TF antibody in a patient in need of such treatment. Other anti-bodies that bind selectively to integrins or integrin subunits, specifically those that bind to the alphaV subunit, are described in US Patents. Nos. 5,985,278 and 6,160,099. Mabs that inhibit the binding of alphaVbeta3 to their natural ligands containing the tripeptide argininyl-glycyl aspartate (RGD) are described in US Pat. No. 5,766,591 and WO 0078815. Other antibodies that prevent integrins containing the alphaV subunit from binding to vitronectin, fibronectin, or other ligands have similar utility in the prevention of angiogenesis. Such antibodies include the antibody known in GEN 095 or CNTO 95 and described in the co-pending application of the applicants published as WO 02012501. In accordance with the invention, other known anti-angiogenic agents such as thalidomide in combination with an anti-TF antibody.

Abbreviations ATCC-American Type Culture Collection C? 2-carbon dioxide DMEM-medium Eagle modified Dulbeccos EDTA-ethylenediamine tetra-acetic acid FBS-fetal bovine serum FVI la-Factor Vlla (activated FVII) FX-Factor X (inactive) FXa -Factor Xa (activated FX) hlg-lg of human 2 mM LNN-L-glutamine, 1 mM sodium pyruvate, non-essential AAs 0.1 mM PBS-pH regulated saline with SQ-subcutaneous phosphate IV-intravenously IP-intraperitoneal TF Tissue Factor Although the invention has been described in general terms, the embodiments of the invention will be further described in the following examples.

EXAMPLE 1 Inhibition of tumor growth in breast carcinoma xenoinings This example demonstrates the ability of the tissue anti-factor IgG antibody to inhibit the tumor growth of MDA-MB-231 breast carcinoma xenografts implanted in nude mice.

Materials and methods. Naked mice (50) of 4-6 weeks of age (Crl: NU / NU-CD1) were obtained from Charles River Laboratories and acclimated for 10-14 days prior to experimentation. The mice were maintained in the animal facility at Centocor, Inc. in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animáis. The human breast carcinoma cell line MDA-MB-231 was obtained from ATCC (Rockville, MD, Catalog # HTB-26). The cells were cultured in DMEM medium supplemented with 25 mM HEPES, 10% FBS and 1% LNN at 37 ° C, 5% C02. The cells were harvested in logarithmic growth phase with trypsin-EDTA and resuspended in sterile HBSS at 5x107 cells / mL.

Antibodies: CNTO 859, CDR-grafted antibody TF8-5G9 is described in WO 96/40921, storage concentration 3.75 mg / mL; hlg, ZLB Bioplasma, AG, Berne Switzerland. Storage concentration 30 mg / mL in sterile USP water). All items for testing were established at a working concentration of 2 mg / mL in sterile HBSS. All test items and controls will have LAL values < 1. 0 EU / g. At day 0, the mice were randomly assigned to each of the 5 groups, 10 mice per group. The cells were implanted subcutaneously within the flank of nude mice at a concentration of 5x106 MDA-MB-231 tumor cells in a volume of 0.2 mL (0.1 mL cells in HBSS mixed with 0.1 mL of Matrigel®). Groups of mice were dosed once a week as proscribed in table 1.

TABLE 1 At day 0 of the study, 50 mice for study were placed in 5 groups (10 mice / group, according to table 1). All animals were injected with 0.2 mL of MDA-MB-231 cell suspension containing 5X106 cells (1: 1 mixture of cell suspension in HBSS with Matrigel®) subcutaneously on the right side of the rib area. At day 0, all animals in groups 1, 2, and 3 received an intraperitoneal injection of 10 mL / kg of the test article or HBSS (Table 1). The animals in groups 4 and 5 received an intraperitoneal injection of 10 mL / kg of the test article on day 14 or an average tumor size of 100 mm3). After intraperitoneal injection initiate all animals received weekly intraperitoneal doses (5 mL / kg) of the test or control article until day 80. The dose of 20 mg / kg for the first dose and 10 mg / kg for the subsequent doses is calculated based on the most recent previous body weight value. The animals were dosed IP on the Monday of each week until day 80 or until the rumors reached a volume of 2,000 mm3. Animal weights and tumor volumes were measured once a week until the end of the study. The animals were weighed starting at day 0 while the tumor volumes were recorded only once they were palpable. Tumors were measured in three dimensions using calibrators and tumor volumes were calculated based on the formula V = (LxWxT) / 2, where L = length, W = width and T = thickness. The completion of the study was planned when the tumors reached an average volume of -2000 mm3, with an option to extend the study as long as the condition of welfare of that animal was not compromised. At the end, the animals were sacrificed via asphyxiation by C02 and the tumors were excised and weighed. Subsequently individual tumors were bisected with one half being rapidly frozen in OCT and the other half fixed in 10% formalin. Serum samples were taken from each animal at term via cardiac puncture.

Results The growth rate of each treatment group was plotted as a function of tumor volume (mm3) versus time (days after implantation) (figure 1 and 2). There was a 100% shooting speed in all groups. The treatment of animals on day 0 (figure 1) or day 14 (figure 2) with control hlg did not significantly impact the relative tumor growth with respect to the group treated with negative control HBSS (P = 0.779, P = 0.979, respectively) . Tumor growth in animals treated with CNTO 859 at day 0 was inhibited by 62% (P < 0.0001) (figure 1). When the treatment with CNTO 859 started on day 14, when the average tumor size was 100 mm3, the tumor growth rates were inhibited by 47% (P <0.0001) (figure 2). The effect of the combined general treatment of CNTO 859 was also significantly relative to the effect of the general treatment of the control hlg group (P <0.0001). The day-to-day importance, defined as P < 0.005, was generally achieved after 17 days of treatment. The results show that CNTO 859 inhibited tumor growth rates by up to 62%. This is the first time that it is demonstrated that an anti-human tissue factor IgG antibody has the ability to inhibit human-derived tumor growth in vivo. Both early (day 0) and late (day 14) treatments with CNTO 859 significantly inhibited tumor growth rates.

EXAMPLE 2 Effect of anti-TF antibody on human breast carcinoma in an orthotopic xenograft model In this example, an orthotopic tumor growth model was used using the human breast carcinoma cell line, MDA MB 231, injected into the mammalian fat pads of SCID / Beige mice to evaluate the anti-tumor effect of CNTO 859. In addition, the effect of variations in the structure of the anti-tissue factor antibody was compared: one differing in the class identity of human CNTO 859 (IgG4) and CNTO 860 (IgGI); and in the modification of the union region FcR CNTO 859 designated CNTO 859ala / wing.

Materials and methods. Four week old SCID / Beige mice were obtained (C.B.-17 / lcrCrl-scid-bgBR) from Charles River Laboratories and acclimated for 10-14 days prior to experimentation. The mice were housed 7-8 / cage in cages with upper filter and were supplied with autoclaved food and acidified water containing Bactrum (0.13 mg / mL trimethoprim / 0.66mg / mL sulfamethoxazole) ad libitum. The animals were identified by individual numbered tags on the ear placed 5 days before the start of the study. The cage records were marked with source, sex, number of animals, animal ID numbers, group number, treatments, study number and IACUC protocol number were fixed to the cages. All animal studies were carried out at the vivarium at Centocor, Inc., Radnor, PA in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animáis. The human breast carcinoma cell line MDA MB 231 was obtained from the cell depository in Centocor and has been considered sterile and free of mycoplasma. The cells were cultured in DMEM medium supplemented with 10% FBS and 1% LNN at 37 ° C, 5% C02. Cells were harvested in logarithmic growth phase with trypsin-EDTA and resuspended at 5x107 cells / mL in serum free DMEM and implanted into the (inguinal Rt # 2/3) mammalian fat pad in a volume of 50 uLs. The test and control antibodies were the following: CNTO 859, 3.75 mg / mL storage concentration; CNTO 859, 10.29 mg / mL storage concentration; CNTO 860, 2.4 mg / mL storage concentration; CNT0859Ala / Ala, C1081, 1 mg / mL storage concentration; Human Ig, ZLB Bioplasma AG, Berne Switzerland, 30 mg / mL storage concentration. The antibodies were supplied at appropriate concentrations in PBS. All control and test items had been evaluated for endotoxin to be < 1 EU / mg and were administered intravenously.

Animals were randomly divided into 7-8 mice / group. At day 0, 2.5x106 MDA MB 231 cells were injected into the mammalian fat pad of the animals in a volume of 50 uLs using a 30g needle. Intravenous antibody therapy began at day 3. The dose and concentration regimens for each of the three studies are detailed in Tables 2, 3 and 4, respectively.

TABLE 2 TABLE 4 The mice were weighed and the tumor volumes were recorded once a week for a period of 8-9 weeks. The tumor volumes were calculated as (LxW2) / 2. The study was completed approximately eight to nine weeks after the inoculation of the tumor cell. In the event that any animal experiences rapid weight loss, respiratory distress or becomes moribund before the termination point, that animal was sacrificed by the study coordinator. The animals were sacrificed via asphyxiation by C02 and then weighed. The lungs and axillary lymphatic modules were surgically removed, rinsed in cold PBS, blotted, weighed and immediately fixed in Bouin's solution. The primary tumors were dissected, weighed and then fixed in BZT solution for histological analysis.

Primary Anti-Tumor Effect. The tumor volumes were monitored and recorded once a week during the study. At the end, the primary tumors were surgically dissected from SCID / Beige mice sacrificed by C02 and weighed. The tumor volumes and the final masses were recorded during the time (figure 3 and 4). Tumor growth was inhibited by 95% when the animals were treated with CNTO 859 in relation to either the animals treated with PBS or the control animals treated with IgG. (p = 0.0039 and p = 0.0126, two-tailed parametric t test, n = 8).

In a second study, the effect of the dose was examined. CNTO 859 inhibited tumor growth at doses as low as 0.1 mg / kg, given once a week. There was a significant reduction in tumor progression such as change in tumor volume (Figure 6) and individual final tumor weights (Figure 7) in animals treated with either 0.1, 1, 5, 10 or 20 mg / kg of CNTO 859 compared with the PBS groups and human control Ig groups. In a comparison study between CNTO 859 and CNTO 860 at three concentrations, it was observed that the IgG1 version of the anti-tissue factor antibody of the inventors was superior in the prevention not only of tumor growth, but also of tumor incidence (FIGS. -eleven). The tumor volumes from each of the respective groups are shown in (Figure 8) as the mean tumor weight in the group during the time (Figure 8) and as weight and individual final means (figure it out) and in.

Effect on tumor incidence. The therapy with CNTO 859 also showed a marked difference in tumor incidence in the treated animals. In the first study, the cells were able to adhere and give rise to other cells in the mammalian fat pad as observed by palpating a nodule at the site of the injection, but were too small to be measured until about the day 38, when a tumor was of measurable size. In contrast, measurable tumors appeared in animals treated with PBS or control treated with human Ig starting on day 17. Figure 5 shows the rate of tumor incidence in animals treated with either PBS, control Ig or CNTO 859. The results from this model of orthotopic tumor growth of MDA MB 231 indicate that CNTO 859 is a highly effective inhibitor of tumor incidence, growth and progression. Compared to a control vehicle or control Ig, CNTO 859 reduced tumor growth by 95% (p = 0.0039 and p = 0.0126, two-tailed parametric t-test, n = 8) and tumor incidence by 87.5% (p = 0.0017 vs PBS and p = 0.0086 vs Ig control of human, two-tailed parametric t-test, n = 8). In the comparison study between CNTO 859 and CNTO 860, it was evident when a dose of 0.1 mg / kg was used, that CNTO860 was able to delay tumor onset in 44 and 37 days compared to the groups treated with PBS and the groups control treated with human Ig. Similarly, CNTO 859 was able to delay tumor onset in 23 and 16 days. In addition, at the end of the study, more than 70% of the animals were tumor free in the CNTO 860 group compared to only 15% in the CNTO 859 group. All animals in the PBS, human Ig and CNTO 859 ala groups / ala had tumors by day 44 (figure 11).

Summary. CNTO 859 and two variants were compared for efficiency in preventing tumor growth and progression in a series of experiments using this xenograft model. In the first study, CNTO 859 was highly efficient in the prevention of tumor growth when it occurs once a week starting at day 3 after tumor implantation at a concentration of 20 mg / kg, resulting in a rate of growth inhibition 95% compared with either the PBS-treated groups or the Ig-treated control groups (p = 0.0039 and p = 0.0126, respectively). The inventors also observed a 87.5% reduction in tumor incidence in animals treated with CNTO 859 compared to either the PBS treatment groups or the Ig treatment control groups (p = 0.0017 and p = 0.0086, respectively). In a second study, CNTO 859 was administered at a series of doses with a range of 0.1 mg / kg to 20 mg / kg once a week. The results show that anti-TF monoclonal antibody therapy with CNTO 859 was highly efficient in slowing the rate of tumor progression, even at a very low dose of 0.1 mg / kg, resulting in tumor inhibition greater than 90 % compared with either the PBS treatment groups or the control groups treated with Ig (p = 0.0012 and p = 0.0106, respectively, t distribution using two Wilcoxon sample tests). The dose of 1, 5, 10 and 20 mg / kg significantly inhibited tumor growth by more than 95%. Finally, in a separate study, the efficiency of CNTO 859 was evaluated against CNTO860, an IgGI version of CNT0859, and the minimized version of ADCC, CNTO 859 wing / wing. Doses of 0.01 mg / kg of either IgGI or IgGI therapeutic antibody were not different from the groups treated with PBS, human control Ig or CNTO 859 Ala / Ala. In contrast, a dose of 1 mg / kg of either CNTO 859 or CNTO 860 was able to inhibit tumor growth by more than 95%. Interestingly, at the dose level of 0.1 mg / kg, the effect of CNTO 859 against CNTO 860 is distinguished as the tumor growth inhibited by CNTO860 by more than 95% even at this low dose while tumors treated with CNTO 859 showed escape signs of therapy, resulting in an inhibition of only ~ 85%. In addition, CNTO 860 was more effective than CNTO 859 in slowing tumor progression when used at 0.1 mg / kg, presumably due to additional ADCC activity.

EXAMPLE 3 Inhibition of tumor growth in pancreatic adenocarcinoma xenoinines In this example, the inventors demonstrate the effect of an anti-tissue factor antibody on the inhibition of growth of the pancreatic adenocarcinoma cell line, BxPC-3 grown on the flank of SCID mice. CNTO 859 was dosed once a week at 10 mg / kg following an initial loading dose of 20 mg / kg. In groups where therapy with CNTO 859 was started 1 day, after tumor implantation, the growth of BxPC-3 tumors was inhibited by 46.9% (p <0.001). In the groups where the treatment was initiated at an average tumor volume of 50-100 mm3, the tumors were slightly inhibited, but no significant statistical significance was observed (p = 0.6280).

Materials and methods. Six- to eight-week-old SCID mice were obtained from Charles River Laboratories (Wilmington) and acclimated for 10-14 days prior to experimentation. The mice were housed 10 / cage in cages with upper filter, and were supplied with autoclaved food and acidified water, which contained Bactrum (0.13 mg / mL trimethoprim / 0.66 mg / mL sulfamethoxazole) ad libitum. The ear tags individually numbered were placed 7 days before the start of the study in the identified animals. Cage records were marked with source, sex, number of animals, animal ID numbers, group number, treatments, study number and IACUC protocol number were fixed to the cages. All animal studies were carried out at the vivarium at Centocor, Inc., Radnor, PA in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animáis. The human pancreatic adenocarcinoma cell line BxPC-3 was obtained from ATCC (Rockville, MD, catalog # CRL-1687). These were evaluated so that they were free of viral contamination or contamination by mycoplasma, and was deposited by Centocor's Cell Biology Services. The cells were cultured in RPMI 1640 medium supplemented with 10% FBS and 1% LNN at 37 ° C, 5% C02. Cells were harvested in logarithmic growth phase with trypsin-EDTA and resuspended inSterile PBS at 1.5x107 cells / mL CNTO 859 produced at Centocor, Inc., was used at a storage concentration of 3.75 mg / mL; hlg, ZLB Bioplasma, AG, Berne Switzerland was used at a storage concentration of 30 mg / mL in sterile USP water), and PBS, pH7.1, Gibco BRL. All test articles were diluted to a working concentration of 2 mg / mL in sterile HBSS. At day 0, the animals were randomly assigned to each of the 5 groups, 10 mice per group. Mice were inoculated subcutaneously with 3x10 6 BXPC-3 tumor cells in the flank in a volume of 0.2 mL of PBS or PBS alone (group 1). The therapeutic regimen is detailed in the table . On day 1, animals in group 1 received 0.2 mL of PBS (ip), group 2 received 20 mg / kg of control antibody hlg, and group 3 received 20 mg / kg of antibody CNTO-859. subsequently dosed once every 7 days at 10 mg / kg. PBS was given once every seven days at 0.2 mL. The animals in group 4 received treatment with 20 mg / kg of CNTO 859 dosed i. p. once the tumors reached approximately 50 mm3 to 100 mm3 and then once every 7 days subsequently to 10 mg / kg. The animals in group 5 received treatment with 20 mg / kg of human Ig dosed i. p. when tumors reached approximately 50 mm3 to 100 mm3 in size and then once every 7 days subsequently to 10 mg / kg.

TABLE 5 Animal weights and tumor volumes were monitored once a week until the end of the study. The animals were weighed at the beginning to day 0 while the tumor volumes were recorded only once they were palpable. The tumors were measured in three dimensions using calibrators and the tumor volumes were calculated based on the formula V = (LxWxT) / 2, where L = length, W = width and T = thickness. The termination of the study was planned when the tumors reached an average volume of -2000 mm3, with an option to extend the study as long as the animal's welfare condition was not compromised.At the end, the animals were slaughtered via asphyxia with C02 and the tumors were excised and weighed, then the individual tumors were bisected, with one half rapidly frozen in OCT and the other half fixed in 10% formalin.Serum samples were taken from each animal at term via cardiac puncture. .

Results The tumor growth rate of each treatment group is shown, plotted as tumor volume (mm3) against time (days after implantation) (Figure 12). An average tumor volume of 40 mm3 was reached at day 29 for mice treated with hlg at day 1. However, in mice treated with CNTO 859 the average tumor volume did not reach -40 mm3 until day 36. The treatment of animals on day 1 with CNTO 859 resulted in a 46.9% inhibition of tumor growth (P <0.0001) (see figure 12). The treatment of tumors at approximately 50-100 mm3 resulted in a mild but not significant inhibition of 28% (P = 0.6280) (see figure 13). It should be mentioned that on day 14 of the group treated with CNTO 859, two of seven mice had to be sacrificed 18 days after the tumor implant, due to ulcerative tumors. The final tumor volume of these animals was 129.97"mm3 and 461.10 mm3, respectively In the hlg treatment group at 14 days, eight animals were reduced to seven at day 18. For these reasons, the late treatment arm of the study was finished on day 25.

Summary CNTO 859 inhibited tumor growth rates by up to 46.9%. As far as the inventors know, this is the first time that an anti-human tissue factor antibody has been shown to have the ability to inhibit the tumor growth of a pancreatic adenocarcinoma. Early treatment with CNT0859 resulted in a significant inhibition of tumor growth rates (P <0.0001) relative to the control antibody hlg. Late treatment with CNT0859 inhibited tumor growth rates by 28%, but was not statistically significant (P = 0.6280).

EXAMPLE 4 Inhibition of angiogenesis induced by pancreatic adenocarcinoma PANC-1 in an angiogenesis model in matrigel In this example, the inventors demonstrate the effect of anti-murine tissue factor antibody in the inhibition of pancreatic adenocarcinoma cell-induced angiogenesis of human PANC-1 in an angiogenesis model in matrigel. Antibodies against murine tissue factor were obtained using the directed selection of a phage library of human antibody sequences which were converted to full length mlgG2a antibodies. Both human anti-TF mouse antibodies, designated PHD 126 and PHD 127, inhibit the activity of mouse tissue factor by a mechanism identical to that of CNTO 859 and the analogues. Specifically, PHD 126 and PHD 127 are competitive inhibitors of FX, and inhibit the formation of a tertiary complex formed by TF, factor Vlla and the enzymatically active factor Xa. Both antibodies inhibit the coagulation promoted by murine TF and are selective for murine TF on human TF.

Materials methods. Naked mice (Nu / Nu CD1) from four to six weeks of age were obtained from Charles River Laboratories (Wilmington) and acclimated for 10-14 days prior to experimentation. The mice were housed in groups (7 / cage) in plastic cages with upper filter and were supplied with food and water subjected to autoclaving. A numbered mark for the ear or tattoo was placed at least 7 days before the start of the study, identifying the animals individually. All animal cages were identified with the IACUC protocol number, study number, numbers and treatment of the animal. All animal studies were carried out at the vivarium at Centocor, Inc., Radnor, PA. The human pancreatic adenocarcinoma cell line PANC-1 was obtained from ATCC (American Type Culture Collection, Rockville, MD). It has been shown to be free of viral contamination or by mycoplasma, and was deposited by Centocor's Cell Biology Services. The complete IgG versions of the antibodies were designed and cloned in Centocor: the PHD126 storage solution was 1.7 mg / ml, the PHD storage solution was 0.62 mg / mL, and the control antibody vVaM was 10.09 mg / ML. all antibodies have been evaluated and have an LAL < 4 EU / mg PANC-1 cells were harvested in a logarithmic phase by trypsinization, then washed once in complete medium and once in HBSS. PANC-1 cells (3.2 x 10 7 cells) were resuspended in 8.0 mL of ice cold, sterile HBSS and mixed with 24 mL of ice-cold MATRIGEL (Becton Dickson) (the final concentration was 1x10 6 cells / mL). The final concentration of Matrigel was 10 mg / mL. The mice were divided randomly into five groups (7 mice / group). The mice were anesthetized with Cetamine / xylazine (90/10 mg / kg, i.p.) and weighed. Mice were injected in each of two sites with 0.5 ml of Matrigel with tumor cell suspension (groups 1 to 4).

Animals of group 5 were injected with Matrigel alone. The injection sites were located on the dorsal side approximately 0.5 inches (1.25 cm) in the caudal direction to the last rib and 0.5 inches (1.25 cm) from the spinal column on each side. Placing a finger on the injection site accelerates the polymerization of the Matrigel and prevents any potential loss. Due to the use of anesthesia and the injection of a cold substance, the body temperature was maintained until the animal was conscious again. Under these conditions, angiogenic factors that are released slowly stimulate the process of angiogenesis and the formation of new blood vessels. The invasion of the gel plug by the blood vessels occurs around 12-48 hours and the neovascularization continues for up to 7-10 days after the Matrigel injection.

The animals were injected with 0.2 ce (10 mg / kg) of each test item or 10 mL / kg of control on days 1 and 5 after the Matrigel / tumor implant.

TABLE 6 All animals were weighed on days 1, 5, and 9 (end of study). At the end, Matrigel plugs were excised and weighed. On day 9, all mice were sacrificed by asphyxia with C02. The animals were transported in a closed container (empty cage with micro-isolator or sealed plastic bag) to the oncology laboratory. While they were out of vivarium the animals were handled in a bell for biosecurity. The bodies were returned in a sealed plastic bag to the vivarium freezer when the work was finished. The plugs were surgically removed and weighed blind. The plugs were photographed and then processed for hemoglobin content and length of the blood vessels. If at any time during the study an animal became moribund (> 15% loss of body weight, dyspnea, ataxia, shivering, etc.) the PI could be notified and the animal was sacrificed. The disposition of the body was at the discretion of Pl.

Summary. Following the analysis of blood vessel density the inventors found that PHD 126 inhibited angiogenesis induced by PANC-1 by approximately 60% (not statistically significant), whereas PHD 127 inhibited angiogenesis by approximately 88% relative to the control antibody (p. < 0.05.) PHD 127 inhibited angiogenesis at speeds up to 66% (p <0.05, one way ANOVA) demonstrating that an anti-tissue factor antibody has the ability to inhibit angiogenesis. Treatment with PHD 126 also inhibited angiogenesis in the same model by approximately 45% although the result was not statistically significant (Figure 14). These data show that the proliferating cells produce a host-mediated TF response from the host that leads to angiogenesis and, therefore, to the creation of permissive conditions for tumor growth.

Claims (16)

NOVELTY OF THE INVENTION CLAIMS

1. - The use of a tissue factor antagonist to prepare a medicament for treating a disease in a mammal characterized by an increase in vascularized tissue.

2. The use claimed in claim 1, wherein the disease is selected from the group consisting of cancer, retinopathy, macular degeneration, rheumatoid arthritis and psoriasis.

3. The use of a tissue factor antagonist to prepare a medicament for inhibiting the growth of a tumor in a mammal.

4. The use claimed in claim 3, wherein the tissue factor antagonist is a monoclonal antibody to the tissue factor or a fragment thereof.

5. The use claimed in claim 4, wherein the anti-code fragment is a Fab, Fab ', or F (ab') 2 fragment or derivative thereof.

6. The use claimed in claim 4, wherein the antibody or fragment thereof prevents the formation of the complex of factor tisulapFactor Vlia: Factor X in plasma.

7. The use claimed in claim 4, wherein the monoclonal antibody or fragment competes with the monoclonal antibody TF8-5G9 for binding to human tissue factor.

8. The use claimed in claim 4, wherein the monoclonal antibody is intravenously administrable.

9. The use claimed in claim 4, wherein the monoclonal antibody is administrable in the amount of 0.05 mg / kg to 12.0 mg / kg of body weight.

10. The use claimed in claim 4, wherein the monoclonal antibody is administrable in a bolus dose followed by an infusion of said antibody.

11. The use claimed in claim 1 or 3, wherein the mammal is a human patient.

12. The use claimed in claim 3, wherein the tumor is a breast carcinoma or a pancreatic carcinoma.

13. The use claimed in any of claims 1-12, wherein the antibody is administrable in combination with a second anti-angiogenic agent.

14. The use claimed in claim 13, wherein the second anti-angiogenic agent is a Mab capable of specifically binding to the adhesion molecules containing alphaV.

15. The use claimed in claim 13, wherein the second anti-angiogenic agent is a mAb capable of functionally binding or blocking other agents involved in cellular signaling pathways that lead to angiogenesis.

16. The use claimed in any of claims 3-12, wherein the antibody is administrable in combination with antibody therapy, radiation therapy, a chemotherapeutic agent, a proteosome inhibitor, or a farnesyl transferase agent.